Absence of functional GABAA receptors in transit-amplifying stem cells of the early postnatal subventricular zone

Cesetti T¹, Obernier K¹, Bengtson P¹, Mandl C¹, Holzl-Wenig G¹, Horsch K², Eckstein V², and Piccolini F¹

¹Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 364, 69120Heidelberg, Germany, ²Department of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany

In the subventricular zone (SVZ) neurogenesis is regulated by the coordinated proliferation and differentiation of different precursor types: slowly proliferating type B cells, rapidly dividing transit-amplifying type C cells and type A neuroblasts. Previous evidence suggests that g-aminobutyric acid (GABA) released by neuroblasts provides a feedback signal inhibiting the proliferation of type A and B cells by activation of GABAA receptors, while the effects of GABA on type C cells are unknown. Here we used levels of EGFR expression and lineage specific markers to purify stem cells and neuroblasts from the postnatal SVZ. Clonal assays and analysis of mice lacking expression of orphan receptor Tlx antigen showed that cells expressing high levels of EGFR (EGFRhigh) directly correlated with stem cell activity in vitro and in vivo. Analysis of antigen expression and BrdU incorporation in vivo revealed that more than 80% of the isolated cells represented putative type C cells. Although isolated EGFRhigh cells and neuroblasts were both PSANCAM positive, they displayed profound functional differences. Neuroblasts but not stem cells expressed GABA and Doublecortin and showed a fast intracellular Ca2+ increase upon acute membrane depolarization or application of GABA. Electrophysiological measurements also revealed different passive membrane properties and resting potentials in the two cell populations. Neuroblasts and stem cells both showed TEA-sensitive outwardly rectifying K+ currents, but at different current densities. Strikingly, only neuroblasts expressed voltage-activated Ca2+ channels and displayed GABAA receptors mediated Cl- currents. Furthermore, selective activation of GABAA and GABAB receptors did not affect stem cells clone forming capability nor their in vitro proliferation rate. Thus, transit-amplifying stem cells derived from the early postnatal SVZ do not express functional GABAA receptors and GABA does not directly modulate their proliferation.

(JSRM Code: 002010700016)

Developmental Potential of Human Mesenchymal Stem Cells derived from Bone Marrow, Cord Blood and Fat Tissue

Brousos N, Bieback K, Lannert H, Muller AM
Institute for Medical Radiation and Cell Research, University of Wurzburg, Versbacherstr.5, 97078
Wurzburg, Germany

 Introduction: Mesenchymal stem cells (MSC) are multipotent adult stem cells. They can be isolated from bone marrow (BM), fat tissue, cord blood (CB) and other tissues. Although these cells gain importance as potential sources for stem cell-based therapies, the developmental potential of MSCs from different origins and from different culture conditions are not well characterised. Therefore the BMBF-funded START-MSC (Standardization for Regenerative Therapy - Mesenchymal Stem Cell) consortium was established to develop standardised procedures for cell preparation and cell cultivation. Furthermore START-MSC aims to characterise the developmental potentials, marker profiles, proteins and signal transductions pathways in MSCs from different origins. Our aim in this consortium is the analysis of the in vivo developmental potential of MSCs by blastocyst injection.

Material and Methods: Fat-MSC and CB-MSC were isolated by plastic adherence, BM-derived MSCs were FACS-sorted for the marker CD271. CD271 is considered a potential marker for BM-MSCs. To analyse the developmental potential of human MSCs (hMSC) from different origins (BM CD271+, CB, fat), we injected 5-10 hMSC of each type into murine embryonic day (E) 3.5 blastocysts and analysed the progeny of injected hMSCs in different tissues on embryonic day E16.5 by human-specific real-time PCR.

Results: We were able to detect progeny of injected hMSCs in several embryonic tissues of E16.5 embryos. Donor positive tissues were detected after injection of all three hMSC types. Positive tissues showed low-level donor cell engraftment (between 2-20 human cells per 10.000 murine cells). Further we observe that CD271+ BM cells generated less frequent engraftment compared to hMSC derived from CB or fat tissue. The quantification of donor signals by real time PCR indicated that the injected cells underwent proliferation.

Conclusion and Discussion: All three MSC types that were analysed showed engraftment in developing murine embryos. So far we could not detect enhanced engraftment of distinct tissues. The injection of pre-selected CD271+ BM cells shows the least frequent donor positive tissues. To further address the question whether donor cells are functionally integrated into recipient tissues we are planning combined immunohistochemistry and in situ hybridisation analyses to characterize the phenotype of donor derived cells.

(JSRM Code: 002010700017)

Spermatogonial stem cells from adult human testis and the
generation of pluripotency in vitro and in vivo

Conrad S1, Renninger M2, Hennenlotter J2, Just L1, Wiesner T1, Sievert KD2, Stenzl A2, and Skutella T1,3
1Institute of Anatomy, Department of Experimental Embryology, Section of Tissue Engineering, Tubingen, Germany
2Department of Urology, University Clinic Tubingen, Germany
3Centre for Regenerative Biology and Medicine (ZRM), Tubingen, Germany

Objectives: Recent literature reported the isolation of spermatogonial stem cells from adult mouse testis and their response to differentiating culture conditions in a pluripotent manner similar to embryonic stem cells. Aim of the present study was to show the possibility of isolation and differentiation of adult spermtogonial stem cells from human testicular parenchyma into the different tissues of all of the three human germ layers.

Materials and Methods: Regular testicular parenchymas from orchiectomy specimens, determined by a pathologist, were collected. Cells were cultured in knockout culture medium with GDNF. After 7-10 days the medium was changed to basic medium with LIF. Clusters formed which contain the spermatogonial stem cells and for higher purification a MACS seperation with CD49f followed. The isolated cells were transposed into specific established culture mediums and protocols for neuronal, pancreatic, osteogenic and myogenic differentiation of embryonic stem cells. The precursor cells were transplanted in utero and adult nude mice for testing the potency of the cells.

Results: Adult spermatogonial stem cells could spontaneously differentiate into derivatives of all three primary germ layers: differentiation towards insulin producing cells, osteogenic cells, smooth muscle cells, and neuronal live cells was confirmed by PCR analysis, ELISA and immunohistochemistry. Transplanted cells showed characteristics of neural and pancreatic tissues.

Conclusions: Adult spermatogonial stem cells seem to keep their pluripotency and plasticity throughout life. Human stem cells from testicular tissue may allow individual cell based therapy without the ethical problems associated with human embryonic stem cells and immunological problems of nowadays transplantation of allografts.

(JSRM Code:002010700018)

Jak/Stat signalling regulates dpp transcription to control germline stem cell maintenance in the Drosophila ovary

Lopez-Onieva L, Fernandez-Minan A, and Gonzalez-Reyes A
Centro Andaluz de Biologia del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide, 41013 Sevilla, Spain

The existence of specialised regulatory microenvironments or niches that sustain stable stem cell populations is well documented in many tissues. However, the specific mechanisms by which niche support cells govern stem cell maintenance remain largely unknown. We have show that the Jak/Stat signalling pathway acts in somatic, support cells of the Drosophila ovary to maintain the adjacent germline stem cells (GSCs) in an undifferentiated state. In addition, we have demonstrated that removal of this pathway in support cells leads to stem cell loss by differentiation. Further, the ectopic activation of Jak/Stat signalling in support cells augments dpp mRNA levels and increases the range of Dpp signalling, a BMP2 homologue known to act as a niche extrinsic factor required for GSC survival and division in Drosophila. As a consequence, the GSC niche is expanded and tumours of stem cells are produced. Our results provide strong evidence for a model in which Jak/Stat signalling in support cells regulates dpp transcription and thus niche size.

(JSRM Code:002010700019)

Fibulin-1 is an adhesion-modulating component of the hematopoietic stem cell niche

Schreiber TD1, Hergeth SP1,4, Aicher WK2, Essl M1, Sasaki T3,5, Klein G1
1 Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tubingen, Germany
2 Center for Medical Research, Department of Orthopedic Surgery, University of Tubingen, Germany
3 Max-Planck-Institute for Biochemistry, Martinsried, Germany
4 present address: Spemann Laboratories, Max Planck Institute of Immunobiology, Freiburg, Germany
5 present address: Department of Biochemistry, Oregon Health and Science University, Portland, USA

In the bone marrow stem cell niche, osteoblasts lining the endosteum are of major importance in supporting hematopoietic stem cell maintenance and controlling self-renewal. Here, we report on the expression of the fibulins, highly conserved calcium-binding glycoproteins that are components of the extracellular matrix of human osteoblasts. We also provide insights into their functional relevance in the stem cell niche. All six members of the fibulin family were detected by RT-PCR. The expression of fibulin-1 and -2 was verified by Western blot analysis. Two-color immunofluorescence staining revealed a co-localization of both fibulins with fibronectin in an extracellular meshwork. Fibulin-2, but not fibulin-1, appeared to be instantly degraded by active metalloproteases present in osteoblast cultures. Fibulin-2 was shown to be a substrate for MMP-2, which was constitutively secreted by primary osteoblasts, as assessed by gelatin zymography. Whereas fibulin-2 exhibited cell binding properties towards the hematopoietic progenitor cell line KG1a, fibulin-1 was capable of modulating adhesion to fibronectin, resulting in the disturbance of fibronectin-induced cell signaling pathways, as analyzed with an anti-phosphotyrosine antibody. Thus, fibulins seem to be important components of the extracellular matrix of osteoblasts, which are likely to influence the adhesive properties of the stem cell niche towards hematopoietic stem cells.

(JSRM Code:002010700020)

Reproducible and complete early separation of stem and feeder cells does not attenuate the differentiation potential of feeder-freed stem cells

Schafer U, Schneider A, Spitkovsky D, Neugebauer E, Hescheler J
Institute for Research in Operative Medicine, University Witten/Herdecke, Germany
Inst. for Neurophysiology, University of Cologne, Germany

Introduction: Mouse or human embryonic stem (mES/hES) cell lines are usually derived and propagated on inactivated feeder cell layers. Human feeder cell layers such as foetal human fibroblasts or post-natal human skin fibroblasts have lately been used in order to eliminate the risk of foreign protein contamination during hES propagation. However, it is impossible to conclusively evaluate the beneficial or detrimental effects of feeder cell layers on propagation and subsequent differentiation of stem cells without the possibility to dissociate feeder and stem cells. Furthermore, feeder cells themselves might have a detrimental effect in clinical application, since feeder cells might be carried along during in vitro differentiation of stem cells. We have therefore developed a methodology that allows for the mechanical separation of stem cell from colonies grown on feeder cell layers and the subsequent differentiation of feeder-freed colonies and cells.

Materials and Methods: Stem cells (D3) were grown on neomycin resistant (nr) feeder cell layers derived from mouse embryos for 5 to 8 days. Stem cells from single colonies were harvested by using an automated cell selection system. Stem cells were aspirated and immediately transferred to a non-adhesive 96 well RT-PCR plates. Aspiration of stem cells but not feeder cells was achieved by carefully pre-defining aspiration pressure. Feeder cell contamination was detected by nested RT-PCR of the nr gene expressed in feeder cells only. Differentiation of feeder-freed stem cells was analysed by marker expression analysis (RT-PCR)

Results: The sensitivity of the nested RT-PCR approach was demonstrated by amplifying nr mRNA from single feeder cells. Feeder cell contamination in aspirated stem cells was not observed, even when stem cells were successively harvested from the same colony. Propagation and differentiation of stem cells under standard conditions resulted in feeder cell contaminations that were still detectable up to 20 days following initiation of differentiation, indicating the possibility of feeder cell contamination in subsequent stem cell based cell-replacement therapies. In a second step the differentiation potential of colony derived feeder-freed stem cells was examined. Embroid body formation of aspirated cells single colonies was allowed for 4-5 days in non-adhesive 96 well RT-PCR plates. >95% of cells or single colonies aspirated developed into single embroid bodies. Embroid body morphology closely resembled the spherical morphology of embroid bodies developed under standard conditions. Subsequently feeder-freed embroid bodies derived from defined single colonies were differentiated into cardiomyocytes or neuronal cells by standard protocols. Early differentiation events were detected by expression analysis of gata6 (endoderm), fgf5 (primitive ectoderm), brachyury (mesoderm). Differentiation into cardiomyocytes was evaluated through analysis of GATA-4, cardiac Troponin C, alpha-MHC expression. Neuronal differentiation was verified by RT-PCR analysis of nestin, TH, GFAP, NSE and neurofilament. The time dependent pattern of expression of stem cell differentiation in neuronal cells or cardiomyocytes was comparable and reproducible between stem cells differentiated under standard conditions and feeder-freed stem cells differentiated from single embroid bodies in PCR tubes.

Discussion and Conclusion: We have therefore developed a method that for the first time allows the complete and reproducible separation from stem and feeder cells at an early time point and the analysis of distinct stem cell colonies. Complete separation from stem and feeder cells is essential in regenerative cell replacement therapies. Separation of stem and feeder cells is also an important prerequisite in the unambiguous analysis of differentiation mechanism.

(JSRM Code: 002010700021)

GMP-Compliant Manufacturing of Autologous Stem and Progenitor Cells For Tissue Repair

Prante C1, Gastens MH1, Prohaska W1, Stratmann B2, Lammers D2, Kirana S2, Tschopev D2, Kleesiek K1
1Institut fur Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitatsklinik der Ruhr-Universitat Bochum, 32545 Bad Oeynhausen
2Diabeteszentrum, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitatsklinik der Ruhr-Universitat Bochum, 32545 Bad Oeynhausen

Many regenerative medicine research efforts focus on the treatment of severe medical conditions with cells derived from the patient’s own bone marrow (BM). The effectiveness of these autologous BM cell therapies is often limited by the number of stem and progenitor cells in the final product that contain tissue regenerating potential. One strategy to address the issue of stem and progenitor cell number is to culture the patient’s BM cells, prior to implantation, in a standardized and reproducible cell manufacturing process. Here we describe a closed, automated Good Manufacturing Practice (GMP)-compliant process that begins with a small bone marrow aspirate from the patient and through a series of process steps generates a patient-specific dose of Tissue Repair Cells (TRCs) that contains a higher number and higher percentage of stem and progenitor cells than the original BM.

Bone marrow mononuclear cells (MNCs) from a small sample of the patient’s BM (30-50 ml) are isolated by Ficoll gradient centrifugation using an automated device to deplete the erythrocyte and granulocyte components of the BM. The resulting MNCs are then inoculated into a cell culture cassette and cultivated for 12 days in a completely closed automated cell manufacturing system that provides a completely computer-controlled culture environment through the regulation of temperature and gas and media exchange. This cell environmental control together with the single-pass perfusion technology, enables the replication of early-stage stem and progenitor cells while preventing their differentiation into mature cells. The resulting cell product shows significantly increased populations of stem and progenitor cells and decreased populations of mature cells. CD90+ mesenchymal stem cells, represent a key cell population that undergoes significant expansion under this perfusion culture and has been observed to increase up to 48-fold in cell number and up to 200-fold in CFU-F forming capability during the manufacturing process. The stem cell content of the product has been proved through the ability of specific sub-populations to differentiate into adipocytes, osteoblasts or cells with endothelial characteristics.

(JSRM Code: 002010700022)

Megadoses of autologous Tissue Repair Cells (TRC) in the treatment of Ischemia induced chronic issue ulcers of diabetic foot patients without option of revascularisation ? preliminary results from Bad Oeynhausen

Stratmann B
Diabetes Center at the Heart- and Diabetes Center NRW, Ruhr University Bochum
Georgstr. 11, D-32545 Bad Oeynhausen

Diabetic foot syndrome (DFS) appears as a concomitant illness of diabetes mellitus. Diabetes is one of the main causes of non-traumatic amputation in Germany due to severe peripheral arterial occlusive disease (PAOD) with chronic critical limb ischemia (CLI) being the most abundant problem. Ulceration occurs as failure of the microcirculation and progressive occlusion of the larger blood vessels exacerbates pre-existing microvascular abnormalities.

For the first time megadoses of pluripotent stem and progenitor cells - Tissue Repair Cells (TRCs) - are used in the treatment of diabetic ulcers to induce revascularisation of the affected limb and to enhance local perfusion. As a concomitant result wound healing, which is highly dependent on perfusion, should occur and lead to preservation of the limb.

DFS patients with chronic limb ischemia and without the option of surgical or interventional revascularisation are being recruited and randomized to either the transplant group or the control group within this clinical trial. Approximately 40 ml of bone marrow is harvested from the iliac crest, followed by preparation and culture of mononuclear cells. Application sites of the enriched mesenchymal bone marrow cells (BMCs) or Tissue Repair Cells (TRCs) are intramuscular (i.m.) into the gastrocnemius of the affected limb or intra-arterial (i.a.) into the femoral artery.

Of the 26 patients have been enrolled in the study. 10 patients have completed the 12 month follow-up, 2 patients were immediately dropped from the study due to low bone marrow quality, 1 patient from the control group and 1 patient from the BMC i.a. left the study to receive major amputations, 1 patient withdrew his consent and the other 11 patients are still in the follow-up period.

To measure the therapeutic effects of the various treatment arms patients were evaluated for ABI, TcpO2, and reactive hyperemia (Laser Doppler and BOLD). Patients also underwent imaging with angiographic methods..All patients treated with BMCs or TRCs, independent of application method, showed tendential improvement of microcirculation (reactive hyperemia) in the affected foot as well as complete primary wound healing. No serious adverse events related to the treatment were noted. In addition, no calcification of soft tissue has been observed.

The transplantation of expanded autologous bone marrow stem cells as well as mesenchymal bone marrow cells in diabetic patients with critical limb ischemia induced chronic tissue ulcers proved to be safe and shows tendential improvement of microcirculation and complete wound healing.

(JSRM Code: 002010700023)

Stem cell-based therapy for the treatment of femoral head necrosis - first clinical experience

Noth U
Orthopedic Clinic, Konig-Ludwig-Haus, University of Wurzburg,
Brettreichstr. 11, 97074 Wurzburg, Germany

Osteonecrosis of the femoral head involves the death of cells in trabecular bone and marrow, fracture of subchondral bone, and often leads to total hip replacement. Retrospective clinical studies have shown that osteonecrosis in 80-90% of affected patients progresses to destroy the femoral head, usually within 2-3 years after diagnosis. There are no effective treatment options for terminating or reversing the disease process.

Reports using autologous bone marrow tissue injected percutaneously into the necrotic area have shown a high rate of success in early stage osteonecrosis. As a more standardized alternative to fresh bone marrow, Aastrom Inc. has developed a proprietary automated process to expand autologous bone marrow cells ex vivo. Bone Repair Cells (BRCs) are a mixture of stem and early progenitor cells including cells of hematopoietic, mesenchymal, and endothelial lineages derived from a small sample of the patient’s own bone marrow. Clinically, the cells have been used to treat over 50 patients with BRCs formulated with osteoconductive matrices. Interim data reported on the first 20 patients who completed the one year follow-up of a now completed 36 patient Phase I/II non-union fracture trial demonstrated excellent safety profile, and >90% callus formation and bone bridging in no-option patients.

A new procedure has been developed to deliver BRC/matrix compositions into the femoral head to treat osteonecrosis. This abstract outlines the clinical formulations, the surgical procedure, safety profiles and clinical outcome of the product in 3 compassionate use patients with necrosis of the femoral head.

(JSRM Code:002010700024)

From unmanipulated bone marrow to custom tailored stem cell grafts ?
novel therapies from bench to bedside

Eyrich M, Wolfl M, Winkler B, Tscherner H, Vahsel B and Schlegel PG
Stem Cell Transplantation Centre, Pediatric Stem Cell Program, University of Wuerzburg, Germany

Transplantation of allogeneic stem cells of bone marrow or peripheral blood origin has proven to be the only curative therapy for a variety of malignant as well as non-malignant diseases. The authors describe recent advances in the field of stem cell selection techniques that have enabled the use of selected cell components for differential clinical applications under GMP conditions. The reconstitution of a functional immune system over time has been carefully monitored in several pediatric cohorts following transplantation of positively selected stem cells by V beta spectratyping of the emerging T cell repertoire as well as by TREC analysis.

(JSRM Code:002010700025)

New avenues of hematopoietic stem cell transplantation

Stuhler G and Einsele H
Stem Cell Transplantation Centre, Medical Clinic II, University of Wuerzburg, Germany 

We here report mayor scientific contributions that have rapidly revolutionized today’s haematopoietic stem cell transplantation strategies: Bone marrow, stimulated apheresis product or umbilical cord blood stem cells- which graft works best in the adult transplant setting? Does HLA disparity really limit transplantation success? CD34 selection, CD3/19 depletion and T cell therapies - how to puzzle the perfect cell graft. The stem cell recipient of increasing age: What is the appropriate conditioning chemotherapy with little toxicity?

(JSRM Code:002010700026)

Neuronal hypoxia: protective effects of mononuclear cord blood cells after direct and indirect application

Reich DM1, Hau S1, Scholz M3, Emmrich F1; 2, Kamprad M2, Boltze J1
1Fraunhofer-Institute of Cell Therapy and Immunology
2Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig
3Institute of Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig

Pathophysiological models developed from animal studies form the basis of our understanding of the development of stroke. In vivo data display a perfusion-related dependency of neuronal cell damage. Nearly total loss of cerebral blood perfusion leads to necrotic cell death in the ischemic core. Residual energy supply in the surrounding penumbra induces apoptosis whose early phases are reversible. Consequently, rescue of the penumbra is the basis of stroke therapy. In vivo administration of external cell fractions recently demonstrated clear therapeutic benefits. However, the complexity of in vivo models has hindered understanding of the mechanisms involved in functional recovery so far.

Based on fully matured neuroblastoma cells (SH-SY5Y) we established a unique in vitro model of neuronal hypoxia that affords the possibility of investigating apoptotic neuronal cell death and neuroprotective cell therapies.

We employed the post hypoxic cell cultures for direct co-culturing with various fractions of stem cell containing mononuclear cells (MNC) from human umbilical cord blood (HUCB). Over the course of 3 days, all applicated MNC-fractions provided significant protection from neuronal apoptosis and also triggered the retaining of neuronal characteristics such as forming networks.
Clear neuroprotection was also detectable when MNC were affixed in impermeable cell-culture-inserts. In direct as well as in indirect co-cultures MNC induced an alteration in cytokine and chemokine concentrations. Our data suggest that the neuroprotective effects of MNC might result from direct cell-cell contacts and/or the adjustment of specific soluble mediators.

(JSRM Code:002010700027)

Alloreactive T-cell trafficking after hematopoietic stem cell transplantation

Beilhack A1,3*, Schulz S2,3, Baker J3, Beilhack GF3, Herman EI3, Baker EM3, Landan G3,Nishimura R3, Butcher EC4, Contag CH5 and Negrin RS3.
1Departments of Medicine II, Wurzburg University, Germany
2Department of Pathology, Technical University Munchen, Germany
Departments of 3Medicine, 4Pathology and 5Pediatrics, Stanford University, Stanford, California, USA

Acute graft-versus-host disease (aGVHD) is the major limitation for a broader application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In aGVHD alloreactive donor T-cells attack the recipient’s gastrointestinal tract, liver and skin. To address this unusual tissue tropism we developed luciferase transgenic (luc+) mice and utilized bioluminescence imaging to track adoptively transferred luc+ T-cells non-invasively in living recipients. Either myeloablative conditioned BALB/c (H-2d, 8Gy) or C57Bl/6 (H-2b, 9Gy) recipient mice were transplanted with allogeneic luc+ T-cells (FVB/N, H-2q) and FVB/N wild type bone marrow. We observed that T-cell proliferation was confined to secondary lymphoid organs until day+3 after allo-HSCT. At this time alloreactive T-cells up-regulated specific homing receptors and subsequently migrated via the blood to aGVHD target tissues. When we blocked T-cell entry to specific lymphoid organs we found a high redundancy of these priming sites. However, by preventing T-cell entry to all secondary lymphoid organs aGVHD was completely averted. In subsequent experiments we isolated in vivo primed alloreactive luc+ T-cells from mesenteric lymph nodes, peripheral lymph nodes or the spleen and transferred these cells into conditioned secondary transplantation recipients. Luc+ T-cells attacked aGVHD target tissues irrespective of the original priming site. In contrast, after secondary transfer into non-conditioned recipients luc+ T-cells preferentially homed to lymphoid organs. These data suggests that not the lymphoid priming sites but instead signals from the aGVHD-target tissues dictate the distinct tissue tropism in aGVHD.
A.B. and St. S. contributed equally to this study.

(JSRM Code:002010700028)

Nucleolin activates expression of CD34 and Bcl-2in CD34-positive hematopoietic cells

Grinstein E, Santourlidis S, Fischer J, Uhrberg M, Wernet P.
Institute of Transplantation Diagnostics and Cellular Therapeutics, Heinrich Heine University Medical Center, 40225 Duesseldorf, Germany
Correspondence: edgar.grinstein@web.de 

Introduction: CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation and reconstitution. Nucleolin is an abundant factor of growing and cancerous cells, involved in regulation of gene transcription and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we investigated the involvement of nucleolin in gene regulation in human CD34-positive hematopoietic cells.

Materials and Methods: Interactions of nucleolin with CD34 promoter region were detected by EMSA and chromatin immunoprecipitation, and activation of the CD34 promoter region - by co-transfections of a nucleolin cDNA expression vector together with CD34 promoter reporter constructs. Levels of gene expression were monitored by immunoblotting, real-time RT-PCR and FACS. Human bone marrow-derived CD34-positive cells KG1, stably transfected with a nucleolin cDNA expression vector, were employed. CD34-positive cells from apheresis collections were enriched using CD34 MicroBeads.

Results: It is shown that, in human CD34-positive hematopoietic cells, nucleolin increases the abundance of the CD34 and Bcl-2 transcripts and proteins, and cell surface CD34 protein expression is enhanced by nucleolin. Nucleolin-mediated activation of the CD34 gene transcription results from sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative PB mononuclear cells. Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region is detected.

Discussion and Conclusions: Our study reveals a novel role of nucleolin for gene regulation in CD34-positive hematopoietic cells and likely for the maintenance of these cells. We report that nucleolin is a crucial activator of the CD34 gene transcription. We suggest that nucleolin may be involved in controlling basic functions of CD34-positive hematopoietic cells, and its deregulation may contribute to the development of acute myelogenous leukemias.

(JSRM Code:002010700029)

Differentiation potential and ectopic cartilage formation capacity of human mesenchymal stem cells derived from bone marrow, adipose tissue and synovium

Dickhut A, Pelttari K, Bischel O, Richter W
Division of Experimental Orthopaedics, Orthopaedic Clinic, University of Heidelberg, Germany

Introduction: Mesenchymal stem cells (MSC) are suitable candidates for the cell-based reconstruction of different tissues including cartilage and have been isolated from different sources such as synovium, adipose tissue and bone marrow. These cells were assumed to be similar with regard to self-renewal, multidifferentiation potential and surface epitopes. For the clinical application of MSCs for articular cartilage repair, functional suitability and phenotypic stability are crucial factors. In this study, we compared the in vitro differentiation capacity of MSC derived from bone marrow, adipose tissue and synovium and their ectopic cartilage formation capacity.

Materials and Methods: MSC were isolated from adipose tissue (ATSC), bone marrow (BMSC) and synovium (SMSC) and were cultured under chondrogenic, osteogenic and adipogenic conditions. Differentiation was evaluated based on histochemical staining, immunohistology and gene expression analysis. After 5 weeks of chondrogenesis, MSC were transplanted subcutaneously into SCID mice. Four weeks later, explants were analysed by histology.

Results: ATSC and SMSC showed reduced chondrogenic differentiation potential compared to BMSC and required combined application of TGF-beta 3 plus BMP-6 for successful chondrogenic differentiation. Chondrogenesis was always associated with up-regulation of hypertrophic markers like Col X. In vivo, explants derived from ATSC and BMSC remained positive for proteoglycans and Col II and underwent calcification. SMSC-derived explants showed a more heterogenic differentiation pattern: some explants were positive for proteoglycans and Col II, while others were positive for proteoglycans but negative for Col II. Remarkably, explants derived from two of five SMSC populations lost the Col II revealed at transplantation time, while proteoglycans persisted. These Col II negative SMSC transplants were negative for calcification.

Discussion and Conclusions: Our findings indicate that adipose tissue, bone marrow and synovium provide heterogenous MSC populations. SMSC, like ATSC, required more stringent conditions for chondrogenesis when compared with BMSC. None of the in vitro differentiated ATSC and BMSC populations were able to form stable ectopic cartilage with resistance to vascular invasion and calcification. Transplants underwent alterations related to endochondral ossification rather than adopting a chondrogenic phenotype typical for articular cartilage. Since calcification was confined to Col II positive areas in SMCS explants, the persistent presence of Col II may be relevant for mineralization.

(JSRM Code:002010700030)

An effective Model for the Expansion of human Mesenchymal Stem Cells

Lindner U, Boehrnsen F, Driller B, Kramer J, Rohwedel J, Schlenke P
Institute of Immunology and Transfusion Medicine, University of Luebeck, Ratzeburger Allee 160, 23558 Luebeck, Germany 

Bone-marrow derived human mesenchymal stem cells (hMSCs) will probably be a valuable tool for regenerative medicine in the future. In-vitro cultured hMSCs show restricted proliferation and differentiation capability. In this study we demonstrate, that age of donors is a main limiting factor for in-vitro expansion and differentiation of hMSCs, but extracellular matrix (ECM) proteins (ECM-gel, Laminin-1, -5, -S, collagen IV, fibronectin), hypoxic conditions (1% and 5% O2), the use of human serum instead of fetal calf serum (FCS), and CD 271+ selection have positive effects on growth rates and differentiation capacity.

Cells were analyzed via histochemical and immunohistochemical staining procedures, telomerase activity detection, different flow cytometry protocols (incl. STRO-1, Oct-3/4 and Alkaline Phosphatase expression), quantitative photometrical and fluorometrical assays, and RT-PCR.

HMSCs, selected from young donors (age 21-38), had significant higher proliferation and differentiation potential, compared to a group of elder (44-89) donators. Basement-membrane ECM proteins lead to a 103 fold higher in-vitro expansion and each protein had defined ascending effects on the process of differentiation; these effects were inhibited by antibodies against receptors of the particular protein. Hypoxic conditions, human serum, and CD 271 positive selection had also direct, significant effects on proliferation and the maintenance of differentiation capacity, and the combination of mentioned factors yielded again in a significant higher cumulation for the contemplated attributes. Cultivated samples were investigated for proliferation behaviour. The subunit of so-called RS-cells (flow-cytometry: light forward and side scatter population; small cells: 7-13 μm diameter) was exclusively responsible for growth of hMSCs cultures and showed better differentiation features. Each detected subpopulation originated from RS cell subunit. At least this in-vitro model was used for the expansion of peripherial blood extracted circulating cells after G-CSF stimulation, and CD 133 positive selection and/or Interleukin-6 (IL-6) treatment. Selected cells showed flow cytometric characteristics of hMSCs and could successfully cultivated, and differentiated into osteoblasts, chondrocytes and adipocytes. Without positive selection or IL-6 usage no cells with hMSCs properties were detected.

(JSRM Code:002010700031)

Integration and tissue specific differentiation of fetal cells into maternal organs in a murine pregnancy model suggests multilineage differentiation potential

Zeng XX, Tan S, Neo BH, Sasajala P, Low D, Udolph G

Institute of Medical Biology, 61 Biopolis Drive, 138673 Singapore, Singapore

Cell trafficking between fetus and mother during pregnancy resulting in a situation of maternal microchimerism is a widespread phenomenon in mammalians including humans. We have investigated the trafficking of pregnancy associated progenitor cells (PAPC) to mother’s brain in a murine pregnancy model. PAPCs can be detected first in mother’s at delivery (P0) and up to 5 month post-partum, the longest time point examined, indicating long-term engraftment potential. Cells are found in different brain areas such as the cortex, striatum, olfactory bulb and hippocampus. We find cells predominantly in the CA1 area and at lower frequencies in the CA2/3 area and the dentate gyrus of the hippocampus. Strikingly, the frequency of PAPCs increases and the cell distribution is changed in Hippocampi of animals with experimental Parkinsonism demonstrating that PAPCs respond to changing homing cues.

In the brain, PAPCs coexpress mature neuronal specific markers indicating neuronal differentiation potential. In contrast glial cell markers are not coexpressed. Some PAPCs were found to express stem/progenitor or immature neuronal markers such as nestin, doublecortin, and PS-NCAM. Further evidence for neuronal maturation comes from an increasing axonal/dendritic complexity over time. PAPC integration into the hippocampus is organotypical with neurons developing axonal/dendritic polarity which is indistinguishable from endogenous hippocampal neurons. In summary, PAPCs migrate to the maternal brain where they integrate and differentiate predominantly towards the neuronal linage. PAPCs undergo a process of maturation and express stem/progenitor or immature neuronal cell specific markers when they are phenotypically immature. Mature neuronal markers are coexpressed when cells are phenotypically more mature. This might indicate that integration and maturation of PAPCs into the brain follows mechanisms similar to that observed during natural adult neurogenesis.

Furthermore, we find PAPC in maternal organs such as pancreas, small intestine as well as kidney where, like in the brain, cells integrate organotypically. In the pancreas we found evidence for the coexpression of exocrine lineage markers which suggests pancreatic differentiation of PACs into acinar cells. We conclude that PAPCs as a population demonstrate multipotent lineage differentiation potential, a feature typical for stem cells. The fetal cell transfer model mimics an autologous transplantation paradigm and as such is an invaluable tool to study the integration, survival, differentiation, maturation and functionality of stem cells and their derivatives in different host tissues.

(JSRM Code:002010700032)

T-cells in stem cell transplants: dissecting the good, the bad and the ugly

Wolfl M1,2, Eyrich M2, Schlegel PG2, and Greenberg PD1
1Fred Hutchinson Cancer Research Center, Seattle, USA
2Pediatric Stem Cell Program, Children’s University Hospital, Wurzburg, Germany

Allogeneic stem cell transplantation for patients with hematologic malignancies has proofed to be one of the most powerful forms of immunotherapy. Studies comparing T-cell depleted versus undepleted grafts convincingly demonstrated the role of T-cells in the graft-versus-leukaemia effect at the cost of increased graft-versus-host-disease. It has been an elusive goal of research to prevent GVHD without dramatically increasing the chance of relapse or major transplant complications. New insights in the regulatory mechanisms and dynamics of various T-cell subsets now open avenues for dissecting the T-cell response adapted to the patient’s requirements: 1) supportive antigen-specific T-cell therapy provides protection against viral and fungal antigens; 2) regulatory T-cells may be used to modulate GVHD; 3) T-cells against minor histocompatibility antigens and leukaemia-associated antigens can provide targeted immunotherapy with a reduced risk of increased GVHD. We have developed methods to prime and expand antigen-specific cells from the naive T-cell repertoire and to specifically select for these T-cells on the basis of their CD137 expression upon stimulation. In consequence various epitopes of the leukaemia-associated transcription factor Wilms Tumor antigen 1 have been described that will be of further use for leukaemia-specific T-cell therapy. Furthermore the use of novel cytokine combinations in vitro results in a T-cell phenotype with a favourable CD28 expression, which increases the chances that adoptively transferred T-cells persist in vivo.

(JSRM Code:002010700033)

Human embryonic stem cells elicit an immune regulatory phenotype by upregulating CTLA-4 and IL-10 expression in T cells

Ajjikuttira P a, Wong SC b, Penzkofer S a*, Ying JY a and Kadereit Sa*
a Institute of Bioengineering and Nanotechnology
b Center for Molecular Medicine, Singapore
* University Konstanz, Germany

Introduction: Human embryonic stem cells (hESC) hold great promise for regenerative medicine. Presumably, however, they would be rejected by the immune system of the recipient, as hESC would be transplanted into an allogeneic setting. Surprisingly, previous reports had shown reduced immune reactivity against hESC, with reduced proliferation and reduced cytotoxicity. Underlying cellular immune mechanisms are however not well defined. Interestingly, the inhibitory effect was shown to be soluble factor-independent, as fixation of hESC did not abrogate this inhibitory effect.

Materials and Methods: We used well established in vitro human co-culture systems to investigate mechanisms underlying the immune-modulatory effect of hESC. Fixed hESC were incubated with human peripheral blood mononuclear cells (PBMC) stimulated with allo- and xenoantigens, as well as with strong T cell receptor (TCR) stimulation in presence of co-stimulation. The latter stimulation provides a strong antigen-independent activation of T cells. We assessed proliferation, apoptosis, presence of regulatory T cells and expression of immune modulators CTLA-4 and IL-10.

Results: We found that the presence of hESC could reduce specifically T-cell proliferation against allogeneic and xenogeneic antigens, as well as against the much stronger antibody-mediated TCR activation. Reduced T-cell proliferation in presence of hESC was neither due to an increase in apoptosis, nor to an increase in FoxP3+ regulatory T cells. Rather, we showed that T cell stimulation in the presence of hESC resulted in an increase in the proportion of surface CTLA-4 expressing T cell. CTLA-4 is a major inhibitor of T cell proliferation. Concomitantly, we also found an increase in IL-10 secreting T cells. IL-10, a major regulator of inflammation, inhibits T-cell proliferation and plays a role in tolerance induction after hematopoietic stem cell transplantation. Moreover, IL-10 has also been shown to be an effector of CTLA-4-mediated immune regulation.

Conclusion: Our results thus strongly suggest that the reducing effect of hESC on T-cell proliferation is mediated through upregulation of these two potent immune-regulatory molecules. Moreover, this effect was mediated by membrane-associated molecules on the hESC, as only fixed cells were used. It will be now interesting to characterize such molecules and to specifically address whether their presence would be maintained throughout terminal differentiation of hESC, as for transplantation no undifferentiated hESC would be transplanted.

(JSRM Code:002010700034)

The stem state: the nature of normal stem cells and the relevance to cancer

Zipori D
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

Stem cells are commonly defined as being undifferentiated and capable of self-renewal. Although indefinite self-renewal is often regarded as an intrinsic and unique stem cell trait, accumulating evidence, points to the possibility that stem cells also arise through processes of de-differentiation. Therefore, the classical presentation of cell populations as being organized in a hierarchical manner, toped by a stem cell that gradually differentiates in an irreversible mode, should be re-examined. If progenitor cells that have already begun to differentiate, may turn back and become stem cell, then stemness is a state rather than a cellular entity. Our recent studies on multipotent stromal cells (MSC), show that such cell populations do not differentiate in a hierarchical manner, and that their differentiation mode is compatible with a phase space model. One example is that the capacity of MSC to support hemopoiesis, which is regarded as one direction of MSC differentiation, may be either a property of the undifferentiated MSC itself, or alternatively a property of fully differentiated osteogenic products of the MSC. An additional issue which is a focus of intense controversy, is the finding that bone marrow-derived stem cells may give rise to cells of other organs and tissues. The same is true for muscle-derived stem cells, neural stem cells and skin stem cells. I recently suggested that pluripotency that entails plasticity, rather than any other trait, is a hallmark of the stem state. Plasticity is thus a state in which the cells harbor a potential to give rise to a multitude of lineages and choose among the options available within the three germ layers. Several studies demonstrated the plastic nature of some adult tissue stem cells, such as the mesenchymal stem cells from the bone marrow, or the skin progenitor cell. These studies strongly suggest that the commonly accepted notion, that adult stem cells should be classified as entities distinct from pluripotent stem cells, is questionable. It is suggested that the underlying properties of cells in the stem state are: being undifferentiated, dependent on their niche for asymmetrical divisions and self-renewal and capable of differentiation into a wide range of cellular entities. These properties are not shared by cells within tumors that are capable of giving rise to new tumors upon transplantation, the tumor initiating cells (TIC). Indeed, unlike stem cells that depend on their niche and are lost following serial transplantation, TIC proliferate indefinitely. Most importantly, TIC have a very limited differentiation potential and are certainly not pluripotent. They therefore represent a biological entity that is biologically different from stem cells.

