State of the Art and New Trends from the 2022 Gism Annual Meeting

The 2022 Italian Mesenchymal Stem Cell Group (Gruppo Italiano Staminali Mesenchimali, GISM) Annual Meeting took place on 20–21 October 2022 in Turin (Italy), with the support of the University of Turin and the City of Health and Science of Turin. The novelty of this year’s meeting was its articulation, reflecting the new structure of GISM based on six sections: (1) Bringing advanced therapies to the clinic: trends and strategies, (2) GISM Next Generation, (3) New technologies for 3D culture systems, (4) Therapeutic applications of MSC-EVs in veterinary and human medicine, (5) Advancing MSC therapies in veterinary medicine: present challenges and future perspectives, (6) MSCs: a double-edged sword: friend or foe in oncology. National and international speakers presented their scientific works with the aim of promoting an interactive discussion and training for all attendees. The atmosphere was interactive, where ideas and questions between younger researchers and senior mentors were shared in all moments of the congress.


Introduction
The 2022 Italian Mesenchymal Stem Cell Group (Gruppo Italiano Staminali Mesenchimali, GISM) annual meeting took place on 20-21 October 2022 in Turin, with the support of the University of Turin and the City of Health and Science of Turin. The novelty of this year's meeting was its articulation reflecting the new structure of GISM, based on six sections, that have been implemented during the last few years to better cover the interests concerning the different aspects of mesenchymal stem/stromal cell (MSC) use. The meeting brought together GISM-Regenerative Medicine with the support of the Cell Factories and researchers close to the clinic, and the GISM-Next-Generation section reflects the willingness of young talented researchers on MSCs. The GISM-Veterinary section, a very active group that reached not only researchers but also clinical veterinarians, introduced research in animal models towards the GISM-Secretome, focused on paracrine mechanisms mediated by soluble molecules and extracellular vesicles of MSCs. The last section, GISM-Oncology, covered the discussed relations between MSCs and tumours as well as the use of MSCs as a tool for drug delivery in cancer.
National and international speakers presented their scientific works with the aim of promoting an interactive discussion and training the youngest researchers.

Session Summary
The meeting was held in six different sessions in order to diversify the various study and research sectors (clinical, cell cultures and extra vesicles, veterinary, oncology, etc.) and to broaden the discussions and experiences of clinicians and researchers in the specific fields of study.
2.1. Session 1: "Bringing Advanced Therapies to the Clinic: Trends and Strategies" The first session started with Graziella Pellegrini (University of Modena and Reggio Emilia, Italy) who presented her work about ex vivo expanded autologous human corneal epithelial cells containing stem cells and their production process for marketing authorisation (Holoclar) [1]. She and her group have been studying Limbal Stem Cell Deficiency (LSCD) since 1997 [2]. Since the regulatory guidelines changed in 2008 [3], their focus was aimed to obtain EMA approval for Holoclar which happened in 2015 [4] with a confirmatory trial of 80 patients. Their follow-up from 1997 to 2008 made it possible to have this court and, therefore, to already have in vivo studies, which allowed the facilitation of the approval. This allowed for real in vivo models as no animal eye can be compared to the human eye. She explained their procedure consisting of the isolation of epithelial cells from a very small biopsy from the healthy eye and their expansion and freezing. After thawing, the cells were transplanted with a special carrier on the damaged cornea [5]. The patients had a fast recovery with re-epithelialization after one week and re-vascularization after a couple of months with restored vision and stability. The final costs showed that Holoclar is less expensive than surgery (O-01).
Following, Stefano Cosma (University of Modena and Reggio Emilia, Italy) spoke about economic aspects and how researchers can bridge the funding gap. Scientific and operational factors, as well as economic factors (such as a lack or inadequacy of funding) represent risks of failure in medical research. Cosma and colleagues collected from the ClinicaTrial.gov database: 12,934 interventional studies were performed in Italy and 1137 in Europe, which were classified by keywords within advanced therapy medicinal product (ATMP) research. Through manual data reprocessing, they highlighted two important features: first, the information about failed and successful projects and their failure causes; second, the classification of the studies according to the funding source. An intersection of these data explained how financial resource allocations are correlated to the success or failure of clinical trials. This intervention was very important and elucidating, showing the point of view of an economist to understand and have indications of the economic aspects of the ATMPs (O-02).
After a break, Nicholas Crippa Orlandi (University of Siena, Italy) presented three cases of different body segments obtained through orthopaedic surgery that benefit from the use of gelled preparations containing autologous bone marrow MSCs. Crippa Orlandi and colleagues optimised the isolation and expansion protocol of bone marrow MSCs using Platelet Rich Plasma (PRP) instead of Foetal Bovine Serum (FBS), characterising cells seeded on the bovine bone matrix (SmartBone) or on a lyophilized acellular matrix, and they increased cell differentiation and osteogenic properties by adding growth factors. On SmartBone, the cells differentiated into osteoblasts and produced collagen. These data suggest that MSCs can provide valid help in complicated orthopaedic surgeries (O-03).
In order to have an overall view of the various sources of MSCs, Ilaria Roato (University of Turin, Italy) compared MSCs isolated from different districts of the oral cavity [6]. Adult Dental Pulp Stem Cells (DPSCs) are less osteogenic than Buccal Fat Pad Stem Cells (BFPSCs) but are more indicated for the pulp-dentin complex regeneration. MSCs isolated from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed no differences in the MSC marker immunophenotype, but SHED and DPSCs had a higher percentage of endothelial precursors than PDLSCs. The capability of MSCs to differentiate into a specific tissue depends also on the different anatomical origins, therefore, MSCs isolated from the oral cavity might be more effective than MSCs isolated from other origins such as adipose-derived stem cells (ASCs). Furthermore, these cells can of OA synovial fluid on the joint-on-chip were conducted and then the effect of MSCs was tested as a possible therapy option. These types of models could allow the personalization of the therapy for patients as the cells can be obtained directly from them and could allow the testing of innovative therapies (O-08).
Afterwards, the word was given to Maria Harmati (Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary) who illustrated the crosstalk routes between tumour cells and stromal cells and vice versa. The crosstalk was conducted through extracellular vesicles tracked with specific dyes. She conducted her experiments not only on human ductal carcinoma cells but also on melanoma and osteosarcoma models. This provided more proof of the crosstalk that occurs and it provided proof on how the crosstalk changes in different tumour models (O-09).
Subsequently, Lucia Ceresa (Charles River Microbial Solutions, Italy) illustrated the suitability of Rapid Microbial Methods as a method to test the quality and safety of new cell-based medicinal products, using an ATP-based luminescent platform such as the Celsis platform which depletes the presence of cellular ATP and also allows fast detection of microbial presence (O-10).
Concluding this session, Silvia Scaglione (React4life startup, University of Genova, Italy) was chosen to discuss her poster as an oral communication. She presented her results on a novel Multi In Vivo Organ (MIVO) on a chip platform and how it could facilitate drug testing on cancer models [8]. Indeed, the models proposed were a 3D ovarian model developed and treated with Cisplatin, and a 3D neuroblastoma cancer model which was used to coculture with immune cells. These studies obtained a relevant disease model that can be used to investigate crosstalk between healthy and pathogenic cells and can also be employed as a drug screening platform (O-11).

