Spindle Shaped Human Mesenchymal Stem/Stromal Cells from Amniotic Fluid Promote Neovascularization

Human amniotic fluid obtained at amniocentesis, when cultured, generates at least two morphologically distinct mesenchymal stem/stromal cell (MSC) subsets. Of these, the spindle shaped amniotic fluid MSCs (SS-AF-MSCs) contain multipotent cells with enhanced adipogenic, osteogenic and chondrogenic capacity. Here, we demonstrate, for the first time, the capacity of these SS-AF-MSCs to support neovascularization by umbilical cord blood (UCB) endothelial colony forming cell (ECFC) derived cells in both in vitro and in vivo models. Interestingly, although the kinetics of vascular tubule formation in vitro were similar when the supporting SS-AF-MSCs were compared with the best vasculogenic supportive batches of bone marrow MSCs (BMSCs) or human dermal fibroblasts (hDFs), SS-AF-MSCs supported vascular tubule formation in vivo more effectively than BMSCs. In NOD/SCID mice, the human vessels inosculated with murine vessels demonstrating their functionality. Proteome profiler array analyses revealed both common and distinct secretion profiles of angiogenic factors by the SS-AF-MSCs as opposed to the hDFs and BMSCs. Thus, SS-AF-MSCs, which are considered to be less mature developmentally than adult BMSCs, and intermediate between adult and embryonic stem cells in their potentiality, have the additional and very interesting potential of supporting increased neovascularisation, further enhancing their promise as vehicles for tissue repair and regeneration.

Amniotic fluid (AF) stem cells, which are reminiscent of adult bone marrow MSCs (BMSCs) in their plastic adherence, expression of such markers as CD90 and their lack of expression of hematopoietic lineage markers, are most frequent in the first trimester of pregnancy [19][20][21][22][23][24] (reviewed in [25][26][27]). In contrast to MSCs sourced post-natally, both these circulating fetal and second trimester AF-stem cell or AF-MSCs are reported to have increased proliferative potential, increased multipotentiality and longer telomeric lengths, but with AF-MSCs at earlier gestational stages expressing higher levels of endodermal and mesodermal markers than those at later gestational stages [21,23,24,[28][29][30](reviewed in [25][26][27]. Thus, the second trimester AF taken during scheduled amniocenteses is a rich source of multipotent MSCs. AF-stem cells or AF-MSCs have been enriched using a variety of techniques, including one and two step cultures, CD117 + selection or short term culture to generate fibroblastoid colonies (reviewed in [26]) [19,21,23,28,29]. Using the latter approach, Roubelakis et al. [29] have identified and enriched for two subsets of human AF-MSCs, the spindle shaped (SS-AF-MSCs) and the round shaped (RS-AF-MSCs), obtained at the time of amniocentesis. These cells have distinctive morphologies, phenotypic differences and differing abilities to differentiate into multiple cell types. The spindle shaped amniotic fluid MSCs (SS-AF-MSCs) express pluripotency markers and have a greater propensity for adipogenic, osteoblastic and chrondrogenic differentiation [29]. Importantly, compared to adult human BMSCs, human AF-MSCs appear to be more stable karyotypically when cultured ex vivo [27,30]. We have shown previously [29,30], using the same SS-AF-MSC cell lines used in this manuscript, that karyotypic abnormalities could not be detected in at least these 6 cell lines when cultured for over  passages. It has been reported previously by some researchers that adult BMSCs can develop chromosomal abnormalities during in vitro culture and that this can lead to their malignant transformation [31][32][33][34][35], although, others have reported that human adult BMSCs do not malignantly transform in vitro and that aneuploidity may occur without transformation [36]. Since ageing is often associated with increased chromosomal abnormalities, increasing cancer rates and the acquisition of or exposure to viral infections, and generally a loss in the proliferative ability and the multipotency of MSCs [37], one advantage of sourcing cells from younger donors or at earlier stages of ontogeny might be that these risks or disadvantages are reduced, thus making them a preferred source of cells. Indeed, in our publications [29,30], we have shown that SS-AF-MSCs can be expanded significantly more in culture than BMSCs and that this occurs without karyotypic changes and with maintenance of their potency for generating osteogenic, adipogenic and chondrogenic cells. In addition, when compared to perinatal or postnatal MSCs, none of the AF-MSC cell lines tested so far has exhibited a susceptibility to transformation in culture nor have they generated teratomas in vivo [11,23], suggesting that AF-MSCs may constitute a potentially safe prospective tool for clinical applications as supported by our recent publication in disease mouse models [37].
We have shown that human dermal fibroblasts (hDFs) and BMSCs support vascular tubule formation from umbilical cord (UC) and umbilical cord blood (UCB) endothelial colony forming cells (ECFC) in vitro [38][39][40][41]. However, the ability of AF-MSCs to support neovascularization has not been addressed. Here, we show, for the first time, that the highly proliferative human SS-AF-MSCs produce a defined profile of angiogenic factors, and that they have similar capacities to support neovascularization by UCB ECFC derived cells in vitro as the best pre-selected vasculogenic supportive batches of adult BMSCs or hDFs. However, in vivo, human SS-AF-MSCs are much more effective than BMSCs in supporting neovascularization in vivo in NOD/SCID mice.