(JSRM Code:002010700035)

Reduction of hypertrophy by PTHrP and bFGF during in vitro chondrogenesis of mesenchymal stem cells from bone marrow

Weiss S, Bock R, Hennig T, Richter W
Division of Experimental Orthopaedics, Orthopaedic Clinic University of Heidelberg, Schlierbacher Landstr. 200 a, D-69118 Heidelberg, Germany

Introduction: Hypertrophic differentiation of mesenchymal stem cells (MSCs) presents a major problem in TGF-b-driven in vitro chondrogenesis. BMPs have been described as inductors of chondrogenesis while PTHrP or bFGF are associated with a reduction of hypertrophy. We aimed to identify chondrogenic culture conditions avoiding cell hypertrophy by analyzing the effect of growth factors alone or in combination with TGF-b3 on MSC pellets cultured in vitro and after transplantation in SCID mice in vivo.

Materials and Methods: Chondrogenic induction of hBMSC spheroid cultures was modified by addition of factors suspected to stimulate or inhibit chondrogenic hypertrophy. Hypertrophic differentiation was assessed by immunohistochemical analysis (collagen type I, -II, -X, alcian blue), RT-PCR (Col1A1, Col2A1, Col10A1, MMP-13) and quantification of ALP activity up to 6 weeks of differentiation. After 6 weeks of culture under chondrogenic conditionsmicromasses were transplanted subcutaneously in SCID mice for 4 weeks and analyzed histologically (alizarin red) thereafter.

Results: Chondrogenic differentiation as confirmed by positive staining of type II collagen and alcian blue was achieved after supplementing chondrogenic medium with TGF-b3. None of the other growth factors (BMP-2, -4, -6, -7, aFGF, IGF-I) led to chondrogenesis, alone, whereas combination with TGF-b results in chondrogenesis without suppressing collagen type X expression. Combinations of TGF-b with PTHrP or bFGF suppressed collagen type X staining and ALP induction. However, they also prevented the differentiation to chondrocyte-like cells when added from day 0. Delayed addition of PTHrP or bFGF rescued chondrogenesis and suppressed ALP activity along with expression of other hypertrophic markers. In vivo, delayed PTHrP or bFGF treatment could not inhibit calcification.

Conclusion: PTHrP and bFGF are attractive anti-hypertrophic factors able to modulate the chondrogenic effect of TGF-b on MSC in vitro. A fine tuning to collagen type II-positive pellet cultures with low content of collagen type X seems feasible.

(JSRM Code:002010700036)

Defining the end of pluripotency in murine embryonic stem cells

Choi SW, Obier N, Dinger TC, Vallabhapurapu D, Muller AM
Institute of Medical Radiation and Cell Research, University of Wuerzburg, Versbacherstr. 5, 97078
Wuerzburg, Germany  

Introduction: Pluripotency describes the ability of stem cells to self-renew and to give rise to all cell types of the developing embryo. Pluripotent embryonic stem cells (ESCs) are established from the inner cell mass of blastocysts and can be maintained in vitro as stable cell lines in the presence of LIF whereas they start differentiation upon LIF withdrawal. To further study molecular processes at the end of pluripotency we analysed ESCs duringearly in vitro differentiation with respect to gene expression and chromatin state.

Materials and Methods: CCE and BL6 ESCs were routinely grown on murine embryonic fibroblasts (MEFs) and E14 ESCs without MEFs on gelatine-coated dishes in the presence of LIF and 15% FCS. Upon FCS reduction (10%) and MEF- as well as LIF-removal differentiation was induced. Loss of pluripotency was measured by CFC assay, for which appropriate numbers of differentiated ESCs were plated onto MEFs and colonies were counted. Gene expression of pluripotency- and differentiation-associated genes was determined via Real Time PCR. Besides Western Blot we established an intranuclear FACS-based analysis to detect global changes of histone H4 acetylation levels during ESC differentiation.

Results: Upon differentiation induction ESCs quickly lose their pluripotent character and their potential to form colonies on MEFs. The expression of pluripotency-associated genes Rex1 and FGF4 was downregulated, whereas the expression of mesodermal or entodermal marker genes brachyury and ?-Fetoprotein was increased during differentiation. Western Blot analysis on whole cell protein lysates revealed that histone H4 acetylation levels in early differentiating ESCs do not change significantly. However, in flow-cytometric studies - implementing two different staining strategies - we constantly observed coordinated patterns of histone H4 acetylation, increasing from day 0 to day 1 and decreasing from day 2 of differentiation induction. In parallel, cell proliferation rates were only marginally reduced, as analysed by CFSE label retention studies.

Discussion and Conclusion: Together, these data indicate that the end of pluripotency is characterized by an unexpectedly low level of heterogeneity within the pool of differentiating ES cells regarding histone H4 acetylation, proliferation and gene expression. Further studies shall address the question whether the end of pluripotency is a defined point of no return or whether there is a time window of ambiguity.

(JSRM Code:002010700037)

Differential expression of HMGA proteins influence HP1 dynamics and myogenic differentiation

Brocher J, Hock R
Department of Cell- and Developmental Biology, Biocenter University of Wuerzburg, Am Hubland
97074 Wuerzburg, Germany

Introduction: HMGA proteins are abundant architectural chromatin proteins. They bind to AT-rich DNA and localize preferentially in heterochromatin. Their acidic C-terminal domain is considered to be relevant in interactions between HMGA and other factors. In addition, HMGA proteins are expressed particularly in early embryos and undifferentiated cells. In differentiated cells they are downregulated and high HMGA expression levels in adult cells correlate with tumor progression and malignancy.

Materials and Methods:. We used C2C12 mouse myoblast cells as a model system to investigate the influence of HMGA1 on differentiation and chromatin plasticity. Therefore, we created C2C12 cells stably expressing HMGA1a-EGFP and performed knock down of HMGA1 by siRNA. Using bimolecular fluorescence complementation (BiFC), we examined protein-protein interaction between HMGA and HP1 in living cells.

Results: We showed for the first time that HMGA1 expression is gradually downregulated after induction of myogenesis. However, persistent HMGA expression inhibited myogenesis. This inhibition can be explained by an HMGA-dependent misexpression of several genes that are required for proper differentiation. RNAi experiments demonstrated that downregulation of HMGA proteins is necessary to restore proper gene expression and to release the myogenic program.

Furthermore, BiFC analyses revealed that the acidic C-terminus of HMGA interacts with the chromodomain of HP1. Photobleaching experiments indicated that the HP1 residence in heterochromatin dependents on the presences of functional HMGA proteins. Moreover, HP1 binding properties during early myogenic differentiation variegate dependent on the expression level of HMGA1.

Conclusions: Together, our data indicate that the differential expression of HMGA proteins and their capacity to interact with HP1 proteins participates in the regulation of heterochromatin maintenance and plasticity during differentiation. We propose that downregulation of HMGA proteins is required to allow chromatin plasticity and remodeling and to enable cells to enter the myogenic differentiation program.

(JSRM Code:002010700038)

Stem cell ageing, replicative senescence, and tumorigenesis

Jakob F
Orthopedic Center for Musculoskeletal Research, University of Wuerzburg, Brettreichstrasse 11,
97074 Wuerzburg, Germany

Ageing of the organism besides functional impairment of mature somatic cells may be a matter of impaired regeneration as a consequence of altered self renewal of stem cells. The latter is caused by endogenous (cellular) and systemic (humoral) factors. These factors can involve cell damage and repair mechanisms, altered differentiation and induction of transdifferentiation towards alternative stem cell differentiation pathways, and epigenetic changes causing silencing of important key signalling genes.

Both stem cells and somatic cells are subjected to genomic and proteomic damage which accumulates and leads to the induction of cellular fail safe mechanisms, e.g. apoptosis and replicative senescence. Thus the suppression of tumour development by such fail safe programs occurs at the cost of proliferative and regenerative capacity. Monogenetic models of progeria suggest that altered DNA repair can cause ageing and progeria syndromes. Both loss of function mutations of DNA repair enzymes as well as enhanced DNA damage due to an increased load of reactive oxygen species (ROS) initiate cellular fail safe programs, but could also promote tumorigenesis, if proliferative stimuli overcome cell cycle control check points. There is recent evidence that quiescent stem cells can also accumulate damage because the quiescent state results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. Thus in spite of sufficient numbers of stem cells their regeneration capacity may be severely impaired. The cellular load of ROS is controlled by environmental and nutritional factors but also by ROS metabolizing enzymes, which also can be targets of polymorphisms and mutations. Thus active control mechanisms in cells are essential to maintain their genomic and proteomic integrity.

Age-related switching into preferred stem cell differentiation pathways can also augment the ageing process of an organism, due to the lack of specific functions of the resulting tissue. Augmented Wnt signalling was reported to drive muscle stem cells towards fibrosis rather than muscle regeneration. Preferred adipogenic differentiation of mesenchymal stem cells and adipogenic transdifferentiation of osteogenic precursors represent another similar scenario. Epigenetic dysregulation may be another hallmark of ageing where the expression of growth and stemness factors can be impaired by DNA methylation processes and may also contribute to alterations of differentiation processes.

Adult and embryonic stem cells are both discussed as tools for cell based therapeutic strategies. Cell damage and ageing processes may occur during the ex vivo / in vitro handling procedures. Thus research on the maintenance of stem cell integrity appears to be extremely important. Strategies of differentially addressing healthy versus presenescent stem could be established to avoid forced proliferation of damaged cell populations. Exciting new data about a protective function of enhanced activity of the Arf/p53 (Cdkn2a locus (Arf) and p53) tumor suppressor pathway against ageing may promise that anti ageing protection can be even engineered in target stem cells to foster the security and minimize the risk of inducing malignancies in regenerative therapeutic strategies.

(JSRM Code:002010700039)

Aldosterone mediated dysfunction of human endothelial progenitor cells - Mechanisms and therapeutic opportunities

Thum T, Schmitter K, Fraccarollo D, Jakob M, Ertl G and Bauersachs J
Julius-Maximilians University, Internal Medicine I / Cardiology, Wurzburg, Germany 

Introduction: Aldosterone is a well known risk factor associated with hypertension and cardiovascular injury. Bone marrow derived endothelial progenitor cells (EPC) play an important role in neovascularization and endothelial repair implicating a possible role as target for the prevention/therapy of vascular diseases.

Materials and Methods: We examined the effects of aldosterone as well as the mineralocorticoid receptor (MR) antagonist eplerenone on EPC number and function in vitro, in vivo and a clinical trial.

Results: Human EPCs expressed the MR both at gene and protein level. Aldosterone treatment of human EPC in vitro impaired cellular function, which could be rescued by MR antagonism. EPC from patients with primary hyperaldosteronism had a significant impairment in migratory potential compared with age-matched healthy controls. Likewise, incubation of peripheral blood mononuclear cells with aldosterone (1-100 nmol/l) in vitro reduced EPC formation and development of colony forming units (CFU) in a concentration dependent manner. Co-treatment with the selective MR antagonist eplerenone alleviated this effect. Aldosterone reduced EPC migratory capacity and increased the intracellular production of reactive oxygen species (ROS), which was attenuated by MR blockade. While the protein kinase (PK) C inhibitor chelerythrine had no effect, co-treatment with the PKA inhibitor H-89 completely alleviated aldosterone effects on EPC migratory capacity and ROS production. Finally, eplerenone treatment improved number and function of circulating EPC in rats with secondary hyperadosteronism due to heart failure.

Discussion and Conclusions: We have shown impaired EPC number and function in both primary and secondary hyperaldosteronism. In vitro, aldosterone impaired EPC formation and function in a MR- and PKA-dependent manner involving ROS formation. In vivo and clinical data additionally show a rescue of EPC function in hyperaldosteronism by specific targeting of the MR receptor. Beneficial effects of MR antagonists in cardiovascular disease prevention and therapy may be mediated in part by improved EPC biology.

(JSRM Code:002010700040)

Local expression of insulin-like growth factor (IGF) signaling components during in vitro aging of mesenchymal stem cells

Schilling T, Schonefeldt C, Zeck S, Meissner-Weigl J, Schneider D, Jakob F, Ebert R
University of Wurzburg, Orthopedic Clinic, Orthopedic Center for Musculoskeletal Research, Brettreichstr. 11, 97074 Wurzburg, Germany

Introduction: Impairment of the proliferation potential of mesenchymal stem cells (MSCs) may account for age-related deficiencies in bone regeneration contributing to bone-related diseases like osteoporosis. As a model for cellular aging, extended in vitro passaging of MSCs results in replicative senescence that is associated with IGF signaling. Therefore, we aimed here to determine the expression of IGF signaling components as well as the effect of IFG supplementation during in vitro aging.

Material and Methods: MSCs were isolated from bone marrow and incubated in standard propagation medium. Additional MSC cultures were supplemented with 2 or 6 ng/ml IGF2 to check for a possible rescue of replicative senescence. For each incubation, monolayers were passaged as soon as they reached confluence and RNA was isolated in parallel with each passaging. Gene expression levels of IGF signaling components and putatively IGF2-responsive genes were evaluated by RT-PCR.

Results: MSC populations of eight different donors were subjected to in vitro aging by cultivation for 6 - 11 passages. Independent from gender, IGF2 expression continuously decreased with progression of in vitro aging in most of the MSC populations. The expression of putatively IGF2-responsive genes like selenoprotein P (SeP) and collagen type 1 (COL1A1) decreased with advanced passaging. Supplementation with 6 ng/ml IGF2 decreased endogenous expression of IGF2, SeP, and COL1A1 already in earlier passages and enabled extended passaging for two out of three different MSC batches.

Discussion and Conclusions: Reproducible down-regulation of IGF2 expression and some IGF2-responsive genes during in vitro aging of MSCs as well as the extension of the in vitro life span by IGF2 supplementation suggest an important role of IGF signaling for maintenance of the self-renewal potential of MSCs. Since IGF1 and IGF2 both mainly signal via the IGF1 receptor, their combined role in this regard remains to be examined. The decrease of endogenous IGF2 expression under IGF2 supplementation indicated an autocrine negative feedback of IGF2 signaling on local IGF2 production in MSCs. Examination of further components of IGF signaling, e.g. IGF binding proteins, will provide insight into the mechanisms regulating IGF2 and IGF2-responsive genes and therefore cellular aging. Prevention of replicative senescence via modulation of the IGF signaling pathway could stimulate MSC proliferation and hence osteogenic differentiation resulting in improved bone regeneration.

(JSRM Code:002010700041)

NOS inhibitors synchronize calcium oscillations in fat-derives mesenchymal stem cells by increasing gap junctional coupling

Sauer H, Hatry M, Steffen P, Wartenberg M
Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany

Fat-derived mesenchymal stem cells are a promising source of stem cells for cell transplantation. In the present study it is shown that undifferentiated human fat-derived stem cells display robust oscillations of intracellular calcium [Ca2+]i which may be associated with differentiation processes. [Ca2+], oscillations were dependent on extracellular calcium and calcium release from intracellular stores since they were abolished in calcium-free medium and in the presence of the store-depleting agent thapsigargin. [Ca2+]i oscillations were apparently regulated by the inositol 1,4,5-trisphosphate (InsP3) pathway and dependent on gap junctional coupling since they were abolished in the presence of the phospholipase C antagonist U73,122, in the presence of the InsP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as in the presence of the gap junction uncouplers 1-heptanol and cabenoxolone. Preincubation with the nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), and diphenylen iodonium (DPI), synchronized [Ca2+]i oscillations between individual cells within the area of inspection, whereas the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) were without effects. The synchronization of [Ca2+]i oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) experiments revealed increased reflow of the fluorescence indicator calcein into the bleached area in the presence of the NOS inhibitor DPI. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca2+]i oscillations in undifferentiated fat-derived stem cells by controlling gap junctional coupling.

(JSRM Code:002010700042)

Self-renewing mesenchymal progenitors in the bone marrow and in other mesodermal tissues

Bianco P
La Sapienza University, Rome, Italy

The original notion that the bone marrow stroma would include a population of multipotent progenitors of skeletal tissues has evolved into the notion of a “mesenchymal stem cells”, which would be found not only in the bone marrow but also in other mesoderm derivatives in the adult organism. Until recently, self-renewal, one of the key defining properties of stem cells, remained undemonstrated, leaving an aura on uncertainty around the very notion of mesenchymal stem cell. Using the Melanoma Cell associated Adhesion Molecule (MCAM, CD146) as a marker suited to follow the fate of explanted clonogenic progenitors through ex vivo expansion and in vivo transplantation, we were able to show that MCAM expressing clonogenic progenitors in the bone marrow stroma do self-renew in vivo, generate multiple skeletal tissues, and regenerate a compartment of subendothelial stromal cells identical to the ones originally explanted as CFU-Fs. A unique feature of bone marrow stromal clonogenic progenitors is their ability to establish the hematopoietic microenvironment (HME) at heterotopic sites, which makes skeletal progenitors in the bone marrow stroma a key component of the hematopoietic stem cell “niche”. MCAM-expressing clonogenic progenitors (CFU-Fs) are found in, and can be purified from, a variety of other mesodermal derivatives, including skeletal muscle. Here, MCAM-expressing CFU-Fs represent a non-canonical, self-renewing myogenic progenitor, distinct from satellite cells, and endowed with an amazing capacity for spontaneous myogenic differentiation. Both in the bone marrow stroma and in skeletal muscle, cells that express MCAM in situ are located to the abluminal side of microvessels (sinusoids in the bone marrow). From all these data, a novel picture emerges in which a system of clonogenic progenitors endowed with tissue specific differentiation potential and self-renewal capacity exist in different tissues, with the same anatomical location and a closely similar surface phenotype. Their nature as microvascular subendothelial cells provides a novel clue as to the developmental mechanisms that lead to the establishment of progenitor/stem cells in post-natal tissues.

(JSRM Code:002010700043)

Short-time zoledronic acid pretreatment stimulates osteogenic differentiation of human mesenchymal stem cells

Ebert R, Krug R, Meisner-Weigl J, Zeck S, Schneider D, Jakob F
Orthopedic Center for Musculoskeletal Research, University of Wuerzburg, Brettreichstrasse 11, 97074 Wuerzburg, Germany

Pluripotent mesenchymal stem cells (MSC) are the source for the regeneration of bone and cartilage. In osteoporosis anabolic and antiresorptive drugs are applied which respectively stimulate the differentiation of MSC to osteoblasts (e.g. PTH and Teriparatide) or inhibit osteoclasts (e.g. bisphosphonates (BP)). The amino-bisphosphonate zoledronic acid (ZA) inhibits the farnesylsynthase of osteoclasts which leads to inhibition of posttranslational prenylation of proteins like Ras and Rho resulting in osteoclast apoptosis. It was shown that a once-yearly infusion of ZA 5 mg during a 3-year period significantly reduced the risk of osteoporotic fractures. Osteoblasts and precursor cells are also targets of bisphosphonates but little is known about short-time and chronic effects of ZA on MSC cultures and their osteogenic offspring.

To clarify the influence of ZA on MSC, cells were treated with 5, 20 and 50 μM ZA for 6 and 16 h and the gene expression pattern was determined by RT-PCR. Additionally, MSC were treated with 5 ? 50 μM ZA for 3 h and the osteogenic differentiation potential of MSC was analyzed after 4 weeks in osteogenic medium. Cells were harvested and osteogenic marker genes were amplified by RT-PCR. Mineralization of the cell monolayer was analyzed by alizarin red staining. For chronic stimulation with BP MSC were treated with 5, 20 and 50 μM ZA for 24, 48 and 72 h and apoptosis and proliferation capacity of the cells were determined.

Stimulation of MSC with 50 μM ZA for 6 to 16 h increased the expression of Dkk1 and runx3 and decreased the expression of L1CAM. Short-time treatment of MSC with 20 and 50 μM ZA for 3 h stimulated osteogenic differentiation capacity after 4 weeks compared to untreated controls which was shown by alizarin red staining. Osteogenic markers as runx3 and osteopontin were upregulated dose-dependently while osteocalcin was downregulated. In contrast, long-time treatment of MSC with 20 and 50 μM for 48 and 72 h led to increased apoptosis and decreased proliferation capacity. After 24 h no effect was detected.

We show here that a 3 h exposure to μM concentrations of ZA is already sufficient to enhance osteogenic differentiation of MSC in vitro while long-time exposure to ZA impairs MSC proliferation and induces MSC apoptosis. In osteoporosis treatment this might be of clinical relevance to better determine the dosing and the upper threshold of BP accumulation in the bone microenvironment when constantly high local BP concentrations start to impair osteogenic effects in bone.

Supported by Novartis

(JSRM Code:002010700044)

Differential expression of ostoblast related genes in mesenchymal progenitor cells induced by serum of patients with active crohn’s disease with and without osteoporosis

1Blaschke M, 1Giesen M, 2Baums M, 1Kopp R, 3Heikens J, 3Salinas-Riester G, 1,4Siggelkow H
1Department of Gastroenterology and Endocrinology, Georg-August-University, Goettingen, Germany
2Department of Orthopedic Surgery, Georg-August-University Goettingen, Germany
3Department of Molecular Cellbiology, Georg-August-University Goettingen, Germany
4Endokrinologikum Goettingen, Germany 

Introduction: Crohn´s disease (CD) is associated with a higher prevalence of osteoporosis, a complication that is increasingly recognized as a significant source of morbidity also in these patients (pts.). Predicting those individuals at high risk remains controversial, yet disease activity is thought to be one of the main contributing factors. We used primary human mesenchymal progenitor cells directed to the osteoblastic phenotype to investigate the effect of serum of seven pts. during the active phase in CD on expression of osteoblast related genes. Four of the investigated pts. had osteoporosis or osteopenia (CD-opo), three showed no obvious bone disease (CD-con).

Materials and Methods: Mesenchymal progenitor cells obtained from a healthy patient were grown up to confluence in DMEM supplied with 10% FCS, glutamine, penicillin/streptomycin. Cells were seeded at 105/ml in 6 well plates. Supplementation was switched to 1% human serum (healthy proband). When reaching confluence, cells were stimulated with 10 mM? β-glycerolphosphate and 10 μM ascorbic acid and human serum was changed to eight different CD sera (four CD-opo, three CD-con, one healthy control). After 3 and 14 days cell were harvested and mRNA was isolated using TRIZOL. Marker expression was determined with real-time PCR and beta-actin was used for normalization.

Results: A clear difference in gene expression profile between sere from CD pts. and from the healthy proband was detectable. Sera from CD-opo pts. induced RANKL gene expression 100 fold compared to healthy proband and 50 fold compared to CD-con. Osterix and RUNX 2 expression increased 2 to 10 fold, respectively, by serum from all CD pts., whereas no difference was seen for alkaline phosphatase and interleukin 6. Collagen and osteocalcin gene expression seemed to be influenced without reaching significant effects.

Discussion and Conclusion: Serum from pts. with CD induced RANKL gene expression reflecting activation of bone resorption. This induction was largest in CD-opo pts. suggesting RANKL as one of the main contributing factors in this condition. Osteoblastic transcription factors osterix and RUNX2 were also induced by inflammatory activity but did not differentiate between CD-opo and CD-con. No effect on late osteoblast differentiation markers was detectable. Our results suggest that factors in the serum of pts. with active CD lead to bone disease mediated by mesenchymal progenitor cells.

(JSRM Code:002010700045)

Molecular Regulation of Adult Hippocampal Neurogenesis

Badde A, Jagasia R, Lie DC
Research Group “Adult Neurogenesis and Neural Stem Cells”, Institute for Developmental Genetics,
GSF National Research Center for Environment and Health, Ingolstadter Landstrasse 1,
85764 Munich Neuherberg, Germany

Neurogenesis persists throughout adulthood in two restricted regions of the mammalian central nervous system: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. The generation of new neurons from neural stem cells is controlled by signals derived from the local microenvironment. We previously showed that Wnt-proteins are key regulators of adult hippocampal neurogenesis. Specifically, we found that Wnt-signaling stimulates neuronal fate determination and proliferation of neuronally committed precursor cells. We have now identified several transcription factors, which are prominently expressed in immature newborn neurons of the adult dentate gyrus. In this presentation, we will discuss the potential function of these transcription factors as well as their potential interaction with Wnt-signaling in adult hippocampal neurogenesis.

(JSRM Code:002010700046)

Neural stem cells and synaptogenesis

Liebau S, Bockers T
University Ulm Anatomy & Cell Biology, Albert-Einstein-Allee 11, 89081 Ulm, Germany

Neural stem cells (NSCs) are found in the developing as well as in the adult brain. They are self-renewing cells that maintain the capacity to differentiate into all major brain-specific cell types, such as glial cells and neurons. However, it is still unclear whether these cells are capable of gaining full functionality, which is one of the major rerequisites for NSC-based cell replacement strategies of neurological diseases. The ability to establish and maintain polarized excitatory synaptic contacts would be one of the basic requirements for intercellular communication and functional integration into existing neuronal networks. In primary cultures of hippocampal neurons it has already been shown that synaptogenesis is characterized by a well ordered, time-dependent targeting and recruitment of pre- and postsynaptic proteins. In this study, we investigated the expression and localization of important pre- and postsynaptic proteins including Bassoon and synaptophysin, as well as proteins of the ProSAP/Shank family, in differentiating rat fetal mesencephalic NSCs.

Moreover, we analyzed the ultrastructural features of neuronal cell-cell contacts during synaptogenesis. We show that NSCs express and localize cytoskeletal and scaffolding molecules of the pre- and postsynaptic specializations, in a well defined temporal order

(JSRM Code:002010700047)

Pro-neural conversion of human bone marrow stromal cells for regenerative therapy of Parkinson’s disease

Habisch HJ, Brenner R, Storch A

Neurology Dep., University of Ulm, Helmholtzstrasse 8/1, 89081 Ulm, Germany

Indroduction: Current pharmacotherapy of Parkinson’s disease does improve symptoms and extends the patients life expectancy, but does not provide a cure for this detrimental disease. Alternative approaches include cell replacement therapy. Human bone marrow stromal cells (MSC) are not capable of neuroectodermal differentiation per se. Though, in recent years several reports have claimed to achieve a neural transdifferentiation of these easily accessible stem cells. Here we report our protocol to epigenetically convert MSC into neural stem cell-like cells (MSC-NSC) using conditions similar to neural stem cell (NSC) culture.

Materials and Methods: Upon trypsination MSC are cultured in suspension in serum-free P48F medium with 5 μM heparin, FGF2, and EGF (20 ng/ml each), under 3% oxygen (instead of 21%).
Results: Initially, we identified changes of gene expression upon conversion of MSC by mRNA gene chip analysis. In respect to their genome-wide transcriptome, both MSC and MSC-NSC were similar to each other, but differed significantly from adult human NSC. On closer examination we could identify several “neural” genes (e.g. EAAT1, GFAP, MAOA, MAP2, MBP, NES, NOT1, NSE, PAX6, PTCH, SNCB) and dopaminergic genes (e.g. Nurr1, TH) being up-regulated in MSC-NSC and confirmed these data by real-time RT-PCR. Further investigations revealed that these changes were due to the specific culture conditions (suspension, serum-withdrawal, and hypoxia). Additionally, several neurotrophic factors (e.g. HGF, VEGFa) are up-regulated on both mRNA and protein level, reaching concentrations significantly higher than in human adult cortex. Though lacking therapeutic effects in a mouse model of amyotrophic lateral sclerosis due to limited survival of intrathecally transplanted cells, we observed a directed migration of MSC-NSC in vivo complying with in vitro experiments.

Discussion and Conclusions: Based on their neural gene expression profile MSC-NSC share noticeable similarities with adult human NSC arguing for a partial transdifferentiation. The converted cells release neurotrophic factors at high concentrations and further investigations will reveal whether the cells synthesize and release dopamine. Presenting a relatively simple epigenetic protocol to induce a cell phenotype with potentially improved neurotherapeutic properties, MSC-NSC might be a valuable resource for future treatment of neurodegenerative diseases by autologous transplantation.

(JSRM Code:002010700048)

Frizzled-4 a potential neuronal marker of the enteric nervous system

Bareiss P, Metzger M, Skutella T, Just L
University of Tuebingen Anatomy, Tissue Engineering, Oesterbergstr. 3, 72074 Tuebingen, Germany

The enteric nervous system (ENS) is part of the PNS and regulates the blood flow and the peristaltic and secretory activity of the gut. There is evidence that the ENS is also involved in the function of the mucosal immune system and influences the epithelial stem cell compartment of intestinal mucosa.

Several groups have demonstrated that multipotent, self-renewing ENS progenitor cells persist in the fetal and postnatal gut of humans and rodents. These neural progenitors can be expanded and differentiated in neurons and glia cells under appropriate cell culture conditions.

An important role for the regulation of stem cells and progenitor cells has been attributed to the canonical Wnt pathway. The Wnt signaling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of Frizzled (Fzd) transmembrane receptors and a specific co-receptor of the family of low-density lipoprotein receptors.

We have recently identified Fzd-4 expressing cells in the ENS of human small and large intestine. We further characterized, positive cells from human perinatal und adult gut samples using RT-PCR, immunohistochemistry and FACS. Additionally, Fzd-4 positive cells were separated by MACS-Sorting to investigate the proliferation and differentiation capacity of selected cells in vitro.
Our results indicate that Fzd-4 could be an interesting marker to isolate and enrich enteric progenitors and neural cells of the enteric nervous system.

(JSRM Code:002010700049)

MicroRNA regulation of osteogenic differentiation

Genever P
Biomedical Tissue Research Group, Department of Biology, University of York, York, YO10 5YW, Great Britain

Understanding molecular mechanisms that control differentiation of multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) is central to the development of novel therapies. Many studies have successfully identified critical factors involved in initiating and maintaining lineage-specific transcriptional changes although the role of post-transcriptional gene silencing during differentiation remains unclear.

In the last decade, it has emerged that RNA may play a major role in the regulation of gene and protein expression in all eukaryotic cells. The transcriptome describes the repertoire of RNAs transcribed from the genome of a particular organism. Gene expression profiling, using defined cell populations, can determine which mRNA species are transcribed, the proteins they encode (though not necessarily translated) and the potential functional consequences. However mature mRNAs represent only about 2% of the total transcriptional output of complex genomes and there is growing evidence that non-coding RNAs profoundly influence gene expression profiles and therefore cellular function.

MicroRNAs (miRNAs) are a superfamily of evolutionary conserved, small non-coding RNAs that bind to the 3’ UTR of mRNAs to inhibit protein translation. Consequently, miRNAs can influence a broad range of biological activities, which are instrumental in conferring tissue identity and function. We have determined the expression function of miRNAs during osteogenic differentiation of MSCs and osteoprogenitors in vitro. Expression was determined by microarray analysis of 984 known and predicted miRNAs using RNA isolated from undifferentiated cells or following differentiation in the presence of osteogenic supplements (dexamethasone, ascorbic acid and beta-gycerolphosphate) over 21 days. Differentiation was confirmed by induction of alkaline phosphatase, von-Kossa positive mineralisation and qRT-PCR, revealing temporally relevant changes in expression of osteogenic markers (cbfa-1, alkaline phosphatase, type-I collagen, osteopontin and osteocalcin). The analyses revealed constitutive and differentiation-dependent expression of different miRNAs. The functional role of miRNAs was determined using anti-mir microRNA inhibitors transfected into progenitors before osteogenic stimulation and bioinformatic analyses using predictive algorithms were used to identify potential miRNA targets. Our evidence suggests an important functional role of miRNAs during osteogenesis which may have pervasive implications for our understanding of MSC biology. 

(JSRM Code:002010700050)

MMP activity is an essential link between mechanical stimulus
and mesenchymal stem cell behaviour

Kasper G1,2, Glaeser JD3,1, Geissler S1,2, Ode A1,2, Tuischer J1,
Matziolis G1, Perka C1, & Duda GN1,2
1 Musculoskeletal Research Center Berlin, Charite - Universitatsmedizin, Berlin, Germany
2 Berlin-Brandenburg Center for Regenerative Therapies, Charite - Universitatsmedizin, Berlin, Germany
3 Free University Berlin, Department of Biology, Chemistry, Pharmacy, Berlin, Germany

Introduction: Mechanical boundary conditions as well as the function of mesenchymal stem cells (MSCs) play a pivotal role during bone regeneration. However, the molecular mechanisms underlying the dependency of the regeneration process on mechanical loading remain unclear. This study has therefore investigated how matrix metalloproteases (MMP) activity is influenced by mechanical stimulation and whether MMP activity affects MSC behaviour.

Methods: MSCs were isolated from bone marrow aspirates and subsequently characterised functionally and by flow cytometry. The influence of mechanical stimulation was analysed in a bioreactor system that aims to resemble the early phase of bone healing (compression of 10 kPa, 1Hz, 3 days) using a fibrin/cancellous bone construct. A furin inhibitor was added to investigate the mechanism of MMP regulation. RNA and protein expression were determined by means of gene arrays and ELISAs. Gelatinolytic activity of MMPs was detected by zymography. Functional assays investigating migration, proliferation and differentiation were supplemented with specific inhibitors for MMP-2, MMP-3 and MMP-13. Three independent experiments using different MSC donors with at least duplicates in each were conducted.

Results: Mechanical stimulation of MSCs led to an upregulation of their extracellular gelatinolytic activity, which was consistent with the increased protein levels seen for MMP-2, -3, -13 and TIMP-2. However, mRNA expression levels of MMPs/TIMPs showed no changes in response to mechanical stimulation. The furin protease was proved to be involved in the regulation of MMP-2. Specific inhibition of MMP-2, -3 and -13 showed MMP-13 to be associated with osteogenic differentiation.

Discussion: The observed independency of mRNA and protein expression levels indicates the involvement of post transcriptional processes for MMP regulation, such as the alteration in MMP activity demonstrated for MMP-2. To summarise, the results of this study suggest that MSC function is controlled by MMP activity, which in turn is regulated by mechanical stimulation of these cells. Thus MMP/TIMP balance seems to play an essential role in transferring mechanical signals into MSC function. In the future, details of this cascade are to be unravelled before such understanding could be translated into stimulation of bone healing even in unloaded clinical conditions.

(JSRM Code:002010700051)

Gender differences in the differentiation and chondrogenic potential of progenitor cells in human osteoarthritis

Koelling S and Miosge N
Zentrum ZMK, AG orale Biologie und Geweberegeneration, Georg-August-Universitat, Robert-Koch-Strasse 40, 37075 Gottingen, Germany

Introduction: Osteoarthritis (OA) is such a widespread complication of age that it is expected to become the fourth leading cause of disability by the year 2020. The prevalence and the incidence rates are significantly higher in women. The healthy, homogeneous population of chondrocytes in articular cartilage changes in the pathogenesis of OA to a heterogeneous mixture of OA chondrocytes, newly emerging elongated chondrocytes and progenitor cells, mainly found adjacent to the OA defect.

Materials and Methods: With the help of fluorescence associated flow cytometry (FACS), micro-array and real time RT-PCR we characterized the different cell types from different patients to evaluate their chondrogenic and differentiation potential according to the gender.

Results: In vivo, elongated chondrocytes from women and men exhibit a more fibroblast-like expression pattern than the OA chondrocytes. Micro-array analysis (Affymetrix Human Genome U133A 2.0) of healthy cartilage tissue and of the cell populations of OA tissues revealed gender specific differences: e.g., SOX-9, collagen type II and COMP turned out to be regulated; whereas fibronectin, integrin alpha 5 and collagen type I expression was unregulated between women and men. FACS analyses demonstrated progenitor cells from late stages of human OA positive for “so called” stem cell markers. Tested for their differentiation potential in vitro, the OA chondrocytes were not able to differentiate even after dedifferentiation on plastic. In contrast, approximately 20% of the elongated chondrocyte population and 75% of the progenitor cell population can be differentiated into chondrocytes, osteocytes and adipocytes.

Discussion and Conclusions: We present evidence that the elongated chondrocytes from late stages of OA originate from the progenitor cell population. These progenitor cells have a strong chondrogenic potential which can be enhanced by suppressing their osteogenic differentiation. Their differentiation potential and their matrix production capacity differ between women and men, which is in line with the differences in the prevalence and the incidence rates of OA. The regulatory influences leading to these differences need further investigations to allow a better understanding of the pathogenesis of OA and hopefully to reveal new aspects for therapeutic interventions. The results support our opinion that the progenitor cells seem to be a promising starting point for new concepts of a cell biological treatment of OA.

(JSRM Code:002010700052)

The mineralization of the extracellular matrix during the osteogenic differentiation of mesenchymal stem cells correlates with increased levels of human xylosyltransferase I

Muller B, Gotting C, Kuhn J, Kleesiek K and Prante C
Institut fur Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Universitatsklinik der Ruhr-Universitat Bochum, 32545 Bad Oeynhausen, Germany

Introduction: Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable to differentiate into osteoblasts. Therefore, they represent attractive cell sources for tissue engineering applications, especially for bone replacement. Proteoglycans (PGs) exhibit a crucial role for matrix assembly and remodeling. Nevertheless, since bone development is a highly dynamic and complex process, the regulation of the extracellular matrix (ECM) formation remains elusive. Consequently, the aim of this study was to investigate the mRNA expression levels of genes involved in PG assembly. Hence, we analyzed mRNA and protein expressions of crucial PG core proteins and key enzymes involved in glycosaminoglycan (GAG) biosynthesis in different stages of osteogenesis.

Material and methods: Human MSCs were induced to differentiate into osteoblasts in appropriate medium. The progress of osteogenesis was confirmed using histological stainings. The mRNA levels of osteogenic marker genes as well as the investigated target genes were analyzed by real-time RT-PCR. The xylosyltransferase activity was monitored in the culture supernatant.
Results: For one of the rate-limiting enzymes in GAG biosynthesis xylosyltransferase I (XT-I), maximal mRNA expression levels (3.89 ± 0.83-fold increase) and elevated enzyme activities (285 ± 17 dpm/μg DNA) were observed 10 days after osteogenic induction, simultaneously to the beginning mineralization of the ECM, whereas the highly homologous protein XT-II showed no specific alterations. The differential expression of chondroitin sulfate, dermatan sulfate and heparan sulfate chains were determined by analyzing the mRNA expression of EXTL2 (α-1,4-N-acetylhexosaminyltransferase), GalNAcT (β-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5-epimerase) as they represent crucial enzymes in GAG biosynthesis. Besides GlcAC5E, all key enzymes showed upregulated mRNA contents (up to 3.6-fold) around day 10. Except for decorin, which exhibited heightened mRNA levels even in the early stages of osteogenesis, we found similar upregulated mRNA contents (up to 14.6-fold) for all investigated PG core proteins.