Session 4: "Therapeutic Applications of MSC-EVs in Veterinary and Human Medicine"
Session 4 of the congress had a focus on the therapeutic applications of MSC-EVs in veterinary and human medicine. Stefania Bruno (University of Turin, Italy) presented the obstacles that can be found during the transfer of therapy with MSC-EVs from the laboratory to the clinic. In fact, MSC-EVs have been shown to have therapeutic effects in preclinical models of several diseases as they have pro-regenerative capacities and are considered therapeutic tools for various pathologies. To this end, EV characterisation is an area of intense investigation. To successfully translate EV research from the laboratory to patients, strategies must be designed to clinically test the safety and efficacy of MSC-EVs. Currently, manufacturing of cells and EVs, and quality controls, are being used for clinical testing of the feasibility of non-industrial processes. However, defining the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting (O-12).
In this regard, Silvia Zia (StemSel srl, Bologna, Italy) presented the Celector ® instrument, useful for ATMPs quality control and standardisation. Since MSCs are a heterogeneous population, the identity/purity of the cell population and its yield are critical issues that may limit their clinical application. To improve the characterisation of MSCs and standardise protocols, it is necessary to develop functional assays that evaluate the biophysical properties, profile, and quality of the cells. The Celector ® instrument is able to analyse, discriminate, and tag free separate a wide range of cells based on their physical characteristics, with high resolution and without damage. She explained the separation method, imaging acquisition, post-processing, and data analysis underlining that Celector@ is able to highlight physical differences related to cell viability and regenerative potential [9] (O-13).
Afterwards, space was given to young researchers to orally communicate their posters.
The first was the researcher Elena Ceccotti (University of Turin, Italy), who presented her work on chronic kidney diseases. In particular, she explained that human liver stem cells extracellular vesicles (HLSC-EVs) can be used as carriers for the transfer of active biological drugs both in vitro and in vivo models of renal ischemic reperfusion injury (IRI) associated with acute kidney injury (AKI) and chronic kidney disease (CKD). In this study, the results showed that in AKI mice, EV treatment attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation, and downregulated expression levels of fibrosis-related genes. In CKD mice, interstitial fibrosis and the expression levels of pro-fibrotic and pro-inflammatory genes were decreased [10]. Thus, the administration of HLSC-EVs immediately after renal IRI protects the kidney from the development of AKI and interferes with the development of subsequent CKD (O-14).
Proceeding with the session, Tarlan Eslami Arshaghi (University of Galway, Ireland) introduced the Aptamer approach: a fluorescence polarisation-based approach for EV quantification. This approach aimed to develop a novel high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific surface component, using fluorescence polarisation (FP) for detection. The method analysed the change in polarisation of the emitted light between unbound and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of bound probes. This property of FP allowed them to use it to quantify the amount of EVs in the solution. She explained the different strategies used to demonstrate the enhanced fluorescence polarisation in response to increasing EV concentration, quantified by NTA and the study of probe-target kinetics (O-15).
Finally, the last speaker, Enrico Ragni (IRCCS-Galeazzi Hospital, Milan, Italy) talked about the MSC secretome for regenerative medicine related to orthopaedic conditions. He characterised adipose-derived MSCs (ASCs) and EV-miRNAs and their modulation after high levels of IFNy preconditioning and mimicking OA. The penetration of ASC-EVs was evaluated in cartilage explants. Bioinformatics tools were used to predict the modulatory effect of the identified molecules on pathological cartilage and to follow and quantify the incorporation of fluorescent EVs into cartilage explants. The ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes thanks to the detected presence of 50 cytokines/chemokines and more than 200 EV-miRNAs, and inflammatory preconditioning or OA-like conditions have been able to increase this ability. The ASC-secretome's ability to stimulate healing and reduce inflammation allows it to be proposed as an ideal candidate for orthopaedic regenerative medicine [11] (O-16).
2.5. Session 5: "Advancing MSC Therapies in Veterinary Medicine: Present Challenges and Future Perspectives" Laura Barrachina Porcar (University of Galway, Ireland) presented her work on MSCs and their immune properties. She explained that the use of allogeneic MSCs presented several advantages with respect to autologous cells as a possibility to have a ready-to-use product. Although cell-based products in the veterinary market are emerging, allogeneic therapy does not come without limitations. At first, MSCs, which were initially considered immune-privileged, can actually induce cellular and humoral immune responses. Based on these observations, she reported several human and animal studies with positive results after allogeneic MSC administration in the absence of adverse effects. A potential explanation for the mixed outcomes often seen was that MSCs can be recognised by the immune system (immunogenicity), but they can also regulate it (immunomodulation). She also explained the different strategies that could be designed to develop safer and more effective allogeneic therapies and how it could allow the creation of 'haplo-banks' of cells from donors. The understanding of the interactions between MSCs and the immune system could be a key to learn which factors we can manage, and how (O-17).
Following, the first oral selected communication was from Barbara Merlo (University of Bologna, Italy) who presented the results of a pilot study concerning the effect of GM18, an α4β1 integrin agonist, on the adhesion properties of equine adipose tissue and Wharton's jelly derived MSCs and on their ability to adhere to GM18-incorporated poly L-lactic acid (PLLA) scaffolds. The use of biomaterials with integrin agonists promoted cell adhesion in tissue repair processes confirming the presence of GM18-containing PLLA scaffolds. In conclusion, GM18 affects equine MSCs adhesion ability with donor-related variability.
These preliminary results suggested that MSCs from Wharton Jelly might be more suitable than MSCs from adipose tissue [12] (O-18).
The second selected oral communication was presented by Gabriele Scattini (University of Perugia, Italy) which was about migrasomes (MG), a particular kind of EVs released by MSCs of different species and tissue sources possibly related to their migratory activity. He reported that MGs were isolated by differential ultracentrifugation of MSC supernatant and were observed by Transmission Electron Microscopy (TEM) as different from other microparticles in size and morphology. Their biogenesis and morphologic features were described in detail compared to the EV characteristics, although he underlined that their different functions are still to be clarified (O-19).
2.6. Session 6: "MSCs: A Double-Edged Sword: Friend or Foe in Oncology?" Roisin Dwyer (University of Galway, Ireland) opened the last session on the dual role of MSCs in oncology with a presentation of her research on the use of EVs isolated from MSCs as therapeutic delivery for cancer. MSC-derived EVs raised interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. The recent work presented by Dwyer's group showed the development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy. Their objective was to develop an innovative approach based on MSC-EVs as a cancer treatment for patients with breast cancer who have limited treatment options. From her point of view, the use of more reflective pre-clinical models of the patient experience has to be developed to test new therapeutic approaches [13] (O-20).
Andrea Papait (Cattolica del Sacro Cuore University, Rome, Italy) demonstrated the MSC role in the tumour microenvironment and their complex interaction network in contact with anticancer agents. In his presentation, he discussed the use of MSCs considered as a double-edged sword: they can serve as a drug carrier while their immunomodulatory properties [14] can participate in tumour initiation, development and progression, and metastasis formation [15]. For him, in the era of immunotherapy, MSCs or their secretome could represent an adjuvant therapy in association with new drugs such as monoclonal antibodies aimed at re-educating the immune response (O-21).
Finally, the meeting ended with the selected presentation by Valentina Coccè (University of Milan, Italy). She presented data on the inhibitory effect of adipose tissue-derived MSCs uploaded by paclitaxel (PTX) on malignant pleural mesothelioma in vitro and in vivo models. In particular, she saw how adipose tissue (FAT) after micro fragmentation (MFAT) was able to exert a dose-dependent inhibition on the growth of the human mesothelioma cell line (MSTO-211H). While, in xenografted Balb/c-Nude mice obtained after subcutaneous injection with MSTO-211H, she observed a reduction of tumour mass volume measurements after treatment with MFAT loaded with PTX similar to that of free PTX. This could be a new therapeutic approach for a difficult to treat tumour [16] (O-22).

Concluding Remarks
After each session, at least 30 min were dedicated to questions from the audience and replies from the speakers, making the discussion very open and engaging for all the presentations and also helping to draw out take-home messages. In particular:

•
The first session about the source of funding for the research projects was very animated and of great interest for all the attendees. • The "GISM-Next Generation" section, which primarily concentrates on improving general skills related to publishing, patent writing, fundraising, and knowledge transferring, provided valuable insights based on the latest technological advancements and opportunities presented by social networks.

•
The third session, dedicated to new 3D culture systems, gave insight on the current 3D strategies that could aid in the creation of personalised therapy options with systems such as joints-on-chip or the MIVO system.