Human Umbilical Cord Blood Endothelial Colony Forming Cell (ECFC) Derived Cells
Umbilical cord blood (UCB) units were sourced and collected with informed written consent from the John Radcliffe Hospital in Oxford with ethical approval for collection from the Oxford Research Ethics Committee and used in these studies with ethical approval from the Berkshire Research Ethics Committee as described previously [17,39,42] and with approval of the NHSBT R&D institutional review committee. UCB mononuclear cells (MNC) were isolated by density gradient centrifugation using Ficoll-Histopaque (density 1.077 g/ml; PAA Laboratories GmbH, Pasching, Austria) and 2610 7 MNC/well were seeded into 6-well plates pre-coated with 50 mg/ml type I rat tail collagen (BD Biosciences, Aylesbury, England) in endothelial growth medium-2 (EGM-2; Lonza Biologics, Tewkesbury, England) containing 10% (v/v) Hyclone fetal calf serum (FCS; Fisher Scientific, Loughborough, England; termed complete EGM-2 medium). After 24 hours of culture, non-adherent cells were removed by washing with EGM-2 medium and adherent cells then cultured in complete EGM-2 medium at 37uC, 5% CO 2 in a humidified incubator [39,42,43]. The medium was changed daily for seven days and then every other day until the first passage. Single colonies derived from ECFCs appeared between 5 and 14 days of culture as well circumscribed cobblestone monolayers as described [40,44]. Those with $50 cells were trypsinized by the end of this 2 week culture period using polystyrene cloning rings (Sigma-Aldrich Company Ltd., Poole, England), and colonies from single UCB units pooled, and plated into 25 cm 2 tissue culture flasks precoated with type I rat tail collagen in complete EGM-2 medium. Cells from this pool when they reached confluency 1-2 days later were termed passage (p) 1 ECFC derived cells.

eGFP-lentiviral Vector Generation, Production and Transduction of ECFC Derived Cells
Where indicated, UCB ECFC derived cells were transduced with the second generation self-inactivating amphotrophic HIVbased lentiviral vector expressing enhanced-green fluorescent protein (eGFP) under the control of the spleen focus forming viral (SFFV) promoter, a kind gift from Professor Adrian Thrasher and as previously described [39]. The eGFP lentiviral vector was propagated and packaged by transient three plasmid (50 mg eGFP lentiviral genome, 17.5 mg vesicular stomatitis virus-G (VSV-G) and 32.5 mg D8.91 plasmids) co-transfection in 293 T cells using Lipofectamine 2000 as recommended by the manufacturer (Invitrogen Ltd., Paisley, Scotland) instead of polyethyleneimine (PEI). The crude vector containing medium was collected from cells 48 h post transfection and titered on 293 T cells. Viral stocks propagated in this way were within the range of 1.5 6 10 6 to 2 6 10 7 Infectious Units (IU)/mL. Optimization of the transduction efficiency was similar to that described for UC ECFC derived cells. UCB ECFC derived cells were subsequently labeled with eGFP by transduction with viral vectors at multiplicities of infection (MOIs) ranging from 7 to 15 with a transduction efficiency of approximately 100% and with no significant loss of cell viability as described [39].