Discussion and Conclusions: The tight synchronized expression profiles demonstrate the coordinated biosynthesis of the PGs and underline their importance for the formation of bone and for the osteogenic stem cell differentiation. Furthermore our results indicate that prominent assembly and remodeling processes occur especially in later stages of osteogenesis and in parallel to the mineralization of the ECM.

(JSRM Code:002010700053)

Embryonic stem cell-derived neurons as a novel cellular model system to study neurodegenerative and neuroregenerative processes in vitro

Bibel M, Lacroix E, Klein C, May-Nass R, Perez-Alcala S, Richter J, Schrenk-Siemens K
Target&Lead Discovery, Neurodegeneration, Neuroscience, Novartis Institutes for BioMedical Research (NIBR), CH-4002 Basel

Introduction: The isolation and culture of embryonic stem (ES) cells has opened the possibility of generating unlimited numbers of any cell type. This is of particular importance in neurobiology as homogeneous cell populations are not available in sufficient quantities. In addition, ES cells can be genetically manipulated or isolated carrying relevant mutations. Thus, wild-type and mutant neurons may be compared and mechanisms causing the loss of specific cell types in neurodegenerative diseases identified.

Materials and Methods: We have devised a neuronal differentiation procedure of mouse ES cells in vitro. Our procedure is novel in that we can differentiate ES cells into a defined neuronal lineage. More precisely, we obtain an essentially pure population of neural progenitors, defined as radial glial cells. These progenitors differentiate subsequently into a uniform population of neurons with characteristics of glutamatergic pyramidal neurons, thus recapitulating a recently described in vivo lineage (Bibel et al., Nat Prot 2:1034, 2007; Bibel et al., Nat Neurosci 7:1003, 2004).

Results: We use this system to analyze signaling pathways in neurodegeneration. In contrast to neuronal cell lines we can address synaptic function and neurite de- and regeneration. For example, using ES cells that we isolated from app-/- aplp2 -/- mice we identified a novel function of APP in glutamate uptake/release that we could confirm in vivo studying EPSP curves in organotypic slice cultures of wild type versus knockout mice. In other examples we introduce disease-relevant mutations in ES cells (huntingtin, synuclein, tau, Aβ) and compare wild-type and mutant differentiated neurons to analyze mechanisms of neuronal cell death applying gene and protein profiling technologies, including epigenetic as well as miRNA profiling.

Discussion and Conclusions: Our studies show the highly predictive value of the differentiation system for the in vivo situation and the unique possibility to identify new targets and gene functions. In this regard, we also have successfully established bi-allelic targeting of genes using Blmtet/tet Es cells. In addition, lentiviral infection of candidate genes in the neural progenitors allows us to study signaling networks in a given mutant ES cell background. Importantly, we have optimized conditions for long-term cultures for neurons to mature with high synaptic activity, form spines and express all isoforms of tau as neurons do in the adult brain, thus we can analyze degeneration processes of mature neurons.

(JSRM Code:002010700054)

Neural differentiation of murine androgenetic embryonic stem cells

Dinger TC1, Eckardt S2, Hornich V1, McLaughlin KJ2, Muller AM1
1Institute for Medical Radiation and Cell Research (MSZ), University of Wurzburg, Versbacher Strase 5,
97078 Wurzburg, Germany
2Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA

Introduction: Zygotes with two genomes from the same sex generated by the exchange of paternal (androgenetic; AG) or maternal (gynogenetic; GG) pronuclei are not competent to develop into viable offspring. Despite the restricted developmental potential of uniparental cells probably reflecting the different roles of maternal and paternal genomes during development, uniparental zygotes develop into blastocyst from which embryonic stem cells (ESC) can be generated. The neural differentiation potential of GG ESCs is well characterised while the developmental potential of AG ES cells is less clear.

Materials and Methods: Our study investigates the potential of murine AG ESCs in comparison to GG and biparental (normal fertilised; N) ESCs to differentiate into neural progenitor/stem cells utilizing an in vitro and an in vivo approach. Firstly, AG, GG and N ESCs were in vitro differentiated into pan-neural progenitor cells and subsequently into neuronal and glial cell types. Secondly, after blastocyst injection of AG, GG and N ESCs and isolation of donor cells from chimeric fetal brains, the multi-lineage neural differentiation potential of donor cells was analysed.

Results: We observe that AG ESCs similar to GG and N ESCs differentiate in vitro into cells with neuronal and glial morphology that express neuronal- and glial-specific markers. In addition, following blastocyst injection and analysis of donor cells in E12.5 chimeric brains we observe similar brain seeding patterns of uniparental and biparental cells. Likewise, FACS-sorted cells from E12.5 chimeric brains show comparable frequencies of neurosphere-initiating and neural multilineage-differentiation potentials.

Discussion and Conclusions: Our data show that uni- and bi-parental ESCs do not differ in their in vitro neural differentiation potentials. Furthermore, uni- and bi-parental ES cells generate similar seeding patterns and neural progenitor/stem cells in E12.5 chimeric brain. These in vivo data suggest that the previously described differences in the in vivo engraftment pattern of uniparental cells in late fetal and adult brains is not due to limitations in the proliferation or differentiation properties of uniparental neural progenitor/stem cells

(JSRM Code:002010700055)

Differential effect of Dlx2 in neural precursors derived from the anterior and hippocampal SVZ

Suh Y, Ciccolini F
Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), Heidelberg University, Germany

Introduction: Neural stem cells (NSCs) are self-renewing multipotent precursors capable of giving rise to both neurons and macroglia. NSCs persist postnatally in the subventricular zone (SVZ) the germinal epithelium lining the lateral ventricle. In the anterior SVZ (SVZa), three main types of precursors drive the process of neurogenesis (type B, C and A) leading to the generation of olfactory inhibitory interneurons throughout adulthood. Type B cells, the primary stem cell type divides rarely giving rise to type C cells or transit amplifying cells that by rapid divisions generate committed migratory neuroblasts also known as type A cells. Although both type B and C cells are clonogenic in vitro they can be distinguished on the basis of expression of glial fibrillary filament protein (GFAP) and distalles homeobox transcription factor Dlx-2 respectively. Neuroblasts also express Dlx-2 however in contrast to type C cells they do not form clones and do not express epidermal growth factor receptor (EGFR). Recent reports have suggested that also the posterior hippocampal SVZ contains a similar cellular organization however the nature of these precursors is poorly understood. Thus, EGFR+ cells derived from anterior and hippocampal SVZ were compared by clonal assay, gene expression level of EGFR and Dlx2 and effect of Dlx2 gene expression.

Materials and Methods: NPCs were proliferated in NSA medium containing EGF (20 ng/ml), FGF-2 (10ng/ml) and B27 (2%). For differentiation, NPCs were cultured on poly-L-lysine (PLL)-coated chamber slides with NSA medium containing 1% FCS and 2 ng/ml FGF-2. Lentivirus was produced in 293FT cell line by cotransfection of lentiviral construct and packaging plasmids for Dlx2 gene delivery to NPCs. EGFR+ cells or infected cells were sorted by FACS. Gene expression level was analyzed by qPCR.

Results and Discussion: EGFR expressing cells were isolated from both the anterior and the hippocampal SVZ of postnatal and embryonic mice at day 18 of embryonic development (E18). Despite they were both capable of forming clones, cells derived from the hippocampal SVZ expressed lower levels of EGFR and Dlx-2 mRNA and were less neurogenic compared to their SVZa counterpart. Forced expression of Dlx2 promoted clone forming capacity, proliferation, and neurogenesis in precursors derived from the SVZa but not in precursors obtained from the hippocampal SVZ. Importantly the effect of Dlx-2 overexpession in SVZa precursors was mediated by activation of EGFR.

Taken together, these data suggest that SVZa and hippocampal SVZ precursors are intrinsically different. Furthermore Dlx2 promotes transit-amplifying properties only in precursors derived from the SVZa

(JSRM Code:002010700056)

Embryonic stem cells in Duchenne muscular dystrophy

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

78 patients aged 3-18 were treated for DMD. Diagnosis was confirmed by the genetic analysis.
Patients were administered mesenchymal, ectodermal, and endodermal stem cells isolated from germ layers of 4-8 weeks old cadaverous embryos’ systems and organs; amounts administered - 0,5-3 ml, cell count - 0,1-100x105/ml.

In the course of the first two months after the treatment, strength of different muscle groups increased by 100-700% and was maintained for 8-15 months. Besides, reported was also ROM increase, within the limits of the patient’s stage: stage II patients reported improved gait quality, ability to step on the heel and walk the stairs, raise from the floor with more confidence, and endure bigger loads during the day. Observed were also decreased pseudohypertrophy and strain of forearms and calves, contractures of knee and ankle joints, subsidence of myocardiopathy manifestations and respiratory insufficiency, especially in stage IVa-IVb patients.

DMD patients require continuous treatment (at least, once every 6-8 months) aimed at aversion of muscular atrophy progression. Termination of the treatment results in further progression of the disease and limitation of physical activity. Transplantation of embryonic stem cells terminates DMD progression, notwithstanding the stage of the process, and results in marked improvements.

(JSRM Code:002010700057)

Nanomaterials and stem cells in skeletal tissue engineering and regeneration

Tuan RS
Chief, Cartilage Biology and Orthopaedics Branch
National Institute of Arthritis, and Musculoskeletal & Skin Diseases
National Institutes of Health
Bethesda, MD 20892, USA
(Email:tuanr@mail.nih.gov)

Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the “regenerate tissue” needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This seminar will present the excitement as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomateriral scaffolds. The biology of adult stem cells, particularly the mechanisms regulating their proliferation versus differentiation into specific lineages, is intricately regulated by cell-cell interactions, signaling by extracellular bioactive factors, and transcriptional and epigenetic activities. More importantly, the extracellular matrix milieu provides critical cues, both architectural and structure-dependent, to guide cell-based tissue morphogenesis. We have developed biomimetic and biodegradable nanofibrous biomaterials to serve as scaffolds for cell-based tissue engineering. Information on the fabrication and biological basis of the scale-dependent bioactivities of the nanofibrous scaffold will be presented. Cell-nanofibrous constructs are currently being tested in animal models for their cartilage reparative potential in vivo. In conclusion, tissue engineering represents a unique, emerging inter-disciplinary research field that is a natural platform for life scientists, engineers, and clinicians working together to advance regenerative medicine.

(JSRM Code:002010700058)

Evaluation of the potential therapeutic use of immature stem cells in a canine model for Duchene muscular dystrophy

Ambrosio CE2, Kerkis I1, Martins DS2, Kerkis A3, Miglino MA2 & Zatz M4
1Laboratorio de Genetica, Instituto Butantan, Sao Paulo, SP, Brasil
2Departamento de Cirurgia da Faculdade de Medicina Veterinaria da Universidade de Sao Paulo, SP, Brasil
3Genetica Aplicada, Atividades Veterinarias LTD, Sao Paulo, SP, Brasil
4Centro de Estudos do Genoma Humano, Departamento de Genetica e Biologia Evolutiva, Universidade de Sao Paulo, SP, Brasil

Introduction: Duchene muscular dystrophy (DMD) is a most severe form of muscular dystrophy, which is inherited as a sex-linked recessive trait and affects 1/3500 of newborn males. Molecular genetic studies indicate that DMD is the result of mutations in the huge gene that encodes dystrophin. In order to confirm the results obtained from mouse model, which did not provide clinical sings of the disease, it has been proposed that muscular dystrophy in the golden retriever dog may be homologous to human to clinical trials. To compare two types of adult stem cells, Umbilical Cord CD 34+ and Dental Pulp Stem Cells (IDPSC), as potential multipotent stem cells for cell therapy use, by the evaluation of their skeletal myogenic potential, migration ability and capacity to restore dystrophin function in skeletal muscle cells of dystrophic young dogs.

Methods: Each cell type was analyzed according to their morphology, ultrastructure (confocal, immunohistochemistry and TE microscopy), and cell culture ability. In vivo tests were carried out to analyze engraftment features after infusion of Dil-labeled cells, without any immune suppression, either into the femoral artery or by intramuscular injection of 30-days-old dystrophic dogs. After 60 days, biopsies were taken for tissue immunostaining with anti-IDPSC antibody developed in our laboratory. Clinical trials were made performing critical analyses of disease evolution.

Results: By TEM, canine umbilical cord progenitor cells had an immature cell structure, differing from all primitive blood components. Cell cultures showed poor proliferation. Also, cells, obtained by density solution and magnetic separation, were used for injections into the biceps femuralis or the femural artery. After 60 days, tissue biopies failed to demonstrate the presence of dystrophin either by imunnohistochemistry or by protein blotting. Conversely, the analysis of tissue biopsies of animals injected with IDPSC showed denser cell engraftment, as indicated by both the presence of DiI-stained cells and anti-IDPSC antibody positive labeling. Clinical aspects were considered relevant, with the demonstration of significant differences depending on the route of injection and cell type.

Conclusion: The efficacy of arterial injection of pulp dental cells to treat muscular dystrophy demonstrated that canine multipotent stem cells have great potential for cell therapy, promising to become a new trend for therapeuthical approaches aiming muscular dystrophy.

(JSRM Code:002010700059)

BMP-12 transduced MSCs in collagen hydrogel for ligament reconstruction

Weber M, Kunz M, Stehle J, Noth U, Steinert A
Orthopaedic Center for Musculoskeletal Research, Division of Tissue Engineering, University of Wurzburg, Brettreichstrasse 11, 97074 Wurzburg, Germany

Introduction: With increased participation in sports, the frequency of anterior cruciate ligament (ACL) ruptures is rapidly increasing. Currently over 100,000 patients rupture their ACL each year in the USA. The ACL fails to heal after rupture, and loss of ACL function leads to knee instability, loss of proprioceptive function, and osteoarthritis in over 60% of patients. Ligament reconstruction with biologic grafts such as autologous patellar tendon or hamstring tendon is the gold standard but does not restore the complex architecture and biomechanics of the ACL. A number of growth factors such as transforming growth factor (TGF-??, insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and respectively, bone morphogenetic proteins-12 and -13 (BMP-12 and -13) have been evaluated for their ability to stimulate different aspects of ligament repair, including outgrowth, cell division, and collagen synthesis. In this study the ligament differentiation of human mesenchymal stem cells (hMSCs) transduced with BMP-12 and embedded in collagen hydrogel was investigated.

Material and Methods: Human MSCs from bone marrow were transduced at confluency with 10 vp/cell of adenovirus encoding BMP-12. Afterwards, genetically modified hMSCs were placed at 3 x 105 cells per 200 μl collagen type I hydrogel (Arthro Kinetics plc, Esslingen, Germany). After 21 days in culture histochemical, immunohistochemical, and RT-PCR analyses of the hydrogel constructs were performed.

Results: Histochemical (H&E, Azan, Masson Goldner, van Gieson) and immunohistochemical analyses revealed elongated fibroblast-like cells embedded in a ligament-like matrix. The genetical modification of MSCs with BMP-12 resulted in a weak staining for collagen type III and V and elastin. In contrast, a strong staining for tenascin and vimentin was detected. Furthermore, specific ligament marker like biglycan, collagen type III and V, decorin, elastin, tenascin, tenomodulin, and vimentin were expressed as shown in the RT-PCR analyses.

Discussion and Conclusion: Gene transfer with BMP-12 in combination with a collagen type I hydrogel lead to the development of a ligament-specific extracellular matrix. However, the use of genetically modified hMSCs could be an effective strategy for the formation of genetically optimized hydrogel constructs for the ligament repair.

(JSRM Code:002010700060)

Electrospun collagen type II-nanofibers for stem cell based cartilage repair

1,2Rackwitz L, 1Li WJ and 1Tuan RS
1Cartilage Biology and Orthopaedics Branch NIAMS, National Institutes of Health,
DHHS, Bethesda, MD 20892, USA
2Orthopaedic Center for Musculoskeletal Research, Konig-Ludwig-Haus, University of Wurzburg, Germany
lrackwitz@gmail.com 

Introduction: Matrix-based autologous stem cell transplantation marks a promising approach for the treatment of chondral lesions, although none of the clinically used biomaterials reflects both biochemical composition and ultrastructural aspects of articular cartilage. Recent findings have shown that electrospun three-dimensional nanofibrous structures provide a biomimetic environment for seeded cells. In this study, we investigated the fabrication of a collagen type II-based nanofibrous scaffold via electrospinning for the potential use in stem cell based cartilage repair strategies.

Materials and Methods: Electrospun collagen type II (bovine) scaffolds were fabricated using different solvents, hexafluoropropanol (HFP) or acetic acid, with varying concentrations of collagen type II. Morphological characteristics of the different scaffolds were assessed by scanning electron microscopy. Optimized nanofiber scaffolds were subject to chemical cross-linking using hexamethylene-diisocyanate to prevent rapid biodegradation. Cross-linked scaffolds were seeded with 40,000 MSCs and cultured for 21 days in basal medium. Cell proliferation and cell mediated contraction was assayed over time.

Results: In general, nanofibers produced using HFP showed a larger diameter (400-3000 nm) when compared to fibers spun from acetic acid (150-650nm), which also appeared more homogeneous. In addition, fiber size increased with higher collagen II concentration for each solvent. Cross-linking of the scaffolds was necessary to stabilize and prevent the structure from rapid biodegradation. An increase in cell number during the culture period was seen for both types of scaffolds, a 2.1-fold increase when using acetic acid, and a 1.41-fold increase when using HFP as solvent. After 21 days, cell-mediated contraction in the acetic acid derived scaffold was 23%, and 12% when using HFP as the solvent.

Discussion: We have successfully electrospun collagen type II using a weak acid solvent to produce collagen type II-based nanofibrous scaffolds. Our results show that fiber sizes are dependent on fabrication parameters. By using cross-linking reagents, the collagen type II nanofibers are structurally stable during the culture period, and support continuous cell proliferation. Taken together, these findings suggest that promising electrospun collagen type II nanofibers have biologically favorable structure and properties and represent tissue engineered scaffolds applicable for cartilage repair.

(JSRM Code:002010700061)

Embryonic stem cells in human sacrococcygeal teratomas: Isolation and characterization of an embryonic stem cell line

Busch C1, Bareiss PM2, Just L2, Drews U1
1Section of Dermatologic Oncology, Department of Dermatology
2Section of Tissue Engineering, Institute of Anatomy, University of Tubingen, Germany

Introduction: Teratomas are benign tumours derived from pluripotent embryonic stem cells and contain a variety of organ structures. Sacrococcygeal teratomas (SCTs) are the most frequent solid tumours of the newborn with an incidence of approximately 1:35.000. To find a novel source for human embryonic stem cells, SCTs were examined.

Materials and Methods: 1. A histological survey was performed on 20 SCTs. 2. Tissue from a SCT of a newborn girl was obtained intra-operatively. After generation of a cell suspension, cells were expanded in selective media to facilitate the cultivation of stem cells contained therein. After a few cell passages, a homogeneous population of stem cells was isolated.

Results: 1. Histological analysis of the 20 SCTs revealed that in particular, caudal tissues (Fallopian tube, vaginal epithelium) and the outgrowth of pancreatic tissue with islets from colonic crypts were encountered in 50% of cases. Stem cells were successfully demonstrated in paraffin sections by using nanog, Oct4, SSEA-4, and nestin antibodies.

2. Immunofluorescence and RT-PCR analyses revealed that the isolated cells were positive for nanog, Oct4, SSEA-4, and Stella, and thus showed traits of embryonic stem cells. The isolated stem cells spontaneously grew as spheroids. First differentiation assays showed that in vitro the cells were able to differentiate into astrocytes and neurons. After transplantation of the spheroids into the neural tube or the brain vesicles of the 2 day chick embryo, the cells integrated into the neural crest and into the embryonic brain, respectively. Further in vivo and in vitro characterization of the stem cells is under way.

Discussion and Conclusions: Sacrococcygeal teratomas represent a novel source for human embryonic stem cells.

(JSRM Code:002010700062)

Desmin enters the nucleus of embryonic stem cells in an amino-terminal dependent manner and interferes with their proliferation

Stary M, Weber W, Puz S, M. Hofner, Hollrigl A, Fuchs C, Weitzer G
Max F. Perutz Laboratories, Medical University of Vienna, Dr.-Bohrgasse 9, A1030 Vienna, Austria 

Introduction: During cell division type III intermediate filament proteins such as desmin and vimentin are disassembled by phosphorylation of their amino-terminal domains. Embryonic stem cells (ESCs) have a high mitotic index, a very short cell cycle and do not synthesize type III IF proteins with the exception of vimentin. We hypothesize that type III intermediate filament proteins, different from vimentin, might not only be dispensable but also detrimental for proliferation or phenotypic maintenance of ESCs, and these effects might be mediated by their amino-terminal domains.

Material and Methods: Mouse ESCs were stable transfected with a constitutively expressed desminect and a mutant desmin?1-48 transgene, respectively. Proliferation of ESCs was analysed and localisation of desmin and vimentin in mouse ESC, in differentiating ESCs in embryoid bodies and in mouse cardioblast like stem cells isolated from embryonic and neonatal heart was studied by confocal scanning immunofluorescence microscopy.

Results: Constitutive expression of desmin led to the immediate death of all but one ESC clone. Low level synthesis of desmin in one surviving ESC line caused severe growth retardation. Constitutive synthesis of desminΔ1-48, lacking the amino-terminal domain, allowed establishing ESC lines which were not hindered in their proliferation. Synthesis of desmin or desminΔ1-48 did not alter the stem cell phenotype and was not sufficient to induce differentiation of ESCs. As vimentin, desmin was diffusely distributed in the cytoplasm and most surprisingly, in the nucleus of a significant fraction of ESCs. Desmin Δ1-48 never entered the nucleus and formed aggregates in the cytoplasm of ESCs. Desmin but not desminΔ1-48 was also found to localise in the cell nucleus of a small fraction of putative precursor cells of cardiomyocytes in embryoid bodies.

Discussion and Conclusion: Desmin influences the cell cycle but not the maintenance of the pluripotent phenotype of ESCs. The amino-terminus of desmin is required for its nuclear localization and for the negative effect of desmin on proliferation and survival of ESCs. Localisation of desmin in the nucleus of cells differentiating to cardiomyocytes provides some physiological significance and supports our previous findings that desmin influences expression of cardiomyocyte specific transcription factor genes and corroborates PETER TRAUB´s numerous data demonstrating a specific interaction of type III intermediate filament proteins with DNA.

(JSRM Code: 002010700063)

Derivation of trophoectodermal cells from rhesus monkey embryonic
stem cells

Schwanke K1, Wunderlich S1, Beskov A2, Chikobava M2, Simon AR1, Lapin BA2, Martin U1
1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Podbielskistr. 380, 30659 Hannover, Germany
2Institute for Medical Primatology, Russian Academy of Medical Sciences (IMP-RAMS), Sochi Adler, Russia

Introduction: Embryonic stem cells (ESCs) of murine and human origin have been shown to be able to differentiate into derivatives of all three germ layers and also into extraembryonic trophoectoderm. ESC-derived trophoblasts may represent an extremely useful in vitro model to investigate placental morphogenesis and implantation events during embryonic development. Therefore, the aim of our study was to investigate, whether monkey ES cells are able to form trophoectoderm in vitro similar to mouse and human ESCs.

Material and Methods: Rhesus monkey ESCs (RESCs) were maintained and passaged using standard protocols. To initiate differentiation, non-differentiated RESC colonies were detached and transferred into agarose-coated culture plates to form embryoid bodies (EBs). After 2d of suspension culture in 80% αMEM, 20% FCS, 1 mM Glutamine, 0.1 mM β-Mercaptoethanol and 1% non-essential amino acids, the EBs were plated onto tissue culture plates coated with 0.1% Gelatin in 80% IMDM, 20% FCS, 1 mM Glutamine, 0.1 mM β-Mercaptoethanol and 1% non-essential amino acids. In the course of 2-3 weeks the expression of trophoblast markers was assessed by means of semi-quantitative RT-PCR, immunofluorescence and electron microscopy.

Results: Based on the experimental conditions used to differentiate human ES-cells into cardiomyocytes, we developed a protocol, aiming at the differentiation of RESCs into trophoblasts. Using an EB-based approach, effective differentiation into trophoblasts was achieved. To characterize the trophoblast-like cells, semi-quantitative RT-PCR analyses were performed. The expression of several trophoblast marker genes including human chorionic gonadotrophin α (αHCG), caudal type homeobox transcription factor 2 (cdx2), eomesodermin (eomes) fibroblast growth factor receptor 2 (FGFR-2) and HAND-1 could be detected. Immunofluorescence staining showed expression of cdx2 and cytokeratin Endo-A (Troma-1), whereas the endodermal marker α-fetoprotein was not expressed. The ultra structural analysis of cystic structures, most likely formed due to the expression of fluid-pumping channels as typical for early trophoblasts, showed that the differentiated cells resembled cells of the trophoectoderm. More detailed analyses of the gene expression and of the ultra structures are ongoing.

Discussion and Conclusion: This is the first study demonstrating the successful differentiation of RESCs towards cells of the trophoblast lineage. RESC-derived trophoblasts represent a valuable tool to investigate differentiation and function of early human trophoblast.

(JSRM Code:002010700064)

A novel primate ES cell line from the common marmoset
(Callithrix jacchus) exhibits germ cell development in vitro

Mueller T, Fleischmann G, Horn PA, Sasaki E, Behr R
German Primate Center, Stem Cell Research Group, Kellnerweg 4, 37077 Gottingen, Germany

Non-human primate embryonic stem cells (ESC) are an invaluable tool for preclinical cell replacement therapy approaches. Moreover, primate ESC can be used to study specific problems of early primate development. Since ES cell lines show slight yet significant differences in their differentiation potential and molecular characteristics, it is important to have several different ES cell lines from one species at the same time allowing comparative studies. Thus, the best cell line for each intended application can be chosen.

We have characterized a novel ES cell line (CJES001) from the common marmoset (Callithrix jacchus), a non-human primate widely used in biomedical research. This ES cell line was successfully cultivated for over 24 months (up to passage 75) and extensively characterized.

Blastocyst collection and immunosurgery were performed as described before by Sasaki et al. (2005). CJES001 cells expressed alkaline phosphatase (AP), stage specific embryonic antigens (SSEA)-1, -3, and -4, tumor rejection antigen (TRA) 1-60, and TRA 1-81, as well as the transcription factors SOX-2 and OCT4, indicating pluripotency. Teratoma formation in immunodeficient mice as well as embryoid body (EB) formation revealed the potential to differentiate into derivatives of all three embryonic germ layers. Karyotyping analysis showed presence of a complete set of female chromosomes at passages >70, and a roughly 5-fold increase of telomerase activity was detected by real time PCR when compared to cultured primary cells of the marmoset.

Interestingly, also strong expression of germ cell specific genes was detected in CJES001 cells at the mRNA level by RT-PCR and at the protein level by immunofluorescence indicating that these cells also develop germ line cells in addition to several types of somatic cells. Due to the fact that different ESC lines exhibit different preferences with regard to germ layer differentiation, we now may have a model cell line available for germ cell formation.

In conclusion, we report the establishment of a novel ES cell line from the common marmoset, which can be used to evaluate the potential of cell replacement therapy in a preclinical nonhuman primate model. Moreover, basic cell and developmental studies can be facilitated by these cells.

(JSRM Code:002010700065)

Chondrogenic progenitor cells and asymmetric cell division

Miosge N
Tissue Regeneration Work Group, Georg-August-Universitaet, Robert-Koch-Strasse 40,
37075 Goettingen, Germany

Osteoarthritis (OA) is such a widespread complication of age that it is expected to become the fourth leading cause of disability by the year 2020. The healthy, homogeneous population of chondrocytes in articular cartilage changes in the pathogenesis of OA to a heterogeneous mixture of OA chondrocytes, newly emerging elongated chondrocytes and progenitor cells, mainly found adjacent to the OA defect. In vivo, elongated chondrocytes show a more fibroblast-like expression pattern than the OA chondrocytes. Micro-array analysis (Affymetrix Human Genome U133A 2.0) of healthy cartilage tissue and of the cell populations of OA tissues revealed differences in the expression profiles of these three cell types. FACS analyses demonstrated progenitor cells from late stages of human OA positive for stem cell markers. Tested for their differentiation potential in vitro, the OA chondrocytes turned out to be unable to differentiate. In contrast, approximately 20% of the elongated chondrocyte population and 75% of the progenitor cell population can be differentiated into chondrocytes, osteocytes and adipocytes. There is evidence that the percentages of differentiable cells in populations sorted by FACS for stem cell markers, e.g. CD29, CD44, CD90, CD105, are highly different. The expression profile of “so called” markers for asymmetric cell division might have an influence on these results. RNA silencing experiments show, that the suppression of genes associated with osteocyte differentiation enhances the chondrogenic potential of the progenitor cells. When seeded on the surface of OA defects the progenitor cells attach and integrate in short time. We present evidence that the elongated chondrocytes from late stages of OA originate from the progenitor cell population. These progenitor cells have a strong chondrogenic potential enhanced by suppressing their osteogenic differentiation. The regulatory influences leading to these differences need further investigations to allow for a better understanding of the pathogenesis of OA and hopefully to unravel new cell biological therapeutic interventions.

(JSRM Code:002010700066)

Stem Cell Recruitment and Migration

Brenner RE
Department of Orthopaedics, Division for Biochemistry of Joint and Connective Tissue Diseases,
University of Ulm, Germany

Local or systemic recruitment of adult stem cells is a central process for tissue regeneration and repair. The respective knowledge concerning non-hematopoetic stem cells, however, is just beginning to be elucidated. Multipotent mesenchymal stromal cells (MSCs) for example have been identified in postnatal bone marrow and various tissues or organs throughout the body and it has been suggested that their presence within the perivascular niche may contribute to tissue distribution. In blood MSCs circulate in very low numbers under physiologic conditions, however, an increase has been observed after trauma. On the other hand systemic application does not only lead to “homing” into the bone marrow but also to sites of tissue lesions.

To address the basic question whether human bone marrow-derived MSCs are able to respond to soluble chemoattractive ligands we analyzed their migratory response to various growth factors released during bone remodeling using a modified Boyden chamber assay. These studies indicated that BMP-2/4, different PDGF-isoforms (most potently PDGF-BB) and IGF-I/II were chemoattractive for human MSCs. Interestingly IGFBP5 had a stimulatory effect itself and enhanced the influence of IGF-I in contrast to IGFBP3. Osteogenic differentiation partly decreased (e.g. PDGF-BB) and partly increased (e.g. IGF-I) the respective chemotactic responses. Since bone formation in vivo also involves angiogenesis we analyzed the effects of several members of the VEGF-family and identified VEGF-A as a candidate for coupling the recruitment of MSCs and endothelial cells. CYR61/CCN1, which is well known to induce migration of endothelial cells also proved to be chemoattractive for human MSCs and may fulfill a similar role. WISP3/CCN6, another member of the CCN-family of matricellular proteins was found to be chemoattractive for bone marrow derived MSCs but differred from CYR61/CCN1 in the involvement of the αvs5-integrin receptor. Besides growth factors several chemokines and their receptors (e.g. SDF-1 and CXCR4) have been implicated in directed cell migration of MSCs. In addition, components of the extracellular matrix like hyaluronan and the CD44 receptor may influence these processes as described in recent studies. In 3-dimensional systems testing chemoinvasion a well coordinated action of matrix-degrading enzymes (e.g. MMP-2 and MT1-MMP) was necessary for MSC migration. These studies indicate that MSCs are able to migrate within matrices and transplantation studies using cell-tracking approaches have demonstrated this potential in vivo. The underlying signalling systems, molecular mechanisms and their connection to early fate decision processes of MSCs are still insufficiently understood. Nevertheless, specific and site-directed recruitment of MSCs might become feasible in the future.

(JSRM Code:002010700067)

Enhanced in vitro endothelial progenitor cell number by treatment with the angiogenic inducer CYR61/CCN1

Schenk R1, Hofmann A1, Eck M2, Schmauser B2, Jatzke S1, Noth U1, Jakob F1, Schutze N1
1 University of Wurzburg, Orthopedic Institute, Orthopedic Center for Musculoskeletal Research, Molecular Orthopedics, Wurzburg, Germany
2Institute for Pathology, University of Wurzburg, Germany

Introduction: Endothelial progenitor cells (EPCs) are a promising tool for applications in tissue engineering, treatment of cardiovascular diseases and cancer therapy. A major limitation until today is the small number of EPCs which can be obtained from one patient. The cysteine-rich protein 61 (CYR61/CCN1) is a matricellular signalling protein which belongs to the CCN-family. Here we describe the improved ex vivo propagation of EPCs obtained from peripheral blood in the presence of CYR61 [0.5 μg/ml].

Materials and methods: EPCs were isolated from peripheral blood of 25 healthy volunteers via ficoll gradient centrifugation. Treatment of the cells with CYR61 occurred in different concentrations (0.05 μg/ml to 1.5 μg/ml) and at different time points. CYR61 treated and untreated control cells were characterised using FACS analysis, immunohistochemistry, RT-PCR, uptake of acLDL and concurrent staining for ulex lectin. Additionally, cell treatment with the CYR61 mutants with defect binding sides for the integrins αvβ3 (B3) and α6β1 (T1) was performed.
Results: EPCs respond to CYR61 treatment in a dose dependent manner. Treatment with 0.5 μg/ml CYR61 resulted in a 7-fold increased cell number within one week, compared to unstimulated control cells. Both CYR61 treated and untreated cells show the same EPC marker expression of CD34, CD133 and KDR in FACS analysis, immunohistochemistry and RT-PCR, respectively. Uptake of acLDL and concurrent staining for ulex lectin proved the EPC phenotype of the cultivated cells. Treatment of EPCs with the CYR61 mutants B3 and T1 showed less but not significant results in cell number compared to CYR61 treatment.

Discussion and conclusions: The marker expression of CD34, CD133 and KDR as well as the uptake of acLDL and concurrent staining with ulex lectin in both the CYR61 treated and untreated cells clearly demonstrates that CYR61 treatment does not affect EPCs phenotype. CYR61 treatment at different time points indicated that the observed effect of improved cell number is a result of increased cell adhesion rather than enhanced cell proliferation. The results of the EPCs treatment with the CYR61 mutants B3 and T1 indicate that the integrins αvβ3 and α6β1 are not involved in the CYR61 effect on EPCs.

We conclude that the angiogenic inducer CYR61 is a promising factor for ex vivo propagation of EPC cell number in short time which represents a major supportive potential for subsequent cell based applications.

(JSRM Code:002010700068)

Double-labeled Mesenchymal Stem Cells for 1H / 19F MR Imaging

Basse-Lusebrink TC1, Kampf T1, Weber M2, Heymer A2, Ebert R2, Noth U2, Bauer WR3, Jakob P1,4, Haddad D4,1
1Department of Experimental Physics 5, University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany
2Orthopedic Center for Musculoskeletal Research, Division of Tissue Engineering, University of Wurzburg, Brettreichstr. 11, 97074 Wurzburg, Germany
3Medical Clinic and Polyclinic, University Hospital, Josef-Schneider-Strasse 2, 97080 Wurzburg, Germany
4Research Center Magnetic-Resonance-Bavaria, Am Hubland, 97074 Wurzburg, Germany

Introduction: Cell-based therapies are a promising approach to face multiple medical questions. Therefore, non-invasive monitoring of those cells is an important issue. Today, even single cells can be monitored non-invasively with MRI using superparamagnetic iron oxide (SPIO) particles as MR markers. In MR images SPIOs cause hypointensities which, unfortunately, can also be induced by other causes, e.g. blood clots. Thus, monitoring of SPIO labeled cells is ambiguous. Recently, perfluorocarbon (PFC) particles have shown their potential as non-ambiguous MR markers for detection of cells in-vivo. Due to the relatively weak fluorine signal 19F-MRI has limits to the minimal number of detectable cells. In order to combine the advantages of both methods, we present a study to monitor SPIO and PFC double-labeled cells in-vitro using 1H/19F-MRI.

Materials and Methods: Cells from the human mesenchymal stem cell line hMSC-TERT were double-labeled. First, cells were labeled with perfluoro-15-crown-5-ether (PF15C) emulsions using electroporation. Subsequently, the PFC labeled cells were co-labeled with SPIO particles (VSOPs) via incubation. 1H/19F-MRI was preformed on a 7 T Bruker Biospec with a homebuilt, double-resonant birdcage-coil. For high resolution 1H-MR images Gradient-Echo sequences were used. 19F Turbo-Spin-Echo experiments were performed with lower resolution to identify/confirm the location of the double-labeled cells. 19F-MRS was used to quantify the intracellular PF15C content. Furthermore, the intracellular iron quantity was measured with mass-spectroscopy. Cell apoptosis and proliferation rates were determined using luminescence tests. Histological analyses of the double-labeled TERT cells were performed with haematoxylin-eosin (H&E) and Prussian blue staining.

Results: The double-labeled cells can be detected easily in-vitro using both 1H- and 19F-MRI. 19F-MRS and mass-spectroscopy allow the quantification of the intracellular marker content (PF15C particles and VSOPs). Apoptosis and proliferation tests show a good survival rate of the labeled cells compared to unlabeled control cells. Histological analysis confirms the double-labeling qualitatively.

Discussion and Conclusion: 1H-MR images and 19F-MR images allow the visualization of the labeled cells and could be acquired in experiments lasting only a few minutes. The labeled cells appear as dark spots or regions in the 1H images and can be distinctly identified using 19F-MRI. Double-labeling thus allows to monitor labeled cells non-ambiguously with high spatial resolution and may lead to quantitative cell imaging in the future using 19F-MRS.

(JSRM Code:002010700069)

Potential sources for adult stem cells within the pancreas

Zulewski H
Division of Endocrinology, Diabetes and Clin. Nutrition, University Hospital Basel, CH-4031 Basel, Switzerland

Stem/progenitor cells with the potential to differentiate into insulin producing cells in vitro and/or in vivo were described in pancreatic islets, pancreatic ducts, among the population of pancreatic acinar cells and within adult or fetal pancreas without further specification. In some instances progenitor cells in pancreatic ducts were thought to expand and differentiate into insulin producing cells in response to specific stimuli. Whereas in the case of acinar cells a de-differentiation appears to be the first step followed by re-differentiation into β-cells. The origin of the bona fide stem cell however remains somewhat elusive and a recent report questioned the entire concept of β-cell stem/progenitor cells with studies using genetic lineage tracing experiments. With this approach it has been shown that pre-existing β-cells rather than adult stem/progenitor cells retained a proliferative capacity and may thus represent the major source of new β--cells in adult life, at least in mice. In this study the authors almost excluded the possibility of stem or progenitor cells to play a role in β-cell replacement in adult life. This extreme position however may not be justified by the data, given the fact that the study was not designed to identify precursor cells per se but rather to provide evidence for or against their participation in β-cell regeneration. And, this study is in conflict with numerous recent in vivo and in vitro studies suggesting the existence of pancreatic stem/precursor cells. Today, it is not evident which of the concepts will pass the test of time.