•
The session on therapeutic applications of MSC-EVs in veterinary and human medicine provided that the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting • The fifth session showed the advancing MSC therapies in veterinary medicine present challenges and future perspectives not only in the veterinary field, but also for translation research. In fact, the MSC therapy in animals represents an excellent approach to have results describe an experimental investigation brochure dossier for an experimental clinical trial with secretome or MSCs • The last session was dedicated to how EVs can be used as drug carrier systems and how they interact with the tumour microenvironment, showing interesting new discoveries especially in breast and mesothelioma. Figure 1 summarises the positive and negative aspects in mesenchymal stem cells utilisation and MSC secretome (microvesicles and exosomes), as raised from the six sections discussed here.
general skills related to publishing, patent writing, fundraising, and knowledge transferring, provided valuable insights based on the latest technological advancements and opportunities presented by social networks.

•
The third session, dedicated to new 3D culture systems, gave insight on the current 3D strategies that could aid in the creation of personalised therapy options with systems such as joints-on-chip or the MIVO system.

•
The session on therapeutic applications of MSC-EVs in veterinary and human medicine provided that the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting • The fifth session showed the advancing MSC therapies in veterinary medicine present challenges and future perspectives not only in the veterinary field, but also for translation research. In fact, the MSC therapy in animals represents an excellent approach to have results describe an experimental investigation brochure dossier for an experimental clinical trial with secretome or MSCs • The last session was dedicated to how EVs can be used as drug carrier systems and how they interact with the tumour microenvironment, showing interesting new discoveries especially in breast and mesothelioma. Figure 1 summarises the positive and negative aspects in mesenchymal stem cells utilisation and MSC secretome (microvesicles and exosomes), as raised from the six sections discussed here. In general, the congress was a great success thanks to lively participation of researchers of all ages, demonstrating that the topics of the congress were very attractive In general, the congress was a great success thanks to lively participation of researchers of all ages, demonstrating that the topics of the congress were very attractive in the scientific community. In fact, more than 100 researchers participated in the meeting and 51 posters were exposed and presented in a dedicated section of the congress.
Overall, the atmosphere was interactive and full of young researchers who felt at ease sharing their ideas and questions with senior mentors in all moments of the congress, including pleasant moments of relationship.
All the abstracts of oral and poster presentations, with written consent for the publication here.

Poster Award
During the 2022 GISM annual meeting, three "Young Investigator Awards" of 500 euros each were assigned. In order to be eligible, researchers had to (a) submit a spontaneous candidature; (b) be the first author of an accepted abstract; (c) be younger than 35 years on 20 October 2022; (d) be present at the Award Ceremony held during the "GISM-Next Generation" section. The three winners of the 2022 GISM annual meeting "Young Investi-and financial needs of the research into ATMPs with a Capital budgeting approach in order to correctly determine the ability to generate (economic and social) value. The paper aims to explore the causes of failure of the various phases of medical research and if and how the current funding schemes or financial partners may affect them. The purpose is achieved with the support of the ClinicaTrial.gov database, which includes privately and publicly funded clinical studies conducted worldwide. As a preliminary study, we focused our attention on the Italian context, collecting information on interventional studies carried out by Italian research groups during the entire period of database coverage (since 1998 to 2021). We collected a total of 12,934 interventional studies, 344 of which we classified by keywords within ATMPs research. The analysis was also extended to a sample of 1137 European studies classified by keywords within ATMPs research. The study builds a complete, general picture of the current funding of clinical research, the current role of Finance and the types of involvement of the public sector and other non-profit partners. Through manual data reprocessing, we were able to highlight two important features of the studies in the sample. First, the information on their current status allowed us to pinpoint failed and succeeded projects, while also understanding failure causes. Second, information on their sponsor was re-elaborated in order to classify the studies according to the source of funding (e.g., industry, university, research centres, hospitals, and so on). Thanks to an intersection of this data, our work provides insights into how the current financial resource allocation may be correlated to the success or failure of clinical trials, with a focus on advanced therapies, revealing not only potential funding gaps but also implications for involved researchers, policymakers, and stakeholders.

Abstract
Orthopaedic surgery can benefit a lot from the use of stem cells. Three case reports drawn from the experience of the authors' Orthopaedic Clinic are illustrated to highlight the benefits of applying this technology. Drawing on the extensive experience gained within the authors' Operating Unit, three cases regarding different body segments have been selected to prove the benefits deriving from the use of gelled preparations containing autologous MSC from bone marrow. A case of humeral shaft non-union, the management of an atypical proximal femur fracture in congenital hip dysplasia, and a case of rupture of the patellar tendon and consequent reconstruction of the extensor apparatus with a cadaveric transplant. The experimental study, whose three cornerstones are mesenchymal stem cells (hMSCs), scaffolds, and growth factors, aims to develop new tissue engineering strategies to be applied in orthopaedic surgery. In the first part of the work, the focus was on the optimisation of the isolation and expansion protocol of the hMSCs, taken from the bone marrow and deposited in the Biobank present inside the laboratory which is part of the Network Telethon (TNGB), evaluating the replacement of the FBS (fetal bovine serum) with autologous PRP (platelet rich plasma). Then, the focus shifted to the characterisation of cells seeded on two types of scaffolds: bovine bone matrix (SmartBone) and lyophilized acellular dermis. The final goal of the work is to develop strategies to increase the osteogenic properties of the support by adding growth factors that enhance cell differentiation on the scaffold. Our technique of applying stem cells to cases of complex orthopaedic surgery has shown excellent results both in a clinical functional objective and subjective evaluations and in radiographic evaluations. The experimental study, currently underway, is providing excellent results. The data suggest PRP as a valid alternative to FBS, it supports the expansion of mesenchymal stem cells without compromising their capacity since cells loaded on SmartBone differentiate into osteoblasts and produce collagen. In our opinion, MCSs are and will become more and more a valid tool to provide the surgeon with important help in cases of great complexity and, therefore, to obtain the tailored care that every patient needs and deserves. compared to buccal fat pad stem cells (BFPSCs). The analysis of the immunophenotype of in vitro expanded stem cells from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed a comparable expression of MSC markers among them and a higher percentage of endothelial precursor subset in SHED and DPSCs than in PDLSCs. Owing to their peculiar origin, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, even though MSCs retrieved from different tissues show comparable immunophenotype and multilineage differentiation ability, their capability to differentiate into a specific tissue depends also on the different anatomical origins. Moreover, often, these cells may be harvested without any burden or additional discomfort for the patient who undergoes a tooth extraction for orthodontic indications.

Objective
An important challenge in regenerative medicine to the regulatory approval and widespread clinical acceptance of cell therapies is surrounding the behaviour of cells after transplant. The distribution, engraftment, viability, and activity of transplanted cells are unclear. This reduces confidence in the safety of cell therapies and makes it difficult to understand the mechanism of action for developing potency assays that will be required for advanced clinical testing and the approval of cell therapy. When Extracellular Vesicles (EVs) are used as therapeutic agents, similar issues arise. Optoacoustic imaging (OAI) is a rapidly developing technology with world-leading capabilities in functional imaging that is uniquely informative and lower cost, convenient, and rapid. Gold-Nanostars (GNs) are suited for use as a contrast medium for optoacoustic imaging that generates a very strong photo-thermal signal in response to light of longer wavelengths. The combination of OAI and GNs offers a class-leading imaging solution for cell therapies in regenerative medicine. In this work after a characterisation in vitro of the complexes, GNs-labelled Mesenchymal Stem Cells (MSC) and EVs were studied for their distribution in different organs after a systemic injection by visualisation with Multispectral Optoacoustic Tomography (MSOT) and tested for the in vivo toxicity.

Materials and Methods
MSCs were isolated from human bone marrow, expanded in Platelet Lysates (PL), and derived from the same cell EVs. The MSC and EVs were labelled with GNs and by in vitro tests with the right concentration of particles, in terms of capability to be visualised by MSOT and no cytotoxicity, was defined. Labelled and unlabelled complexes were injected into the caudal vein of male and female Nu/Nu mice. Blood tests and histological analysis of the organs were performed at different time points to test the in vivo toxicity.

Results
No alterations were observed in terms of biochemical and blood markers. MSOT analysis showed that after one day, GNs reached all organs and remained for all monitoring periods without any alteration as revealed by histological analyses. In particular, MSOT analysis showed that GNs cannot overcome Blood-Brain Barrier (BBB).