Amniotic Fluid Mesenchymal Stem/Stromal Cells (AF-MSCs)
Cultured AF-MSCs were isolated from 3 human AF samples, collected during scheduled normal pregnancy amniocentesis between the 15 th and 18 th weeks of gestation, as described previously [29,30,37,45]. All second trimester amniotic fluid samples were obtained with written informed consent, approved by the Ethical Committee of the Alexandra Hospital and the Bioethics Committee of the University of Athens School of Medicine. All samples used were derived from the excess volume of amniotic fluid taken for prenatal diagnosis. Using a 22G needle and under ultrasonographic control, 10-15 ml of amniotic fluid was aspirated for each sample. The procedure-related spontaneous abortion rate ranges from 0.06 to 0.5% [46,47]. Each sample was centrifuged at 1,300 rpm for 10 min. The pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich Company Ltd.) supplemented with 20% (v/v) FCS (Gibco-BRL, Paisley, Scotland) in a 25 cm 2 tissue culture-treated flask and incubated at 37uC in a 5% humidified CO 2 chamber for approximately 8-12 days, when the first colonies appeared. Spindle shaped (SS) colonies were selected at passage 0 and cells were subcultured as described previously [29]. SS-AF-MSCs were used by passages 5-12.

Bone Marrow Mesenchymal Stem/Stromal Cells (BMSCs)
Bone Marrow mesenchymal stem/stromal cells (BMSCs) were purchased from Lonza Biologics at passage 2 and cultured in mesenchymal stem cell growth medium (MSCGM) according to manufacturer's protocols [17,38,39]. Selected batches of BMSCs, which optimally supported vessel formation in vitro, were used at passages 5-6 in all experiments.

Proliferation Assays in Presence of SU6668 Inhibitor
UCB ECFC derived cells, SS-AF-MSCs, BMSCs and hDFs were plated at a density of 1.5610 4 /ml in a 96 well plate and were cultured for 3 and 8 days in the presence of DMEM (10% (v/v) FCS) or DMEM (10% (v/v) FCS) supplemented with 5 mM or 10 mM SU6668 inhibitor (Calbiochem, Merck Chemicals Ltd., Nottingham, England) in 5 replicates for each concentration. After 3 and 8 days, CellTiter 96 AQueous One Solution cell proliferation assays (MTS) (Promega Ltd., Madison, WI, USA) were performed. The absorbance was recorded at 490 nm using a microplate reader (ELX 800, Biotek Instruments Inc, VT, USA). Results were expressed as the percentage of proliferation increase, calculated using the following formula: [(OD dayx -OD-day0 )/(OD control -OD day0 )x100], where OD control corresponded to the absorbance measured in non treated cells on day x. All assays were performed in triplicates and the mean of each experiment was calculated. Statistical analysis was performed using Student's t test.

Proliferation Assays in Presence of Conditioned Media Containing IL-8 and PDGF-AB/BB Neutralizing Antibodies or MMP9 Inhibitor
For the preparation of the conditioned media (SS-AF-MSC-CM), 1610 6 SS-AF-MSCs were cultured until 80% confluent in DMEM with 20% (v/v) FCS, and then the medium was replaced with EBM-2 medium containing 0.5% (v/v) FCS to prevent protein aggregation. Cells were cultured for a further 24 hours and the CM was collected and concentrated approximately 25-fold using ultra filtration units with a 3-kD cut-off (Millipore Ltd.). Sham medium comprised EBM-2 medium containing 0.5% (v/v) FCS which had been incubated for 24 hours in the absence of cells. For proliferation assays, UCB ECFC derived cells were plated in complete EGM-2 medium in 96-well plates (Corning Life Sciences Ltd.) and incubated overnight at 37uC in a humidified air 5% CO 2 incubator. The following day, media were replaced by conditioned or sham media diluted in EGM-2 medium (1:1). On day 3, MTT (Sigma-Aldrich Company Ltd.) was added to each well and absorbance at 570 nm was then measured. For blocking experiments, polyclonal rabbit anti-human IL-8 (Peprotech EC Ltd., London, England, 5 mg/ml) and PDGF-AB/BB mouse antihuman PDGF-AB/BB (BD Biosciences, 5 mg/ml) antibodies (Abs) or MMP-9 inhibitor (Santa Cruz Biotechnology, Inc Santa Cruz, CA, USA, 15 mM) were added in the SS-AF-MSC-CM at day 0.