(JSRM Code:002010700070)

Comparison of stemness and endocrine differentiation potential of human pancreatic islet derived and human bone-marrow derived stromal cells

Limbert C1,2, Ebert R, 1 Path G2, Niu X2, Bretzel G3, Seufert J2, Jakob F1
1University of Wurzburg, Orthopedic Center for Musculoskeletal Research, Stem Cell Biology, Wurzburg, Germany
2 University Hospital of Freiburg, Division of Endocrinology and Diabetology, Department of Internal Medicine II, Freiburg, Germany
3University Hospital of Giessen and Marburg, Department of Internal Medicine III, Giessen, Germany

Introduction: Cell-based therapy of type 1 diabetes mellitus resides on beta cell replacement and islet regeneration strategies. Various cell types of embryonic, foetal and adult origin have been investigated. So far, the most adequate alternative cell source is still to be defined. Human islet pancreatic derived cells (hIPC) have been demonstrated to differentiate in vitro into beta cell phenotypes. In previous work we have shown that stromal hIPCs display mesenchymal stem cell features. Here, we compared the stemness character of hIPCs and human bone-marrow derived mesenchymal stem cells (hMSC) as well as the in vitro potential of these two cell populations to differentiate into insulin producing cells.

Materials and methods: Gene expression pattern was analysed in hIPCs and hMSC by means of cDNA microarray hybridization (Affymetrix U133 Plus 2.0 chip) and stem cell marker genes were confirmed by RT-PCR. hIPCs and hMSC were expanded and cultivated in specific pancreatic endocrine promoting conditions (high glucose serum free medium, Activin-A, s-cellulin, hHGF, exendin-4 and nicotinamid). After 5 days in differentiation medium, islet gene markers were investigated by means of RT-PCR and immunocytochemistry in both cell populations.

Results: Both cell types expressed key genes of a cluster of stemness-associated genes (Oct1, Hmgb1, Meis2, Hoxb5, cbfa1 and cbfa2) though with varying expression signal levels. In addition, both hIPCs and hMSCs could undergo an endocrine differentiation pathway shown by isl-1, Ngn3, Pax4, NeuroD1, Pdx-1, Glut2, insulin, glucagon, somatostatin (SST) and pancreatic polipetiyde (PP) expression, although to a lower extend in hMSC. Mature insulin and c-peptide proteins were identified in both cell populations.

Conclusion: Our results provide evidence for similar stemness character of the two stromal populations. Both, hIPCs and hMSCs can be induced in vitro to differentiate towards a pancreatic endocrine phenotype. However, the more extended endocrine differentiation capacity of hIPCs suggests an endocrine “imprinting” in these cells compared to hMSCs, supporting the hypothesis that hIPCs represent an intermediate phenotype with epithelial-mesenchymal commitment. Further studies are needed to elucidate if stromal cells can be induced to replace beta cells or to support islet regeneration and which population constitutes the most adequate cell source in the context of cell-based approaches in type 1 diabetes.

(JSRM Code:002010700071)

Human pancreatic islet-derived precursor cells display mesenchymal stem cell features and differentiation capacity

Limbert C1,2, Jakob F2, Ebert R2, Path G1, Niu X1, Bretzel G3, Seufert J1
1University Hospital of Freiburg, Division of Endocrinology and Diabetology, Department of Internal Medicine II, Freiburg, Germany
2University of Wurzburg, Orthopedic Center for Musculoskeletal Research, Stem Cell Biology, Wurzburg, Germany
3University Hospital of Giessen and Marburg, Department of Internal Medicine III, Giessen, Germany

Introduction: Strategies for cell based-therapy of type 1 diabetes mellitus are based on pancreatic islet replacement and islet regeneration. Human islet-derived precursor cells (hIPC) expressing nestin and c-met have been investigated as an additional source of beta-cells. These cells have been demonstrated to differentiate in vitro into insulin producing cells and are assumed to be of endodermal origin. In continuation of previous work we provide further evidence that hIPCs share a common phenotype with human bone marrow derived mesenchymal stem cells (hMSC) and, in addition, bear the potential to differentiate along mesenchymal maturation pathways.

Materials and methods: hIPC and hMSC phenotyping was perfomed by FACS and immunocytochemistry. Gene expression was examined by cDNA array analysis (Affymetrix U133 Plus Chip). hIPCs were expanded and subjected to osteogenic, chondrogenic and adipogenic differentiation media. Differentiation markers were analysed by RT-PCR and immunocytochemistry.

Results: Both cell types express nestin and c-met. hIPCs display a mesenchymal immunophenotype (SH3+, SH2+, CD29+, CD44+, CD54+, CD90+) and a gene expression pattern similar to hMSC. Moreover, hIPCs could be induced to differentiate in vitro towards the osteogenic (osteocalcin, alkaline phosphatase, day 28), adipogenic (LPL, PPARgamma, day 14) and chondrogenic (collagen IX, X, day 21) lineages.

Conclusion: Our results demonstrate that hIPCs and hMSCs share common phenotypes and similar mesenchymal differentiation capacities supporting the occurrence of endodermal to mesodermal transition in epithelial precursor cells. Understanding the molecular mechanisms that enable these cells to cross the traditional germ layer boundaries will help to develop effective strategies for in vitro generation and differentiation of specific phenotypes for the use in cell therapeutic approaches.Moreover, if hIPCs represent a population of pancreatic cells with stem cell/ regeneration potential, these cells could be induced in vivo to differentiate into insulin secreting cells or to provide regeneration of beta-cells in the injured diabetic pancreatic islet.

(JSRM Code:002010700072)

Stem cells and heart tissue engineering

T. Eschenhagen
Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Martinistrase 52, 20246 Hamburg, Germany

Over the past decade methods have been developed to engineer spontaneously contracting, force-generating 3-dimensional cardiac muscle tissues, both as a target validation model in drug development and as material for cardiac replacement therapy. The present techniques depend on primary cardiac cells from newborn rat or mouse or embryonic chick that have no significant capacity for cell division. Consequently, cardiac tissue engineering at present is essentially a mean to reassemble a heart tissue in vitro that had been dissociated before. Primary cardiac cells are not suitable for potential human applications and their usefulness as a medium- to high throughput target validation assay is limited for practical and ethical reasons. Our research therefore focuses on 3 goals ? (i) to optimize engineered heart tissue from newborn rat (EHT) for cardiac repair, (ii) to miniaturize and automatize the EHT procedure for target validation and drug screening purposes and (iii) to generate EHT from embryonic stem cells (ES cells). To this end, size and contractile force of EHTs could be increased by weaving several circular EHTs to one multi-loop EHTs and culture them under insulin-supplementation, 40% oxygen and auxotonic load. Implantation of multi-loop EHTs onto infarcted hearts of immunosuppressed rats demonstrated survival, formation of a layer of heart muscle tissue and vascularization of the implanted tissue. This was accompanied by undelayed anterograde impulse propagation over the scar and diastolic and systolic improvement of cardiac function. In the second approach, we aim at medium-throughput screening for drug effects on cardiac rhythm, force of contraction and cardiac toxicity and have developed prototypes to do the experiments in a multi-well format. ES cells have an unlimited capacity of self-renewal and generate spontaneously contracting embryoid bodies that display functional properties of immature cardiac myocytes. Over the past years we have developed methods to generate EHTs both from mouse and human embryonic stem cells (ES cells). These constructs beat coherently and develop contractile force. Histologically, ES cell-EHTs are cell rich with strands of cross striated, actinin-positive muscle tissue and show functional characteristics of immature myocardium with typical drug responses. Human ES cell-EHTs will offer the unique opportunity to perform target validation and drug screening on human heart muscles in a relatively simple, cheap and instructive assay.

(JSRM Code:002010700073)

Global transcriptomic analysis of murine embryonic stem cell-derived cardiomyocytes

Doss MX1, Winkler J1, Chen S1, Hippler-Altenburg R1, Sotiriadou I1, Halbach M1, Pfannkuche K1, Liang H1, Schulz H2, Hummel O2, Hubner N2, Rottscheidt R3 Hescheler J1, Sachinidis A1.

1Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne,
Robert Koch Str., 50931 Cologne, Germany
2Max-Delbrueck-Center for Molecular Medicine - MDC, Robert-Rossle Str., 13092 Berlin, Germany
3Institute for Genetics, Department of Evolutionary Genetics, University of Cologne, Zulpicher Str.,
50674 Cologne, Germany

BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.

Materials and Methods: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, expressing puromycin resistance and enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes.

RESULTS: Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway.

CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

(JSRM Code:002010700074)

Cardioprotective Potential of Thymosin s4 in a Preclinical Pig Model of Ischemia/Reperfusion Injury

Hinkel R1, Aouni C El 1, Bock-Marquette I2, Hatzopoulos A3, Boekstegers P1, Kupatt C1
1Klinikum Groshadern, Munich, Germany
2University of Texas, Dallas, Texas, USA
3Vanderbilt University, Nashville, Tennessee, USA

Prolonged myocardial ischemia results in myocardial dysfunction, even after successful revascularisation. We have reported that retrograde application of embryonic EPCs, derived from murine d 7.5 embryos (Tie-2+, c-Kit+, Sca-1+, flk-1 low, MHC-1-), provide rapid cardioprotection via soluble factors. Now, we investigated the role of Thymosin beta 4 (TB4) as paracrine factor mediating the cardioprotective potential of eEPCs.

Methods: In vitro, neonatal rat cardiomyocytes (NRCMs) were subjected to hypoxia (4h)/reoxygenation (1h) in the absence or presence of eEPCs with or without TB4 shRNA transfection, which decreased of TB4 mRNA levels to 20% of control cells. In vivo, pigs (n=6 per group) underwent percutaneous LAD occlusion for 60 min. After 55 min of ischemia either saline solution or eEPCs (5 * 10 6 DiI labeled cells) ± TB4 shRNA, or TB4 protein (15mg/animal) were applied via selective pressure-regulated retroinfusion into the anterior interventricular vein. Infarct size and functional reserve (sonomicrometry at 150/min atrial pacing) were determined after 24h reperfusion. Myeloperoxidase (MPO) levels were obtained to analyze inflammatory cell invasion.

Results: In vitro, survival of NRCMs was increased from 32 ± 4 to 90 ± 2%, when eEPC were present.TB4 shRNA, but not control shRNA, abolished this effect (45 ± 7%), whereas TB4 protein restored it (85±3%). In vivo, eEPCs significantly decreased infarct size compared to control group (36±3% vs. 58±5% of AAR, p<0.05), but not in the presence of TB4-shRNA (48 ± 7% of AAR). Moreover, TB4 protein retroinfusion alone was as effective as eEPC application (36±4%). Functional reserve of the infarcted area was enhanced after eEPC (33±4 SES, % of RCx) or TB4 protein application (39±5% SES) compared to control (10±3%), except eEPCs transfected with TB4 shRNA (19±5% SES). Retroinfusion of eEPCs or TB4 protein significantly reduced MPO levels in the ischemic tissue (1996±546, 1455±197 U/g tissue MPO) compared to control (3323±388 U/g tissue MPO) or TB4 shRNA treated eEPCs (5449±829 U/g tissue MPO).

Conclusion: Our findings reveal that early cardioprotection after ischemia may be achieved by embryonic EPC retroinfusion, unless TB4 levels are largely reduced. Consistently, TB4 protein suffices to provide significant cardioprotection after ischemia and reperfusion.

(JSRM Code:002010700075)

PPARα stimulation induces cardiomyogenesis in mouse embryonic stem cells

Sharifpanah F.1, Wartenberg M.2, Arnold B.1, Sauer H.1
1Department of Physiology, Faculty of Medicine, Justus-Liebig-University, Giessen, Germany
2Department of Internal Medicine I, Cardiology Division, Friedrich Schiller University Jena, Jena, Germany

Stem cell therapy holds great promise for the replacement of damaged or dysfunctional myocardium. Peroxisome proliferators-activated receptors (PPARα, -β and ??)) are nuclear receptors which have been shown to participate in cell differentiation. The aim of this study was to investigate the role of PPARα in cardiomyogenesis during the differentiation of mouse embryonic stem (ES) cell-derived embryoid bodies (EBs). When EBs were treated with PPARα agonists (WY14643, GW7647 and Ciprofibrate) a significant increase in cardiomyogenesis was observed. In contrast, the PPARα antagonist MK886 decreased the number and the size of beating foci. The effect of the PPARα agonists was abolished when EBs were pre-incubated with the free radical scavengers Vitamin E (trolox) and N-(2-mercapto-propionyl)-glycine, indicating the involvement of reactive oxygen species (ROS). Furthermore, we observed an increase in ROS when EBs were treated with PPARα agonists, and consequently a decrease in intracellular ROS when EBs were treated with MK886. The effect of PPAR? agonists on intracellular ROS was attenuated by the NADPH-oxidase inhibitor DPI and Apocynin, indicating the involvement of NADPH oxidase. Also, we observed a significant increase in mRNA expression of fetal cardiac genes and membrane potential of mitochondria after treatment with PPARα agonists. In summary our data indicate, that PPAR? stimulation induces cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase.

(JSRM Code:002010700076)

In vitro expansion and redifferentiation of adult human islet cells

Efrat S
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, 69978 Israel 

Beta-cell replacement represents the ultimate cure for type 1 diabetes, however it is limited by availability of organ donors. Adult human islets are difficult to propagate in culture, and efforts to expand them result in loss of beta-cell phenotype. We have recently shown that cells from adult human islets of multiple donors can be significantly expanded in tissue culture in a simple medium for at least 16 population doublings, without a change in replication rate or noticeable cell mortality, representing an expansion of over 65,000-fold. Microarray studies revealed extensive changes in gene expression in the expanded cells, compared with normal islets. Indirect evidence suggested that a significant fraction of the replicating cells were derived from dedifferentiated β cells. However, genetic lineage-tracing studies in mouse models failed to document expansion of cultured beta cells. We are currently developing lineage-tracing tools for studying cultured human beta cells. In a screening of compounds capable of inducing redifferentiation of the expanded cells we have shown that betacellulin restores β-cell gene expression and insulin content in cells from part of the donors. These methods may allow transplantation of functional islet cells from single donors into multiple recipients.

(JSRM Code:002010700077)

Bone marrow derived mesenchymal stem cells isolated from patients with diabetes mellitus type 1 are able to induce a pancreatic endocrine genes in vitro

Sebok D1, Eberhardt M1, Barbero A1, Linscheid P1, Timper K1, Martin I1, Keller U1,2, Muller B1,2 and Zulewski H1,2
Dept. of Research1, Div. of Endocrinology, Diabetes and Clinical Nutrition2, University Hospital, Basel, Switzerland 

Background: The shortage of human islets as well as allograft rejection hampers islet transplantation for treatment of type 1 diabetes. Recent findings suggest that bone marrow derived mesenchymal stem cells (MSC) have the capacity to differentiate into a variety of cell types including endocrine cells of the pancreas, which could provide an abundant source of autologous cells for this procedure. Our aim was to investigate if MSC isolated from different patients with type 1 diabetes harbour the potential to adopt a pancreatic endocrine phenotype in order to accelerate a bench-to-bedside approach regarding the future use of bMSC for transplantation purposes.

Material and Methods: MSC were isolated from three patients with diabetes mellitus type 1 and expanded in DMEM with 10% FBS and FGF (5ng/ml). For induction of differentiation, cells were incubated for three days in serum free differentiation medium (DMEM/F12) supplemented with factors known to enhance β-cell differentiation. Total RNA was extracted on days 0, 1, 2 and 3 and subjected to quantitative real time RT-PCR. Immunocytochemistry (ICC) was performed with undifferentiated and differentiated cells from day 3 using an anti human c-peptide antisera.

Results: Upon induction of differentiation, the formation of islet-like clusters was observed and quantitative RT-PCR analysis revealed thatvarious key transcription factors needed for the development of pancreatic endocrine cells were either constitutively expressed (Isl-1, Pax6) or up-regulated (Ngn-3, Ipf-1) during the 3 days differentiation period. Additionally, an induction of the islet protein genes (insulin, somatostatin, glucagon and glut-2) was observed. Furthermore, ICC indicated the presence of C-peptide positive cells in differentiated cells.

Conclusions: bMSC isolated from patients with diabetes mellitus type 1 are able to differentiate into a pancreatic endocrine phenotype in vitro by sequential expression of key developmental genes. The yield of islet hormones however was very low, indicating that the current strategies for isolation and differentiation need substantial improvements.

(JSRM Code:002010700078)

Embryonic stem cells in new inset type 2 diabetes mellitus

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

In the course of 1-5 years, observed were18 patients (6 women and 12 men) with diabetes mellitus (DM) complicated by hyperinsulinism and dyslipidemia, mean age being 43.5±5.8 years, duration of the disease ? 1.2±0.5 years. 40% of these patients managed their condition by diet alone, others were undergoing monotherapy with minimal dosages of sulphonylureas. All patients presented with lasting morning hyperglycemia, absence of glycosuria, above normal C-Peptide (5.4±1.3 ng/ml), HbA1C concentration ? 8.1±1.5%. All patients showed changed blood lipid profiles with elevated cholesterol level, in the mean by 35%, increased LDL and VLDL concentration by mean 30%. The symptoms of the disease were minimal and manifested in the form of functional disturbances and psychophysiological reactions.

Patients were treated by transplantations of hematopoietic and non-hematopoietic mesenchymal and endodermal embryonic stem cells isolated from growth zones of 4-8 weeks old cadaverous embryos’ internal organs. Suspensions were administered intravenously, in the amount of 0.5-3.0 ml, cell count ? 0.1-100x105/ml.

In the course of the first post-transplant week, 44% of patients reported elevation of glycemia by 15-25%, then decrease, without additional glycemic therapy. Within a month, 84% of patients reported decreased weakness and better mood. Decrease of morning hyperglycemia began after 2-3 months, and was accompanied by lowered C-Peptide. Normalization of the above indices, as well as lowering of HbA1C concentration, was reported in 5-7 months after the initial treatment; at the same time period observed was positive tendency in normalization of lipid metabolism. Lasting normalization of carbohydrate and lipid metabolism in 78% of patients was reported after 10-12 months, and was accompanied by discontinuance of hyperglycemic medications. Further 2-year observation of 60% of the above patients confirmed sustainability of the effects.

(JSRM Code:002010700079)

Murine embryonic stem cell differentiation into cardiomyocytes requires L-type Ca2+ channel activity

Nguemo F1, Fleischmann BK2, Kamanyi A3, Duan 1, Huamin L, Malan D2,4, Saric T1, Bloch W, 4 Schunkert H5, Hescheler J1, Reppel M1,5*
1Institute of Neurophysiology, University of Cologne, Cologne, Germany
2Institute of Physiology I, Life & Brain Center, University of Bonn, Bonn, Germany
3Department of Animal Biology, Faculty of Sciences, University of Dschang, Dschang, Cameroon
4German Sport University, Cologne, Germany
5Medizinische Klinik II, University of Schleswig-Holstein, Campus Lubeck, Lubeck, Germany
Corresponding Authors: akp72@uni-koeln.de

Aims: Ca2+ is believed to serve as an important mediator of differentiation. We therefore investigated the role of L-type Ca2+ channels during directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes in vitro using nifedipine.

Methods: The murine ES cell line D3, engineered to express EGFP under control of the a-myosin heavy chain promoter, was cultivated according to standard protocols. Embryoid bodies (EBs) were differentiated into spontaneously beating cardiomyocytes either under control conditions or under presence of 10 μM nifedipine, a known L-type Ca2+ channel blocker. Differentiation was monitored using fluorescence microscopy, immunocytochemistry and flow cytometry. The expression of cardiac genes was studied applying the reverse transcription-polymerase chain reaction (RT-PCR). The classical whole cell configuration of the patch clamp technique was used for action potential (AP) and L-Type Ca2+ current measurements.

Results: EBs cultured in the presence of nifedipine exhibited a markedly decreased number of EGFP-positive cardiomyocytes from 3.16±0.3% in control, n=5 and 0.36% in nifedipine-treated conditions, n=5 (flow cytometry data). This inhibitory effect was related to reduced gene expression of a-cardiac MHC, ANF and MLC-2V. With patch clamp experiments we found after extensive nifedipine washout a prolongation of the APD90 (66.8±0.8 ms in nifedipine-treated EBs (n=10) as compared to 43.1±0.4 ms in control conditions, n=29). We have also observed a significantly decreased of L-type Ca2+ current amplitude (~ 75%), a right shift of the current-voltage (I-V) relationship (-11±1.07 pA/pF at 0 mV (maximum peak) in 6 control cells; -versus -3.45±0.44 pA/pF at 10 mV (maximum peak) in 5 nifedipine-treated cells) and activation curve with no significant effects on steady-state inactivation curves (channel availability). Moreover, time constants of fast, Ca2+ dependent, L-type Ca2+ channel inactivation were significantly decreased (5.9±1.5 ms, n=8 ms in control as compare to 11.08±2.1 ms, n=6 in nifedipine-treated cardiomyocytes).

Conclusion: Blockage of L-type Ca2+ channels inhibits differentiation of ES cells into cardiomyocytes.

(JSRM Code:002010700080)

Alloreactive T-cell trafficking after hematopoietic stem cell transplantation

Beilhack A1,3*, Schulz S2,3, Baker J3, Beilhack GF3, Herman EI3, Baker EM3, Landan G3, Nishimura R3, Butcher EC4, Contag CH5, Negrin RS3.
1Departments of Medicine II, Wurzburg University, Germany
2Department of Pathology, Technical University Munchen, Germany
Departments of 3Medicine, 4Pathology and 5Pediatrics, Stanford University, Stanford, California, USA

Acute graft-versus-host disease (aGVHD) is the major limitation for a broader application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In aGVHD alloreactive donor T-cells attack the recipient’s gastrointestinal tract, liver and skin. To address this unusual tissue tropism we developed luciferase transgenic (luc+) mice and utilized bioluminescence imaging to track adoptively transferred luc+ T-cells non-invasively in living recipients. Either myeloablative conditioned BALB/c (H-2d, 8Gy) or C57Bl/6 (H-2b, 9Gy) recipient mice were transplanted with allogeneic luc+ T-cells (FVB/N, H-2q) and FVB/N wild type bone marrow. We observed that T-cell proliferation was confined to secondary lymphoid organs until day+3 after allo-HSCT. At this time alloreactive T-cells up-regulated specific homing receptors and subsequently migrated via the blood to aGVHD target tissues. When we blocked T-cell entry to specific lymphoid organs we found a high redundancy of these priming sites. However, by preventing T-cell entry to all secondary lymphoid organs aGVHD was completely averted. In subsequent experiments we isolated in vivo primed alloreactive luc+ T-cells from mesenteric lymph nodes, peripheral lymph nodes or the spleen and transferred these cells into conditioned secondary transplantation recipients. Luc+ T-cells attacked aGVHD target tissues irrespective of the original priming site. In contrast, after secondary transfer into non-conditioned recipients luc+ T-cells preferentially homed to lymphoid organs. These data suggests that not the lymphoid priming sites but instead signals from the aGVHD-target tissues dictate the distinct tissue tropism in aGVHD.

A.B. and St. S. contributed equally to this study.

(JSRM Code:002010700081)

PTH treatment after myocardial infarction in mice attenuates late ischemic cardiomyopathy: impact of bone marrow derived versus cardiac stem cells

Huber B1, Zaruba MM1, Brunner S1, Deindl E2, David R1, Fischer R1, Assmann G3,
Mueller-Hoecker J3, Franz WM1
1Medical Department I, Klinikum Grosshadern, Ludwig Maximilians University (LMU), Munich, Germany
2Institute of Surgical Research, LMU, Munich, Germany
3Institute of Pathology, LMU, Munich, Germany

Introduction: Bone marrow (BM) derived stem cells improve cardiac function after myocardial infarction (MI). Recently, parathyroid hormone (PTH) was shown to regulate the stem cell niche in the bone marrow. Therefore, in a murine model of MI we analyzed the influence of PTH treatment on survival, functional parameters as well as stem cell homing to the heart.

Material and Methods: 12-24 h after MI, PTH was injected daily for two weeks. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Heart tissues were investigated by immunhistochemistry and RT-PCR. Cardiac Homing was studied by flow cytometry.

Results: PTH treatment resulted in a significant improvement of post MI survival and myocardial function associated with less reduction of LV wall thickness and smaller infarct size. These effects were mediated by an enhanced homing of CXCR4 enriched CD45+/CD34+ BM derived stem cells into the ischemic heart facilitated by a PTH mediated upregulation of SDF-1a. However, infiltrated inflammatory CD45+/CD34- cells revealed a strongly reduced expression of CXCR4. In contrast to the BM niche, myocardial ischemia alone or after PTH treatment did not promote proliferation of resident cardiac CD45-/CD34-/c-kit+ and even decreased the number of CD45-/CD34-/Sca-1+ stem cells in the heart. Immunohistologically, PTH treated hearts revealed an increased neovascularization of CD31+ capillaries in the borderzone, which could be explained by an upregulation of VEGF-A and VEGF-receptor1 mRNA. Furthermore, PTH treatment enhanced expression of IGF-1 receptor protein primarily localized on cardiomyocytes in the borderzone, which was related to a reduced number of apoptotic cells.

Conclusion: PTH treatment after MI exerts beneficial effects on survival and myocardial function associated with an augmented homing of CXCR4 enriched CD45+/CD34+ stem cells towards an increased SDF-1a gradient in the myocardium. Paracrine effects of these cell populations correlated with an improved neovascularization and cell survival via VEGF and IGF-1 dependent pathways. Since PTH is already used in patients with osteoporosis our findings may have direct impact on the initiation of clinical studies in patients with ischemic heart disease.

(JSRM Code:002010700082)

ATF5, a possible regulator of osteogenic differentiation in adult mesenchymal stem cells

Leong DT, Abraham MC, Chew FT, Lim TC, Hutmacher DW
QUT Chair in Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology.60 Musk Avenue, Kelvin Grove QLD 4059.
Email: Dietmar.hutmacher@qut.edu.au

Regulatory pathways of embryonic stem cells in maintaining pluriopotency are partially known. However, the regulatory pathways governing adult stem cells and their stemness are even less known. Activating transcription factor 5 (ATF5) was downregulated significantly in osteogenically differentiated adipose derived stem cells amongst a randomized human donor cell population. Knockdown with RNAi showed an increased sensitivity to osteogenic induction. These evidences pointed towards an ATF5-regulated differentiation pathway in adipose derived stem cells.

Work on cell populations obtained from adipose tissue points towards confirming the hypothesis that there are resident adult stem cells in adipose tissue. These adipose derived cells (ADSC) were able to differentiate to cells that expressed osteogenic, chondrogenic and neurogenic characteristics [1]. Cowan et al showed the potential of using these cells to repair critical sized calvarial defects [2]. Despite the wealth of data on the differentiation capability of ADSC, there is no knowledge on how the regulatory mechanism of ADSC or any adult stem cells maintained their stemness.

To gain an insight into the regulatory transcriptional mechanism of stemness in ADSC, we compared differentiated and undifferentiated transcriptome profiles of ADSC from human donors undergoing cosmetic abdominal liposuction. When ADSC differentiates from an uninduced state to an induced state, we reasoned that the elusive “stemness” genes would be downregulated and it would be a transcription factor. Stem cells and commited cells have drastically different genetic regulatory processes that resulted in their distinct phenotypes even though they essentially have the same genetic code.

We focused on osteogenic differentiation of 20 randomly selected donors’ ADSC and screened for significantly changed genes between the differentiated and undifferentiated states using the Affymetrix U133 Plus 2.0 array system. We first normalized the data before comparing the sample. In a box plot, the median for each sample was centered around ~1 by using the data from all 65 samples and their corresponding 54,675 genes.

Next we filtered out background expressions thereby reducing the number of genes from 54,675 to 11,148 by accepting genes with t-test p-value < 5×10-6. Further analyses were carried with these 11,148 genes. Working from this expectation of stemness genes, we focused our analysis on genes that are downregulated from the uninduced to the induced state.

Preliminary filtering of genes with at least 1.7 fold-change differences between induced (I+) and non-induced (I-) groups narrowed down the list to 73 genes. By testing only these 73 genes for all the samples within the I+ and I- groups for statistical significance (one-way ANOVA test with variances not assumed to be equal, p-value cutoff at 0.05 and Bonferroni correction) further reduced the list to 46 genes. We found that activating transcription factor 5 (ATF5) was downregulated in 19 of the 20 donor sample paired groups through the course of osteogenic differentiation and validated with real time PCR assay. It is interesting that a human pre-osteoblastic cell line did not show a similar trend of decrease when subjected to the same osteogenic differentiation regime. This showed that ATF5 was important in regulating the osteogenic induction of ADSC or even non-osteoblastic cell types. After knocking down ATF5 levels, ADSC were more responsive to at least a tenth of the osteogenic induction concentration, which otherwise would not have resulted in a significant osteocalcin expression. We repeated the knockdown experiments on three other donors’ ADSC (not entered into the initial microarray screen) and observed the same phenomenon.

The ATF family members are transcription factors that recognized the consensus ATF/CRE site “TGACGTCA” [8] and have been implicated in stress responses [3], physiological processes like proliferation [4] and silencing machinery of genes [5], osteo-matrix formation [6]. To our knowledge this is the first report on data that point towards a novel role of transcription factors in regulating osteogenic differentiation in adult stem cells. ATF5 was initially discovered in a yeast two hybrid pull down system as an interacting partner of Cdc34 [7]. Its murine counterpart ATFx shares a 55% homology to ATF4 thereby ATF5 is classified as a subgroup of ATF4 [8].

Interestingly, ATF4 is a substrate of ribosomal serine/threonine kinase 2 (RSK2). Collagen type I expression is regulated by phosphorylation of ATF4 by RSK2. Without ATF4, the appearance of the bone trabeculae is delayed. This delay in osteogenesis suggested that ATF4 is required to mediate some aspects of RUNX2-initiated osteoblast differentiation. Osteocalcin, an important osteo-protein found in mature bone matrix, is also a target gene of ATF4 in vivo [9]. ATF4 transcript is ubiquitously expressed in all major tissues but its protein expression is limited to bone tissues [10]. The similarity between ATF4 and 5 reinforced the role of ATF5 in osteogenic differentiation of cells of non-osteoblastic origin.

However, it seemed possible that ATF5 role is not limited to the osteogenesis but also neurogenesis as neural stem cells in the developing brain expressed high levels of ATF5. Conversely, fully differentiated neurons, astrocytes and oligodendrocytes did not show any detectable expression of ATF5 in vivo [11]. Constitutive expression of exogeneous ATF5 was able to maintain neural stem cells in their undifferentiated state, conversely knockdown or loss of function studies of ATF5 accelerated the differentiation of neural stem cells to form mature neurons and glial cells [11-13]. In addition, another closely related ATF family member, ATF7 was upregulated in Caco-2 cells, a human colonic adenocarcinoma cell line as they undergo spontaneous function differentiation into intestinal epithelia [14].

ATF5 is an interesting target for further studies elucidating its roles in osteogenic differentiation of mesenchymal stem cells. Finding its binding partners and its exact DNA binding domain in the context of mesenchymal stem cells differentiation will open up a new window in understanding and more precisely controlling the differentiation regulation of adult stem cells. Further down the road knowledge may bring clinical applications of adult stem cells to a reality.

References

(JSRM Code:002010700083)

In vitro comparison of collagen based MACTs produced from ovine chondrocytes and bmMSCs

Schulz Ra, Zscharnack Ma, Hanisch Ia, Geiling Ma, Hepp Pb, Bader Aa
aDepartment of Cell Techniques and Applied Stem Cell Biology, Center of Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany
bDepartment of Trauma and Reconstructive Surgery, Medical Faculty, University of Leipzig, Liebigstrasse 20, D-04103 Leipzig, Germany

Over the past few years matrix-coupled autologous chondrocyte transplantation (MACT) has been developed and applied in clinical practice. Here, proliferated chondrocytes are seeded into biomechanical stable 3D-matrices which act as the scaffold and provide a temporary structure for the cellular organization. Clinical application of MACT is one of the main surgical procedures for repairing cartilage defects; however, limitation of chondrocyte availability and their decreased proliferative potential have been an issue. Over the last few years the research on bone marrow derived mesenchymal stem cells (bmMSC) has become a subject of extensive research because of their ability to differentiate into multiple lineages of tissues in vivo and in vitro. A major area of interest is the application of bmMSC in cartilage tissue engineering. Few studies have applied stem cell derived cartilage in small animal models such as the mouse or the rabbit. However, these animal models are unsuitable for the preclinical study of a focal cartilage defect because of their thin cartilage layer. The objective of the in vitro experiments is to investigate the behaviour of ovine bmMSC based collagen (CaReSR, Arthro Kinetics plc, Esslingen) hydrogels under TGF-s3 addition compared to fully autologous cultivated primary chondrocyte scaffolds with respect to the composite material properties, the cell viability and the deposition of extracellular matrix components.

During 4 weeks of autologous culture, aggregate properties were quantitatively verified, cell viability and the expression of cartilage markers were assayed. Different microscopic techniques (CLSM, SEM) indicated a subdivision of bmMSC based scaffolds into a central construct region with uniformly distributed stem cells with low levels of apoptosis, and peripheral layers of proliferative cells, which undergo chondrogenic differentiation. Immunohistochemical staining and quantitative measurements of sulfated glycosaminoglycans (s-GAG) of bmMSC hydrogels showed a significant increase in matrix deposition, mainly in outer areas. Furthermore, semi-quantitative RT-PCR of bmMSC specimens reflected a constant collagen type I activity with enhanced collagen type II, aggrecan and Sox9 expression which would suggest hyaline-like cartilage formation. We propose the application of bmMSC seeded collagen hydrogels for the repair of a focal articular cartilage defect with a diameter of 5 mm in the medial femoral condyles of adult Merino-sheep.

(JSRM Code:002010700084)

Growth hormone treatment improves markers of systemic nitric oxide bioavailability via the insulin-like growth factor-1
Importance for endothelial progenitor cells

Thum T, Fleissner F, Klink I, Tsikas D, Stichtenoth DO, Ertl G, Bauersachs J
Julius-Maximilians University, Internal Medicine I / Cardiology, Wurzburg, Germany
Medical School Hannover, Clinical Pharmacology, Hannover, Germany

Introduction: Growth hormone (GH) treatment of GH-deficient patients improves endothelial function and reduces overall cardiovascular risk. Mechanistically, the GH-dependent insulin-like growth factor-1 (IGF-1) stimulates activity of the endothelial NO synthase, which regulates in part vasoprotective endothelial progenitor cells (EPC). Low IGF-1 and EPC concentrations in patients have recently been correlated with impaired vascular function and development of coronary artery disease.

Materials and Methods: Here we tested our hypothesis, that treatment with recombinant human (rh) GH increases NO bioavailability and subsequently augments circulating EPC levels in healthy male volunteers (mean age 57.4 ± 1.4 years; n=16). We measured markers of NO bioavailability, such as cyclic guanosine monophosphate (cGMP), vascular endothelial growth factor (VEGF), nitrate, nitrite and asymmetric dimethylarginine (ADMA) as well as EPC numbers and blood pressure before and after a ten day treatment of 0.4 mg rhGH/per day.

Results: rhGH treatment increased levels of IGF-1 by 2-fold (p<0.0001) and of the IGF-1 binding protein 3 by 19%±2.7% (p<0.001). Concentrations of the sensitive NO mediator cGMP (12.69±1.04 vs 14.79±1.00 μM cGMP/ mM creatinine; p=0.03) and of VEGF (19.0±1.0 vs 21.4±1.4 pg/ml; p<0.05) were increased after rhGH treatment. In contrast, plasma concentrations of the endogenous NOS inhibitor ADMA were reduced (p<0.05), whereas plasma nitrate levels tended to be increased. The rise in NO bioavailability was closely correlated with a 2-fold increase in circulating CD133+/VEGFR2+ EPC. These findings were also observed in mice treated with GH for 7 days. Importantly, blocking of the IGF-1 receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability.

Discussion and Conclusions: In healthy middle-aged subjects rhGH treatment enhances overall nitric oxide bioavailability and subsequently EPC numbers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-1 plasma levels. Our proof-of-principle study warrants further clinical trials to test a potential therapeutic effect of recombinant human GH in patients with various cardiovascular diseases, impaired NO bioavailability and EPC.

(JSRM Code:002010700085)

Chondrogenesis in co-culture: An intensive interaction between mesenchymal stem cells and primary chondrocytes

Csaki C1, Matis U3, Mobasheri A2, Putz R1, Ye H1, Shakibaei M1
1Researchgroup Musculoskeletal System, Institut for Anatomie, LMU Muenchen, Pettenkoferstr. 11,
D-80336 Muenchen
2Division of Veterinary Medicine, University of Nottingham, Sutton Bonington Campus,
LE12 5RD, United Kingdom
3Clinic of Veterinary Surgery, LMU Muenchen, Veterinarstr. 13, D-80539 Muenchen 

Introduction: Chondrogenic defects in articular joints, such as the knee, are common and a wide spread problem with little regenerative potential. Although various attempts have been made to regenerate articular cartilage through mesenchymal stem cells (MSC), our understanding of the mechanisms involved in chondrogenesis is still limited. However, for successful tissue engineering a better understanding is required. Therefore, the aim of this study was to examine in vitro the possible interaction and communication that occurs between MSC and primary chondrocytes (PCH) in order to gain more insight into the complicated mechanism of chondrogenesis.

Materials and methods: Bone marrow MSC were cultured in a ratio of 1:1 with autologous PCH. To be able to evaluate the cell interaction, MSC were membrane labelled with a red fluorescent dye and PCH with a green fluorescent dye and monolayer co-cultures monitored in vitro over a period of seven days. High density co-cultures were evaluated with light microscopy, electronmicroscopy and immuno-electronmicroscopy

Results: In high density co-culture an even distribution of MSC/PCH is observed. After 7 days cartilage-nodules form. Already after 1 day in monolayer co-culture, both cell types extend long directed pseudopodia, intensively searching for the other cell type. A few MSC/PCH engage already in close cell-cell contacts. It is noticeable that PCH proliferate strongly in the vicinity of MSC. At day 3, PCH proliferate even more actively around the MSC. Many cell processes can be observed and the intensity of cell contacts increases further. A few PCH now contain small red vesicles and some cells are bicoloured suggesting cell-fusion. At day 4 the number of PCH strongly increases. Furthermore, PCH without contact to MSC now contain red vesicles suggesting the uptake of soluble factors produced by the MSC.