Conclusions
The results showed that the GNs can reach the various organs and that they persist there for some time, but although they persist, they cannot overcome BBB and does not lead to any type of alteration. This approach holds promise for tracking cell distribution in the tissue repair process. Abstract "Scientific Articles" are not the final goal but a necessary intermediate step for worldwide progress. "Scientific journals" are the right place to communicate discoveries to society. "Editors and Reviewers" are important people for evaluating the results. "Impact Factor" is one of the most common words in the life of a researcher. These terms are just a few of many behind the real important things in research including "Hypotheses and Results". However, a large part of the working life of a researcher is spent writing scientific articles and finding the right journals for them. In this presentation, we will discuss how to speed up the publication process, define the best journal, and increase the chances of publication by exploiting opportunities that technology and social networks today provide us.

Abstract
Technology transfer in the Life Science field is a demanding but very stimulating challenge for both researchers and technology transfer experts. The sector has peculiarities that differentiate it from most other technological fields and make the exploitation of research results even more challenging. The results obtained by universities and research hospitals, while promising, are often premature and require a long development path to be commercialised and brought to the clinic. It is well known that very long research and development times and high costs are required to bring a medical device or drug to the market. This gap between proposals from the research world and needs from industry is well known by the technology transfer experts and often discourages the researchers who do not see their research reaching the patient, although a strong clinical need is well identified. Many factors are crucial to fill this gap, not just the need for dedicated funding. The design according to the correct principles is fundamental to allow the industrialisation of the prototype, the scale up of the processes, and the subsequent certification phases. Last but not least, the protection of industrial property (IP) is crucial. Indeed, a company would be unlikely to invest millions of euros in a new life science project without the certainty of exclusivity on the results that would guarantee a competitive advantage over its competitors. The patent is the main tool to obtain this exclusivity, together with the copyright, the design and the trade secret. The proper protection of the research results is an essential step in the technology transfer process. Researchers can have support from the Technology Transfer Offices (TTO) of their institutions, which have the fundamental role of scouting the results, assisting researchers in protecting IP and then activating virtuous paths for the development of the Technology Readiness Level (TRL) of the projects. These paths have to be walked together with several stakeholders such as corporates, start-up incubators, business angels, venture capitalists, etc. The activation of co-development processes with companies and grants dedicated to the development of the Proof of Concept of the patented technologies are key steps in making the project attractive for a company that can licence it or in creating a startup to develop and commercialise the new technology.

Abstract
The musculoskeletal system is composed of different tissues and organs, i.e., bones, muscles, tendons, and joints. Several pathologies can affect its homeostasis including traumatic injuries and inflammatory diseases such as osteoarthritis (OA). In this context, mesenchymal stem cells have been considered either as building blocks to fabricate biological substitutes for damaged musculoskeletal tissues due to their differentiation potential towards bone and cartilage or as a possible therapy due to their anti-inflammatory properties. On the other hand, 3D in vitro models are increasingly being considered a powerful tool to investigate pathological mechanisms and therapeutic efficacy, allowing to overcome the excessive simplification of standard in vitro models and species-specific differences of animal models. Thus, we developed 3D in vitro models of joints to test the potential of mesenchymal cells as anti-OA therapy and of the muscle-tendon-bone junction, based on differentiated mesenchymal cells. We generated a microfluidic joint-on-a-chip model including cartilage, synovial membrane and synovial fluid, based on patient-matched synovial fluid and cells embedded in gels mimicking the composition of cartilage ECM. The injection of OA synovial fluid allowed for the reproducing of the main hallmarks of early OA as the production of degradative enzymes and an increase in inflammatory cytokines. Furthermore, we were able to detect the effects of mesenchymal cell injection, measuring MMPs and inflammatory cytokine release, on a patient basis, opening new possibilities for the establishment of personalised treatments. We also fabricated a 3D bioprinted muscle-tendon-bone model embedded in a microfluidic chip, allowing the fabrication and culture of three different connective tissues in their specific culture media. Computational simulations were performed to design the chip, keeping the culture media separated during perfusion. The chip also allowed to apply compression to the bone and stretch to muscle and tendon in physiological and pathological ranges, simulating traumatic conditions. The 3D bioprinting procedure resulted in three separated cell compartments, maintaining a good shape fidelity and high cell viability. In conclusion, biofabricated 3D in vitro models could help foster the application of mesenchymal cells as anti-inflammatory therapy and could benefit from their potential as starting material for the reconstruction of musculoskeletal tissue units.

Objective
The evolutionary process of solid tumours highly relies on the extracellular vesicle (EV)mediated crosstalk between malignant cells and stromal cells in the tumour microenvironment (TME). In this study, we aimed to establish a multicellular three-dimensional (3D) tumour model system for tracking the EV communication network of different tumour tissues under physiological conditions and cytostatic treatments.

Materials and Methods
Human ductal carcinoma, melanoma, and osteosarcoma models were established via co-culturing the respective tumour cell line (T-47D/A375/MG-63) with MRC-5 fibroblasts and EA.hy926 endothelial cells on flat-or U-bottom plates after staining with Cell-Tracker dyes (Orange CMTMR, Deep Red, Green CMFDA). To mimic chemotherapeutic stress, low dose doxorubicin was used and the 2D and 3D cultures were imaged daily by a PerkinElmer Operetta High Content Screening System and a Leica SP8 Digital LightSheet microscope, respectively.

Results
Preliminary experiments showed that CellTracker dyes can be used for in-cell labelling of EVs, allowing the quantitative monitoring of EV crosstalk, i.e., EV routes between each cell type and in both directions. The three types of tumour models showed differences in their 3D structure, EV crosstalk activity, and drug-induced effects as well. We could observe distinct temporal kinetics in the development of the EV communication network in 2D and 3D, also priorities of the investigated EV routes varied between the two co-culture systems.

Conclusions
The developed 3D model system is suitable for live tracking of EV crosstalk in the TME, which enables the comparison of the primary EV communication routes in different tumour types and drug treatments. Further data will help (i) to identify potential targets of EVblocking therapies, which may increase the efficacy of chemotherapies, and (ii) to predict the drug-induced changes of the communication activity in different tumour tissues.

Lucia Ceresa, Senior Technology and Market Development Manager Microbial Solutions, Charles River, Italy
Abstract New and emerging cell therapies and medicinal products present new challenges in the assurance of quality and safety in terms of end-product testing. While traditional pharmaceutical drug products have long-established standards for sterility assurance, these established processes are not optimised for cell therapies. For example, the Compendial Sterility Test requires more than fourteen days of incubation for a reliable result, making it a rate limiter in the distribution of therapies to patients. Rapid Microbial Methods (RMM's) offer reliable alternatives to ageing microbiological methods to solve the problem of reducing cycle time in cell therapy manufacture. ATP Bioluminescence is a matured technology that is used in the quality testing of various product samples in different industries. Detection of microbial ATP using the luciferin-luciferase reaction allows for the detection of microbes before they can be cultured to visual detection levels on microbial media. Until recently, ATP bioluminescence was not a viable contamination detection option for cell-based products because these samples also contain cellular ATP. The established ATP Bioluminescence platform, Celsis ® , was further developed to address this limitation. A sample cell lysing procedure allows for the extraction and, more important, the depletion of "non-microbial-ATP", while leaving microbial ATP intact. A case study on tests performed on different cell lines demonstrate the detection of the "slow growing" C. acnes as well as a wide panel of other typical and critical microbial species. Studies performed using the Celsis ® platform and the Celsis Adapt™ complimentary technology demonstrated the successful depletion of cellular ATP from samples, while also allowing fast detection of microbial presence contamination for superior microbiological contamination control.

Objectives
The human disease modelling for basic research and drug testing purposes is currently carried out through 2D cell culture in static conditions and in vivo xenografts or genetically engineered animal models, but predictability, reliability, and complete immune compatibility remain important challenges. For this aim, novel 3D, fully humanised in vitro tissue models have been recently investigated by adopting emerging technologies such as bioprinting and microphysiological systems, also named organ on chips. A novel Multi-In Vitro Organ (MIVO) organ on a chip platform has been recently developed to culture 3D clinically relevant size tissues under proper physiological culture conditions.