Transwell Migration Assay-in vitro Blocking Experiments
In vitro motility assays were performed as previously described [48]. In brief, UCB ECFC derived cells at passage 2-6 were transferred at 2610 4 /100 mL density to the insert of a transwell plate with 8 mm pore size (Corning Life Sciences Ltd.) in EBM-2: EGM-2 (4:1) medium. Cells were then allowed to migrate for 6 h across the pore membrane, toward Sham medium or SS-AF-MSC-CM. After the 6 h incubation period, the non-migrated cells were removed from the top of the insert with a wet cotton swab. The migrated cells were then fixed with 4% (w/v) paraformaldehyde on the membrane and stained sequentially with eosin and hematoxilin (all from Sigma-Aldrich Company Ltd.). Migration was quantified by counting the nuclei that passed through the filter. Photographs of the stained nuclei were taken from a minimum of 10 fields of view (20X) for each membrane using an inverted TE300 microscope (Nikon Ltd., London, England) fitted with a cooled CCD camera and Simple PCI software (Digital Pixel, Brighton, England) and experiments were performed repeated twice. Statistical analysis was performed using Student's t-test. For the in vitro blocking experiments, IL-8 and PDGF-AB/ BB were blocked in SS-AF-MSC-CM with the respective neutralizing Abs described above at a concentration of 5 mg/ml for 10-30 min at 4uC. CM was also incubated under the same conditions with irrelevant control Abs. For MMP-9 blocking experiments, the MMP-9 inhibitor-I (15 mM) was added in SS-AF-MSC-CM. Fresh EGM-2 medium and CM derived from HEK-293 T cells were used as control media. In addition, recombinant IL-8 (Peprotech EC Ltd.) or PDGF-AB/BB (Peprotech EC Ltd.) were added, both at 1 mg/ml, in EGM-2 medium supplemented with 0.5% (v/v) FCS.

Vascular Tubule co-culture Assay
For tubule formation, 3 batches of UCB ECFC derived cells at p4-6 were seeded with SS-AF-MSCs, and pre-selected batches of BMSCs or hDFs known to be good supporters of vessel formation in vitro in a coculture assay. In initial experiments, SS-AF-MSCs, BMSCs and hDFs (ranging from 1610 4 to 4610 4 per well) were plated with UCB ECFC derived cells (ranging from 1610 3 to 16 per well) in 48-well collagen-I coated plates to determine the best ratio and cell numbers required for optimum tubule formation. A ratio of 1:4 ECFC derived cells: MSCs or hDFs was estimated as the optimum for the co-culture experiments. Subsequently, SS-AF-MSCs, BMSCs (p5-6) or hDFs (p5-6) were plated in complete EGM2 medium in 48-well plates [38,41], the culture plate gently swirled to evenly distribute the SS-AF-MSCs, BMSCs or hDFs and cells were incubated for 2 to 24 h at 37uC and 5% CO 2 . eGFP labeled UCB ECFC (p4-6) were detached with trypsin-EDTA, counted, resuspended in complete EGM-2 medium and added to the wells with the preplated SS-AF-MSCs, BMSCs or hDF. The plates were gently swirled, incubated at 37uC and 5% CO 2 , the medium was changed every 2-3 days and the cells were cultured for up to 14 days at which point the cells were fixed, and eGFPexpressing tubules were photographed at 46 magnification on an inverted TE300 microscope (Nikon Ltd.) fitted with a cooled CCD camera and Simple PCI software (Digital Pixel). Images were processed in Adobe Photoshop 7 and tubule numbers, lengths and number of junctions were quantified using Angiosys software (TCS Cellworks, Buckingham, England). To test the sensitivity of vascular tubule development to anti-angiogenic factors, SS-AF-MSCs, BMSCs or hDFs were co-cultured with UCB ECFC derived cells and SU6668 inhibitor (at 5 and 10 mM in DMSO; Calbiochem) [42] was added to co-cultured cells in triplicate wells in complete EGM-2 medium at the initiation of the culture. Fresh SU6668 was also added at each medium change (3-4 day intervals) for the duration of the assay over 14 days.  [42]. Plates were incubated for 20 h at 37uC before photomicroscopy. After incubation, media were removed, plates washed with distilled water (PAA Laboratories GmbH) twice, and cells were fixed using 100% (v/v) ice-cold methanol (Sigma-Aldrich Company Ltd.). Each image from each well was taken at 4 magnification using a Nikon Eclipse TE2000-U microscope (Nikon Ltd.).