Conclusions: We could show that there are active interactions between MSC and PCH leading to adequate communication. This intensive interaction through the co-culture might be an important stimulus for the chondrogenic differentiation of MSC and opens up new exciting possibilities for cartilage tissue engineering.

(JSRM Code:002010700086)

Influence of standardized 3D-cell-culture on multilineage differentiation of human adipose tissue derived stem cells

Bohrnsen F, Gadallah A, Lindner U, Schlenke P, Rotter N, Meier M, Rohwedel J, Kramer J
Department of Medical Molecular Biology, University of Lubeck, Ratzeburger Allee 160, 23538 Lubeck, Germany

Introduction: Adult stem/progenitor cells have been isolated from different tissues such as bone marrow, adipose tissue, muscle and peripheral blood. Adipose tissue-derived stem/progenitor cells have the potential to differentiate in vitro into osteogenic, chondrogenic and adipogenic cell types. They may therefore prove to be an easily accessible and abundant source of stem/progenitor cells.

Materials and Methods: We isolated and characterized adult stem/progenitor cells from human cervical subdermal fatty tissue, which was obtained during tracheostomy, and analysed their adipogenic, osteogenic and chondrogenic differentiation in vitro. The isolated stem/progenitor cells were characterized using (a) fluorescence activated cell sorting, (b) histochemical- and immunhistochemical staining as well as RT-PCR analysis and, (c) determination of their proliferation rate during passaging. Asking the question whether close cell-cell interactions favor human stem/progenitor cell differentiation prior to lineage specific induction, osteogenic, adipogenic and chondrogenic properties of the isolated stem/progenitor cells were analyzed using the standardized three-dimensional mesenchymal microsphere (MMS) -model of stem cell cultivation.

Results and Discussion: Using the standardized MMS-model of stem cell differentiation as well as classical monolayer protocols, we were able to demonstrate that subdermal adipose tissue derived stem/progenitor cells differentiate along the osteogenic, adipogenic and chondrogenic lineages. These results were confirmed by the expression of tissue-specific genes like osteopontin, osteocalcin, PPARγ, aP2, C/EBP, collagen type II, and aggrecan. Immunhistochemical staining revealed cells to be positive for osteopontin, bone sialoprotein as well as collagen type II and collagen type X. Comparison of MMS-cultivation to monolayer and micro mass body protocols demonstrated MMS cultivated stem/progenitor cells to exhibit tissue-related attributes favoring multilineage differentiation prior to lineage specific induction. Standardized MMS differentiation might therefore offer a model of three-dimensional adult stem/progenitor cell differentiation, considering cell-cell interactions as well as multilineage differentiation.

(JSRM Code:002010700087)

Low oxygen during expansion of MSC iIncreases the following chondrogenic differentiation in pellet culture and collagen gel

Zscharnack M1, Schnepp C1, Marquass B2, Bader A1
1 Professorship of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine (BBZ), Medical Faculty of Leipzig, Germany
2Department of Trauma and Reconstructive Surgery, Medical Faculty of Leipzig, Germany

Introduction: One important parameter for bone marrow mesenchymal stem cell (bmMSC) fate including stemness and differentiation is the oxygen environment. The oxygen tension inside the bone marrow in vivo is 5% on average. We investigated the influence of low oxygen tension during expansion of bmMSC on following chondrogenic differentiation in standard pellet culture and in clinical applied collagen gel (Arthro Kinetics plc). The differentiation was performed at commonly used 21% ambient oxygen conditions.

Materials and Methods: Ovine bmMSCs were expanded at ambient 5% pO2 and 21% pO2 as control. The proliferation potential was analysed by colony forming unit ? fibroblast (CFU-F) assay.
The second passage cells were used for chondrogenic differentiation in pellet cultures and collagen gels. Differentiation was accomplished at normoxic conditions with supplementation of 10ng/ml TGF-β3 for up to 21 days. Chondrogenic differentiation was assessed by immunhistochemical staining (aggrecan, type II collagen), measurement of sulfated glycosaminoglycans (sGAG) and quantitative real-time RT-PCR (type I collagen, type II collagen, type X collagen, aggrecan).

Results: Ovine bmMSC expanded at low oxygen showed an approximately two times higher CFU-F number compared to normoxic conditions.

After expansion at 5% pO2, pellets showed an increased chondrogenic differentiation compared to 21% pO2 as shown by immunhistochemistry, production of sGAG, and gene expression. The amount of sGAG in pellets was 35% ± 6% higher after hypoxic proliferation. The cells in pellet culture had a more rounded shape and produced distinct more collagen type II compared to cells expanded at 21% pO2.

Discussion and Conclusions: Our results suggest that maintaining ovine bmMSCs in vitro at low oxygen environment is more physiological and increases the following chondrogenic differentiation.

These findings contribute to the knowledge of stem cell cultivation, and they might be a potent approach to improve chondrogenic differentiation of cartilage tissue engineering from bmMSCs.

(JSRM Code:002010700088)

Isolation, characterization and spontaneous differentiation of human umbilical cord-derived mesenchymal stem cells

Fischer J 1, 2, Jahnen-Dechent W 2, 3, Rosewick S 1, 3, Knuchel R 1, Neuss S 1, 3
1Institute of Pathology
2Institute for Biomedical Engineering, Biointerface Group
3Interdisciplinary Centre for Clinical Research, IZKF "BIOMAT"

RWTH Aachen University, Aachen, Germany

Introduction: Bone marrow is the most commonly used source for the isolation of human mesenchymal stem cells (hMSC). Similar cells can also be isolated from various other tissues including adipose tissue, dental pulp and umbilical cord. Umbilical cord-derived stem cells are believed to have a higher proliferation and differentiation capacity due to their primitive developmental stage. We isolated hMSC from bone marrow and the Wharton’s Jelly of umbilical cords and compared the cells regarding their surface epitopes, proliferation and differentiation.

Materials and Methods: HMSC were isolated from bone marrow according to standard protocols (Pittenger et al., Science, 1999) and from the Wharton’s Jelly of umbilical cords. To this end, 2 cm pieces of umbilical cords were opened lengthwise with a scalpel. The matrix was scraped off and incubated with 2 mg/ml collagenase for 16 h, trypsinized for 30 min, washed and seeded on T75 culture flasks. Proliferation was analyzed using XTT tests. The differentiation into adipocytes, osteoblasts and chondrocytes was performed according to standard protocols (Pittenger et al., 1999). Surface epitopes were analyzed by flow cytometry. Spontaneous 3D aggregation and differentiation within cellular aggregates was investigated by histological staining and immunohistochemistry.

Results: The flow cytometric analysis of the surface epitopes CD51, CD54, CD73, CD90 and CD105 showed that the isolated cells from bone marrow and umbilical cord displayed an hMSC phenotype. Cells could be readily differentiated into adipocytes, osteoblasts and chondrocytes. Interestingly, the umbilical cord-derived cells spontaneously formed 3D aggregates when cultured under post-confluent conditions. All cells of these aggregates were viable and spontaneously differentiated into several specialized cell types akin to the well-known differentiation of embryoid bodies.

Discussion and Conclusion: The umbilical cord-derived hMSC resemble bone marrow-derived hMSC and additionally show a spontaneous 3D aggregation and differentiation in vitro. These results indicate that umbilical cord-derived hMSC are a valuable tool for cell-based therapy, but due to their spontaneous differentiation capacity, teratoma formation might be possible.

(JSRM Code:002010700089)

Coculture systems of CD34+ cells with hepatocytes as models for induction of hepatic differentiation

Wulf-Goldenberg A, Eckert K, Fichtner I
Max-Delbruck-Center fur Molekulare Medizin, Robert-Rossle-Str.10, 13125 Berlin-Buch
annika.wulf@mdc-berlin.de

Adult human haematopoietic stem cells possess high plasticity that was demonstrated with respect to their differentiation into organ specific cell types. Using differential experimental design, studies have indicated that adult stem cells can differentiate into hepatocytes-like cells which differ in hepatic marker expression profiles and functional properties. However, methods are still being developed for induction of stem cell differentiation and the contribution of direct cell-cell-contact or soluble factors is under investigation. For hepatic differentiation of CD34+ cord blood cells we investigated models with respect to contributions of direct stem cell - hepatocyte ? interactions and secreted factors from hepatocyte culture.

CD34+ stem cells were cocultured with different cell types as murine hepatocytes line AML-12, human hepatocarcinoma HepG2 and murine hepatocytes. Flow cytometric analysis using dye transfer technique showed that stem cells get in contact with hepatocytes. Video imaging revealed a high CD34+ motility. FACS and Western Blot techniques demonstrated that CD34+ cells express Connexin43 and the hepatocytes Connexin32. Through these gap junctions cells may communicate and perhaps support differentiation into hepatic-like cells. Futhermore, conditioned cell culture media from HepG2 cells induced early endodermal differentiation (SOX17, FOXA2, AFP) of CD34+ stem cells, suggesting the involvement of secreted soluble factors.

Conditioned hepatocyte media and coculture of CD34+ cells with hepatic cells represents a valuable model for studying hepatic differentiation processes.

(JSRM Code:002010700090)

Inducible differentiation of medakafish spermatogonial stem cells to various somatic cell types

Thoma E, Wagner TU, Schartl M
Physiological Chemistry I, Biozentrum, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

Introduction: Embryonic stem cells are characterized by pluripotency, the ability to form every cell type of the body. This feature gives them a great potential in regeneration therapy. However, the mechanisms that regulate and control the process of differentiation into a defined cell lineage are still poorly understood. The application of embryonic stem cells for research or clinical therapy is linked to several ethical and immunological problems enhancing the need for other approaches. One alternative is the use of spermatogonial stem cells that have recently been shown in mice to be pluripotent. We have established a system for induced directed differentiation using a spermatogonial cell line from medakafish.

Methods: The spermatogonial cell line SG3 was obtained from testis of adult medakafish and is stable in culture in the presence of a complex mix of growth factors. Directed differentiation was induced by chemical treatment, modulation of culture conditions or transfection with expression vectors for presumed or known lineage specific masterregulators of differentiation. Status of differentiation was investigated by morphological analysis and by examination of gene expression patterns.

Results: Our experiments demonstrate that SG3 cells are capable of spontaneous differentiation and of formation of other cell types than sperm. Attempts to induce directed differentiation resulted in the formation of cells showing morphological features of melanocytes, neurons, osteoblasts, and adipocytes. We also obtained cells capable of phagocytosis that therefore seem to be macrophages.

Discussion and conclusions: The use of stem cells for regeneration therapy requires at first methods to generate specific cell types from stem cells. Our results give indications for directed differentiation by chemical treatment, modulation of culture conditions, and transfection. Moreover, this study demonstrates that medaka spermatogonia are pluripotent and therefore may be ascribed an equal status to embryonic stem cells. This finding, together with compatible data published for mouse spermatogonia, strongly suggests the possibility for human pluripotent spermatogonia.

(JSRM Code:002010700091)

Influence of extracellular calcium concentration on the bone matrix synthesis and mineralization in cultures of human primary osteoprogenitor cells

Majore I, Jager V*, Rohde M*
Institute of Technical Chemistry, University of Hannover, Callinstrase 3, D-30167 Hannover, Germany
*Helmholtz Centre for Infection Research, Inhoffenstrase 7, D-38124 Braunschweig, Germany

Introduction: The proper ratio of organic and inorganic components within the bone matrix is an extremely important factor that both influences development and functionality of bone tissue. Focusing on the influence of extracellular calcium concentrations on the bone matrix formation and mineralization we were looking for suitable methods to better control the process of human bone tissue generation in vitro.

Materials and Methods: For the experiments primary human osteoprogenitor cells were used which were derived either from a heterotopic ossification or from trabecular bone. Cells were grown in ZKT-I medium (in-house formulation*) containing 0.89 mmol/l Ca2+ or in ZKT-I modifications with twice and four times increased Ca2+ concentrations. All media were additionally supplemented with human serum, dexamethasone, ascorbate-2-phosphate, and β-glycerophosphate. Cell growth and vitality, type I collagen synthesis, alkaline phosphatase (AP) activity, Ca2+ consumption and Ca2+ accumulation in the extracellular matrix (ECM) were monitored over a period of 31 days.

Results: Increased Ca2+ concentrations in the media resulted in an accelerated collagen matrix mineralization and a higher mineralization level. Scanning electron microscopy carried out in the middle of cultivation showed different stages of mineral crystal formation dependent on the Ca2+ concentration in the extracellular environment. Moreover, the increase of Ca2+ content in culture media caused reduction of collagen I synthesis and lowering of AP activity. Therefore, bone-forming cells produced more ECM with a lower degree of mineralization using medium with a low Ca2+ content. Less ECM with a higher degree of mineralization was generated in medium with a Ca2+ content of about 1.8 mmol/l. An even higher extracellular Ca2+ concentration of 3,3 mmol/l was associated with non-physiological mineralization or calcification as well as a decline of cell growth and intensification of apoptosis.

Discussion and Conclusions: Ca2+ concentrations in culture media have a substantial impact on the ECM synthesis and mineralization. We believe that the reduction of collagen matrix formation and decrease of AP activity are two of numerous regulatory reactions which counteract the non-physiological mineralization. In the context of bone tissue engineering media with lower or slightly increased Ca2+ content may be used to influence and to improve the ratio of organic and inorganic components of artificial bone tissue.

(JSRM Code:002010700092)

A completely serum-free differentiation protocol facilitates the search for key factors which enhance the generation of alveolar type-2 epithelial cells from murine embryonic stem cells

Mauritz C, Schmeckebier S, Menke S, Martin U
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Podbielskistrase 380, 30659 Hannover, Germany

The alveolar epithelium of the lung is composed of two cell types, alveolar type-1 epithelial cells (AT1 cells) and alveolar type-2 epithelial cells (AT2 cells). AT2 cells are known for their ability to produce pulmonary surfactant and to serve as progenitor cells for AT1 cells. Transplantation of murine embryonic stem cell (mESC)-derived AT2 cells would provide a new therapeutic option to treat pulmonary injuries and disorders which are associated with loss of AT2 cells and impaired surfactant production, respectively.

Based on our recently established completely serum-free differentiation protocol we are searching for factors which efficiently enhance the generation of mESC-derived AT2-like cells. In this context the keratinocyte growth factor (KGF) is a promising candidate. KGF is a member of the fibroblast growth factor family and has been shown to be a strong growth factor for AT2 cells in vitro and in vivo. We hypothesize that KGF either supports the differentiation of AT2-like cells from mESCs and/or induces the proliferation of mESC-derived AT2-like cells.

Studies concerning the appropriate concentration and the right point in time for the application of KGF during the differentiation process are ongoing. Furthermore, proteomic analyses of culture supernatants will help in the identification of further AT2 cell inducing factors.

(JSRM Code:002010700093)

Characterization of signal transduction pathways in Muller Glial cells and retinal progenitor cells

Ernst W, Florian C, Langmann T, Weber BHF, Morsczeck C
Institut fur Humangenetik der Universitat Regensburg, Franz-Josef-Straus-Allee 11, 93053 Regensburg, Germany

Introduction: Recent studies demonstrate that Muller glial cells (MGCs) are capable of dedifferentiation into retinal progenitor cells (RPCs). Therefore we isolated MGCs and RPCs and characterized the activation state of selected pathways by quantitative real-time reverse transcription (qRT) PCR.

Materials and Methods: RPCs and MGCs were isolated from retinas of C57/BL6 mice between postnatal days P0 to P10 (for RPCs) and P8 to P12 (for MGCs). Isolated cells were cultured and used for analysis after passage four. We performed a reverse transcription (RT)-PCR for Nestin, Musashi 1, Sox 2 as RPC specific markers and for the gene expression of GFAP and Vimentin as MGC specific markers. To analyze mRNA profiles, a qRT-PCR array was established for signal transduction pathways Wnt, FGF, Hedgehog, Notch and TGF-β.

Results: We proved successful isolation of RPCs and MGCs. Gene expression was determined for specific markers for the Wnt, FGF, Hedgehog, Notch and TGF β signal transduction pathways. However gene expression levels for WNT5, Gli3, Shh, Dll1, Hes5, BMP2 and BMP7 were upregulated in RPCs compared to MGCs, but no differentially gene expression was observed for MGCs.

Discussion and Conclusion: We demonstrate successful isolation of RPCs and MGCs from the postnatal murine retina. All investigated signal transduction pathways seem to be activated in RPCs and MGCs. Interestingly enough, gene expression of selected signal transduction pathways were increased in RPCs in comparison to MGCs. These data may suggest that MGCs have more restricted capacities for cell differentiations than RPCs.

(JSRM Code:002010700094)

Human dermis-derived cells demonstrate strong features of controlled mesodermal differentiation

Tiede S1, Danner S 3, Tronnier V 2, Paus R1, Zechel C 2
1Department of Dermatology, Allergology and Venerology, UK-SH, Campus Lubeck, Lubeck, Germany
2Department of Neurosurgery, UH-SH, Campus Lubeck, Lubeck, Germany
3IBMT, Fraunhofer-Institute, Lubeck, Germany

Multiple tissue niches in the human body are now recognized to harbour adult stem cells. Here, we have examined a panel of putative stem cell/progenitor markers on progenitor cells derived from mechanically and enzymatically processed adult human scalp skin, which may serve as a convenient and abundant source of adult human progenitor cell populations.

After removal of the epidermis, adherent growing cells of a purposely heterogenous mixture of intradermal cells (including sweat glands, fibroblasts, and hair follicle-derived cells), could be propagated for more than 20 passages in serum-supplemented medium. Cells stained positive for Vimentin, and weak expression of nestin, GFAP or neuronal proteins could be occasionally observed in a very small subfraction of the culture.

When grown to confluency a small subfraction of the cells (less than 1%) within the culture began to express low levels of SMA. These Sma+ cells did not undergo significant morphological changes.
The cells could be also maintained in media containing FCS and the mitogens bFGF, EGF or PDGF AB. Interestingly, human skin-derived stem/progenitor cells obtained in this manner exhibited a strong commitment to the mesodermal lineage, since a large subfraction of them expressed high levels of a-SMA (about 50%) and underwent dramatic morphological changes.

Replacement of serum by the supplement BIT9500 affected cell growth in two ways: Firstly, growth as monolayer cultures was changed towards a growth pattern that resembled formation of foci. Secondly, proliferation was impaired and eventually stopped after 2 to 3 weeks. Adding back FCS during the first two weeks restored normal growth pattern and proliferation, indicating that the culture had not undergone terminal growth arrest or differentiation.

Growth supports such as fibronectin and laminin or otherwise coated flasks and glass coverslips did not affect growth or marker expression.

The diverse cell populations generated in this manner were then tested for spontaneous differentiation markers by immunocytochemistry and RT-PCR.

This intriguingly differentiation potential of progenitor cells contained in normal, aging human scalp skin (mean age: 50+/- 5 years) warrants systemic follow-up, since it raises the possibility that adult human skin-derived progenitor cells can serve multiple cell-based therapy purposes for regenerative medicine.

(JSRM Code:002010700095)

Isolation and characterization of dental follicle precursor cells (DFPCs)

Vollner F1, Driemel O2, Reichert TE2, Morsczeck C1
1Institute of human genetics, University of Regensburg, Franz-Josef Strauss Allee 11, 93053 Regensburg, Germany
2Department of Oral and Maxillofacial Surgery, University of Regensburg, Franz-Josef Strauss Allee 11,
93053 Regensburg, Germany

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Previously our group reported the isolation of precursor cells (DFPCs) derived from the dental follicle of human third molar teeth, which are fibroblast-like, colony forming and plastic adherent cells (1). DFPCs are unique undifferentiated lineage committed cells residing in the periodontium prior or during tooth eruption. For DFPCs the differentiation capacity was demonstrated under in vivo and in vitro conditions (1,2). Long-term cultures with DFPCs containing dexamethasone produced compact calcified nodules or appeared as plain membrane structures of different dimensions consisting of a connective tissue like matrix encapsulated by a mesothelium-like cellular structure. Our work demonstrated also differentiation of DFPCs with an insulin-based protocol (2). However, mechanisms for osteogenic differentiation are not known in detail. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro (3). Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures (3). We suppose that molecular mechanisms in dental follicle precursor cells during osteogenic differentiation are different from those in bone marrow-derived mesenchymal stem cells. In recent times, we test different conditions for the isolation and proliferation of DFPCs before we investigate for example their differentiation capacities into osteoblast-like cells or neural-like cells.

References:
1. Morsczeck C, Gotz W, Schierholz J, et al. (2005). Matrix Biol. 24(2),155-65
2. Morsczeck C, Moehl C, Gotz W, et. al (2005). Cell Biol Int. 29(7),567-75
3. Morsczeck C. (2006). Calcif Tissue Int. 78(2):98-102

(JSRM Code:002010700096)

Transcriptional profiling of in vitro erythroid progenitors

Lazar-Karsten P, Dorn I, Driller B, Schlenke P
Institute of Haematology and Transfusion medicine, University of Luebeck, Luebeck, Germany

Introduction: The mechanisms of nuclear extrusion, the unique ability of Erythrocytes, are poorly researched. For a better understanding of erythroid differentiation and enucleation process, we performed an oligonucleotide microoarray analysis of human CD34+ cells during cytokine-induced in vitro erythropoiesis. Several genes were found to be differentially up- or down-regulated during erythropoiesis belonging to different functional categories.

Material and Methods: Human CD34+ cells were isolated and cultured over 21 days in a three-phase liquid assay (d1-7: SCF, TPO, FLT3-L; d8-14: SCF, EPO, IGF-1; d15-21: EPO). Cell growth and differentiation was evaluated by flow cytometry and cytospin preparations. Cells were harvested on days 7, 10, 14 and 17 and RNA was isolated. Gene expression was analyzed by Miltenyi Biotech using topic-defined PIQR Stem Cell Microarray. The measurements were compared to day 0 as a control. A pathway analysis was performed to identify genes which are involved in erythroid differentiation, such as genes involved in pro- and anti apoptotic and enucleation processes.

Results: A total of 4 experiments were performed for the gene arrays. At the end of the culture more than 95% of the cells were glycophorin A positive and therefore clearly of erythroid nature. According to cytospins, cells showed morphological characteristics of normoblasts (43%) and reticulocytes (52%).

Several genes and gene families were identified as being differentially regulated during erythropoiesis. Genes belonging to ribosome cluster were significantly down-regulated during erythropoiesis with 18 genes including DDX21. Other annotations which were enriched were mitosis and stress with 14 genes (including BUB1B) and 42 genes (including NFκ-family), respectively.

Discussion and Conclusion: Associated with an appropriate in vitro erythropoiesis assay, we were able to identify a large amount of genes (about 73) which were significantly up- or down-regulated. RT-PCRs of mRNA were performed to reproduce our observation. Further experiments should confirm our preliminary observations during RBC development.

(JSRM Code:002010700097)

Steering embryonic stem cell fate towards osteoblasts with downstream non-canonical wnt targets in vitro

Taube S, zur Nieden NI
Fraunhofer Institute for Cell Therapy and Immunology, Group Applied Stem Cell Technologies,
Deutscher Platz 5e, Leipzig, Germany

Introduction: Embryonic stem cells (ESCs) have gained significant attention over the last few years. Due to their unlimited proliferation potential and pluripotency they represent a cell population that could be potentially used in cellular therapies for the treatment of otherwise incurable diseases, such as degenerative bone diseases. The classical non-canonical wnt molecule wnt5a has been associated with bone development, but three sub-pathways exist that vary depending on the cell context. Here, we have characterized the downstream targets of wnt5a in ESCs during the course of osteoblast differentiation.

Material & Methods: We have used murine ESCs as a model to investigate how wnt5a affects normal osteogenic development thereby identifying molecular processes that could be used to drive differentiation more efficiently in vitro. Previously, we have successfully shown that recombinant wnt5a protein can support osteogenesis when administered during the time of osteoprogenitor commitment during ESC culture. In the current study, we used a reporter wnt5a::GFP ESC line as readout to characterize prospective downstream targets of wnt5a. Specifically, inhibitors to PKC and CamKII were administered to ESC cultures.

Results: Proliferation, cAMP levels and gene expression was measured in early osteoblasts and could be directly correlated to the degree of mineralization in mature osteoblasts. Specifically, proliferation of osteoprogenitors seems to be regulated by PKC, whereas osteoblast-specific gene expression is controlled through CAMKII.

Discussion and Conclusion: Eventually, our results suggest that the non-canonical wnt signaling pathway regulates osteoprogenitor maturation through PKC and CamKII.

(JSRM Code:002010700098)

Analysis of STAT3-activity in embryonic stem cells and during early development of Oryzias latipes

Kraussling M, Wagner TU, Schartl M
Physiological Chemistry I, Biozentrum, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

Introduction:, In order to gain insights in the mechanisms of “stemness”, an important aspect in stem cell research is, how the maintenance of the potential for self-renewal and the prevention of spontaneous differentiation in stem cells is achieved. In murine embryonic stem (mES) cells, one member of the family of signal transducers and activators of transcription proteins, STAT3, is needed to keep mES cells in the ?stemness“-status. The latent transcription factor STAT3 is activated by the leukaemia inhibitory factor (LIF) and consequently imported into the nucleus. In contrast to mES-cells, activated nuclear STAT3 is neither necessary nor sufficient to keep human embryonic stem (hES) cells in the undifferentiated status.

In this work, we analyzed the localization and thereby the activity of STAT3 in embryonic stem cells of medaka fish (MES) and during early embryonic development of medaka.

Material and Methods: The embryonic stem cell line MESI was obtained from blastulae of medaka fish (Oryzias latipes) and remains undifferentiated in cell culture in presence of a complex mix of growth factors. STAT3-activity in MESI-cells and early embryos of medaka fish was investigated via immunofluorescence.

Results: Ectopic expression of an eGFP-tagged STAT3 resulted in dominantly cytoplasmic and thus inactive localization in MESI.

Analyses in medaka embryos of blastula-stage demonstrated that STAT3 is inactive in all cells.
Investigations on embryos before 1024-cell-stage revealed that STAT3 has a wave-like import into the nucleus between 64-cell-stage and 512-cell-stage with a peak at 64-cell-stage. No increase of nuclear STAT3-levels was detected before 64-cell-stage or after 512-cell-stage.

Discussion and Conclusions: The assays on MESI cells and blastula-staged medaka embryos demonstrated that STAT3 is not activated in medaka stem cells. Consequently, these data indicate that activated STAT3 is not necessary for stem-cell status in medaka. Hence, the mouse-system is the only vertebrate stem cell system depending on active STAT3.

Furthermore, the import-wave of STAT3 spanning 64-cell-stage to 512-cell-stage medaka embryos argues for a specific process in medaka development which is depending on nuclear presence of activated STAT3.

(JSRM Code:002010700099)

Engineering and differentiation of stem cell sheets

Semenov OV1,2, Voros J2, Zisch AH1
1Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland
2Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH-Zurich, Zurich, Switzerland

Introduction: Cell Sheet Engineering is emerged as a novel technique which allows creating of replacement three dimensional tissue structures without the use of biodegradable scaffolds. It was already showed, that cell culture surfaces based on temperature responsive polymer films allow for the none enzymatic harvesting of viable Adult Cell Sheets, which can be used for tissue reconstruction and transplantation surgery.

Materials and Methods: The experimental method is based on growing confluent sheets from Human Placenta Mesenchymal Stem Cells [HPMSC] on conducting substrates, for example [ITO] coated with polyelectrolyte multilayer films consisting of Poly-L-Lysine [PLL] and Hyaluronic Acid [HA]. After reaching confluence intact dense stem cell sheets will be either directly differentiated into adult cell types and then detached from the surfaces of substrates or alternatively firstly detached from the surfaces of substrates by applying of potential and then differentiated as intact three dimensional structures.

Results: Dense viable stem cell sheets were created on the experimental conducting substrates coated with polyelectrolyte multilayer films of different compositions. Experimental substrates were stratificated by analysis of viability, vitality, grade of confluence, amount, and morphology of stem cells. The ability of confluent viable stem cell sheets to be differentiated into adult cell types, for example Chondrocytes, Osteocytes or Cardiomyocytes will be analyzed.

Discussion and Conclusions: Cell Sheet Engineering is a new promising and universal methodology that allows obtaining intact assemblies of stem cells which can be harvested from the substrates and differentiated into three dimensional adult tissue structures destined for transplantation. This method can also be applied for construction of cell sheets from the adult cell types to obtain three dimensional tissue structures for assembling of complex tissue models, such as an in vitro model of adult angiogenesis, or for drug screening applications.

(JSRM Code:002010700100)

In vitro induction of endothelial cell differentiation of adipose tissue derived stem cells (ADSC) and vascularisation in dermal skin equivalents

Lorenz K1, Rupf T1, Bader A1
1Department of Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine, University Leipzig, Germany

OBJEKTIVE: The wound coverage of extensive burn injuries and other deep skin defects represents a major problem in dermatology. The main problem is set by low availability of autologous material provided by the patient for covering the site of injury. Therefore the development of skin equivalents for transplantation consisting of dermal and epidermal components to support the regeneration of skin is of high interest. The aim of our work is to support the engraftment of skin equivalents by implanting endothelial vessels in the dermal layer of skin transplants.

METHODS: ADSC were immobilised in a bioartificial collagen-elastin matrix and cultured under dynamic flow conditions in the presence or absence of VEGF (Vascular Endothelial Growth Factor). Analyses of numbers and deepnesses of vessels were performed by cLSM (confocal Laser Scanning Microscopy). Furthermore, mRNA profiles for KDR (VEGF receptor 1), FLT-1 (VEGF receptor 2), VE-Cadherin (Vascular Endothelial) and VCAM (Vascular Cell Adhesion Molecule) were analysed. The respective proteins were also detected as cell-surface endothelial specific proteins and proved by histological immunfluorescence techniques.

RESULTS AND DISCUSSION: Our results show that the flow rate and the VEGF concentration have a high impact on the development of vessels formed by ADSC in the matrix. The vessels formed by ADSC under flow rates of 1.2ml/24h and VEGF concentration of 50ng/ml were about 100μm to 130μm deep and stable for several days. Most important, ADSC differentiated into endothelial cells and formed vessel like structures under these conditions.

This very successful in vitro vascularisation of dermal skin equivalents with endothelial differentiated ADSC may offer the possibility to support the engraftment of skin transplants by advancing the neovascularisation in vivo.

(JSRM Code:002010700101)

Co-culturing of in vitro erythropoiesis with extra cellular matrix proteins

Meyer G, Lazar-Karsten P, Lindner U, Driller B, Dorn I, Schlenke P
University of Luebeck, Immunology and Transfusion Medicine, Ratzeburger Allee 160, 23538 Luebeck, Germany

Objective: Erythropoiesis proceeds in so called hematopoietic microenvironments or niches, in which stroma cells produce extra cellular matrix molecules. To investigate the influence of these molecules on proliferation and differentiation we used an established in vitro Erythropoiesis system in co-culture with different matrix proteins.

Material and Methods: Human CD34+ cells were isolated and cultured over 16 days in a two-phase liquid assay (d1-d8 SCF, EPO, IGF-1, steroids; d9-d16 EPO, insulin) in 6 well plates coated with Laminin, Collagen, Fibronectin or Extra Cellular Matrix (ECM). An uncoated plate was used as a negative control. Cell viability was determined by trypan blue staining and microscopic examination. To measure cell proliferation, vital cells were enumerated using a Neubauer counting chamber. Cell growth and differentiation was evaluated by flow cytometry and cytospin preparations on each day of analysis.

Results: A total of 5 experiments were performed. At the end of the culture more than 95% of the cells of the control culture were glycophorin A positive and therefore clearly of erythroid nature. According to cytospins, cells showed morphological characteristics of normoblasts (50%) and reticulocytes (45%). The cells cultured with ECM showed the best vitality (91%) compared with the other cultures. The proliferation rate assessed in different culture conditions showed no significant difference. On the last day of culturing the control culture showed the highest amount of erythroid cells compared to cells cultured on proteins.

Conclusion: The hematopoietic niche or microenvironment provides in vivo the optimum condition for proliferation and differentiation of erythroid precursers. Our aim was to optimize the in vitro Erythropoiesis by co-cultivating the erythroid precursor with extra cellular matrix proteins. However our experiments showed that the in vitro Erythropoiesis could not be optimized by adding matrix proteins to the culture.

(JSRM Code:002010700102)

Human dermis-derived cells demonstrate strong features of controlled mesodermal differentiation

Tiede S1, Danner S 3, Tronnier V 2, Paus R1, Zechel C 2
1Department of Dermatology, Allergology and Venerology, UK-SH, Campus Lubeck, Lubeck, Germany
2Department of Neurosurgery, UH-SH, Campus Lubeck, Lubeck, Germany
3IBMT, Fraunhofer-Institute, Lubeck, Germany

Introduction: Multiple tissue niches in the human body are now recognized to harbour adult stem cells. Here, we have examined a panel of putative stem cell/progenitor markers on progenitor cells derived from mechanically and enzymatically processed adult human scalp skin, which may serve as a convenient and abundant source of adult human progenitor cell populations.

Material and Methods: After removal of the epidermis, adherent growing cells of a purposely heterogenous mixture of intradermal cells (including sweat glands, fibroblasts, and hair follicle-derived cells), could be propagated for more than 20 passages in serum-supplemented medium. The diverse cell populations generated in this manner were then tested for spontaneous differentiation markers by immunocytochemistry and RT-PCR.

Results: When grown to confluency a small subfraction of the cells (less than 1%) within the culture began to express low levels of SMA. These Sma+ cells did not undergo significant morphological changes.

The cells could be also maintained in media containing FCS and the mitogens bFGF, EGF or PDGF AB. Interestingly, human skin-derived stem/progenitor cells obtained in this manner exhibited a strong commitment to the mesodermal lineage, since a large subfraction of them expressed high levels of a-SMA (about 50%) and underwent dramatic morphological changes.

Replacement of serum by the supplement BIT9500 affected cell growth in two ways: Firstly, growth as monolayer cultures was changed towards a growth pattern that resembled formation of foci. Secondly, proliferation was impaired and eventually stopped after 2 to 3 weeks. Adding back FCS during the first two weeks restored normal growth pattern and proliferation, indicating that the culture had not undergone terminal growth arrest or differentiation.

Growth supports such as fibronectin and laminin or otherwise coated flasks and glass coverslips did not affect growth or marker expression.

Discussion and Conclusion: This intriguingly differentiation potential of progenitor cells contained in normal, aging human scalp skin (mean age: 50+/- 5 years) warrants systemic follow-up, since it raises the possibility that adult human skin-derived progenitor cells can serve multiple cell-based therapy purposes for regenerative medicine.

(JSRM Code:002010700103)

Cholinesterases and cholinergic system in embryonic stem cell regulation: data on gene expression and functions

Paraoanu LE1, Boutter J1, Landgraf D1, Barth M1, Wessler I2, Layer PG1
1Institute of Zoology, Darmstadt University of Technology, D-64287 Darmstadt, Germany
2Institute of Pathology, University of Mainz, Germany

Introduction: How about cells that are ontogenetically completely immature and far from being “neuronally” differentiated, do they have a cholinergic system, and if so, what does this mean functionally? It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independent from innervation. Here, we analysed the expression and possible functions of the cholinergic system in the embryonic stem cells (ESCs).

Materials and Methods: In order to better define the onset of cholinergic markers expression during development, we examined their expression in two murine embryonic stem cell lines (CGR8 and D3) and in differentiating embryoid bodies (EBs) by HPLC, RT-PCR, histochemistry and enzyme activity measurements; their functions were analysed by acetylcholine (ACh) supplementation, by inhibition of muscarinic and nicotinic ACh receptors and of ChEs and by overexpression of AChE.

Results: ESCs expressed all cholinergic components, including ACh and its receptors. Amounts of pmol ACh could be detected in both stem cells lines and increased during EB differentiation. Acetyl- (AChE) and Butyrylcholinesterase (BChE) mRNA and activity were present already in undifferentiated ESCs; BChE was always higher than AChE. Along with differentiation into EBs, AChE mRNA and activity increased. A significant increase in cell viability and proliferation was observed when ACh was added to the medium, the effect being concentration-dependent; specific inhibition of cholinesterases led to the same results.

Discussion and Conclusions: This suggests that ACh functions as a signal molecule in ESCs, controlling basic cellular processes like cell proliferation and cholinesterases act as regulators of its concentration. With these results we could show that mouse embryonic stem cells, a non-neuronal tissue, express a cholinergic system; its functions are not related to synaptic transmission, but to developmental processes.

(JSRM Code:002010700104)

Studies on improving the embryonic stem cell test for an assessment of neuroembryotoxic effects

Miechielsen N+, Merz F+, Heselich A+, Kunz S*, Heinzel-Wieland R+, Pollet D+
+Department of Chemical Engineering and Biotechnology, University of Applied Sciences, Darmstadt, Schnittspahnstrasse 12, 64287 Darmstadt, Germany
*RCC Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Introduction: The Embryonic Stem Cell Test (EST) represents a validated in vitro system for the classification of chemical compounds according to their embryotoxic potential. It is based on the evaluation of beating cardiomyocytes in embryoid body (EB) outgrowths in comparison to cytotoxic effects on D3 murine embryonic stem cells and 3T3 fibroblasts. To improve the predictive potential of the EST and to avoid false-negative classifications, further cell type-specific endpoints of differentiation have to be established. Recent attempts employ RT-PCR methods or FACS analyses based on antibody staining of neuronal markers, but these techniques are cost-intensive and need a lot of hands-on time. Both aspects are disadvantageous in future routine applications. Hence we tested the feasibility of detecting neuronal differentiation by using a simple fluorescent staining of Nissl substance, which is specific for neuronal cells and can therefore be used to distinguish them from non-neuronal cells.

Materials and Methods: EBs were produced from D3 suspension cultures within 4 days. Neuronal differentiation was induced by incubating EBs for 4 days with retinoic acid (RA). Then EBs were transferred to TC-dishes and further cultured for at least 9 days. To detect neuronal cells in EB outgrowths, NeuroTrace? fluorescent Nissl stain was used together with DAPI. To confirm the specificity of staining, a coculture of embryonic neuronal cells from chicken retina with a human Nissl-negative keratinocyte cell line was used. Additionally, RT-PCR was performed to monitor the expression of other neuronal markers such as tyrosine hydroxylase.

Results: RA treatment of EBs led to a pronounced spot-like staining of the Nissl substance similar to that usually found in fully differentiated neuronal cells. A few neuronal cells spontaneously developed also in RA-untreated EBs and could clearly be detected by Nissl staining. In monolayer cocultures, a weak homogeneous background staining of nuclei and cytosol could also be observed in HaCaT keratinocytes, but chicken embryo retina cells displayed a significantly stronger but still homogeneous staining of cells.

Discussion and Conclusion: A reliable identification of neuronal cells by fluorescent Nissl staining is possible in combination with morphological criteria. For future applications in routine testing, image analysis employing threshold-based algorithms or FACS analysis should be used to standardize the evaluation procedure.