Material and Methods
Biologically relevant cancer samples (up to 5 mm), and patient biopsies of scaffold-based tissues have been cultured within the MIVO chamber, while either testing molecules or human immune cells (e.g., Natural Killer cells, NK) can circulate in the OOC mimicking the blood capillary flow. The cell proliferation, viability, and migration within 3D matrixes were investigated in such dynamic cell culture conditions. When systemic drug administration was simulated within the OOC, the anticancer drug efficacy was tested and compared to the animal model. When NK cells were placed in circulation, their extravasation through a permeable barrier resembling the vascular barrier and infiltration within the cancer tissue were analysed.

Results
A human 3D ovarian model was developed and treated with Cisplatin in static conditions within MIVO and in the xenograft model. Similar tumour regression was observed in MIVO and in mice, while the static culture displayed an unpredicted chemoresistance, due to unreliable drug diffusion. A human 3D neuroblastoma cancer model with proper immunophenotype was optimised to develop a complex tumour/immune cell co-culture as a paradigm of an immune-oncology screening platform. Importantly, a tumour-specific NK cell extravasation was observed with a tumour-specific NK cell infiltration within 3D tumour tissue and cancer cells apoptosis induction.

Conclusions
We generated a relevant human disease mode, through the adoption of the MIVO device, that can be efficiently employed as a drug screening platform, but also for better investigating crosstalk among immune cells and other healthy/pathological tissues.

Department of Medical Sciences, University of Turin, Italy
Abstract Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to have therapeutic effects in pre-clinical models of different diseases. EVs derived from various types of MSCs have pro-regenerative capabilities comparable to cells of origin and are considered promising tools for the treatment of a variety of acute and chronic pathologies. To this end, the characterisation (size, phenotype, molecular content, etc.) of EVs and the evaluation of their biological effects are areas of intense investigation. To successfully translate EV research along the path from the laboratory to the patients, strategies should be designed to reach the aim of clinical testing of MSC-EVs safety and efficacy. Currently, there are emerging strategies for manufacturing cells and EVs and quality controls for practicability of clinical testing of the EVs, mainly related to non-industrial processes. Moreover, the identification of the mode of action in the therapeutic approach in the different diseases remains a major challenge to the translation path of EVs from the laboratory toward the clinical setting.

Abstract
Cell therapy represents innovative medical approaches in many clinical fields such as osteo-articular reconstructive surgery, tissue engineering, and cancer. Advanced therapy medicinal products (ATMPs) are cell and tissue products that are considered new types of drugs. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials. Besides their regenerative properties, hMSCs have also shown high immunomodulatory potential. The ATMP products should follow requirements including sterility, identity, purity, viability, potency, and reproducibility. Because stem cells are a heterogenous population that can differ depending on the origin and manipulation of the starting material, the identity/purity of the final cell population and its yield is still a critical issue which can limit clinical application of MSC-based products. New approaches for the standardisation of cell-based protocols may allow for the development of new highefficiency drug systems. To improve MSC characterisation, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. In this work, we present a new technology, Celector ® , for the quality control of the cell population. Celector ® has been shown to be able to tag-less analyse, discriminate, and separate a wide size range of cells based on their physical characteristics, with high resolution and throughput and with total maintenance of native properties. The separation is obtained in a short time (around 15 min) in a rectangular shape capillary device, through to the combined action of gravity, acting perpendicularly to the flow and opposing lift forces that depend on the morphological features of the sample. A micro-camera is connected as a detector and specifically designed software was developed for image acquisition, post-processing and data analysis, and the fingerprint of the biological sample. Celector ® is able to highlight physical differences that can be correlated to cell viability and regenerative potential. In addition, cells with stable and reproducible doubling time analysis can be collected and used as standardised systems for the development of high-quality clinical protocols.

Objective
Renal ischemia reperfusion injury (IRI) is the major cause of acute kidney injury (AKI), and it increases the risk of progression to chronic kidney disease (CKD). Human liver stem cells (HLSCs) are a mesenchymal stromal cell (MSC)-like population isolated from adult liver biopsy. HLSCs share with MSCs the same phenotype, gene expression profile, and differentiation capabilities. As shown in previous studies, HLSCs improved the recovery in different experimental models of liver and kidney injury. HLSC-derived extracellular vesicles (HLSC-EVs) have been studied as vehicles for transferring active biological materials both in vitro and in vivo. In this study, we set up an in vivo murine model of IRI-AKI, which subsequently developed into IRI-CKD and we investigated the potential therapeutic effect of HLSC-EVs.

Materials and Methods
EVs were purified from HLSC supernatant by ultracentrifugation. They were analysed through flow cytometry and Western Blotting to detect the expression of the main mesenchymal and EV surface markers. Transmission electron microscopy was performed to analyse EV size and morphology. Male BALB-c mice were subjected to 30 min ischemia followed by reperfusion. HLSC-EVs were intravenously administered immediately after the surgery and three days after. To evaluate AKI, mice were sacrificed two and three days after the surgery; while to assess the development of CKD, mice were sacrificed two months after. The histological analyses were performed on tissue sections stained with hematoxylin and eosin and Masson's trichrome for collagen detection. Expression of specific markers of fibrosis development (alpha-Smooth Muscle Actin (alpha-SMA), collagen I and transforming growth factor-beta) and inflammation (interleukin-1 beta, interleukin-6, and tumour necrosis factor-alpha) were evaluated at mRNA and protein levels.

Results
In AKI mice, the EV treatment-attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation. We also noticed the downregulation of the expression levels of fibrosis-related genes. In CKD mice, EVs effectively reduced the development of interstitial fibrosis at the histological level and reduced the expression levels of pro-fibrotic and pro-inflammatory genes.

Conclusions
The administration of HLSC-EVs immediately after renal IRI protects the kidney from AKI development and interferes with the development of subsequent CKD. Objective Extracellular vesicles (EVs) have attracted wide interest in recent years due to their potential applications in regenerative medicine, as biomarkers for disease diagnosis, and their role in cell-cell interactions. However, EV isolation, quantification, and characterisation remain challenging in terms of purity and specificity as well as time-and cost-effectiveness. This work aims to develop a novel and high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific EV surface component, using fluorescence polarisation (FP) for detection. The method analyses the change in the polarisation of emitted light, between unbound, and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of the bound probe. This property of FP allows us to use it to quantify the amount of EVs in the solution.

Materials and Methods
Two distinct strategies have been investigated, with probes targeting (i) specific EV surface markers (Tetraspanins, e.g., CD63) for EV sub-population quantification or (ii) the EV phospholipid bilayer membrane for total EV quantification. Commercially available fluorescently labelled CD63 binding aptamers and proteins and lipophilic dyes have been evaluated. EVs derived from HEK and MSC cultures have been purchased or isolated through PEG-precipitation and ultracentrifugation, and particles quantified through Nano Tracking Analysis (NTA) or Imaging Flow Cytometry. Each probe candidate was incubated with EVs, and FP was measured over time using a Spark Cyto plate reader.

Results
Tetraspanin specific (Anti-CD63) aptamer probes and lipophilic dyes have demonstrated increased fluorescence polarisation in response to increasing EV concentration quantified by NTA.

Conclusions
This initial proof of concept supports the use of FP as a high throughput EV detection and quantification method, with the ability to provide both total particle and CD63 +ve particle numbers. Further investigation is required to demonstrate the specific binding of each probe to its target to benchmark the FP assay against currently available methods. For this purpose, Bio-Layer Interferometry (BLI) assay is under examination to study the probe-target kinetic.

Objective
In regenerative medicine approaches related to orthopaedic conditions, mesenchymal stromal cells (MSCs) showed positive outcomes due to the secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. MSCderived factors may be modulated by both culturing and in vivo conditions. Nevertheless, a homogenous and comprehensive fingerprint in the frame of orthopaedic applications is missing. The aim of this work was to characterise adipose-derived MSC, (ASC)-secreted factors, and EV-miRNAs and their modulation after high levels of IFNγ preconditioning, as proposed for clinical-grade production of secretome with improved potential or low levels inflammatory conditions, mimicking osteoarthritis (OA) and synovial fluid (SF). In addition, ASC-EVs penetration in cartilage explants was scored.