Tubule Analysis Using Angiosys Software
Quantitation of the vascular tubules was performed by photographing eGFP-expressing tubules at 46 magnification on an inverted TE300 microscope (Nikon Ltd) fitted with a cooled CCD camera and Simple PCI software (Digital Pixel). Images were processed in Adobe Photoshop 7 and image analysis of tubule numbers, lengths and number of junctions were quantified using Angiosys software (TCS Cellworks) as described [41,42]. For quantification, cells were cultured in triplicate, then 4 central fields/well were analyzed from each assay well. Results for the different groups assayed are expressed as means 6S.E.M.

Proteome Profiler Arrays of Conditioned Media (CM)
For the preparation of the CM, 1610 6 SS-AF-MSCs, BMSCs, UCB ECFC or hDFs were cultured until 80% confluent in their respective media, and then the media were replaced with EBM-2 medium containing 0.5% (v/v) FCS to prevent protein aggregation. The cells were cultured for a further 24 hours and the CM was collected and concentrated approximately 25-fold using ultra filtration units with a 3-kD cut-off (Millipore Ltd. Ltd.), and analyzed for specific proteins using proteome profiler arrays for angiogenesis growth factors (Catalog # ARY007, R&D Systems Inc., Minneapolis, USA) according to the manufacturer's instructions. Quantitation of the detected spots was performed using the Quantity One Software 4.4.1 (BioRad Laboratories Inc., Amersham, England). Three different samples for each cell type were tested, each in two replicates. Results for the different growth factors assayed are expressed as means 6S.E.M and statistical analysis was performed using Student's t test. week old NOD-SCID mice in a slightly modified protocol to those described [49]. Six animals per group were used. For the negative control, 200 ml of matrigel was injected without added cells. The animals were sacrificed 2 weeks later and the matrigel mass was excised. Ten to twenty minutes before sacrifice 3 animals per group were intravenously (tail vein) injected and perfused with 200 ml biotinylated tomato lectin (1:20) (Vector Laboratories, Peterborough, England).

Immunohistochemistry, Vessel Quantitation and Diameter Estimation
Matrigel implants were removed at two weeks after implantation, fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich Company Ltd.) and sectioned as 5 mm cryosections. Sections were incubated with mouse anti-human CD31 monoclonal (DakoCytomation) or rat anti-mouse CD31 monoclonal (BD Biosciences) primary antibodies. Sections were then incubated with biotinylated goat anti-rat (BD Biosciences) or goat anti-mouse secondary antibodies (DakoCytomation) for 30 min, followed by ABC-complex-HRP (DakoCytomation) for another 30 min. Reactions were developed for 5 min in DAB (Vector Laboratories), counterstained in Gill's hematoxylin (Sigma-Aldrich Company Ltd.), subsequently dehydrated, mounted and studied by light microscopy. Alternatively, hematoxylin and eosin (H&E) staining was performed to examine the presence of vascular structures containing red blood cells. Images were taken with a Leica CTR MIC microscope and then the number of vessels per mm 2 and the diameter of each vessel (in microns) were estimated using the Image J 1.386 software (http://rsb.info.nih.gov/ij/). Statistical analysis was performed using Student's t test, (*p,0.05). A minimum of 15 fields of view (at 406 magnification) was analyzed from each photograph. Error bars indicate S.D. of the mean for 10 photographs from each group.

Cell Characterization
In the present study, we used batches of SS-AF-MSCs and BMSCs which had been characterized previously in terms of their phenotype and ability to give rise to adipogenic, chrondrogenic and osteogenic lineages [17,29,30]. The SS-AF-MSCs and BMSCs used in these experiments expressed high levels of CD73, CD90, CD105, CD29, CD44 and CD166 surface markers, but lacked hematopoietic surface markers such as CD45 and CD14 and hematopoietic/endothelial markers such as CD31 when analyzed by flow cytometry [17,29,30], thereby confirming their MSC phenotype. The endothelial lineage phenotype of UCB ECFC derived cells was confirmed as described in the Supplementary data with cells lacking CD90, as well as the hematopoietic markers, CD45 and CD14, but expressing CD31, CD73, CD105, CD144 and CD146 ( Figure S1).