(JSRM Code:002010700105)

Embryonic stem cell test for assessing embryotoxic effects in vitro

Merz F+, Haus A+, Kunz S*, Pollet D+

+Department of Chemical Engineering and Biotechnology, University of Applied Sciences, Darmstadt, Schnittspahnstrasse 12, 64287 Darmstadt, Germany

*RCC Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Introduction: To obtain information on the embryotoxicity of compounds, testing is performed in vivo using pregnant rats or in vitro using cultured embryos or cells. Taking advantage of the potential of embryonic stem cells to differentiate in culture, the embryonic stem cell test (EST) with a permanent murine embryonic stem cell line (D3) has been developed. This test is based on the determination of several indicators of embryotoxicity as the inhibition of differentiation is combined with the study of differences in the cytotoxic response of embryonic and adult cells.

Materials and Methods: The differentiation assay with D3 cells was performed after adding several concentrations of test compounds according to the validated EST protocol, published by ECVAM, 2002. After 10 days of exposure, the number of EBs with beating myocards was assessed microscopically. In parallel, cytotoxicity assays were performed with embryonic D3 cells and 3T3 fibroblasts, representing adult tissue.

Results: 4 model compounds were examined: penicillin G (non-embryotoxic in vivo), LiCl (weakly embryotoxic), retinoic acid and 5-fluorouracil (both strongly embryotoxic). The results obtained with these agents were in line with previously published data. However, the concurrent cytotoxicity tests for discriminating between cytotoxic and specifically embryotoxic effects turned out to be a confounding factor. The addition of the test compounds shortly after passage of the cell cultures (as described in the ECVAM protocol) strongly increased their apparent cytotoxicity. This was particularly the case with t-chlorobutanol, another compound tested. It is widely used as a drug preservative but its embryotoxicity is still under investigation [Smoak, 1993].

Discussion and Conclusions: The observed increase in cytotoxicity presumably is either the result of an interference of the agent with cell adhesion and spreading or of a much higher sensitivity of freshly passaged cells due to sublethal plasma membrane damage. Either way, this effect resulted in an overestimation of cytotoxicity for some test compounds thereby giving rise to an erroneous underestimation of embryotoxicity. Thus, in routine testing this confounder would increase the risk of generating false-negative results regarding the embryotoxicity of test compounds. We propose that this particular aspect of the present EST protocol should be subjected to further investigations.

(JSRM Code:002010700106)

Expression of parathyroid hormone receptor 1 (PTH1-Rc) in bone marrow derived mesenchymal stem cells (MSC).

Bahar H1,2, Benayahu D1, Shefer G2, Yaffe A1, Binderman I 1
1Department of Oral Biology, Maurice and Gabriela Goldschleger School of Dental Medicine
2Department of Cell and Developmental Biology, 'Sackler' Faculty of Medicine, Tel Aviv University, Israel
3Haddasah School of Dental Medicine, Hebrew University, Jerusalem, Israel

PTH is an effective anabolic agent of bone in patients with osteopenia. Recently, the presence of PTH1-Rc was demonstrated during early stages of antler bone regeneration. We also found that PTH1-Rc is present in mesenchymal cells derived from fresh marrow transplanted into demineralized bone matrix cylinder (DBM) in a rat ectopic bone regeneration model, during early stage of endochondral bone formation. Elevated expression of PTH1-Rc was detected at days 3 and 7 where mesenchymal cells from bone marrow are adhering on the DBM surface, proliferate and produce osteo and chondro progenitors. At later stages, the level of PTH1-Rc expression is relatively reduced. We further studied the temporal expression of PTH1-Rc in marrow derived MSC in culture. Bone marrow was obtained from 2 month old DA rat femurs and plated at 9x105 cell/cm2. After 4 days in culture, 10-8M dexamethasone was added to half of the dishes. Immunostaining of PTH1-Rc was performed after 7, 14 and 21 days in culture. We observed that at day 7 most of the cultured cells expressed PTH1-Rc. At 14 and 21 days, PTH1-Rc was expressed mostly in small, round cells while in the more differentiated fibroblast-like cells the receptor was not detected. In dexamethasone treated cultures the expression of PTH1-Rc declined. Taken together, we propose that PTH and PTHrP increase the pool of osteo-progenitors by activating the PTH1-Rc on MSCs.

(JSRM Code:002010700107)

Engineering and differentiation of stem cell sheets

Semenov OV 1,2, Voros J 2, Zisch AH 1
1Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland
2Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH-Zurich, Zurich, Switzerland

Introduction: Cell sheet engineering has emerged as a novel technique for creation of artificial, three-dimensional model tissues without the need for biodegradable scaffolds. Others could show that cell culture on temperature-responsive polymer films allows non-enzymatic harvesting of intact mesenchymal stem cell sheets, which can be further surgically transplanted for tissue reconstruction (Miyahara et al. Nat Med 2006; 12:459-65). We have begun to explore new methodology that builds on the use of electric charge to accomplish non-enzymatic harvest of cell sheets.

Materials and Methods: We used charge-sensitive arrangements of polyelectrolyte multilayer films (PEMs) that were obtained by piling up to 8 alternating layers of poly-L-lysine (PLL) and hyaluronic acid (HA). Some PEM layers were additional formed with a top layer of PLL-PEG-RGD or adsorbed fibronectin. Tests were performed with placenta-derived mesenchymal stem cells (HPMSC). The cells were cultured for 48hrs, then examined for morphology (phase microscopy), viability (life/death stain) and vitality (WST-assay).

Results: Stratification of nine different surfaces revealed following principal requirements (1) HPMSC adhered and grew out on HA but not on PLL surfaces (2) Fibronectin precoating, or alternatively, PLL-PEG-RGD layers, are necessary to mediate efficient adhesion and outgrowth.

Discussion and Conclusions: These are first steps into development of new technology for cell sheet engineering. We showed that the charge-sensitive arrangements of PEM cell do indeed function as substrates for human mesenchymal stem cells. Next steps are whether stem cell sheets grown on PEMs can be differentiated prior to transplantation which is under current investigation.

(JSRM Code:002010700108)

Gene expression profiling of murine ES cell-derived endothelial cells

Mariappan D, Winkler J, Chen S, Schulz H, Hescheler J, Sachinidis A
Institute of Neurophysiology, University of Cologne, Cologne, Germany

Purification of endothelial cells and identification of genes involved in endothelial differentiation are of great interest as it could be beneficial for therapeutic applications. Mouse embryonic stem (ES) cells serve as a potential source of endothelial cells (EC) for transcriptomic analysis. ECs were isolated by immuno-magnetic separation using platelet endothelial cell-adhesion molecule-1 (PECAM1, also known as CD31). When plated on matrigel, CD31+ cells were able to form capillary-like structures. Immunostaining also revealed the presence of endothelial-specific markers like CD31, VE-cadherin and the characteristic ability of the endothelial cells to uptake DiI Ac-LDL. Quantitative and semi-quantitative PCR analysis further confirms the presence of endothelial markers along with hematopoietic markers. In order to explore the known and novel transcripts involved in vasculogenesis and angiogenesis, large-scale microarray analysis were performed to determine the expression profiles of CD31+ cells which have been compared with murine endothelial cell line (1G11 cells). Several differentially expressed genes were identified which includes some well-known genes important for endothelial differentiation, angiogenesis and hematopoiesis. Moreover, our gene ontology (GO) analysis from the complete transcriptome have identified several genes involved in the signalling transduction pathways and the biological processes that are enriched in CD31+ endothelial cells giving an insight into the cellular and molecular mechanisms underlying the vascular development in mouse ES cells. Further investigation in this field can be extended by gain-of-function and loss-of-function analysis that may allow examining the role of genes in vascular development.

(JSRM Code:002010700109)

Fibroblast differentiation: Relevance of the myofibroblast for tissue regeneration

Oberringer M, Jennewein M, Metzger W, Bubel M, Holstein J, Pohlemann T
Clinic of Trauma-, Hand and Reconstructive Surgery, Saarland University, 66421 Homburg, Germany

Introduction: Fibroblasts represent a cell source having the potential of differentiating into other cell types apart from the fibroblast line. Fibroblasts are among those adult cells, which are easily to acquire and a variety of cell culture and tissue engineering methods are already available. Our research activities focus on a distinct differentiation step -from the fibroblast to the myofibroblast (MF)- due to the high clinical relevance. Because the MF is a key player during the development of tissue fibrosis (i.e. liver fibrosis, arteriosclerosis, excessive scarring after wounding) on the one hand, and is believed to support tissue regeneration (organ- and wound healing) on the other hand, it is a promising approach to develop molecular and biochemical methods, enabling the characterization and handling of MF. The detailed knowledge of the process of MF differentiation (MFD) is a prerequisite for subsequent strategies aiming at the development of tissue engineered soft tissue substitutes.

Materials and Methods: Differentiation of fibroblasts to MFs can easily be assessed after cell culture by the detection of α-smooth-muscle-actin among fibroblasts either by FACS or fluorescence immunocytochemistry.

Human wound explants were cultured and the presence of MF among other cell types was characterized and correlated to the healing status (n=10).

In addition the effect of human dermal microvascular endothelial cells (HDMEC) on MFD was investigated in 2D- (n=10) and 3D- (matrigel; n=3) co-cultures.

Our experiments aiming to actively control MF differentiation included the application of transforming growth factor β1 (TGFβ1) in a soluble (n=8) as well as in a covalently immobilized form. Cell culture substrates were two kinds of aldehyde- (n=5, n=6) and epoxy-functionalized (n=6) culture slides.

Results: Enhanced MFD was observed in cultures from human wound explants compared to normal human dermal fibroblasts.

Areas, where fibroblasts were in contact with HDMEC in co-culture showed an enhanced differentiation potential, which was likely influenced by the detected secretion of endothelin-1 by HDMEC. The 3D-growth of HDMEC in matrigel -manifesting in elongated bundles- seemed to be supported by accompanying MF.

Soluble TGFβ1 was most effective in triggering MFD at a concentration of 1 ng/ml. Covalently immobilized TGFβ1 also remained biologically active leading to enhanced MFD.

Discussion and Conclusions: The data show that the characterization of the MFD rate of human wounds is a promising diagnostic tool in the future. Furthermore it is possible to control MFD actively by soluble and covalently immobilized TGFβ1. Investigation of the MFD process also reveals basic differentiation phenomena and strategies transferable to differentiation strategies of stem cells from other origin.

(JSRM Code:002010700110)

Mechanichal stimulation of adipose tissue derived mesenchymal stem cells

Diederichs S1,2, Weyand B3, Kasper C1, Scheper T1, van Griensven M2
1Institut fur Technische Chemie, Universitat Hannover, Callinstrase 3, 30167 Hannover, Germany
2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrase 13,
A-1200 Vienna, Austria
3Klinik fur Plastische, Hand- und Wiederherstellende Chirurgie, Medical School Hannover, Podbielskistrase 380, 30659 Hannover, Germany

Introduction: Mesenchymal stem cells (MSCs) are a widely used cell source in tissue engineering and regenerative medicine due to their ability to be expanded and differentiated easily in vitro as well as in vivo. MSCs can be obtained from a variety of tissues including adipose tissue. This is easy to obtain and it is capable of yielding cell numbers substantial enough to obviate extensive expansion in culture.

Static cell culture with the aim of tissue engineering applications has been proven to be disadvantageous since constructs grown in static cell culture lack the mechanical stability native tissue exhibits. Mechanical stimulation has therefore become a substantial tool in tissue engineering to accustom cells to their future physically active environment. Moreover, differentiation of stem and progenitor cells has been proven to be supported by mechanical strain.
Material and Methods: Adipose tissue derived mesenchymal stem cells (adMSCs) were subjected to a cyclic mechanical strain with frequency of 1 Hz and an elongation of 5%. Time schemes of strain were varied and included 15 min, 60 min, 2 h, 4 h and 8 h and a repetitive strain of thrice 15 min, 60 min, 2 h, 4 h and 8 h with each having an intermission of double the strain time (i.e., 30 min, 60 min, and 4 h, 8 h and 16 h respectively). Osteogenic marker expression (collagen I, cbfa1/runx2, bone sialoprotein 2, Osteopontin, bone morphogenetic proteins 2 and 4, osteocalcin, alkaline phosphatase) after strain was monitored via reverse transcriptase polymerase chain reaction. Collagen III expression was monitored as indication for repair mechanisms induced by the cells after cell damage.

Results: Osteogenic marker expression was unique after each applied time scheme. Considerable differences of osteogenic marker expression compared to control cells were determined after thrice 15 min and once 60 min of strain. Repetition of strain resulted in lower collagen III expression compared to singular strain indicating cellular accustomization.

Discussion and Conclusions: Mechanical strain was shown to have distinct effects on the osteogenic differentiation status of adipose tissue derived mesenchymal stem cells. Different time schemes were applied reaching from singular short time strain to a repeated long time strain. It was shown that strain duration has to be considered carefully for each experimental setup keeping in mind that mechanical load effects cellular processes including differentiation.

(JSRM Code:002010700111)

Comparative study on differentiation characteristics of CD34-negative and CD34-positive human adipose tissue-derived stem cell subpopulations

Peters K1, Salamon A1, Elmaouhoub A1, Adam S1, Rychly J2
1Junior Research Group, Department of Cell Biology, Biomedical Research Centre, Schillingallee 69, 18057 Rostock, Germany
2Department of Cell Biology, Biomedical Research Centre, Schillingallee 69, 18057 Rostock, Germany

Adipose tissue-derived stem cells (ASC) have multilineage differentiation capacity. Therefore, ASC may have the potential to be used for various clinical applications, including bone, cartilage, and cardiac tissue regeneration. Recent studies revealed a cell population within freshly isolated ASC showing a distinct expression of CD34, a well-known marker of hematopoietic stem cells. These cells are able to perform osteogenic and adipogenic differentiation after specific stimulation.

An important step during regeneration of tissues is the vascular supply. It is induced by pro-angiogenic factors (e.g. vascular endothelial growth factor/VEGF) released by tissues requiring blood vessels.

Since ASC are an interesting cell type for tissue engineering applications and since the characteristics of ASC-subpopulations are rather unidentified, we have compared CD34-negative and CD34-positive human ASC regarding their capacity of adipogenic and osteogenic differentiation. Furthermore, the release of VEGF was analyzed in both adipogenic and osteogenic differentiation.

In principle, both ASC-subpopulations performed adipogenic and osteogenic differentiation (quantified by oil red O and alkaline phosphatase staining). Significant variations in the differentia-tion degrees were not detectable. Interestingly, adipogenic stimulation did not lead to significant deviations in VEGF release (assayed by ELISA) whereas osteogenic stimulation induced a sig-nificant downregulation of VEGF release in both ASC-subpopulations by ca. 50%.

Since blood vessel formation is crucial in tissue development and repair, the role of VEGF downregulation during osteogenic differentiation in vitro remains unclear. One explanation could be an intricate regulatory control of VEGF release during the different phases of osteogenic differentiation. Thus, knowledge about the regulation of angiogenesis-inducing factors in ASC-subpopula-tions and osteogenic differentiation of stem cells in general remains fragmentary and needs further investigation.

This work is financially supported by the European Union and the Federal State Mecklenburg-Vorpommern.

(JSRM Code:002010700112)

Microarray analyses and bioinformatic evaluation of the plasticity between human mesenchymal stem cell-derived osteoblasts and adipocytes

Schilling T1, Kuffner R2, Klein-Hitpass L3, Zimmer R2, Jakob F1, Schutze N1
1University of Wurzburg, Orthopedic Clinic, Orthopedic Center for Musculoskeletal Research, Molecular Orthopedics, Wurzburg, Germany
2Ludwig Maximilians University Munich, Institute of Informatics, Department of Bioinformatics, Munich, Germany
3University of Duisburg-Essen, Institute of Cell Biology (Tumor Research), Essen, Germany

Introduction: With aging, the gain of fatty tissue in the bone marrow and the concomitant loss of bone mass are inversely related. Transdifferentiation of (pre-)osteoblasts is likely to contribute to the fatty degeneration. Elucidation of the hitherto unknown molecular mechanisms initiating this process could help to address novel therapeutic targets that inhibit adipogenesis and enhance osteogenesis. After establishment of a cell culture system of human mesenchymal stem cells (MSCs) that allows for transdifferentiation of (pre-)osteoblasts into adipocytes and vice versa, we aimed here to identify gene products that initiate transdifferentiation.

Material and Methods: Isolation of MSCs, adipogenic and osteogenic (trans)differentiation, cytochemical staining and mRNA expression analyses were performed using standard methods. RNA was isolated 3 h and 24 h after initiation of adipogenic transdifferentiation of pre-osteoblasts as well as of osteogenic transdifferentiation of adipocytes and used for Affymetrix microarray analyses. Duplicate microarray analyses were evaluated by signal log ratios and change p-values and re-evaluated by RT-PCR.

Results: Reproducible and often reciprocal regulation patterns for adipogenic and osteogenic transdifferentiation were observed for 414 and 922 genes, respectively. A combined evaluation of all 16 GeneChips was enabled by the development of a bioinformatic scoring scheme that ranked regulated genes according to their potential relevance for initiation of transdifferentiation taking reproducibility, high degree of regulation and distinct reciprocity between both transdifferentiation directions into account. Members of Wnt, IGF and FGF signaling were detected amongst the highly ranked gene products. Functional examination of FGF1 as one of the highly ranked potential key factors confirmed its inhibitory effect on adipogenic transdifferentiation as expected by microarray results.

Discussion and Conclusions: The identification of potential key molecules associated with transdifferentiation of osteoblasts and adipocytes was enabled by the combination of Affymetrix software and further bioinformatic analysis. The principle usability of our ranking was proven by the positive functional testing of FGF1. Further functional examination of individual, highly-ranked genes with reciprocal regulation in the different transdifferentiation directions could uncover novel signaling pathways and factors, which initiate these processes

(JSRM Code:002010700113)

Ensuring defined oxygen levels in stem cell cultivation - a novel approach using DO online monitoring in multiwell plates

Arain S, John GT, Krause C
PreSens Precision Sensing GmbH, Josef-Engert-Str. 11, 93053 Regensburg, Germany

At cultivation of stem cells, defined in-vitro conditions are important. Differentiation largely depends on the biological environment and growth conditions. Therefore, monitoring of essential parameters like oxygen and pH is of vital importance and enables systemic optimization of culture conditions for complex differentiation processes.

Differentiation of haematopoietic and neuronal stem cells needs exact hypoxic conditions to guarantee reproducible results. The SDR SensorDishR Reader enables for the first time non-invasive online monitoring of dissolved oxygen (DO) and pH in 24-well multidishes. Optical sensors for DO or pH are located at the bottom of each well and read out through the bottom of the multidish by a small and robust 24-channel reader. This reader can be placed in an incubator or on a shaker for continuous monitoring during the whole period of cultivation.

The application of the SensorDish Reader is demonstrated with examples for cultivation of human embryonic stem cells and haematopoietic stem/progenitor cells (HSPC). The cells were cultivated directly in the OxoDishR for online oxygen monitoring. Different oxygen levels of the gas phase were applied and the dissolved oxygen in the medium was measured for the whole time of the cultivation. The influence of opening the incubator, lifting the lid of the multidish, and of media change was also investigated as well as different cell concentrations.

With the SDR SensorDishR Reader, negative effects can be detected and resolved in time. Effects which had not been considered before can now be obtained and regarded, e.g. too high oxygen content in the medium can be avoided by media change under hypoxic incubator atmosphere. The non-invasive method of the system guarantees continuous monitoring during the whole cultivation and reduces the risk of contamination.

(JSRM Code:002010700114)

Phenotypic characterization of human umbilical cord stromal cells (UCSC) and their differentiation potential

Schultz U, Low R, Thomsen A, Thoma S.
CellGenix Technologie Transfer GmbH, Am Flughafen 16, 79108 Freiburg, Germany

Introduction: The identification of appropriate cell types is necessary to establish cell-based therapies for muscular dystrophy. From the applicative point of view, the ideal cell population should be: easy to obtain, expandable in vitro to the large number of cells presumably required for systemic treatment (1 x 109 or more), able to reach skeletal muscle through a systemic route, and should be able to differentiate into skeletal muscle cells in vivo with high efficiency. In this context, the resemblance of umbilical cord stromal cells (UCSC) with pericytes, a cell type recently identified as myogenic precursor, prompted us to determine the surface antigen expression and to investigate the differentiation potential of UCSC.

Materials and Methods: Perivascular and stromal cells were isolated from 15 human umbilical cords and expanded in culture until they reached senescence. Cells were characterized by flow cytometry and their differentiation potential was investigated under suitable conditions.

Results: The isolated cell populations consisted of adherent cells with fibroblastoid morphology which could be cultured for >20 passages equivalent to >50 population doublings. Flow cytometric analysis revealed that the cells express mesenchymal stem cells markers (CD73, CD90, CD105) at high levels but do not express hematopoietic/leucocyte antigens (CD34, CD45, CD117, HLA-DR). When properly stimulated, UCSC differentiated into the osteogenic, chondrogenic and adipogenic lineage. Furthermore the cells express markers also expressed by pericyte-like cells (desmin, smooth muscle actin, alkaline phosphatase, CD146). Preliminary results indicate that UCSC can be induced to express myogenin. In addition, they express a number of integrins and adhesion molecules (CD29, CD49a-e, CD51, CD61, CD44).

Discussion and Conclusions: UCSC show an extensive in vitro proliferation which indicates that cells from a single umbilical cord may generate enough cells to treat a paediatric patient with muscular dystrophy. The cells express some of the proteins that enable leucocytes to adhere and cross the endothelium which is a prerequisite for diffusion into the interstitium of skeletal muscles. In addition, UCSC could not only be differentiated into the osteogenic, chondrogenic and adipogenic lineage but seemed to contain precursors with myogenic potential. Because of these features, UCSC might be considered as a valuable source for the isolation of potent cells for future cell therapies of muscular dystrophy.

(JSRM Code:002010700115)

Stem cell markers of the human hair follicle- characterization of a tissue engineered reconstruct based on plucked hairs compared to human scalp

Hartmann A, Derow E, Schuler G
Department of Dermatology, University of Erlangen, Germany

Objectives: In patients suffering from therapy-resistant wounds, transplantation with split-thickness skin grafts is hampered because of the limited availability of donor skin as well as mechanical long-term stability of the grafts. Alternative tissue engineered concepts (EpiDexO) use the hair follicle as origin for the transplantation of follicular keratinocytes which are presumed to correspond to stem cells of the outer root sheath, however; had not yet been characterized upon their stem cell properties.

Material and Methods: Biopsies from human scalp were characterized for the expression of stem cell markers, previously identified in the murine hair follicle. Keratinocytes were isolated and expanded from human plucked hair follicles, according to the EpiDexO-protocol. The expression of keratinocyte precursor cells as well as differentiation markers were analyzed immunohistochemically in multilayered sheets as well as in primary and serial cultures of follicular keratinocytes. By clonality assays, clonogenic potential of serial cultures was investigated morphologically as well as quantitatively.

Results: We could identify cytokeratin 15 (CK15), follistatin, and CD200 as markers for bulge-stem cells of the scalp hair follicle (however, not CD34, nestin, and cytokeratin 19), transferrin and p63 as markers for transient amplifying cells. In EpiDexO-sheets expression of CK15, follistatin, s1-integrin, transferrin, and p63 could be identified according to normal human epidermis. However, during serial culture, number of holoclones diminished and CK15 was almost completely lost within the secondary passage.

Conclusions: Follicular keratinocytes seem to be an appropriate origin to transplant keratinocyte precursor cells. However, mechanisms should be identified to enhance stem cell pool of such transplantation concepts.

(JSRM Code:002010700116)

Generation of organized Lacrimal gland cell spheroids by simulated microgravity

Schrader S*, Kremling C*, Klinger M**, Laqua H*, Geerling G***
*Department of Ophthalmology, University of Luebeck, Germany
**Department of Anatomy, University of Luebeck, Germany
***Department of Ophthalmology, Julius-Maximilian-University Wuerzburg, Germany 

Introduction: A Rotary Cell Culture System (RCCS) allows the creation of a microgravity environment of low shear force, high-mass transfer and 3-dimensional cell culture of various cell types. Aim of this study was to evaluate the growth pattern and the secretory function of rabbit lacrimal gland acinar cells in a microgravity environment using a RCCS.

Methods: Lacrimal gland acinar cells from New Zealand White rabbits of both sexes were isolated and cultured in a RCCS up to 28 days. Cells were analysed by light and electron microscopy at day 7, 14, 21 and 28. Secretory function was tested by measuring the s-hexosaminidase activity.

Results: After seeding to the RCCS, the lacrimal gland cells formed spheroidal aggregates. The acinar cells inside the spheroids retained their histotypic features, but in the center of the spheroids groups of necrotic cells became more abundant during the culture period. The evaluation of the secretory function showed a response to stimulation with carbachol until day 7.

Conclusion: Acinar lacrimal gland cells can be successfully cultured in a RCCS up to 28 days, with a secretory response to carbachol up to 7 days. A simulated microgravity environment allows to maintain long-term cultures of lacrimal gland acinar cells and promises opportunities for further applications in basic and applied cell research on lacrimal gland cells.

(JSRM Code:002010700117)

Amniotic membrane as a matrix for lacrimal gland tissue engineering

Geerling G***, Wedel T**, Kremling C*, Laqua H*, Schrader S*
*Department of Ophthalmology, University of Luebeck, Germany
**Department of Anatomy, Christian-Albrechts-University Kiel, Germany
***Department of Ophthalmology, Julius-Maximilian-University Wuerzburg, Germany 

Introduction: The secretion of the lacrimal gland provides 95% of the aqueous tears, which are essential for lubrication, nutrition and protection of the ocular surface. Long term studies of acinar lacrimal gland cells in-vitro are complicated by low proliferation rate and fast loss of cell function on plastic. Aim of this study was to evaluate the growth pattern and the secretory function of lacrimal gland acinar cells on amniotic membrane in a rabbit model.

Methods: Lacrimal gland acinar cells from Chinchilla Bastard- and New Zealand White rabbits of both sexes were isolated and cultured on denuded amniotic membrane. Cells were analysed by light and electron microscopy. Secretory function was tested by measuring the s-hexosaminidase activity.

Results: Three days after seeding to the amniotic membrane, the acinar cells had attached to each other and formed small cluster. Cell clusters consisted of 2-5 cell layers and the cells showed fine granulation in their cytoplasm, typical for secreting cells. Between day 7 and 14 cell clusters increased in size and acini-like structures with a central lumen were found. Cells showed polarity, with a basal nucleus and apical secretory granules. Between day 21 and 28 still acini-like structures were found inside the cell clusters. Accumulation of secretory material in the central lumen and desmosome formation connecting the apical cell structures was frequently evident. However, the number of cytoplasmatic granules decreased and on parts of the AM cell morphology changed to flat, spindle shaped cells with a small nucleus. Stimulation with carbachol showed a strong s-hexosaminidase release until day 7, with a decreasing secretory function detectable until day 21.

Conclusion: Acinar lacrimal gland cells can be successfully cultured on amniotic membrane up to 28 days, with a secretory response to carbachol up to 21 days. This model may be used for further experimental work, to elucidate cellular mechanisms in normal and diseased lacrimal tissue.

(JSRM Code:002010700118)

Umbilical cord blood-derived cells with embryonic phenotype ? a population of cells with great potential for future clinical applications

Koestenbauer S1, Vanderzwalmen P2, Dohr G1, Zech NH3
1Institute of Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Harrachgasse 21, A-8010 Graz, Austria
2Institute of Reproductive medicine and Endocrinology, Romerstrase 2, A-6900 Bregenz, Austria
3Reproductive Genetics Institute, 2825 North Halsted, 60657, Chicago, Illinois, USA

Introduction: Hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) can easily be collected without harming the patient. Former studies showed that these cells can be turned into cells which share characteristics with embryonic stem cells. We are aiming at optimizing this method using a population of naive cells from UCB for further expansion, differentiation and transplantation in the field of cell based therapies.

Material and Methods: Isolated HSCs from UCB were cultured in-vitro by using a mixture of the cytokines LIF, FGF and TGF. By comparing different culture strategies such as using different media, different amount of additives (Glutamine, FCS) and different mixtures of the mentioned cytokines we study efficient ways to propagate and expand cells with embryonic phenotype. Up to now, cells were analyzed by flow cytometry, immunohistochemistry and RT-PCR using hematopoietic markers and embryonic stem cells markers.

Results: Critical factors in this method are the basic media and the cell concentration. IMDM supplemented with 2 mM Glutamine, 10-20% FCS, and a cell concentration of 5x105/ml showed optimal results. For cell expansion the isolated HSCs were treated with LIF for several days. To promote the expression of embryonic cell markers additional cytokines were introduced. To generate suitable cells for transplantation we also work on strategies to avoid any animal substances in our culture system.

Discussion and Conclusions: It is desirable to set up an easy method to generate a homogeneous population of embryonic-like cells derived from HSCs of UCB in order to use them in future clinical cell based therapies. It seems that our cells express stem cells markers such as SSEA-4 and are expandable. However, a lot more work needs to be done to assure that these cells (and/or their differentiated offspring's) will not cause more harm than good after clinical use (e.g. transplantation).

(JSRM Code:002010700119)

Malfunction of endothelial progenitor cells and endothelial function in patients with Fabry disease

Dietrich B, Karpinski N, Jakob M, Weidemann F, Breunig F, Wanner C, Ertl G, Bauersachs J and Thum T
Julius-Maximilians University, Internal Medicine I / Cardiology, Wurzburg, Germany

Introduction: Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of the enzyme alpha-galactosidase resulting in progressive intracellular accumulation of glycosphingolipids. Deposition of glycosphingolipids in the vascular system may lead to alterations in vascular function and endothelial progenitor cells (EPC).

Materials and Methods: In patients with Fabry disease (n=23; 38.9±12.4 years) and in healthy age-matched controls (n=21; 40.8±8.9 years), vascular function was assessed by peripheral arterial tonometry (PAT) based on finger plethysmography. Vascular function was defined as the ratio of pulse wave amplitude (PWA) during reactive hyperemia relative to baseline. In addition, the duration of increased PWA until return to baseline levels was determined as an additional marker for vasodilatory capacity. The augmentation index was calculated by an automatized determination of the reflected pulse wave as a marker for arterial stiffness. Left ventricular wall thickness was assessed by echocardiography. Vasoregenerative capacity was estimated by investigating number and function of circulating EPC.

Results: Patients with Fabry disease had an increased PAT hyperemia ratio (2.44±0.13 vs 1.96±0.1; p=0.008), whereas the duration of increased PWA during hyperemia was significantly reduced compared with healthy controls (222.4s±54.3s vs 300.5s±52.2s; p<0.01). Fabry patients had a significant increase in arterial stiffness with increasing age, whereas this was not observed in healthy controls. Duration of increased PWA was inversely correlated with increased left ventricular end-diastolic posterior wall thickness (standardized coefficient -0.58, p=0.029). In contrast, PAT hyperemia ratio, basic blood parameters, glomerular filtration rate, gender and age were not correlated with left ventricular hypertrophy. Finally, migratory potential of circulating EPC was impaired by 46±12% (p<0.05) in Fabry patients compared with healthy age-matched controls. Underlying molecular mechanisms and therapeutic options are currently investigated.

Discussion and Conclusions: The present data point to an early pathologic involvement of the vascular system including impairment of the vasoregenerative reserve in Fabry disease. This was may aid our understanding of the progressive development of cardiovascular disease in patients with Fabry disease.

(JSRM Code:002010700120)

Age-related differences in osteogenic differentiation of mesenchymal stem cells in vitro

Bertram H, Wang X, Richter W
Division of Experimental Orthopaedics, Orthopaedic University Hospital Heidelberg, D-69118 Heidelberg, Germany

Introduction: Mesenchymal stem cells (MSCs) have the capacity to proliferate and differentiate into chondrocytes, osteoblasts and adipocytes. For this reason, MSCs are considered to be of great clinical potential in cell based regenerative strategies for the skeletal system. Questions arise to what extent MSCs are subject to age related changes and whether MSCs based strategies should be limited to patients of selected age groups.

The aim of this study was to elucidate the effect of age on MSC-frequency, cell proliferation and differentiation capacity in vitro.

Materials and Methods: Fifteen donors from 7-85 years were involved in this study. The age range was covered by three groups of 5 donors each. Group 1 ranged from 7-12 years, group 2 from 20-55 years and group 3 included 5 donors from 60-85 years. Mononuclear cells from bone marrow were isolated and CFU-F number was determined. Subcultures were prepared to assess single cell cloning efficiency and proliferation rate.

Chondrogenic differentiation was assessed by glycosaminoglycan (GAG) quantification and histologic/immunohistologic evaluation of Alcian blue and Collagen Type II. For osteogenesis, cells were evaluated for membrane bound alkaline phosphatase (ALP) and Alizarin Red S. For adipogenesis, lipid accumulation was quantified by Oil Red O staining.

Results: No correlation was found between CFU-F-frequency or proliferation rate of MSCs and donor age. A negative correlation of single cell cloning efficiency with age was, however, observed (r2= -0,604; p<0,01). While no correlation was obvious between adipogenic differentiation capacity of MSCs and age, activity of bone related ALP (r2=0,413; p<0,05) and calcium deposition (r2=0,721; p<0,001) showed a positive correlation with age. Furthermore we observed significant correlations between ALP activity and calcium deposition (r2=0,6; p<0,01) and between proliferation rate and lipid accumulation (r2=-0,422; p<0,05).

Discussion and Conclusions: MSC proliferative potential and differentiation capacity to adipogenic lineage did not change with age. Osteogenic capacity correlated positively with increasing donor age, which sheads a promising light on the potential application of cell based bone remodelling strategies also in patients of older age. It seems promissing to further study MSC populations from donors of older age to gain a better understanding about the molecular mechanisms regulating age-related changes in connective tissue biology and their possible consequences for regenerative medicine.

(JSRM Code:002010700121)

Personalization by criteria of blood plasma influence on cell population: what can stem cells and their derivatives meet with after their introduction into an organism

Kordium V, Ruban T, Sukhorada O, Gulko T, Likhacheva L, Lysenko S, Deryabina O
Institute of Genetic and Regenerative Medicine, Kiev, Ukraine

Introduction: In different variants of cell therapy with stem cells (SC) and their derivatives, both allogeneic (e.g., obtained from cord blood) and autologous materials are used. And though the last one is “own”, after its multiplication ex vivo for reaching sufficient amount for therapeutic effect, the cells obtained differ from the native cells and therefore are taken by the organism in somewhat other manner. The reaction of an organism on the introduction of allogeneic and natively autologous (but multiplied ex vivo) cells is realized both by direct cell and humoral interactions. Such reaction significantly determines success or failure of the treatment. On the other hand, many factors are involved into the organism response, and it is different in various individuals. To evaluate such response, its personalization is necessary.

Materials and methods: Due to restricted access to the biological material of an individual, we chose influence of blood plasma on cell growth as the most informative from the possible accessible variants. Model objects were rabbits. As the most difficult patients for cell therapy are aged groups, model objects were chosen from the population of healthy rabbits more than 4 years old. Blood serum was testes in 3 variants: native, heated at 56oC during 40 min and filtered through nitrocellulose membrane with pore diameter 0,22 mkm. The last variant actually represented adsorption: plasma volume for filtration was not more than 1 ml and nitrocellulose in non-specific absorber.

The prepared plasma was added to DMEM to the final concentration 0,3 ? 5% with twofold dilutions. Controls were the same concentrations of fetal calf serum (FCS) and medium without any serum. Test culture for control and experimental samples was cell line RK-13 (rabbit kidney). Culturing was performed in standard conditions during 72-96 h. Evaluation was based on growth rate and character as well as on cell morphology.

Results: These showings varied significantly both among different individuals and for the same animal in various time intervals. The diversities varied from complete cell death to proliferation, morphology changing, growth character and attachment to substrate. Growth with FCS was always standard in all experiments.

Conclusion: As plasma concentration in human organism is at least 20 fold higher in comparison with the experiments presented, even moderate differences in SC and their derivatives from the native cells can meet any reaction of the host during their introducing. Even personalization made not directly before introducing, is not always adequate.

(JSRM Code:002010700122)

Impact of donor age on functional behaviour and protein expression pattern of mesenchymal stem cells

Kaper G*#, Mao L+, Geisler S*#, Duda GN*#, Klose J+#
*Musculoskeletal Research Centre Berlin, Charite-Universitatsmedzin Berlin, Germany
+Department for Human Genetics, Charite-Universitatsmedzin Berlin, Germany
# Berlin-Brandenburg Centre for Regenerative Therapies

Bone is a unique and highly regenerative tissue in vertebrates. The process of bone healing and regeneration is thought to be influenced by the action of mesenchymal stem cells (MSCs). It is known from clinical and animal studies that the regeneration potential of bone decreases with age. Therefore, the aim of this study was to analyse the relationship between donor age and the functional behaviour of MSCs. In addition, the underlying molecular mechanisms were investigated by proteome analysis.

MSCs were obtained from 3 weeks (yMSCs) and 12 month (oMSCs) old male Spraque Dawley rats. Cells were isolated from bone marrow of the femoral and tibial bones, and selected by plastic adherence. MSCs were characterised by flow cytometry (CD44+; CD73+, CD90+ and CD45-). The numbers of colony forming units (CFUs) were determined four days after isolation. The migration capacity was measured in a modified Boyden chamber assay. Senescent cells were identified by their s-galactosidase activity. The osteogenic differential potential was analysed by measurement of alkaline phosphatase (AP) and the matrix mineralization (Alizarin Red) after incubation in osteogenic medium. The proteomes of yMSCs and oMSCs after cultivation in expansion media and after an osteogenic stimulus were investigated by high resolution 2D-PAGE followed by mass spectrometric protein identification as described by Klose and colleagues.

No difference in the MSCs typical surface marker profile was observed between yMSCs and oMSCs. In contrast, total number as well as number of AP-positive CFUs was significantly decreased in old animals. The migration capacity of oMSCs was lower than that of yMSCs. Proliferation and differentiation capacity of MSCs were independent of donor age. Higher passage cultures of oMSCs contained higher numbers of senescent cells. The proteome analysis of low passage oMSCs and yMSCs demonstrated differential expression of 34 proteins (expansion medium) and 64 proteins (osteogenic medium). According to functional annotation clustering, age related molecular function were associated with cytoskeleton organization and antioxidant defence. Notably, potential target proteins were found which are known to be associated to bone physiology.

In summery, we could show that MSC number, function as well as protein expression changes with the donor age. Results might be relevant for the development of innovative therapies.