Material and Methods
ASCs were cultured with and without IFNγ (1 ng/mL) or TNFα (5 pg/mL) + IL1β (10 pg/mL) + IFNγ (40 pg/mL) mimicking OA-SF. First, 200 secreted factors were assayed by ELISA. Second, 754 miRNAs were searched by qRT-PCR in ultracentrifuge-purified EVs. Bioinformatics tools were used to predict the modulatory effect of identified molecules on pathologic cartilage and synovial macrophages. Time-lapse coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence were used to follow and quantify fluorescent EVs incorporation into cartilage explants.

Results
Data showed that more than 50 cytokines/chemokines and more than 200 EV-miRNAs could be identified. The vast majority of molecules are involved in extracellular matrix remodelling and homeostasis of inflammatory cells. Inflammatory priming and synovial fluid-like conditions were able to further increase the capacity of the secretome to stimulate healing and inflammation reduction. Eventually, EV penetration was monitored as a fast process, starting in a few minutes, and reaching 30-40 µm depth after 5 h and plateau at 16 h in both cells and matrix of the cartilage explants.

Conclusions
Due to the portfolio of soluble factors and EV-miRNAs, the ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes. Inflammatory preconditioning or OA-like conditions were able to increase this ability. Eventually, microscopy data supported the capacity of MSC-EVs to influence the chondrocytes embedded in their native ECM by active interaction and eventual therapeutic cargo release. analysis of MG generated by Mesenchymal Stromal Cells (MSC) of different species was performed.

Material and Methods
EVs were isolated by differential ultracentrifugation. Briefly, the conditioned medium was centrifuged at 300× g to pellet cells. The supernatant was centrifuged at 2000× g (2 K EVs fraction), at 10,000× g (10 K EVs fraction), and finally at 100,000× g (100 K EVs fraction). EVs suspensions were placed on formvar-coated copper grids, contrasted with 2% uranyl acetate, and observed under a Philips EM 208 electron microscope (TEM) equipped with a digital camera. MSC monolayers were also fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated, and resin embedded. 80 nm thick sections were contrasted with 2% uranyl acetate and observed at TEM.

Results
Ultrastructural analysis revealed that MSC produces a previously unrecognised kind of vesicles, referred to as "migrasomes" that originate from the cell surface. The biogenesis and morphologic features of these vesicles are completely different from typical EVs. In fact, they are very large in diameter (500-2000 nm) and contain a variable number of luminal vesicles. MG were also detected in the 2 k fraction obtained by supernatant centrifugation.

Conclusions
Migrasomes are a special kind of EVs that are released by migrating cells. Determining their molecular content and signalling potential clearly need extensive research. The most crucial things to comprehend are how MG works, which signals are triggered by coming into contact with or ingesting MG, what messages they transport between cells, and so on. The discovery of these EVs raises many new questions for future research. In fact, although the heterogeneity of EVs has become obvious, as highlighted by the International Society for Extracellular Vesicles, specific tools to distinguish EVs of different origins are still lacking, and thus different functions are probably not correctly evaluated at the moment.

Róisín Dwyer
University of Galway, Ireland

Abstract
Despite improvements in treatments for breast cancer, when patients are diagnosed with metastatic disease that has spread to distant sites there are limited treatment options and no cure available. Extracellular Vesicles (EVs) hold immense potential as cancer therapeutics due to their small size, biocompatibility, and potential for manipulation of content and surface characteristics. Tumours actively recruit stromal cells including Mesenchymal Stromal Cells (MSCs) into the tumour microenvironment. The tumour-targeted tropism of MSCs is thought to be due to high local concentrations of inflammatory chemokines and growth factors. This tumour tropism combined with the apparent immunosuppressive characteristics of the cells raised remarkable interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. MSC-derived EVs (MSC-EVs) will potentially retain the tumour targeting and immune privilege associated with MSCs while overcoming challenges associated with the use of cells. Our recent work in development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy will be discussed. The impact of human-and murine-derived MSC-EVs on the immune system and the potential for scale up of EV production will also be highlighted. An approach to support sustained delivery of EVs over time would be very beneficial to prevent cancer resurgence. The use of pre-clinical models that are more reflective of the patient experience will be critical for testing novel approaches to breast cancer treatment. Progress and challenges in the development of MSC-EVs as cancer therapeutics will also be addressed. While we require further understanding of factors mediating EV content, persistence, and uptake, this exciting approach holds tremendous potential for patients with limited existing treatment options.

Materials and Methods
MFAT was prepared according to standardised methods using Lipogems device. MSCs were obtained by enzymatic digestion of MFAT. The in vitro effect of MFAT on MSTO-211H cell proliferation was analysed using transwell inserts and measuring the absorbance by a crystal violet assay. PBS were used as negative control. For in vivo experiments, Balb/c-Nude female mice were subcutaneously injected with 10 6 MSTO-211H cells suspended in Matrigel/PBS. Mice were randomised into 4 groups: control, paclitaxel (PTX), MSCs and MFAT. After a week from injection (time 0), vehicle alone (control group) or PTX (20 mg/kg) were administered intraperitoneally (IP) and MSCs (5 × 10 5 ) or MFAT (200 µL) were subcutaneously injected close to the tumour. On days 0, 7, and 14, the size of tumour nodules was measured and on day 20, nodules were collected. Morphometric evaluation of xenograft composition was performed on Masson's trichrome-stained sections.

Results
The in vitro exposure of MSTO-211H cells to MFAT produced a dose-dependent inhibition of cell proliferation. In the in vivo study, the measures of volume of growing tumour mass indicated that the in situ treatment with MFAT produced an important inhibition similar to those obtained in mice treated with the anticancer drug PTX. A trend of inhibition, but not significant, was also observed in mice treated with free MFAT derived MSCs. The morphometric analysis of the tumour xenograft did not show significant differences among groups.

Conclusions
Our results show that MFAT, injected in situ, produced a significant (p < 0.05) inhibition of the MSTO-211H growth both in vitro and in vivo, and was even comparable to IP PTX treatment. Interestingly, the treatment with free MSCs (5 × 10 5 ), at a similar amount contained in around 1 mL of MFAT, exerted only a little anticancer activity.

Objective
Mesenchymal Stem/Stromal Cells (MSCs) have been studied and applied as therapeutics in regenerative medicine based on their peculiar properties. Although several papers demonstrate the efficacy of MSC-based therapy in preclinical models, clinical applications are still limited due to doubts about the safety of treatment with viable cells. MSCs secretome is a cell-free product that maintains a large part of cells' therapeutic properties providing soluble and insoluble bioactive molecules involved in cellular crosstalk. Recent data support an antibacterial activity for both MSCs and their secretome. In this work, canine Lyosecretome (c-Lyo), a freeze-dried secretome prepared from adipose tissue-derived MSCs, has been tested to evaluate its antimicrobial activity against some of the most common canine pathogens involved in infections of the gastrointestinal tract, skin, and ears. Pathogens were subjected to a Minimal Inhibitory Concentration (MIC) assay to evaluate the amount of c-Lyo necessary to inhibit bacterial/fungal growth. Materials and Methods c-Lyo was resuspended in 500 µL of sterile saline (0.9%) and tested at a concentration range of 20-0.04 mg/mL. Nine replicates for each assay were performed. Tested reference bacteria and yeasts were E. coli, S. Typhimurium, S. aureus, Methicillin-Resistant S. aureus (MRSA), S. pseudintermedius, P. aeruginosa, and M. pachydermatis. Strains were amplified for 18-24 h at 37 • C to bring them into logarithmic growth phase and added to the plate at 5 × 10 5 CFU/mL and incubated at 37 • C overnight. MIC plate reading was performed with a reverse mirror. Mannitol was used as an internal control.