SS-AF-MSCs have a Similar Capacity to BMSCs and hDFs in Supporting Vascular Tubule Formation in vitro
We initially examined the ability of SS-AF-MSCs, to support vascular tubule formation in vitro, comparing these to pre-selected batches of BMSCs and hDFs which optimally supported vessel formation. Figure 1 (a to d) shows that eGFP-labeled UCB ECFC derived cells co-cultured with SS-AF-MSCs formed vascular tubule networks over the course of 2 weeks of culture. When UCB ECFC derived cells were plated together with SS-AF-MSCs (Figure 1 a to d) or optimal batches of hDFs (Figure 1 f) or BMSCs (Figure 1 e), they initially adhered to collagen 1 coated dishes. By day 4, vascular tubule development was obvious and the tubule network formation was characterized by intense sprouting over the next 6 days of culture (Figure 1 a to c; days 4 to 10; data not shown). This was followed by consolidation and the formation of an anastomosing network of tubules (Figure 1d; day 14).
Quantitation of vascular tubule formation by UCB ECFC derived cells in the presence of supporting stromal cells (Figure 1 g to i) revealed that, over the course of 14 days of co-culture, the dynamics of vascular tubule formation by UCB ECFC derived cells in the presence of SS-AF-MSCs did not significantly differ from that observed in the presence of the optimal batches of BMSCs or hDFs based on the number of junctions or tubules formed, or the total tubule length. SS-AF-MSCs supported the formation of networks with 254616 junctions, 445631 tubules and a total tubule length of 311826728 microns, compared to 267655 and 235632 junctions, 490680 and 494658 tubules, and total tubule lengths of 2970262107 and 3057161452 microns for BMSCs and hDFs respectively (p.0.05, SS-AF-MSCs compared to BMSCs or hDFs). We next chose to add SU6668, a competitive angiogenesis inhibitor, which disrupts the integrity of the association of stromal cells or pericytes with endothelial cells [42,[50][51][52][53] to our UCB ECFC-AF-MSC co-cultures in vitro to confirm the efficacy of vascular tubule formation in vitro. SU6668 (5 mM and 10 mM) when added from the start of and then chronically throughout the co-cultures severely disrupted vascular tubule formation in vitro in a concentration dependent manner, with the number of vascular junctions, tubules and total tubule length being significantly different with SU6668 addition than without it (p,0.05 Student's t test; n = 3 independent experiments; Figure S2). This SU6668 treatment did not affect the proliferation of SS-AF-MSCs, BMSCs, hDFs or UCB ECFs as shown by the MTS proliferation assay 3 and 8 days after treatment ( Figure S3).

SS-AF-MSCs Support in vivo Vessel Formation more Effectively than BMSCs
We next evaluated the potential of SS-AF-MSCs to support vessel formation in vivo. For this reason, we conducted experiments based on earlier studies [49,54]  Vessel density in the implants was observed by staining sections for human and mouse CD31. Human CD31 was expressed ubiquitously in the matrigel area ( Figure S4), whereas mouse CD31 was primarily found at the edge of the matrigel implant, where the human and mouse vascular network was connected. A similar vascular network was confirmed in the matrigel implants containing eGFP tagged UCB ECFC derived cells and SS-AF-MSCs (Figure 2 f). Vessels containing human CD31 expressing cells were not detected in any of the implants containing SS-AF-MSCs only (Figure 2 g), whereas, in implants containing UCB ECFC derived cells only (Figure 2 h), just a few superficial vessels were detected. The connection of human vessels to the murine vasculature is not only supported by the ability to perfuse the resultant human vessels with tomato lectin after its i.v. injection (Figure 2 d), but also by the demonstration of murine erythrocytes within human vessels positive for human CD31 antigen (Figure 3 a (i-iii) ). The numbers of vessels formed and the vessel diameters for single or combined cell populations transplanted in vivo in comparison with BMSCs and hDFs are shown in Figure 3 b and c. There was a statistical difference in vessel formation by UCB ECFC derived cells in the presence (59.1612.7/mm 2 ) compared to the absence of SS-AF-MSCs (11.160.4/mm 2 ) (p,0.05 Student's t test; n = 3, independent experiments), but no statistical difference in the diameters of the vessels which formed (11.761.9 and 13.462.6 microns respectively for the presence and absence of SS-AF-MSCs) (p.0.05 Student's t test; n = 3 independent experiments). Notably, there was a statistically significant difference in vessel formation by UCB ECFC derived cells in the presence of SS-AF-MSCs (59.1612.7/mm 2 ) compared to BMSCs (42.264.9/mm 2 ) (p,0.05 Student's t test; n = 3, independent experiments). The average number of vessels formed in the presence of hDFs (47.866.4/mm 2 ) differed from that in the presence of SS-AF-MSCs but this did not reach statistical significance (p = 0.12 Student's t test; n = 3, independent experiments).