(JSRM Code:002010700123)

Nanotube diameter directs stem cell fate

Park J, Bauer S, Schmuki P, Schlegel K, Neukam F, von der Mark K,
Erlangen-Nuremberg University, Nikolaus-Fiebiger Zentrum, Glueckstr. 6, 91054 Erlangen, Germany

We generated, on titanium surfaces, self-assembled layers of vertically orientated TiO2 nanotubes with defined diameters between 15 and 100 nm and show that adhesion, spreading, growth and differentiation of mesenchymal stem cells are critically dependent on the tube diameter. A spacing less than 30 nm with a maximum at 15 nm provided an effective length scale for accelerated integrin clustering / focal contact formation and strongly enhances cellular activities compared to smooth TiO2 surfaces. Cell adhesion and spreading were severely impaired on nanotube layers with a tube diameter larger than 50 nm, resulting in dramatically reduced cellular activity and a high extent of programmed cell death. Thus, mesenchymal stem cells seems to be very sensitive to nano-scale microenvironment such as a lateral spacing geometry representing 30-50 nm of nanotube diameter as a critical borderline for cell fate.

(JSRM Code:002010700124)

What is the true hypoxic environment for bone marrow mesenchymal stromal progenitor cells?

Buravkova LB, Anokhina EB, Grinakovskaya OS
Institute of Biomedical problems, RAS, Moscow, Russia

Participation of mesenchymal stromal progenitor cells (MSC) in tissue reparation may occur under reduced oxygen tension, the degree and duration of which may determine MSC functional characteristics. We showed that 96h hypoxia (5% O2) had a stimulating effect on rat bone marrow MSC which manifested itself in reduced heterogeneity within MSC cultures, an increase in cell proliferation, a decrease in percentage of damaged cells, maintenance of cells immunophenotype and initial stages of differentiation. Moreover MSC stood long-term hypoxia with no increase in the amount of damaged cells and capability of further differentiation. This may be accounted for the fact that 5% oxygen level simulates in vivo oxygen tension and can be regarded as an approximation of physiological conditions rather than hypoxic impact. The aim of this study was to evaluate the effects of anoxia on rat bone marrow-derived MSC.

Cells used in the experiments were at 1-4 passages and cultivated either in normoxia (95% air + 5% CO2), hypoxia (95% gas mixture of 95%N2 + 5%CO2 and 5% O2) or anoxia (95% N2 + 5% CO2). MSC morphology and proliferation were evaluated using videomicroscopy. MSC viability and surface markers expression were assessed by flow cytometry. Osteogenic and adipogenic differentiation MSC was determined as to alkaline phosphatase (AP) activity or lipid droplets formation within cells respectively in response to differentiation stimuli.

96h anoxia didn’t lead to changes in MSC morphology, proliferation rate and immunophenotype; didn’t increase significantly the percentage of damaged cells despite some activation of apoptosis; didn’t inhibit the initial stages of stimulated adipogenic and osteogenic differentiation despite the suppression of AP activity showing that rat bone marrow MSC is a population resistant to a significant decrease in oxygen tension. Further cultivation of MSC in anoxic environment led to their progressive damage mainly by necrosis in contrast to apoptosis as the main damaging mechanism under 96h anoxia and antiapoptotic effect of hypoxia. It may come in line with the data that proapoptotic effect of low oxygen tension depends on HIF stabilization occurring mostly at less than 5% oxygen tension while antiapoptotic hypoxia effect is provided regardless of HIF.

(JSRM Code:002010700125)

Molecular characterization of human immature third molars: dental stem cells and their niches.

Schoenebeck B1, Hartschen HJ1,2, Schindel M1, Degistirici O1, Siemonsmeier J2, Goetz W3, Thie M1
1Stiftung Center of Advanced European Studies and Research (caesar), Bonn, Germany
2 Zahnklinik Medeco, Bonn, Germany
3Poliklinik fur Kieferorthopadie Bonn, Germany

Introduction: Tooth development is driven by a complex crosstalk between epithelium and neural crest-derived mesenchyme via signalling molecules, transcription factors and influences of the extracellular matrix. Despite knowledge of tooth development in rodents, the knowledge on gene expression important for a comprehensive tooth development in humans remains fragmentary. The aim of this study was to gain more insight into the molecular mechanisms that control differentiation of human tooth, and to clarify whether different compartments (niches) show inherent differences in their expression levels of stemness markers due to different cell populations.

Materials and Methods: Human impacted third molars were divided into the operculum, periodontal ligament, developing pulp and ? as novel ? the pad like tissue beneath the pulp. We characterized the expression level of all compartments by real-time PCR of 16 different genes. In addition we performed whole genome expression arrays to clarify whether the coding of stem cells derived from different compartments is maintained in vitro.

Results: The expression of Msx2 and HNK1 in all compartments confirms their ectomesenchymal origin. With regard to markers for ectomesenchyme and tooth development every single compartment held its own signature of gene expression. The expression of stemness markers such as nanog or Oct4 pointed to multipotent / pluripotent features. The differences in relative gene expression turned out to be dynamic along the progress in tooth development. In vitro, cell cultures derived from dental pulp and pad like tissue showed substantial differences in their respective gene expression profiles on a whole genome scale.

Discussion and Conclusion: Concerning a set of 16 genes leads to the impression of dynamic opposed by rather quiescent compartments. Differences observed in cell cultures derived from pulp and pad like tissue may account to the fact that both compartments share in general the same cell populations but vary with respect to their relative abundance. On the other hand, it is conceivable that cells of both compartments are committed due to instructive signalling of their respective niches. Is has to be explored in further experiments whether this is reflected by differences in e.g. differentiation capacities to other tooth derived stem cells.

(JSRM Code:002010700126)

STAT3 and BMP-receptor 1a functionally interact in different stem cell types

Wagner TU, Meierjohann S, Reiss C, Federov L, Kraeussling M, Thoma E, Kneitz B, Schartl M
Biozentrum, Physiological Chemistry I, University of Wuerzburg, am Hubland 1, 97074 Wuerzburg, Germany

Introduction: Stem cell status is maintained and ended by mixtures of signals received and interpreted by the cells. The often referred to stem cell niche is such a mixture of signals and varies strongly for most stem cell types. Our approach to identifying the signaling mechanisms underlying these different effects include inter- as well as intraspecies comparison of signal-cascade statuses in stem cells.

Materials and Methods: Using an array of different stem cell types from the lower vertebrate medaka (Oryzias latipes) and the mouse, we have compared the signalling activity as well as the signal-complex make-up and its consequences on upkeep of stemness versus differentiation. We used approaches such as co-immunoprecipitation, HPLC fractionation, immunofluorescence and real-time PCR as well as live-imaging of fluorescently tagged signal-mediators such as STAT3.

Results: We were able to demonstrate that the pluripotency of medaka stem cells as well as medaka blastula embroys are independent of STAT3 activity. Furthermore, biochemical analyses of the signalling complexes downstream of BMP2 revealed the presence of STAT3. Next, we found STAT3 modified and re-located to the cytoplasm as a result of ligand-engagement of BR1a in embryonic stem cells. Alteration of the composition of the complex also changed differentiation behaviour of neural stem cells.

Discussion and Conclusion: While our comparison between pathways necessary for pluripotency in both mouse and medaka revealed strong differences both in vivo and in vitro, all the systems analysed thus far share a common, huge signal-integrator complex. Amongst other proteins, this complex includes the BMP-receptor 1a and STAT3. Treatment of stem cells with different BMP ligands results in re-localisation and secondary modification of STAT3. The presented data a hence first evidence for a functional crosstalk between these pathways.

(JSRM Code:002010700127)

Interactions of leukemic stem- and progenitor cells with embryonal stroma environment

Trupschuch S, Pramanik K, Henschler R, Muller AM.
Institute of Medical Radiation and Cell Research, Department of Stem Cell Biology, University of Wuerzburg, Versbacher Str. 5, 97078 Wuerzburg, Germany 

Introduction: Addressing the hypothesis that the murine embryonic microenvironment can induce differentiation in human tumour cells, we developed an in vitro coculture system for established human AML cell lines in combination with a variety of stromal cell lines derived from different murine embryonic hematopoietic sites. Coculture-induced effects contain decreased proliferation and colony formation capabilities, further AML cells show differentiated morphologies and the up-regulation of myelo-monocytic lineage markers. Our aim now is to uncover mechanistical details of molecular and cellular interactions between leukemic cells and their stroma, which may lead to the identification of new paradigms for active control and reprogramming of leukemic cells.

Materials and Methods: Irradiated murine stroma cells were plated to confluence. On the next day, human leukemic cells were seeded onto stromal cells. After 3 days AML cells were harvested from the feeder layer and analysed for the expression of myelo-monocytic lineage markers by FACS. In Flow Chamber experiments as well as by adding neutralising antibodies directed against VLA-4 and VCAM-1 to the coculture system, the influence of these cell adhesion molecules on differentiation status was analysed. Also, cytokine profiles of differentiation-supporting and non-supporting stroma cell lines were prepared and differentially expressed cytokines (e.g. TGF-b) were analysed for their involvement in differentiation induction by supplementation to human AML cell cultures.

Results: Flow Chamber experiments show strongly reduced adhesion of AML cells to the stromal layer when VCAM-1 is blocked on stroma cells. On the other hand, blocking of VLA-4 on AML cells seems not to be effective. Regarding the effect of soluble factors, first results indicate, that TGF-b, though per se not sufficient to induce differentiation in human AML cells, is able to increase coculture-induced effects on differentiation. For other cytokines like TNF-a, RANTES, IL-6 or LIF we could not show any effects so far.

Discussion and Conclusions: Our preliminary results suggest that VCAM-1 but not VLA-4 is involved in adhesion of human AML cells to murine embryonic stromal cells. That poses the question whether VCAM-1 is necessary for the coculture-induced differentiation. The cytokine studies have to be repeated with a broader panel of cytokine concentrations. In addition analyses of participating signal transduction pathways are planned to give more information about differences in signaling in human AML cells upon coculture.

(JSRM Code:002010700128)

Dormant cancer stem cells hibernate in the mammalian brain

Siebzehnrubl FA, Jeske I, Muller D, Buslei R, Hahnen E, Kaesbauer J, Corbeil D,
Huttner HB, Appl T, von Horsten S, Blumcke I
Departments of Neuropathology, Neurology and Experimental Therapy, University of Erlangen-Nuremberg, Germany
Institute of Human Genetics; Institute of Genetics, and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany
Tissue Engineering Laboratories (Biotec), Technical University of Dresden, Germany
Center for Human Genetics, Regensburg, Germany

We generated two tumorigenic cell lines from neurosphere cultures of rodent SVZ stem cells. These cell lines, termed R2303 and R2902, express stem cell markers (such as Nestin, CD133, Musashi-1 and Sox2) and can be differentiated into neuronal and glial phenotypes. However, expression of stem cell markers and proliferation continues when these cells are differentiated. Transplantation of R2303 cells into syngenic, immunocompetent rats demonstrated the potential to form malignant tumors. These tumors showed streak necrosis, invasion into surrounding brain parenchyma and expression of undifferentiated (Nestin) as well as neuronal (Map2) markers. Karyotyping revealed a high grade mosaicism and massive structural and numeric chromosomal aberrations. We identified the platelet-derived growth factor receptor alpha as potential candidate for the continued proliferation of R2303 and R2902 cells, as this receptor is not downregulated during differentiation and is also prominently expressed within tumors. Moreover, RNAi knockdown of the PDGF receptor alpha resulted in growth reduction of R2303 cells. These cell lines may provide a link between brain and cancer stem cells. Furthermore, rapid in vitro expansion of adult stem cells (e.g. for stem cell therapies) needs to be closely monitored for the occurrence of aberrant cell types

(JSRM Code:002010700129)

Embryonic stem cells in rheumatoid arthritis

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

Under observation (9 years) was a group of patients with 1.5 ?20-year history of confirmed rheumatoid arthritis (RA) presenting drug intolerance or inefficiency of routine methods, and marked limitation of functional capacity. For treatment, used were cryopreserved embryonic stem cell (ESC) suspensions containing hematopoietic and non-hematopoietic mesenchymal stem cells. ESC suspensions were prepared from organs of 4-8 weeks old embryonic cadavers obtained from legal abortions. Total nucleated cell count ? 10-400x106/ml, mononuclear cell count ? 10-100x104/ml. After the treatment, all patients reported the Syndrome of Early Post-Transplantation Improvements manifested by decreased general weakness, subsidence of pain, improved and more optimistic mood, improved appetite and normalized sleep formula. This method of treatment allowed for the decrease of the degree of the inflammatory activity, from high (14 points) to minimal (3 points), and achievement of clinical remission in 85% of cases.

Within 1 year, patients under observation reported decrease of pain, joint, and inflammatory indices - main clinical criteria of RA, functional capacity increase manifested by the ability of performing everyday activities impossible prior to this treatment, improved gait, ability to use public transport and continue full-time work. Subsequent transplantations were performed in 62% of cases: 80% of them were aimed at functional capacity and life quality improvement, and only 20% were performed for the decrease of the disease activity.

Immunocorrecting effect of ESC was observed in all the patients. Reported was increase of T-suppressor count (CD8+); helper/suppressor ratio CD4+/CD8+ decreases over a period of 360 days. Prior to the treatment, rheumatoid factor titer amounted to 3.2 U, and within 360 days, it decreased to 1.02 U.

Bioethical principles on application of embryonic cell suspensions were strictly adhered to at all the stages of the research.

(JSRM Code:002010700130)

Comparison of three dimensional scaffolds for bone engineering constructs using human jaw periosteal cells

Alexander D1, Hoffmann J1, Munz A1, Friedrich B2, Geis-Gerstorfer J3, Reinert S1
1Department of Oral and Maxillofacial Surgery
2Department of Internal Medicine IV
3Department of Prosthodontics, Section Medical Materials and Technology, University Hospital Tubingen,
1,3Osianderstr. 2-8, 2Otfried-Muller-Str. 10, 72076 Tubingen, Germany 

Introduction: The variety of commercially available biomaterials for tissue engineering is continually expanding. For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We compared collagen (Coll), open-cell polylactic acid (OPLA) and calcium phosphate (CaP) 3D-scaffolds for their suitability and osteoinductivity of human JPC.

Material and Methods: Human JPC were seeded onto Coll, OPLA and CaP scaffolds (BD Biosciences). JPC proliferation was analyzed by a colorimetric assay. Cell adhesion and spreading was visualized by scanning electron microscopy (SEM). Live-measurements of oxygen consumption within the cell-seeded scaffolds showed maximal time period of in vitro culturing. For further osteogenic differentiation experiments only OPLA scaffolds were used. Gene expression of JPC during osteogenesis was examined by quantitative PCR and element analysis of formed nodules by EDX spectrometry.

Results: Maximal proliferation rates were detected within OPLA scaffolds. 12 days after in vitro culturing we detected the highest oxygen consumption through proliferating JPC within the scaffolds. 15 days after in vitro culturing oxygen concentrations were increasing again, probably due to cell death by apoptosis. SEM clearly showed a greater and multilayered JPC growth and the formation of specific nodules under osteogenic conditions. EDX spectrometry revealed that nodules containted of calcium with or without phosphorus.

Discussion and Conclusions: Polylactid acid seemed to be the most beneficial biomaterial for JPC growth among the analyzed scaffolds. The time period for in vitro culturing should not exceed 14-15 days because of possible transplantation of apoptotic cells. We demonstrated that JPC growing within OPLA scaffolds were able to mineralize in vitro. We concluded that analyzed OPLA scaffolds provide a promising environment for bone substitutes using human JPC.

(JSRM Code:002010700131)

Phenotypic characterization of canine bone marrow stromal cells

Lopez PCM 1,2, Machado MFC1,2, Zamprogno H1,2, Andrade L2, Takiya CM2, Balduino A1,
Duarte MEL1
1National Institute of Traumatology and Orthopaedics, Rua Washington Luiz 61, 20230-020
2Federal University Rio de Janeiro, Av. Brig. Tropowvsky s/n, 21740-940 Rio de Janeiro, Brazil

Introduction: Bone marrow stromal cells (BMSCs) are the major cellular component of the marrow stroma and include the mesenchymal progenitors. Their in vitro adherence ability allows isolation and expansion of multipotent colonies (CFU-F). BMSCs display osteogenic commitment, which can be modulated by culture conditions. The aim of this study was to evaluate the expression of osteogenic phenotypic traits in BMSCs to assess their potential use in tissue-engineering strategies.

Materials and Methods: BMSCs were isolated from canine marrow aspirates after plating at a density of 6-9 x 107 cells per 150-cm2 flasks. After 24 hours medium was replaced and changed three times per week until cultures became ~90% confluent. The number of progenitors was quantified on day 14 by counting CFU-Fs with ? 50 cells. The master gene of osteogenic commitment CBFA1, type I collagen, alkaline phosphatase (ALP) and gene products characterizing the osteoblastic phenotype, such as osteonectin were quantified by RT-PCR. The expression of fibronectin, type I collagen, decorin, biglican, osteonectin, osteopontin and bone sialoprotein (BSP) was investigated on days 7, 14 and 21 by immunocytochemistry and ALP by cytochemistry. BMSCs were also incubated for 21 days under osteogenic conditions to induce Ca deposition that was further analyzed by nuclear fast red staining, scanning electron microscopy (SEM) and by energy dispersive X-ray analysis (EDXA).

Results: BMSCs obtained from CFU-F expansion displayed spindle shape morphology with ~30% of the colonies positive for ALP on day 14. On passaging, the levels of mRNA expression of osteoblast-related markers decreased while CBFA-1 levels remained constant. The expression of osteopontin, BSP, type I collagen and fibronectin did not change substantially over time in culture. Decorin and biglycan were variably expressed. Calcium deposits, detected by nuclear fast red staining as amorphous structures, were confirmed by EDXA analysis and were identified on SEM as 10 μm spheroid structures encased by fibers and extracellular matrix.

Discussion and Conclusions: A number of in vitro methods are employed to determine the differentiation ability and commitment of BMSCs with the osteogenic phenotype. Our data demonstrate that canine BMSCs, after ex-vivo expansion express CBFA-1 and other osteoblast-related gene products that can be taken as evidence of the osteogenic commitment. This biological property strongly supports the use of BMSCs for the reconstruction of bone defects as well as in general veterinary regenerative medicine.

(JSRM Code:002010700132)

Influence of platelet rich plasma (PRP) on chondrogenic differentiation and proliferation of chondrocytes (CC) and mesenchymal stem cells (MSC)

Drengk Aa, Zapf Ab, Sturmer EKa, Sturmer KMa, Frosch KHa
aDepartment of Trauma Surgery, Plastic and Reconstructive Surgery
bDepartment of Medical Statistics, Georg-August-University Goettingen, Germany

Introduction: Our clinical aim is to develop a one-step procedure for autologous chondrocyte transplantation, i.e. harvesting, isolation and reimplantation of CCs performed in one surgical procedure. Consequently there wouldn’t be an increase in cell number. The use PRP, a mitogenic agent, might compensate for this. The aim of this study was to test the influence of PRP on proliferation and differentiation of freshly isolated CCs. In parallel, we submitted MSC to the same experimental set-up.

Materials and Methods: Cartilage from adult sheep was collected and CCs isolated via digestion. After removal of undigested cartilage, cells were directly submitted to the experiment. MSCs were obtained using a standardized protocol, expanded and dealt with analogous to CCs. PRP extracts were produced according to published protocols. We had six different treatment groups: (1) Cells cultured as a micromass (MM); (2) same as (1) but with the addition of activated PRP; (3) cells suspended in a fibrin sealant; (4) cells suspended in a PRP clot; (5) cells in monolayer (ML); (6) same as (5) but with addition of activated PRP. Chondrogenicity was assessed via quantification of collagen type II mRNA (Col_II_mRNA) and immunohistochemical analysis.

Results: MM culture best conserved the chondrogenic phenotype of CCs. Addition of PRP diminished significantly the amount of expressed Col_II_mRNA. Culturing freshly isolated CCs in ML led to a significant reduction of Col_II_mRNA compared to MM; here, addition of PRP had only a weak influence on chondrogenicity. For MSCs, MM culture showed the highest level of Col_II_mRNA. Addition of PRP reduced expression rate. Still, a 3D-growth together with PRP led to a higher expression level of Col_II_mRNA compared to ML.

For both cell types a proliferative effect of PRP was observed

Discussion and Conclusions: Freshly isolated CCs combined with PRP lose their chondrogenic phenotype during in vitro culture. Loss of chondrogenicity seems to be connected with an increase in proliferation. According to our results it might be possible that MSCs combined with PRP could improve the healing of cartilage defects in vivo.

(JSRM Code:002010700133)

Strategies for regenerative mesenchymal stem cell therapies of skeletal disorders: pathways to tendon formation

Gross G, Hoffmann A, Shahab S, Gazit D, Haupl T
Helmholtz-Zentrum fur Infektionsforschung (HZI), Signalling and Gene Regulation, Inhoffenstr. 7, 38124 Braunschweig, Germany

Murine mesenchymal progenitors (C3H10T?) stably expressing BMP2 and the constitutively active SMAD8ca, a member of the family of mediators transferring signals of TGF-beta/BMP growth factors undergo differentiation into cells with tendon and ligament morphology in vitro and in vivo. In addition, adenovirally modified progenitors expressing both Smad8ca and BMP2 generate entire ectopic tendon?bone insertions with an osteotendinous junction exhibiting a fibrocartilage enthesis. Adenovirally modified progenitors expressing Smadca alone develop ectopic tendons without bony elements. These systems were used to repair a partial rat achilles tendon defect.

We also evaluated the capacity to target mesenchymal progenitors to degenerated cartilage surfaces. Chimeric receptors with the ScFv-collagen type II recognition domain as extracellular part and the transmembrane and cytoplasmic domains of BMP receptors type IA and II as signalling domains were hypothesized to initiate chondrogenic differentiation upon cartilage binding. Indeed, in murine C3H10T? the presence of chimeric BMP-receptors is sufficient to induce a massive burst of chondrogenesis which is dependent on the presence of both chimeric receptor types I and II expressed at a high level. Similarly, in a 3D culture system of human mesenchymal stem cells (MSCs), the combination of both chimeric receptors (II+IA) induces chondrogenic differentiation. Further studies will assess the therapeutic potential for these receptors to bring about cartilage regeneration upon cartilage-binding.

These studies are funded in part by the European project STEMGENOS and the European Integrated Project GENOSTEM.

Literature:
Hoffmann, A. et al. (2006). Neotendon Formation Induced by Manipulation of the Smad8 Signaling Pathway in Mesenchymal Stem Cells. J. Clinical Investigation, 116, 940-952

(JSRM Code:002010700134)

Integrational behavior of tissue engineered chondrocyte/MSC-laden hydrogel constructs in an in vitro bovine cartilage defect model

Rackwitz L1,2, Djouad F1, Janjanin S1, Tuan RS1
1Cartilage Biology and Orthopaedics Branch NIAMS, National Institutes of Health,
DHHS, Bethesda, MD 20892, USA
2Orthopaedic Center for Musculoskeletal Research, Konig-Ludwig-Haus, University of Wurzburg, Germany
lrackwitz@gmail.com

Introduction: The success of matrix-based autologous cell implantation for the treatment of articular cartilage defects is dependent on the lateral integration and the deposition of cartilage specific extracellular matrix components. Due to the limited availability of healthy chondrocytes and dedifferentiation during expansion culture, the need for an alternative cell is obvious. In this study, using a bovine in vitro integration model, agarose constructs laden with MSCs, differentiated and dedifferentiated chondrocytes were compared concerning their applicability in matrix-based cartilage repair strategies.

Materials and Methods: Circular native cartilage rings with a centred 4mm hole were fabricated from knee cartilage of calves. In the first group cell-laden agarose constructs (10x106cells/ml) seeded with either de-/differentiated chondrocytes (0, 2, 5 or 8 population doublings), or MSCs were implanted into the cartilage rings. These hybrid constructs were cultured for 28 days in a chemically defined, serum free medium (CM-). In a second group, constructs were initially kept as free-swelling culture in CM- supplemented with TGF-s3 (CM--+) for 21 days and then implanted. Four weeks after implantation lateral integration, biomechanical properties and accumulated glycosaminoglycans/bulk collagen were assessed within the constructs.

Results: Untreated scaffolds seeded with dedifferentiated chondrocytes or MSCs revealed significant lower integration compared to differentiated chondrocytes. In comparison, in the pre-treated group MSC-seeded cell-carriers showed stronger integration into the surrounding native cartilage than those seeded with de-differentiated chondrocytes. Remarkably, pre-treated MSC-seeded constructs showed distinct matrix deposition, mechanical properties and integration that were significantly higher compared to constructs seeded with dedifferentiated chondrocytes.

Discussion: These results show that chondrocytes lose their ability to deposit cartilage specific extracellular matrix components within the implant with increasing number of population doublings, resulting in inferior mechanical properties and integration. In addition, de-differentiated chondrocytes exhibit inferior responsiveness to anabolic factors, such as TGF-s3. MSCs are able to undergo chondrogenic differentiation during the pre-treatment with CM+ and show superior accumulation of cartilage specific extracellular matrix after implantation, supporting their applicability in matrix-based cartilage repair strategies.

(Support: NIH NIAMS Intramural Research Program ? Z01 AR41131)

(JSRM Code:002010700135)

Murine femoral defect model for evaluation of local bone regeneration approaches

Hagedorn GM, Lauer G
Department of Oral and Maxillofacial Surgery, Medical Faculty Carl Gustav Carus,
Fetscherstr. 74, 01307 Dresden, Germany
manolo.hagedorn@uniklinikum-dresden.de

Introduction: Therapeutical concepts of local gene therapy for bony defects need to be verified in vivo. Since permanent genetic alterations, i.e. transgenic animals are realised most easily in mice, an in vivo model for bone healing should be a mouse model, too. Existing mouse fracture models are either externally fixed osteotomies with very narrow bony gaps or internally fixed non reproducible fractures that will always heal spontaneously. This pilot study aimed to establish a mouse model for the investigation of bone healing in a critical size bony defect of the femur.

Materials and Methods: 25 BL/6 mice underwent femoral osteotomy creating externally fixed bony gaps of 3.5mm. Bone healing was studied radiographically and histologically for up to 6 weeks. Fixateur design was optimised to allow for highest reproducibility, widest extension of the bony defect, sufficient mechanical stability, and for stereotactical injection of cell containing material into the defect.

Results: For the established model critical size of the bony defect could be demonstrated for a period of a least 6 weeks. The constructed external fixateur proved to be sufficiently mechanically rigid. The surgical procedure appeared to be safe (loss of one out of 25 mice perioperatively). Injection of bone marrow stromal cells into the defect led to partial ossification within 3 weeks.

Discussion and Conclusions: Standardised critical size bone defects are necessary to evaluate bone healing using gene therapy based therapeutical strategies. With the proposed external fixation bone healing in a highly reproducible critical size bone defect of the murine femur may be analysed for a least 6 weeks. Stereotactical injection into the bone defect of e.g. genetically manipulated cells with increased osteogenic competence is easily possible and qualifies the models for a great variety of experimental settings.

(JSRM Code:002010700136)

Isolation and characterization of multilineage progenitor cells from the anterior cruciate ligament

Steinert AF1, Heymer A1, Barthel T1, Noth U1, Evans CH2, Murray MM3
1Orthopaedic Center for Musculoskeletal Research, Orthopaedic Clinic, Konig-Ludwig-Haus, Julius-Maximilians University Wurzburg, Brettreichstrasse 11, D - 97074 Wurzburg, Germany
2Center for Molecular Orthopaedics, Brigham and Womens Hospital, and 3Department of Orthopaedic Surgery, Childrens Hospital of Boston, Harvard Medical School, Boston, MA, USA

Introduction: In this work, we specifically wished to characterize the differentiation potential of the cells that migrate out of the anterior cruciate ligament (ACL) compared to marrow derived mesenchymal stem cells (MSCs) in order to gain insights how to stimulate a more effective healing response of the ACL cells after injury.

Materials and Methods: Cell isolation: ACL fibroblasts cells were isolated by explant cultures and MSCs were recovered by adhaerent culture of bone marrow aspirate which (informed consent and IRB approval). The recovered cells were placed in monolayer cultures in complete DMEM media. 2nd passage cells were used for all experiments. Cell surface antigens: FACS analyses have been performed using monoclonal antibodies for: CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105, CD106, CD133. Multilineage differentiation cultures: Osteogenesis was induced by monolayer cultures in complete DMEM supplemented with dexamethasone, s-glycerophosphate, ascorbate, and recombinant BMP-2 (25 ng/mL). The adipogenic phenotype was induced by supplementation with dexamethasone, insulin, indomethacine and IBMX in monolayer culture. Chondrogenesis was induced by aggregate cultures maintained in serum-free medium containing dexamethasone, ascorbate, proline, sodium pyruvate, and recombinant TGF-s1 (10 ng/mL). Negative control cultures were also maintained in the respective media without supplements, and all cultures were maintained for three weeks. Phenotype characterization: Lineage specific differentiation was analyzed by RT-PCR (COL I, II, IX, X, SOX-9, ALP, OC, Cbfa1, LPL, PPARg2) and histology and immunohistochemistry (H&E, Alcian Blue, ALP, Oil Red O, COL I, II, X).

Results: FACS analyses revealed positive staining for the markers CD 29, 44, 90, 105, 106 for both cell types, but to a lesser extend in the ACL cells. All other markers were negative for both cell types. Following three weeks of differentiation culture, the ACL cells revealed a strong chondrogenic, adipogenic and osteogenic differentiation potential, such as the MSCs, as shown by the respective histological, immunohistochemical and RT-PCR analyses. In contrast, the respective negative control cultures for the ACL cells and MSCs, which were maintained without any media supplements, were negative for the tissue specific markers.

Discussion: The study was designed to characterize the cells migrating out of the bulk ACL tissue. Our study revealed an almost similar surface antigen expression profile compared to marrow derived-MSCs and an equivalent multilineage mesenchymal differentiation potential of these cells. This might be used in order to augment a favourable healing response toward healing of ACL defects by using e.g. growth factors. Global gene expression analyses (Affymetrix HG-U133_Plus) are underway to further characterize the stemness of the ACL cells on a molecular level.

(JSRM Code:002010700137)

Adult multilineage mesenchymal progenitor cells isolated from the bursa subacromialis

Steinert AF, Heymer A, Kunz M, Jakob F, Noth U, Gohlke F
Orthopaedic Center for Musculoskeletal Research, Orthopaedic Clinic, Konig-Ludwig-Haus, Julius-Maximilians- University Wurzburg, Brettreichstrasse 11, D - 97074 Wurzburg, Germany

Introduction: The subacromial bursa plays key roles in the gliding mechanism of the shoulder and can be efficiently used to augment repair procedures after rotator cuff tears. In this work, we specifically wished to characterize the cells that can be isolated from subacromial bursa tissue and to evaluate their potential to differentiate into the various mesenchymal lineages compared adult marrow-derived mesenchymal stem cells (MSCs).

Materials and Methods: Bursa cells were isolated from 5 bursae subacromiales by collagenase digestion and marrow-derived MSCs were recovered by adhaerent culture of bone marrow aspirate from 5 different donors. Tissues and aspirates were retrieved from patients undergoing rotator cuff repair surgery and total hip arthroplasty after informed consent and local IRB approval. Monolayer cultures were analyzed by flow cytometry (CD34, CD53, CD73, CD90, CD105, CD106, CD133, CD144, CD166, and Stro-1). Osteogenesis was induced by supplementation with dexamethasone, s-glycerophosphate, ascorbate, and recombinant BMP-2 (25 ng/mL), and adipogenesis was induced by supplementation with dexamethasone, insulin, indomethacine and IBMX in monolayer culture. Chondrogenesis was induced by pellet cultures in serum-free medium containing dexamethasone, ascorbate, proline, sodium pyruvate, and recombinant TGF-s1 (10 ng/mL). Negative control cultures were also maintained in the respective media without supplements, and all cultures were maintained for three weeks. The lineage differentiation was analyzed by RT-PCR (COL I, II, IX, X, SOX-9, ALP, OC, Cbfa1, LPL, PPARg2), histology and immunohistochemistry (H&E, Alcian Blue, Alizarin red, ALP, Oil Red O, COL I, II and X).

Results: Light microscopy and flow cytometry revealed a mainly fibroblastic appearance and similar positive stainings for the markers CD 73, 90, 105, 106, 133, 144, 166 for both cell types. Following three weeks of differentiation culture, the bursa cells and MSCs revealed a strong chondrogenic, adipogenic and osteogenic differentiation potential, as shown by the respective histological, immunohistochemical and RT-PCR analyses. In contrast, the respective negative control cultures for the bursa cells and MSCs, which were maintained without any media supplements, were negative for the tissue specific markers.

Conclusion: Cells retrieved from subacromial bursa tissue reveal an almost similar surface antigen expression profile compared to marrow derived-MSCs and an almost equivalent multilineage mesenchymal differentiation potential for all lineages observed. Therefore bursa cells have to be considered adult multilineage progenitor cells. This knowledge might be used in order use these cells to augment of rotator cuff repairs. Global gene expression analyses (Affymetrix HG-U133_Plus) are underway to further characterize the stemness of the bursa-derived cells.

(JSRM Code:002010700138)

Cell-based therapy of fatty degeneration after rotator cuff tears

Wilms A1, Weber M1, Haddad D2, Heymer A1, Basse-Lusebrink T3, Steinert A1,
Jakob P2,3, Noth U1, Gohlke F, Rolf O1
1Orthopaedic Centre for Musculoskeletal Research, Division of Tissue Engineering, University of Wurzburg, Brettreichstrasse 11, 97074 Wurzburg, Germany
2Research Center Magnetic-Resonance-Bavaria, Am Hubland, 97074 Wurzburg, Germany
3Department of Physics, EPV, University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany

Objective: The success of surgical reconstruction of rotator cuff tears is limited due to the lack of ability to heal spontaneously, the degree of muscular atrophy, and the fatty degeneration. Injecting autologous myogenic progenitor cells or multipotent mesenchymal stem cells (MSCs) into the area of fatty infiltration could be a crucial factor in improving the so far unsatisfactory outcome of surgical reconstruction.

Methods: Myogenic progenitor cells and MSCs were isolated using protocols described by Blau & Webster (1981) and Noth et al. (2002), respectively. Both cell types were labelled with very small superparamagnetic iron oxide nanoparticles (VSOPs). The stability of labelling during cell expansion was determined with iron specific histochemical staining (Prussian blue). VSOP labelled, as well as non-labelled control cells were injected into a muscle specimen from a rabbit’s rotator cuff, and magnetic resonance imaging (MRI) was performed using a 7 T high-field MR spectrometer. In the MRI experiments 2D FLASH-sequences with echo times of TE = 8 ms and repetition times of TR = 689 ms were employed. A nominal spatial resolution of 137 x 137 μm2 was achieved with a slice thickness of 1 mm.

Results: Intracellular uptake of iron oxide particles after incubation with VSOPs was shown with Prussian blue staining. Neither the myogenic progenitor cells nor the MSCs showed any loss of labelling within the first 6 cell divisions. Also, the proliferation capacity of both cell types was not influenced by the labelling. After the injection of VSOP labelled cells into a muscle specimen, the cells could be detected successfully with MR imaging.

In a long term study, the VSOP labelled MSCs embedded in a tissue equivalent (collagen type I hydrogel) could be detected for more than 20 weeks using MRI.

Conclusion: Both, myogenic progenitor cells and MSCs were successfully labelled with iron oxide particles and used for MR cell tracking. Running experiments with the rotator-cuff defect model will show if the labelled cells will integrate themselves into the area of fatty degeneration and contribute to the regeneration of the muscle.

(JSRM Code:002010700139)

Iron oxide labeling of human mesenchymal stem cells in collagen type I hydrogels for cellular MR imaging

Heymer A1, Haddad D2,3, Weber M1, Jakob P2,3, Noth U1
1Musculoskeletal Research Center, Orthopedic Department, University of Wuerzburg, Brettreichstrasse 11,
97074 Wuerzburg, Germany
2Research Center Magnetic-Resonance-Bavaria, Am Hubland, 97074 Wuerzburg, Germany
3Department of Physics, EPV, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany

Introduction: The non-invasive monitoring of cell behavior in vivo is still one major issue in cell-based therapies in orthopedic surgery. In particular, detailed information about the localization, proliferation, and differentiation of the transplanted cells in the target tissue is of utmost importance. In this study, magnetically labeled human mesenchymal stem cells (hMSCs) were embedded in collagen type I hydrogels and visualized in vitro using high-resolution magnetic resonance imaging (MRI).

Materials and Methods: Very small superparamagnetic iron oxide nanoparticles (VSOPs) were used to label hMSCs. Particle incorporation was demonstrated using fluorescent dye-labeled VSOPs. Stability of labeling during expansion of cells was determined with iron specific histological staining (Prussian blue). To assess whether the iron oxide particles are incorporated into the cells or only attached to the cellular surface, transmission electron microscopy (TEM) was performed. The influence of VSOP-labeling on the differentiation ability of hMSCs was determined histologically and on the mRNA level. For in vitro MR imaging, different concentrations of both magnetically labeled and unlabeled hMSCs were embedded in collagen type I hydrogels (Arthro Kinetics AG, Esslingen). MRI was performed at different time points using a Bruker 11.7 T MR spectrometer.

Results: After incubation of hMSCs with VSOPs, fluorescent dye-labeled particles could be detected within the cells. This was confirmed with iron specific Prussian blue staining and TEM. During expansion in monolayer culture, a distinct reduction of VSOP-labeling appeared after 5 to 9 cell divisions, based on the dilution of label upon cell division. Compared to unlabeled cells, no inhibition of adipogenic, osteogenic, and chondrogenic differentiation of magnetically labeled hMSCs could be detected. Both, VSOP-labeled and unlabeled cells showed a specific matrix production and similar expression levels of according genes. After embedding of magnetically labeled hMSCs in collagen type I hydrogels, where no proliferation takes place, the cells could be detected with a MR spectrometer over at least 20 weeks. Controls with unlabeled cells were also visible in the MR images, but didn’t exhibit the typical signal intensity decrease conditioned by VSOP.

Discussion and Conclusions: The results of our study show that the incorporation of VSOPs in hMSCs leads to an efficient cellular label with no adverse effect on their stem cell function. VSOP-labeled hMSCs can be successfully detected in a three-dimensional construct in vitro using high-field MRI. Further investigations have to reveal how long labeled cells can be visualized and monitored reliably in vivo.

(JSRM Code:002010700140)

The stability of cartilage phenotype in human bone marrow stromal cells and chondrocytes

aSonius P, aDe Cocq van Delwijnen WS, bKoevoet JLM, aWeinans H, a,bVan Osch GJ.
aDept. of Orthopaedics, bDept. Of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam,
The Netherlands

Introduction: To repair cartilage lesions, modern cell-based treatments focus on the employment of various cell types like Bone Marrow Stromal Cells (BMSCs) and chondrocytes. To be successful on the long term, stability of the cell phenotype in the generated cartilage is required. Recent studies show that besides BMSCs, chondrocytes are also multipotent. In this study we analyze the stability of the chondrogenic phenotype of BMSCs, and primary and dedifferentiated chondrocytes. To induce a chondrogenic phenotype the 3 cell types are pre-cultured in chondrogenic medium. To test the phenotypic stability of these cells an assay was used where chondrogenic medium was switched to adipogenic or control medium. Finally, we evaluate and compare the phenotype of the different cell types by RT-PCR and histology.