Results
A fair inhibitory activity of c-Lyo against both Gram-positive and Gram-negative bacteria was observed. Growth inhibition was higher for Gram-positive (2.2 < MIC < 9.8 mg/mL) but positive results were obtained even on Gram-negative bacteria (10.5 < MIC < 26.1 mg/mL). c-Lyo demonstrated an inhibitory activity also against the yeast M. pachydermatis (MIC = 13.3 mg/mL) supporting a possible efficacy in skin infections. Conclusions MIC data suggest that canine c-Lyo exerts inhibitory activity against various bacterial and yeast pathogens. The availability of an off-the-shelf, ready-to-use MSCs secretome acting as an antibacterial agent could help in replacing/supporting traditional antibiotics therapy, decreasing their use in veterinary medicine, as requested to control the spread of antibiotic resistance.

Materials and Methods
We first calculated the PTX-IC50 in 3 MSC batches, then, we isolated PTX-MSC-Secr by loading MSCs with PTX at the concentration of 15 µg and we analysed its cytotoxic effect on MG63 and SJSA after treatment for 5 days using MTT test. We also analysed the size distribution, particle concentration, and Zeta potential of EVs present in PTX-MSC-Secr by Nanoparticle Tracking Analysis (NTA) instrument. The secretome ability to have EVs with encapsulated PTX was analysed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results PTX-IC50 for MSCs was a mean of 25.1 ± 3.6 µg and for the OS cell lines was, respectively a mean of 17.5 ± 1.0 µg for MG63 and 30 ± 3.1 µg for SJSA. PTX-MSC-Secr induced a decrease of the viability of cells 43 ± 11% and 36 ± 22%, respectively in SJSA and MG63 after five days of treatment. This effect was dose-dependent because scalar dilutions of PTX-MSC-Secr reduced the cytotoxic effect. Cell viability did not decrease after treatment with Secr isolated from CTRL-MSCs (viability of 87 ± 5% in SJSA and of 91 ± 12% in MG63). The dimensional analyses, the result of three independent experiments, indicated EVs mean sizes of 175.8 ± 13 nm (CTRL) and 165.7 ± 11 nm (PTX loaded). EVs Zeta potentials of both CTRL and PTX loaded, as expected, were found to be negative with mean values of −40 ± 2 mV and −38 ± 5 mV.

Conclusions
We demonstrated the cytotoxic effect of PTX-MSC-Secr in OS cell lines. The NTA analyses showed a typical mean size of EVs and that no significant differences between the secretome batches and between experimental conditions (CTRL and PTX loaded) were observed. The experiments we performed have provided promising preliminary data that need further investigation but the EVs ability to encapsulate PTX allows us to propose the EVs-PTX isolated from the MSCs as ideal candidates for drug delivery as innovative paediatric sarcomas treatment.

Objective
The present study proposes the design and sterile manufacturing of 3D-printed polycaprolactone (PCL) scaffolds enriched with mesenchymal stem cell (MSC)-secretome for bone tissue regeneration and evaluates their in vitro biological efficacy.

Materials and Methods
Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Porous parallelepiped-shaped PCL scaffolds (10 × 10 × 3 mm) were prepared by co-printing PCL with an alginate hydrogel (10% w/v) containing lyosecretome (0.25 mg). Scaffolds were tested for sterility and microbiological tests, as indicated in the EuPh 2.6.27 and 2.6.1 chapters. In addition, the scaffold colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy, and dosage of osteocalcin by ELISA). Scaffolds without lyosecretome were used as a control.

Results
Sterile scaffolds have been obtained and lyosecretome enhanced their colonisation by MSCs: MSCs showed a spread morphology with the initial formation of filopodia, with more frequent and complex cellular processes, overall indicating the cytocompatibility of the scaffold. Lyosecretome also sustained MSC differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralised matrix detected by alizarin red was significantly higher for lyosecretome scaffolds. Likely, the amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with lyosecretome scaffolds after 14 and 28 days.

Conclusions
Overall, these results prove the role of MSC-secretome, co-printed in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC-secretome are now available for in vivo preclinical tests of bone regeneration.

Objective
The present study investigates the capacity of the mesenchymal stem cell (MSC)-secretome, formulated as a ready-to-use and freeze-dried medicinal product (the lyosecretome), to promote the osteoinductive and osteoconductive properties of titanium cages.

Materials and Methods
Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Lyosecretome was added to titanium cages (1 × 1 × 0.3 cm in size) kindly provided by MT Ortho and manufactured by an additive manufacturing technology called electron beam melting. The cages colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy and dosage of osteocalcin by ELISA). Cages without lyosecretome were used as a control.

Results
After 14 days, in the presence of lyosecretome, significant cell proliferation improvement was observed. Scanning electron microscopy revealed the cytocompatibility of titanium cages: the MSCs seeded showed a spread morphology and the initial formation of filopodia. After seven days, in the presence of lyosecretome, more frequent and complex cellular processes forming bridges across the porous surface of the scaffold were revealed. Moreover, after 14 and 28 days of a culture in an osteogenic medium, the amount of mineralised matrix detected by alizarin red was significantly higher when lyosecretome was used. Finally, improved osteogenesis with lyosecretome was confirmed by confocal analysis after 28 and 56 days of treatment and demonstrating the production by osteoblast-differentiated MSCs of osteocalcin, a specific bone matrix protein.

Conclusions
Overall, these results confirm the role of MSC-secretome in combination with titanium cages in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing.

Objective
The present study combines biohybrid bone substitute scaffolds (SB) with lyosecretome, a freeze-dried MSC-secretome formulation containing proteins and extracellular vesicles and evaluates the osteoinductive and osteoconductive in vitro.

Materials and Methods
Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining Lyosecretome (0.1 × 10 6 cell equivalents/mg). Each SB scaffold (1 × 1 × 0.3 cm in size) was loaded with 16 × 10 3 cell equivalents of Lyosecretome by an absorption method, obtaining SBlyo. 1 × 10 6 MSCs were seeded onto the upper surface of SB in an osteogenic medium, and after 14 and 60 days of cultures, gene expression for osteocalcin (OCN), alkaline phosphatase (ALP), and collagen 1 (COLL-1) was evaluated. After 60 days, a high-resolution X-ray microtomography was used to identify the newly formed mineralised tissue after cell colonisation on SB and SBlyo. Moreover, SB and SBlyo were fixed, decalcified, dehydrated, cut into thin sections, and stained with hematoxylin and eosin (H&E) for morphological analyses. Immunohistochemical analysis was also performed using antibodies for osteocalcin and TGF-β.

Results
After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. For SB, on the other hand, the process was still present, but tissue formation was less organised at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralise after 60 days. SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralised tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.

Conclusions
Overall, these results confirm the role of MSC-secretome loaded on biohybrid bovine matrix in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing.

Objective
Feline Chronic Gingivostomatitis (FCGS) is a severe inflammatory oral disease characterised by painful mucosal lesions, oral discomfort, inappetence, reduced grooming, weight loss, and hypersalivation, seriously affecting the patient's quality of life. The current standard of care is invasive full/near-full mouth tooth extraction and long-term pharmacological treatments, with a high rate of relapse. Since FCGS is probably immune-mediated, Mesenchymal Stromal Cells (MSCs) represent a promising tool for this disorder. Different studies have reported the efficacy of systemic administration of adipose-derived MSCs (Ad-MSCs) in cats with FCGS, while a pilot study reported a lack of efficacy when the treatment is performed prior to full-mouth tooth extraction. This study aims to determine the efficacy of local and systemic administration of Ad-MSCs in cats with FCGS, with or without teeth.

Materials and Methods
Eleven client-owned cats with FCGS and with long-term pharmacological clinical history, with or without teeth, were treated with a double application of autologous Ad-MSCs at 30-day intervals. The cats were enrolled in two groups: one was treated with local injections of 5 × 10 6 autologous Ad-MSCs and the other was treated with local injections associated with systemic infusions of 2 × 10 6 /Kg autologous Ad-MSCs. An oral examination with photographs and oral biopsies was performed at the enrolment and 30 days after each treatment. A SDAI (Stomatitis index) scoring was calculated at the same intervals, in addition to a brief owner questionnaire and a veterinarian scoring. Furthermore, a complete blood count, blood immune cell phenotyping, and biochemical profile were planned on day zero and three months after the first treatment.