SS-AF-MSCs Possess Distinctive Secretome Profiles Compared with BMSCs and hDFs
To define the profile of the molecular mediators of angiogenesis/vasculogenesis secreted by SS-AF-MSCs, we examined the conditioned medium (CM) derived from SS-AF-MSCs and directly compared this with BMSC-CM and hDF-CM using Proteome Profiler Human Arrays (see Materials and Methods and  Table S1). Both 24 hour CM and sham media described above were incubated with arrays to allow detection of 55 secreted factors (Table S1). The most interesting factors detected were categorized into 6 groups according to information from the manufacturer and their known functions ( Figure 5 Table S1). However, the commonly shared factors more often occurred in differing amounts in CM from each of the two cell types and more often at significantly higher levels in hDF-CM than in SS-AF-MSC-CM ( Figure 5; Table S1). Indeed, almost 70% of the secreted factors, including VEGF-C, EG-VEGF, PIGF, angiostatin, angiopoietin-2, FGF-4, FGF-7, acidic and basic FGFs, GM-CSF, MIP-1a, IGBP-1, thrombospondin -2, leptin, ADAMTS-1, DPPIV, TIMP-4 and MMP-8, were found at relatively higher concentrations in hDF-CM than in SS-AF-MSC-CM (p,0.05 for all). In contrast, SS-AF-MSCs secreted endothelin-1, angiogenin, MMP-9, MCP-1, serpin-E1, TIMP-1 and thrombospondin-1 at relatively higher levels than hDFs (p,0.05 for all). The BMSC secretome was more limited than that found for SS-AF-MSCs and hDFs. Indeed, less than half the angiogenic factors detected in the SS-AF-MSC-CM and hDF-CM were found in human BMSC-CM (20 factors only) (Figure 4 and Table S1). Of note for the commonly secreted factors, activin-A, angiogenin, FGF7, HB-EGF, IGFBP-1, -2 and -3, pentraxin-3, PIGF, serpin-E1, TIMP-1 and VEGF were detected at significantly higher levels in BMSC-CM than in both SS-AF-MSC-CM and hDF-CM (p,0.05 for all; Figure 4, 5 and Table S1).
Therefore, we selected one factor from each of the three categories, angiogenesis/vasculogenesis, inflammatory response