Materials and Methods: Human BMSCs were expanded for 3 passages. Primary chondrocytes from human knee joints were used immediately, or dedifferentiated by expansion for 2 passages in monolayer. The 3 cell types were pre-cultured for 10 days on chondrogenic medium, both in 2-d monolayer and 3-d alginate beads. For the following ten days cells remained on chondrogenic medium or switched to adipogenic or control medium (no chondrogenic factors). At day 20, histology and RT-PCR analysis were used to evaluate the effects of the medium switch of the cells.

Results: Collagen 2 (COL2) expression confirmed that chondrogenesis was induced on chondrogenic medium in all cell types after 10 days and even more obviously after 20 days. When chondrogenic medium was switched to adipogenic, a tempering of chondrogenic phenotype (COL2) in all cell types was observed. Furthermore, primary and dedifferentiated chondrocytes showed decreased expression of the hypertrophic marker collagen 10 in the adipogenic medium, while BMSCs did not. Furthermore the switch to adipogenic medium gave elevated aP2 expression in all 3 cell types. This occurred most clearly in the BMSCs, which was confirmed by histology, as BMSCs were able to create lipid vacuoles when switched to adipogenic medium, while primary and dedifferentiated chondrocytes were not.

Discussion and Conclusions: These results indicate that the chondrogenic phenotype is not stable after 10 days of chondrogenic culture. BMSCs seem less stable than primary and dedifferentiated chondrocytes. Future plans include creating a more stable cartilage phenotype in BMSCs, via switching experiments with a longer chondrogenic pre-culture time.

(JSRM Code:002010700141)

Embryonic stem cells in ALS

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

61 patients, mean age 48,4±16,3, were treated for ALS. Duration of the disease: up to 1 year - 10 patients, 2 years - 18, 3 years - 29, over 3 years ? 4. Course of treatment envisaged administration of 4 cell suspensions containing mesenchymal, ectodermal, and endodermal stem cells obtained from growth zones of 4-8 weeks old cadaverous embryos’ systems and organs; amounts administered varied from 0,5 to 4 ml, cell count - from 0,1 to 100x105/ml.

During the first course of treatment, 67% of patients reported decreased weakness, improved appetite, decreased fasciculations, decreased spasticity, and higher spirits. In the course of 2 months, 34% of patients observed increased range of motions in the extremities, decreased muscular rigidity, lower reflexes, decreased number of fasciculating muscle zones, better endurance of daily loads, less expressed dysphagia and dysarthria. In 3-6 months, 58% of patients reported aggravation of condition and reversion of relevant symptoms. II treatment was performed in 24 cases, III course ? in 8 cases, IV course ? in 3 cases.

Conclusions:

(JSRM Code:002010700142)

Embryonic stem cells in multiple sclerosis

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

Treated and observed (2-8 years) were patients (14 women, 10 men) with 2-3-year history of relapsing-remitting multiple sclerosis, mean age being 31.2±3.8, duration of remissions ?3.4±1.2 months. Most often, observed were pyramidal and sensory disturbances, and ophtalmological symptoms. Indications for stem cell treatment were as follows: ineffective medicinal therapy, Interferon intolerance, development of the new foci; aggravation of neurological symptoms.

For treatment, used were embryonic stem cell suspensions (ESCS) containing stem cells of mesenchymal and ectodermal origin obtained from active growth zones of 4-8 weeks old embryonic cadavers’ organs. Suspensions were administered in the amount of 1-3 ml, cell count being 0.1-100x105/ml. In the course of treatment, applied were 2-4 different suspensions, mode of administration being intracavitary, intravenous, and subcutaneous. After treatment, syndrome of early post-transplant improvement was observed in 70% of patients, its main manifestations being decreased weakness, improved appetite and mood, decreased depression. In the course of first post-treatment months, positive dynamics was observed in the following aspects: nystagmus, convergence disturbances, spasticity, and coordination. In such symptoms as dysarthria, dysphagia, and ataxia, positive changes occurred at much slower rate. In general, the treatment resulted in improved range and quality of motions in the extremities, normalized muscle tone, decreased fatigue and general weakness, and improved quality of life. Forth, 87% of patients reported no exacerbations, no aggravation of neurological symptoms, and no further progression of disability. MRI performed in 1-2 years after the initial treatment, showed considerable subsidence of focal lesions, mean by 31%, subsidence of gadolinium enhanced lesions by 48%; T2-weighted images showed marked decrease of the foci’s relative density.

(JSRM Code:002010700143)

Influence of growth factors, neurotrophic factors and proteins of the extracellular matrix on expansion and differentiation of adult neural stem cells

Zechel C, Moeckel S, Stoerchel P, Pawlak E, Tronnier V
Universitaetsklinikum Schleswig-Holstein Campus Luebeck, Department of Neurosurgery, Ratzeburger Allee 160,
23538 Luebeck, Germany

Introduction: The subgranular zone of the hippocampal dentate gyrus and the subventricular zone adult of the adult mammalian brain harbour neural stem cells (adult NSCs), which provide a lifelong source for neurons and glial cells. To elucidate their potential usage in neuro-regenerative medicine, several technical problems need to be solved, including the development of optimized protocols for cell expansion and differentiation.

Materials and Methods: rNSC-1b, a subclone of an NSC cell line established from the brain of an adult rat, was grown in defined media containing a serum supplement and growth factor cocktails. Cocktails of the growth factors EGF, bFGF, PDGF and LIF and neurotrophic factors, as well as several growth matrices were analyzed for their effects on cell expansion and differentiation. Immunocytochemistry, RT-PCR and Western blot analysis were performed to monitor gene expression during expansion and differentiation of rNSC-1.

Results: Underlining their neural stem cell character, rNSC-1 cells co-express Nestin, GFAP and S100, as well as Oct4 and Sox2. Moreover, rNSC-1 cells express the pro-neuronal factors HES5 and prox-1 reflecting their commitment to the neural lineage. In addition, they express REST, an inhibitor of the neuronal phenotype which parallels the fact that neuronally differentiating cells are absent in rNSC-1 cultures at this stage. During expansion rNSC-1b cultures exhibit a high proliferation rate and preferentially grow as neurospheres. Adherent growth can be triggered by poly-ornithin, matrix proteins such as fibronectin, laminin or Matrigel, but also by the application of cyclic AMP or FCS. Presence of increasing concentrations of FCS resulted in increasing numbers of cells that differentiated into astroglia. Adherent cell growth per se, however, did not induce neural differentiation. Presence of bFGF, but not of the other growth factors was absolutely required for rNSC-1 expansion. Neural differentiation could be efficiently induced by addition of a mix of cAMP and retinoic acid and led to the generation of cells expressing glial or neuronal markers.

Discussion and Conclusions: Stable in vitro expansion of adult rat stem cells committed to the neural lineage required the presence of bFGF but not of EGF, PDGF or LIF. Neural differentiation was inducible by exposure to cAMP/retinoic acid and resulted in the production of neuronal and glial derivatives.

(JSRM Code:00201070014)

Neural differentiation of human dental follicle precursor cells

Vollner F1, Driemel O2, Reichert TE2, Weber BHF1, Morsczeck C1
Institute of Human Genetics; Department of Oral and Maxillofacial Surgery, University of Regensburg, Germany 2

Introduction: Dental follicle precursor cells (DFPCs) are plastic adherent cells, which are capable of differentiation toward dental cells, like cementoblasts and periodontal ligament cells. These ectomesenchymal cells express neural stem cell markers Nestin and Notch-1 that specify DFPCs also as potential neural progenitor cells. We investigated the gene expression pattern of typical neural and glial cell markers in DFPCs and investigated their neural differentiation potential.

Materials and Methods: DFPCs were isolated from dental follicles and cultured until passage five. For different stimulations of neural differentiation a serum replacement medium (SRM) was used in combination with various mixtures of commercially available supplements B27, N2 and G5 and growth factors fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF). Total RNA was isolated from DFPCs before and after neural differentiations. Gene expression was determined by quantitative reverse transcription (qRT)-PCR for neural and glial cell markers beta Tubulin III, Neurofilament, Nestin, MAP2 and GFAP.

Results: Investigated neural and glial cell markers were found to be expressed in undifferentiated DFPCs. After induction of neural cell differentiation DFPCs became either long drawn out shaped cells or formed neurosphere-like structures after cell detachment from the cell culture surface. Gene expression increased for neural cell markers tested.

Discussion and Conclusion: Previous studies have suggested that DFPCs may also be able to differentiate into neural prescusor cells. Here, we demonstrate that DFPCs express typical neural cell markers and differentiate toward neural like cells after treatment with SRM based cell culture media. This supports the earlier suggestions and shows that DFPCs indeed are capable of differentiation into neuroectodermal cells.

(JSRM Code:002010700145)

Embryonic stem cells in metabolic syndrome

Smikodub O
National Medical University and Embryonic Tissues Center EmCell, Cell Therapy Clinic, Solomenskaya Str. 17, POB 80 UKRAINE, 03110 Kiev

Observed were 12 patients, 7 men and 5 women (mean age ? 58±6.3), with clinical manifestations of metabolic syndrome. All patients presented stage I-II hypertension, carbohydrate metabolism disturbances: impaired glucose tolerance (IGT) ? 8 and mild type II diabetes mellitus (DM) (morning fasting hyperglycemia, aglycosuria) ? 3, with hyperinsulinemia and elevated C-Peptide (4.7±1.6 ng/ml). Lipid metabolism disturbances were reported in all the patients: elevated cholesterol, triglycerides, increased concentration of low density lipoproteids (LDL), very low density lipoproteids (VLDL), decreased concentration of high density lipoproteids (HDL), and abdominal obesity (BMI?30.8±1.6 kg/m2), waist ? relatively W-94.8±2.3, M-105.7±2.9 cm).

Transplanted were embryonic hematopoietic and non-hematopoietic mesenchymal and endodermal stem cells obtained from germ layers of 4-8 weeks old cadaverous embryos’ internal organs and sorted thereafter. 1-3 ml of cell suspensions were administered intravenously, cell concentration ? 0.1-100x105/ml.

Patients were observed for 1-5 years. In 1-2 months, 83% of patients reported gradual BP decrease with subsequent stabilization in the course of the next 2-3 months ? 135-140/80-90 mm Hg, with parallel 1.5-2-fold decrease of hypertension medications. In 7-9 months, glucose tolerance test (glycemia level 2 hours after glucose load was 7.2±0.3 mmol/l) revealed the improvement of carbohydrate metabolism in IGT patients. Decrease of morning hyperglycemia in type II DM patients usually started in 2-3 month, normalization was observed in 5-7 months. C-Peptide concentration gradually decreased, beginning from after 2-3 months, and normalized within 6-9 months. Normalization of blood lipids was reported in 75% of patient after 9-12 months, with parallel decrease of triglycerides, cholesterol, LDL, VLDL, and HDL increase.

(JSRM Code:002010700146)

MR based Detection of Microcapsules for Cell Therapies and Theranostics

Krieg Robert W1, Requardt M1, Ruff J1, Aravind A2, Bulte Jeff WM2,
1Siemens Medical Solutions AG, Allee Rothelheimpark 6, 91050 Erlangen, Germany
2Johns Hopkins Medical Institutes, Blalock 644, 600 N. Wolfe St., Baltimore, MD21212, USA  

Introduction: Medical imaging ideally serves a 2 fold purpose in the application of cell therapies. It facilitates the delivery of the cells and allows monitoring of the therapy over time. Practically many problems need to be still overcome. For example direct cell labeling affects the proliferation of the cells. Immunoresponse limits usually the survival of cells and thereby the efficiency of the therapy. Protective Microcapsules avoid direct cell labeling and provide immunoprotection. In our study we examined the imaging properties of Fe or Flourine labeled Microcapsules.

Materials & Methods: Imaging was performed on a standard MAGNETOM Avanto with 1.5T and a MAGNETOM Trio with 3T field strength. Microcapsules of ~300μm size were synthesized and loaded with 80.9 ± 4.9ng Fe per capsule. Alternatively a second sample of Microcapsules where labeled with Fluorine. The Fe loaded capsules were embedded in a grid shaped Gel phantom starting with different concentrations. The Fluorine labeled capsules were embedded in Gel tubes. Imaging was done with standard balanced Gradient Echo sequences (TrueFISP) and off Resonance Imaging approaches. For Fluorine imaging simple Tx/Rx loop coils with 4cm and 8cm diameter for 1.5 and respectively 3T have been used.

Results: Both types of capsules could be imaged without significant problems. Iron labeling allowed the detection of a single capsule. The fluorine label permitted detection of a cluster of 6 capsules. S/N with Fe labels was significantly higher. Encapsulation of pancreatic islet sustained viability of the cells equally to pure cells. The porosity of the capsules allowed the cells to take up their normal function of insulin production.

Discussion & Conclusion: This work demonstrates the feasibility of new concepts for cellular therapies based on image guidance and therapy monitoring. The approach has the potential to overcome major limitations of cell therapies.

(JSRM Code:002010700147)

Telomerase-immortalized human mesenchymal stem cells (hMSCTERT) can be directed towards an endocrine differentiation pathway with insulin production

Limbert C1,2, Path G1, Ebert R 2, Kassem M3, Jakob F2,Seufert J 1
1University Hospital of Freiburg, Division of Endocrinology and Diabetology, Department of Internal Medicine II, Freiburg, Germany
2University of Wurzburg, Orthopedic Center for Musculoskeletal Research, Stem Cell Biology, Wurzburg, Germany
3University Hospital of Aarhus, Department of Endocrinology and Metabolism, Aarhus, Denmark 

Introduction: Adult stem cells are investigated as alternative source for beta-cell replacement therapy. So far, no consistent differentiation capacity for insulin producing cells has been shown in human bone marrow-derived mesenchymal stem cells (hMSC). Here we investigated the ability of hMSC-TERT cell line to differentiate into insulin-producing cells under regulation of hNgn3 and hPdx-1.

Materials and methods: hMSC-Tert endocrine progenitor potential was analysed. Subsequently, stably overexpressing hNgn3 and/or hPDX-1 cell lines were generated (hMSC-TN, hMSC-TP and hMSC-TN/P). Islet gene regulation and protein synthesis were analysed by RT-PCR, Western Blotting, reporter gene assays and immunocytochemistry. Insulin content and secretion were evaluated by ELISA.

Results: hMSC-Tert expressed progenitor cell markers, nestin and c-met and displayed pancreatic endocrine gene expression under specific culture conditions. Generated cell lines highly overexpressed the ectopic genes along with regulation of multiple islet genes, including insulin. In hMSC-Tert, Ngn3 induced expression of endogenous Pdx-1. hMSC-TP revealed direct activation of insulin gene. Coexpression of Ngn3 and Pdx-1 did not show synergistic effect on insulin expression efficiency. Insulin was expressed, produced and stored under regulation of hNgn3 and/or Pdx-1. However, no glucose dependent insulin secretion was observed in these cells.

Conclusions: In a human system of MSCs: introduction of key endocrine transcription factors is able to induce differentiation towards insulin-producing phenotypes; hNgn3 is able to trigger pancreatic endocrine differentiation cascade, lying upstream of Pdx-1; higher endocrine maturation must be achieved, in order to obtain functional hMSC that are suitable to the cell-based therapy of type1 diabetes.

(JSRM Code:002010700148)

Stem cell chemoattractant gene expression was upregulated by intramyocardial injection of Epoetin-α in a rat myocardial infarction model

Klopsch C*1, Furlani D*1, Gabel R1, Wagner K2, Wang W1, Ong LL1, Li W1, Nizze H3, Titze U3, Ma N1, Steinhoff G1

1Department of Cardiac Surgery, University of Rostock, Rostock, Germany

2Departement of Anaesthesia, Klinikum Sudstadt, Rostock, Germany
3Department of Pathology, University of Rostock, Rostock, Germany
*Both authors contributed equally to this work.
nanma001@med.uni-rostock.de 

Emerging evidence suggests that Erythropoietin (EPO) protects the myocardium from ischemic injury and promotes beneficial remodelling. However, the role of EPO and its receptor (EPO-R) in mediating cardiac regeneration remains unclear. We hypothese that stem cell homing and proliferation modulated by EPO could contribute to its cardio-protective effects. After permanent left ventricular myocardial infarction (MI), Epoetin-α (3000 U/kg) was injected along the infarction border. At six weeks after MI, cardiac functionality was measured by pressure-volume loops in left and right ventricles. Infarction size, angiogenesis and pathologic effects were evaluated. Gene expressions of EPO-R, SDF-1, CXCR-4, c-kit, eNOS, TNF-α, IL-8, Integrin-β and CdK4 were analyzed by RT-PCR at different time points of the first week (24h, 48h, 96h and 7 days). Our findings indicated improved left ventricular function both at baseline levels and under Dobutamine stress (dp/dt maximum and minimum, tau, cardiac output, stroke work, ejection fraction n=11-14, p<0.05) and decreased right ventricular wall stress (maximum and endsystolic pressure n=5-8, p<0.05). Infarction size was reduced from 27.8±3.4% to 20.1±2.8% (n=6-8, p<0.01). Capillary density was enhanced from 257.7±24.5 to 338.5±35.9 (vessels per square mm, n=6-8, p<0.05). Mortality was decreased from 29.0% to 22.2% (n=53-69). No thrombosis was observed in the intramural myocardium. EPO-R was down regulated in infarcted, peri-infarcted and non infarcted areas at all time points (n=7, p<0.05). Cardiac SDF-1?, CXCR-4 and eNOS expressions were increased at 24 hours. C-kit was up regulated significantly at 48 hours compared to 24 hours in the EPO treated hearts. In untreated hearts, c-kit expression remained constant. Proinflammatory cytokines (TNF-α, IL-8 and Integrin-β)? were down regulated. Cell cycle re-entry marker (CdK4) was increased at 24 hours in non infarcted zones. In conclusion, we demonstrate intramyocardium Epoetin-α injection induces an earlier up regulation of stem cell chemoattractants, reduces inflammation, enhances angiogenesis and restores heart function after MI.

(JSRM Code:002010700149)

Stem cell transplantation and cardiac regeneration after myocardial infarction in SCID mice: Human MAPCs are effective and safe

Groebner M*1, Deindl L*2, Dimomeletis I1, Zaruba MM1, Kostin S3, Deutsch MA1, Assmann G4, X, Mueller-Hocker J4, Feuring-Buske M5, Franz WM1
1Medical Department I, Klinikum Groshadern, Ludwig Maximilians University (LMU), Munich, Germany
2Insitute of Surgical Research, LMU, Munich, Germany
3Max Planck Institute Bad Nauheim, Germany
4Institute of Pathology, LMU, Munich, Germany
5Medical Department III, Klinikum Groshadern, LMU, Munich, Germany
*These authors contributed equally to this work 

Introduction: Clinical studies suggest that transplantation of total bone marrow (BM) after myocardial infarction (MI) is feasible and potentially effective. However, focusing on a defined BM-derived stem cell type may enable a more specific and optimized treatment. Multilineage differentiation potential makes BM-derived multipotent adult progenitor cells (MAPCs) a promising stem cell pool for regenerative purposes. We analyzed the cardioregenerative potential of human MAPCs in a murine model of myocardial infarction.

Materials and Methods: Human MAPCs were selected by negative depletion of CD45+/Glycophorin+ BM cells and plated on fibronectin coated dishes. In vitro, stem cells were analyzed by RT-PCR. In vivo, we transplanted hMAPCs (5x105) by intramyocardial injection after MI in immunodeficient SCID beige mice. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Heart tissues were analyzed immunhistochemically.

Results: RT-PCR experiments on early hMAPCs passages evidenced an expression of Oct 4, a stem cell marker indicating pluripotency. In later passages, cardiac markers (Nkx2.5, GATA4, MLC-2v, MLC-2a, ANP, TnT, TnI, Cx 43) and smooth muscle cell markers (SMA, SM22α) were expressed. Transplantation of hMAPC into the ischemic border zone after MI resulted in an improved cardiac function at day 6 (EF 26 vs. 20%) and day 30 (EF 30 vs. 23%). Co-localisation of hMAPC marker vimentin and SMA in immunhistochemistry demonstrated that hMAPCs integrate into vessels in the peri-infarct region and show smooth-muscle-like characteristics. Furthermore, engrafted hMAPCs formed cardiomyocyte-like cells expressing cardiac proteins Nkx 2.5 and MHC. The proliferation marker Ki67 was absent in immunohistochemistry, and teratoma formation was not found indicating no tumorous potential of transplanted hMAPCs in the tumor-sensitive immunodeficient SCID model.

Conclusions: Transplantation of human MAPCs after MI ameliorates myocardial function, which may be explained by hMAPCs` potential to integrate into vessels and myocardial structures in the borderzone and form smooth-muscle-cell-like as well as cardiomyocyte-like cells. Lack of evidence of tumorous potential in the tumor-sensitive SCID model indicates, that hMAPC may deliver not only an effective, but also safe stem cell pool for the treatment of MI.

(JSRM Code:002010700150)

Recent patents on stem cell transplantation in cardiovascular medicine

Jakob M, Bauersachs J, Thum T
Julius-Maximilians University, Internal Medicine I / Cardiology, Wurzburg, Germany 

Introduction: Heart failure is a leading cause of morbidity and mortality worldwide. Coronary artery disease (CAD) is currently one of the most common identifiable cause of heart failure. Mortality after myocardial infarction depends on the time when reperfusion of the infarct-related artery is performed. If this is not done rapidly, necrosis, scar formation and left ventricular remodelling occur. Today, cell-based therapy either by direct application or mobilisation has gained a lot of interest and is believed to improve left ventricular function after myocardial infarction.

Aim: We screen various available patent data bases to give an overview on different patent applications of cell-based therapies in cardiovascular medicine.

Results: The first patents presented are about application of stem cells for heart failure or for inducing neoangiogenesis. Patents have been filed that differ between stem cell therapy alone or stem cell therapy in combination with other factors such as growth hormones. Stem cell therapy alone mostly consists of various steps: 1) isolation of cells from a patient. This is done by muscle or bone marrow biopsy, lipoaspirate or taking circulating blood from the patient. 2) Cultivation and/or expansion of the favoured cell type. These include lineage negative (Lin-) cells (Freyman, US7097833), adipose tissue-derived stem cells (Strem, MX5009044A), bone marrow-derived stem cells (Furcht et al., US7015037) or others. 3) Transplantation of the cell type. This is solved either by direct injection into the specific organ or intravenous administration. Stem cell therapy in combination with other factors requires an additional step. In this setup, accessory factors, like growth hormones (PDGF, FGF, PDGF) or other factors like adrenomedulin are given either to the isolated cells or are directly given to the patient. Other authors are using the administration of growth hormones or other factors like nicotine or nicotine-receptor agonists alone to mobilize stem cells from the bone marrow (Cooke et al., US6720340). We also present an overview about current patent applications of cardiomyocytes generation from embryonic or adult stem cells. Various cell types can be isolated and used to generate myocytes, e.g. cells from the endometrial membrane of the uterus or mesenchymal stem cells obtained from menstrual blood (Umezawa et al., WO06078034A1), TVEM-expanded CD34+/CD38- cells from the peripheral blood (Rudd, WO 06093860A3) or myogenic stem cells from adipose tissue (Geng et al., WO06017320A3). In addition, mesenchymal or myogenic stem cells used for myocyte generation need to be taken into culture before administration to the patient. Culture conditions for cardiac cells differ from those for other cell types. Isolated cells are cultured, e.g. in rotating bioreactor vessels (Rudd, WO 06093860A3) or engrafted on a histoengineered scaffold material of thermoplastic resin having a porous three-dimensional network and application of a pulsatile flow (Nakayama et al., JP2006246770A2). After a certain period of cultivation, myoblasts are then re-transplanted into the patient. We finally report patents in the field of cell culture methods for human stem cells that are used for generation of different types of cardiovascular cells.

Conclusions: A systematic overview on the current patent situation about use of stem cells in cardiovascular medicine enables optimized future decision making in the development of novel strategies for improved regenerative medicine.

(JSRM Code:002010700151)

Characterization of an endothelial progenitor like cell in cardiomyocyte cultures

Weeke-Klimp AH, Plantinga JA, Harmsen MC, Van Luyn MJA
University Medical Center Groningen, University of Groningen, Department of Pathology and Laboratory Medicine, Medical Biology section, Hanzeplein 1, 9713 GZ Groningen, The Netherlands

Introduction: Tissue engineering of cardiac tissue requires the concerted action of appropriate scaffolding biomaterials and functional cardiomyocytes together with a well-developed vascular network to ensure adequate oxygenation. In this study we describe the origin and characterization of a progenitor like cell that is present in cardiomyocyte cultures after isolation of the cardiomyocyte population of neonatal murine hearts.

Materials and Methods: Cardiomyocytes (CM) were cultured from enzymatically digested neonatal murine hearts after depletion of the majority of fibroblasts by adherence to plastic. The cardiomyocyte enriched cell population was characterized by immunohistochemistry, FACS analysis and transmission electron microscopy (TEM). Cell proliferation was measured by quantitating BrdU incorporation. CD31+ cells were isolated/depleted by magnetic cell sorting (MACS).

Results: Freshly isolated CM enriched cells contained 90% cells positive for the cardiac markers troponin and α-actinin, and 10% cells positive for CD31, which is commonly expressed on endothelial cells. During culture of the CM-enriched cells, cobblestone areas were observed. Due to high proliferative activity of the cobblestone forming cells, these cobblestone areas increased rapidly in size and number after 7 days of culture. Further characterization of these areas showed that they were positive for CD31, and partly positive for uptake of DiI acetylated LDL. This indicated an endothelial phenotype of the cobblestone forming cells. Remarkably, TEM of the cobblestone areas also showed the presence of progenitor like cells and immature cardiomyocytes. Depletion of CD31+ cells resulted in a strong decrease in the number of cobblestone areas after culture, which implies that the cobblestone areas originate from CD31+ cells or a CD31+ subpopulation.

Discussion and Conclusions: Overall, these data show that the neonatal heart contains a CD31+ cobblestone forming cell that may have the capacity to differentiate into both endothelial cells and cardiomyocytes. Therefore, these cells may have clinical potential in cardiac regeneration.

(JSRM Code:002010700152)

Strategies to induce cardiac differentiation of Mesenchymal Stem Cells

Armbruster C*, Tevaearai H*, Kleber A°, Carrel T*, Giraud MN*
*Clinic for Cardiovascular Surgery, Inselspital Bern, Switzerland
°Physiology Institute, University of Bern, Switzerland

Introduction: Loss of cardiomyocytes during myocardial infarction leads to decreased heart function. Replacing the injured myocardium with a muscle tissue created in vitro has gained increasing attention. Yet, the optimal cell source to engineer a functional and contractile cardiac tissue has to be defined. In this regards, mesenchymal bone marrow stem cells (MSCs) present numerous advantages: easy accessibility, large expansion capacity, high plasticity and aptitude to differentiate into cardiomyocytes. In the present study we investigated different strategies to induce the differentiation of rat MSCs into cardiac-like cells.

Methods: Isolated from tibial and femoral rat bone marrow, MSCs were separated from hematopoietic stem cells by their capacity to adhere to plastic. MSCs were cultured for 14 days with 20% FBS and FACS analysis were performed. To induce their differentiation into cardiomyocytes, different strategies were evaluated: (1) chemical treatments with 5-azacytidin, TSA or ascorbic acid; (2) culture with cardiomyocytes (CM)-conditioned medium. Phenotype characterization was evaluated by immunostaining for specific cardiac proteins: cTnT, cMHC, alpha-actinin sarcomeric and titin, and by RT-PCR for specific transcription factors (GATA4, Nkx2.5) and structural proteins (cTnT, beta-MHC).

Results: After 14 days, at least 98% of the cell population was positive for CD90, CD29, and CD44, negative for CD109, CD31, and about 40% were positive for CD45. Cells rapidly spread in culture and punctuated localization of alpha-actinin was detected by immunofluorescence in pseudopods and at the periphery of spreading cells. We detected an increased gene expression of cTnT, beta-MHC, desmin and Nkx2.5 after 4 weeks compared to initially seeded cells. There was no difference between non-treated cells, chemical and conditioned medium treatments. This expression decreased after 12 weeks. The immunostaining revealed only a weak presence of related proteins in a small number of cells.

Conclusion: Our results showed that cardiac differentiation of MSCs was initiated independently of the treatments and concerned only a sub-population of the heterogeneous MSCs population. Further investigation will be undertaken to define this sub-population with cardiac progenitor characteristics. Differentiation of cells toward cardiocytes was mainly observed at the gene levels. Additional stimuli such as mechanical stretch may be necessary to promote further cell differentiation and maturation.

(JSRM Code:002010700153)

Combined treatment of intractable ischemic heart disease with TML and endothelial progenitor cells-preliminary results of a therapeutic concept

Babin-Ebell J, Sievers H, Depping, Marxsen J, Jung F, Wagner K
University of Luebeck Cardiac surgery, Ratzeburger Allee, 23538 Luebeck, Germany

Objective: Ischemic heart disease as the most frequent cardiovascular disease still implies a major health burden to western countries. In a certain amount of patients, conventional revascularization therapy is unsuccessful leaving these patients with disabling angina pectoris or congestive heart failure. Theoretically combining TMLR for signalling induction and pain relief and stem cell injection for regeneration support offers a promising treatment strategy for there otherwise intractable patient suffering from ischemic heart disease.

Methods: Five patients with TML and stem cell therapy were evaluated. The pelvic bone was punctured and 300 ml of crude bone marrow were aspirated. Patients were operated “off pump”. Between 11 and 25 laser channels were created. In between the channels the stem cells were applied with 6 to 12 injections. Cell selection was carried out using magnetic cell separation. The patient follow up was 14.2+6.9 (mean+SEM).

Results: NYAH classification improved significantly between preoperatively (mean 3.4) and three (mean 1.,8) and six month (mean 1.8) postoperatively. When patients were asked to rank their complains on a scale from 1 to 10, all patients described an immediate improvement postoperatively (7.4 preoperatively, 3.6 at three month and 3.2 at six month). An improvement in left ventricular ejection fraction was as well observed (48.8+13 preoperatively, 59.6+13 at three month (p=0.028) and 61.6+17 at six month (p=0.038 to preoperatively). The effect on LVEDV varied between the patients and did not reach statistic significance.

Conclusions: In summery, the presented investigation demonstrated an increase in LVEF and better clinical performance by intramyocardial CD 133+ stem cells injection and TML therapy. The reason remains uncertain and may be explained by paracrine mechanism. It is still uncertain how long the observed effect will last, or if the better cardiac performance will lead to better life expectancy.

(JSRM Code:002010700154)

EPO treatment after myocardial infarction in mice improves cardiac function by enhanced homing of bone marrow-derived cells

Brunner S, Winogradow J, Huber B, Fischer R, Assmann G, Muller-Hocker J, Franz WM
Ludwig-Maximilians University, Klinikum Grosshadern, Medical Department I, Munich, Germany
Ludwig-Maximilians University, Institute of Pathology, Munich, Germany

Objective: Bone marrow derived stem cells either transplanted or mobilised by cytokines improve cardiac function after myocardial infarction (MI). Besides of its classical function in erythropoiesis recent reports have shown additional effects of EPO like antiapoptotic effects and stem cell mobilisation. These effects are known to improve myocardial regeneration after MI. Therefore, we analysed in a murine model of surgically induced MI the influence of EPO treatment on survival, functional parameters as well as stem cell mobilisation and homing. Methods: Human EPO (3000 IE/kg) was injected intraperitoneally immediately after ligation of the left anterior descendens (LAD) as well as on the two consecutive days (1000 IE/kg) ? a dose which did not significantly affect erythropoiesis. 6 and 30 days after the surgical procedure, pressure volume relationships were investigated in vivo. Cardiac tissues were further analysed by histology. To show the impact on stem cell mobilisation and homing as well as serum cytokine levels FACS and ELISA was performed.

Results: EPO treated animals showed a significant improvement of survival post MI (62% vs. 36%). FACS data demonstrated mobilisation of CD31, c-kit and Sca-1 positive stem cells and homing of Sca-1 and CXCR4 positive stem cells was enhanced after EPO treatment. Serum levels of G-CSF were significantly increased after EPO administration, whereas SDF-1 levels were decreased and VEGF remained unchanged. Histology of EPO treated hearts showed less reduction of LV wall thickness and a smaller size of infarction at day 30 (22% vs. 42%). In addition, myocardial function of PTH treated mice was improved (EF: 23% vs. 15%) and elasticity demonstrated a less degree of infarct remodelling (Elasticity: 21.0 mmHg/μl vs. 8.1 mmHg/μl).

Conclusion: We have shown that EPO application after MI ameliorates myocardial function. This may be explained - beside direct effects via the EPO receptor - by mobilisation and homing of bone marrow-derived stem cells and a change in the serum cytokine pattern, which may lead to improved neovascularization and cell survival. Therefore, EPO treatment presents a promising non-invasive approach to ameliorate heart failure post MI.

(JSRM Code:002010700155)

Stem cell markers in thoracic aortic aneurysm

Mohamed SA
Department of Cardiac Surgery, University Clinic of Schleswig-Holstein Campus Luebeck, Luebeck, Germany 

Background: Aortic aneurysm is a common disease among elderly people and is usually found in patients with bicuspid aortic valve (BAV) a decade earlier compared with those having normal aortic valve. So far, only the surgical therapy of aortic aneurysm is available and the need for preventative measures is evident. This study investigated the differentially expressed markers in vascular smooth muscle cells (VSMCs) isolated from diseased aorta.

Methods: Diseased aorta removed directly after surgery treated over night with 0.5% collagenase solution and resuspended in smooth muscle cell growth medium. Dissolved cells grown at 37°C in an atmosphere of 5% CO2 in air. 25cm2 cell culture flasks with 8x106cells/ml were harvested and were spreaded on chamber slides for 24h. Immunohistochemical techniques were used to detect alpha- smooth muscle actin, beta-tubulin, tie-2, VEGF R1, and glial fibrillary acidic protein (GFAP).

Results: There was 2 to 3-fold decrease in the GFAP in aneurysmatic tissues compared to nonaneurysmatic (p = 0.002). However, the expression of alpha- smooth muscle actin was increased in aneurysmatic tissues (4.8±0.5 vs 6.9±1.2, p = 0.002)

Conclusions: Stem cell markers were expressed in diseased aorta, the potential strategies to promote the regression of thoracic aneurysm will be here discussed.

(JSRM Code:002010700156)

SC1/PRDM4 is a critical mediator for cell death, mitosis and differentiation of neural stem cells

Orel N, Sendtner M
Institut for Clinical Neurobiology, University of Wuerzburg, Josef-Schneider-Str., 11, 97080, Wuerzburg, Germany

Introduction: PR domain containing 4 (PRDM4) is the human and mouse homolog of rat Schwann cell factor 1 (SC1) and belongs to the PRDM gene family of transcriptional repressors. The PR domain defines a family of transcription factors involved in cell differentiation and tumorgenesis. SC1/PRDM4 is a cytoplasmic interaction partner of the p75NTR receptor, and it is activated by ligand bindung to this receptor. It shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with inhibition of mitosis. It has been shown that SC1/PRDM4 acts as a transcriptional repressor which requires the presence of its zinc finger domains. The PR domain and zinc finger domains are also necessary to direct SC1’s nuclear localization. Last, SC1/PRDM4 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1/PRDM4.

Materials and Methods: NSCs obtained from mouse forebrain embryos at E12 were grown as free-floating neurospheres, dissociated by mechanical trituration and subsequently plated on polyornithin-laminin-coated cover slides. Chimeric lentiviral vector plasmid pGJ3p containing SC1/PRDM4 cDNA and the expression plasmid VSV-G were transfected into 293T cells for virus production. The supernatant containing different lentiviruses was applied to NSCs. For PRDM4/SC1 siRNA, short RNA hairpins were cloned into the self-inactivating lentiviral vector pSIH1-H1-copGFP shRNA.

Results: Overexpression of SC1/PRDM4 in mouse embryonic neural stem cells inhibits mitosis and induces apoptosis. Absence of p75NTR did not protect NSCs from apoptosis caused by SC1/PRDM4 overexpression. Apoptosis cannot be prevented by neurotrophic factor signalling, but it is absent in Bax-/- neural stem cells. Knockdown of SC1/PRDM4 expression by siRNA lentiviruses in NSCs significantly increases the number of pH3 positive cells, thus providing further evidence for the role of SC1/PRDM4 in the regulation of cell cycle. PRDM4/SC1-siRNA treatment also modulated the number of arrested cells with apoptotic nuclei in comparison to control snapshot treatment.
Discussion and Conclusions: Our data suggest that SC1/PRDM4 acts as a key regulator of neural stem cells differentiation and mediates neurotrophin effects via p75NTR and possibly also other signals that determine cell cycle fate during neural differentiation In addition, these data provide evidence for the molecular basis of a link between cell cycle exit and cell death in neural differentiation. Next steps will be to investigate SC1/PRDM4 function in mouse models for further exploration of its role in neural stem cell renewal and neurogenesis in the peripheral and central nervous system.

(JSRM Code:002010700157)

Stem cell regulation and lineage commitment in mammalian skin:
A role for the Wnt/β-catenin signalling pathway

Petersson M, Schettina P, Niemann C
Center for Molecular Medicine Cologne, University of Cologne,
Institute of Pathology, Joseph-Stelzmann Str.9, 50931 Cologne, Germany 

Introduction: Mammalian skin is a highly dynamic tissue and constantly renewed throughout adult life by a population of stem cells capable to produce daughter cells that differentiate along multiple lineages. Stem cells within the epidermis have the ability to regenerate the differentiated cell types of the interfollicular epidermis, hair follicles and sebaceous glands. We are interested in how lineage commitment is regulated in normal skin tissue and during skin tumourigenesis.

Previously, we and others have shown that canonical Wnt/β-catenin signalling plays a crucial role for skin development, stem cell maintenance, and lineage selection. In skin morphogenesis, the level of β-catenin activity determines lineage choice: activation of the pathway stimulates hair-type differentiation, whereas inhibition promotes differentiation of interfollicular epidermis and sebocytes.

Aims of the project: Important questions are if skin tumour formation originates in epidermal stem cells and if pathways regulating these cells also determine the differentiation process of tumour cells.

Results and Discussion: Aberrant activation of Wnt signalling occurs frequently in a range of tumours. What we found is that repression of Wnt/β-catenin signalling in mouse epidermis not only changes lineage commitment but also leads to development of sebaceous tumours and that the level of Wnt/β-catenin activity indeed specifies the tumour type. This is confirmed by other mouse models, where activation of the pathway induces skin tumours with hair differentiation. Taken together, the data indicate that similar cellular mechanisms and signalling pathways are applied for normal differentiation of skin progenitor cells and for directing the differentiation program of tumour cells. We present an inducible mouse skin tumour model which allows us to investigate early steps of tumour formation and to analyse potential progenitor cells that give rise to skin tumours.