Discussion
Recent studies have reported that human BMSCs and adipose tissue (AT) derived MSCs can support human blood vessel formation in vivo in animal models when they are co-transplanted with human blood ECFC derived cells [54][55][56]. In the studies presented here, we show that SS-AF-MSCs have the added value of also supporting new vessel formation both in vitro and in vivo, but notably they provided better support for angiogenesis/ vasculogenesis in an in vivo NOD-SCID murine model than BMSCs did, which had been pre-selected for their ability to optimally support vascular tubule formation in vitro.
There are several potential explanations for these in vivo observations. The first is that MSCs at earlier stages of ontogeny have increased potency compared to adult MSCs in dampening a host versus graft response and/or in supporting stable vessel formation in vivo. It has, for example, been shown that fetal MSCs, such as those derived from second trimester amniotic fluid, exert stronger immunomodulatory effects in terms of transplantation tolerance than adult MSCs [57]. The enhanced immunomodulatory properties of SS-AF-MSCs may thus permit better vessel formation by human UCB ECFC derived cells when compared with BMSCs in our NOD-SCID mouse xenograft model, which lacks T and B lymphoid cells, but not NK cells [58]. These mice are known to be a less efficient model for reconstituting human hematopoiesis than NSG (NOD/Lt-scid/ IL2Rgamma (null)) mice, which lack NK cells and demonstrate other defects in innate immunity not observed in the NOD-SCID mouse strain [58,59]. However, as the NSG xenograft model has not to our knowledge been tested in vasculogenesis assays in vivo, future studies would need to determine if NSG mice provide a more efficient in vivo readout of vasculogenesis than those currently in use (athymic nu/nu, SCID or NOD-SCID mice) and which are not NK cell deficient [59]. While the use of the NOD- SCID in vivo model and enhanced immunomodulatory capability of fetally derived MSCs might partially explain why SS-AF-MSCs are more efficient in promoting vasculogenesis in vivo than BMSCs, this does not explain why the hDFs more closely resemble SS-AF-MSCs in supporting more vessel formation in vivo than BMSCs do. While SS-AF-MSCs and BMSCs both possess immunomodulatory activities (reviewed in [5,9,26]), this is not the case for hDFs and these latter cells would therefore be expected to be less well tolerated in in vivo NOD-SCID xenografts.
An alternative explanation and perhaps a more significant contributory effect for achieving more efficient in vivo vasculogenesis could relate to the balance of angiogenic factors secreted by the supporting hDFs or MSCs. Indeed, our studies demonstrate that, although SS-AF-MSCs, BMSCs and hDFs all promoted vessel formation, significantly more vessels formed in vivo in the presence of SS-AF-MSCs and hDFs than in the presence of BMSCs, and each displayed unique secretome profiles. Notably, BMSCs secreted less than half the angiogenic factors detected in conditioned media derived from SS-AF-MSCs and hDFs, while SS-AF-MSCs and hDFs secreted many factors in common. Those factors present in both SS-AF-MSC-CM and hDF-CM, but not detectable in BMSC-CM, included IL-8, FGF-4, angiopoietin-2, HGF, PD-ECGF and MMP-9, while SS-AF-MSCs also secreted PDGF-AB/BB. Each of these factors has defined roles in regulating angiogenesis and vasculogenesis [53,[60][61][62][63][64][65][66][67][68][69], and our studies confirmed that IL-8, MMP-9 and PDGF-AB/BB secreted by the SS-AF-MSCs were functional in promoting UCB ECFC derived cell migration, while IL-8 also promoted UCB ECFC derived cell proliferation. Interestingly, recent studies from Kachgal and Putnam [61] demonstrate that adipose tissue (AT) MSCs resemble human lung fibroblasts more closely than BMSCs in their angiogenic factor profile, with uPA and MMPs in particular playing key but distinct roles in promoting vessel formation and integrity. Other studies identify leptin as an angiogenic factor in AT-MSCs [65]. As with our own studies, this suggests that different MSCs/perivascular cells promote vessel formation by differing mechanisms and that MSCs derived from bone marrow may differ significantly in their angiogenic profiles from those found in amniotic fluid or adipose tissue. Although beyond the scope of this study, understanding these mechanisms in more detail should prove beneficial not only for our understanding of vessel formation during development and within specific microenvironmental niches, but also for sourcing cells or defining combinations of factors for vascular repair, for revascularizing damaged tissues or organs and for promoting neovascularization in engineered tissues.
In our previous studies, we successfully isolated and expanded over many passages (at least 40) MSCs from human second trimester amniotic fluid [29,30]. Indeed, we and others have demonstrated that SS-AF-MSCs can be expanded and produced rapidly to a clinical scale while retaining their immunosuppressive properties [30,70]. Such AF-MSCs exhibit chromosomal stability with no karyotypic abnormalities detected even at high passages, express pluripotent markers, possess long telomeres and do not form tumours in vivo [11,21,23,29,30]. As putative intermediates in terms of their potentiality between human embryonic or induced pluripotent stem (ES/iPS) cells and postnatal MSCs and without the inherent difficulties that ES/iPS cells have in forming spontaneous tumors in vivo or the lower proliferative and hence expandability difficulties of adult BMSCs [8,26], the AF-MSCs constitute a very interesting and promising population of cells for clinical revascularization. One disadvantage of these AF-MSCs is their collection at the time of amniocentesis, which is not routinely performed in all pregnancies and which can rarely lead to fetal loss. However, where amniotic fluid is collected for routine prenatal diagnosis and by taking advantage of protocols developed from our work and our preclinical studies [29,30,37,45], excess amniotic fluid that is normally discarded can be used to source SS-AF-MSCs, which can then generate millions of cells in culture in a relatively short period of time, enough for prospective clinical applications. Thus, amniotic fluid may prove a valuable source of MSCs for future autologous or allogeneic clinical applications related to tissue revascularization and repair.