Results
At the time of writing, eight cats have been treated with double MSCs application. Seven cats have completely suspended any pharmacological treatment after the first application. The clinical assessment at day 60 showed a marked clinical improvement reported by the owners, except for one patient that showed the maximum SDAI score at the enrolling who improved only in the body weight parameter. A statistically significant difference was observed in the SDAI between day 0 and 60 for seven cats, two with a complete resolution of the oral inflammation (p < 0.05).

Conclusions
Immunohistochemical analysis and blood immune cell phenotyping are needed to confirm the observed clinical improvement.

Objective
Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose-derived Stem Cells) show great potential for degenerative disease treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validate a new commercially available kit able to explore changes in gene expression in human ADSC subjected to osteogenic, adipogenic, and chondrogenic differentiation.

Materials and Methods
As the primary outcome of the study, we selected better indicators of trilineage differentiation using the third passage of cultured ADSC isolated from the stromal vascular fraction (SVF) by enzymatic digestion. ACAN, FABP4A, and Col11a1 were selected as indicators of chondrogenic, adipogenic, and osteogenic differentiation, respectively based on statistically significant results. As a secondary outcome of the study, an in vitro ageing test was performed from passage 2 to 6 to evaluate the highest differentiation potential. Total RNA extraction from differentiated and control ADSC were performed. Relative quantifications of mRNA expression of selected genes was completed according to rt-PCR kit protocol.

Results
ACAN detection, a test for chondrogenic differentiation, revealed equivalent ∆∆Ct values between P3 and P6. These were considered equivalent passages for induction differentiation tests. FABP4 detection, an assay for adipogenic differentiation, showed similar results for all cell passages tested so they can all be considered suitable in differentiation assay induction; on the contrary, only passage P6 for Col11a1 was suitable for osteogenic differentiation.

Conclusions
In conclusion, we validated a new real-time rt-QPCR protocol able to evaluate osteogenic, chondrogenic, and adipogenic ADSC differentiation in vitro.

Objective
Ovarian cancer is the seventh most common cancer and sixth most common cause of cancer death for women globally. Nowadays, surgical resection followed by chemotherapy is the standard of care. However, a number of patients are faced with recurrence due to tumour dissemination and acquired chemoresistance. Therefore, the novel alternative approaches targeting ovarian cancer cells are urgently needed to improve the standard of care for patients. With this regard, mesenchymal stromal cells (MSC) constitute a compelling therapeutic option. Of particular interest, MSC isolated from the amniotic membrane of the human term placenta (hAMSC) are of therapeutic interest and present noteworthy advantages when compared to MSC from other sources, such as their ease of recovery from biological waste. In addition, we previously reported that the hAMSC secretome has antiproliferative effects in vitro when co-cultured with different tumour cells.

Material and Methods
We decided to evaluate the possible anti-proliferative effects of the secretome (conditioned medium, CM) from hAMSC in 2D and 3D models of ovarian cancer. In parallel, we evaluated the CM from the intact amniotic membrane (hAM) to see if antiproliferative effects could be maintained without the need to perform MSC isolation.

Results
Both CM-hAMSC and CM-hAM inhibit the proliferation and migration of ovarian cancer cells (HEY and SKOV-3) in 2D scratch assays. Moreover, both CM-hAMSC and CM-hAM affect the apoptotic process in HEY and SKOV-3. In the SKOV-3 spheroid 3D model, CM-hAMSC and CM-hAM significantly decrease the spheroid area inducing also a change in spheroid morphology.

Conclusions
The data so far collected indicated that CM-hAMSC and CM-hAM impact the growth and activity of ovarian cancer cells. Further experiments are needed to better understand their inhibitory capacity on ovarian cancer cells in 2D and in 3D models and clarify their use as a potential adjuvant therapeutic strategy able to target tumour cells. Objective The central nervous system (CNS) has only a limited capacity to regenerate, hence, after injury, a progressive loss of neurons, due to homeostatic imbalance, leads to neurodegenerative pathology. The homeostasis process critically depends on the interaction between neurons and glial cells. Novel treatment suggestions for neurodegenerative disorders consider the use of cellderived products, relying on the beneficial paracrine effects of the applied products. Extracellular vesicles (EVs) recently emerged as versatile messengers in CNS cell communication. These nanoparticles, defined by a phospholipid bilayer, can convey signals by triggering surface receptors, activating second messenger signalling cascades or delivering their cargo, such as proteins, nucleic acids, and small molecules. In this work, we considered an organotypic in vitro model of spinal injury treated with EVs derived by Wharton Jelly Mesenchymal stromal cells.

Materials and Methods
Isolated spinal cords from rat foetuses were cut into small pieces and cultured on a bed of Matrigel. Organotypic spinal cord (oSpC) were treated with EVs soon after the beginning and after 24 h of culture. To monitor the early effect of the EVs on neural axon sprouting, samples were analysed 48 h after the seeding.

Results
Our results showed that neural axon sprouting was significantly increased in EV-treated samples when compared to untreated oSpCs. Moreover, the neural protein TujI/TUBB3 expressed in the developing neurons and the glia marker GFAP, that identified new astrocytes, were differently detected in the presence of EVs. Fluo-4 imaging revealed a more controlled calcium flux in oSpC treated with EVs at the axonal projection compared to the untreated SpC. Real-time PCR on neural miRNA highlighted how the EVs can be responsible for miRNA mediator of neural regeneration.

Conclusions
Our results suggest a possible role of perinatal EVs in promoting neural axon sprouting, opening new perspectives for their application in neural regeneration, and as new exciting signalling modalities that add a new dimension to the interaction between neurons and glial cells.

Objective
Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and Ulcerative Colitis (UC), are chronic relapsing-remitting disorders characterised by inflammation of the gut. Different factors contribute to IBD development, such as deficiencies in epithelial integrity, immune response mechanisms, and mucosal barrier function, whose complexity is difficult to reproduce in experimental conditions. The need to overcome the typical limits of cell lines, animal models, and organ culture, led to the development of a 3D culture system capable of maintaining the characteristics of the target organ in the long term. We thus explored the feasibility of this tool to evaluate the therapeutic potential of extracellular vesicles derived from mesenchymal stromal cells (MSC-EVs) whose immunomodulatory activity is the object of increasing interest.

Materials and Methods
MSC-EVs were isolated from human umbilical cord MSCs by ultrafiltration (100 kD cutoff), quantified by TRPS and characterised for established markers by MACSPlex. Colon organoids were derived from human colon samples' extracted crypts and seeded in Matrigel beads. Inflamed (IBD) organoids, in particular, were obtained from the stimulation of control organoids with a pro-inflammatory cocktail (IL-1β, TNF-α, and IL-6, at 100 ng/mL) on days 3, 5, and 7 to reproduce a chronic inflammatory state. To evaluate the effect of MSC-EVs, IBD organoids were then treated on day 7 with a dose of 1 × 10 9 MSC-EVs/mL at different time points (3 h, 6 h, 24 h, and 48 h). As readouts, we evaluated the effect on proliferation, differentiation, inflammation, barrier, and growth by immunofluorescence and molecular biology.

Conclusions
In conclusion, we have provided preliminary evidence of the therapeutic effect of MSC-EVs in inflamed intestinal organoids. These results suggest that the present 3D model could represent a useful experimental tool closely reproducing some critical features of human IBD.

Objective
Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) of neural crest origin. High availability, multipotency, and plasticity make them a promising source of patient-specific MSCs for personalised regenerative medicine strategies. However, their clinical translation still faces many challenges due to a lack of deep understanding of their niche microenvironment, biology, and functionality in vivo. By recapitulating the complex in vivo-like microenvironment, three-dimensional (3D) multicellular spheroids open to new opportunities for MSCs translation in clinical and preclinical settings. In this context, the development of human DPSC multicellular spheroids as organotypic 3D in vitro models was assessed. Material and Methods 2 × 10 4 DPSCs at passage IV were used for spheroid creation by hanging drop technique and transferred to 96 ULA plates after 24 h. Spheroid morphology, viability (FDA-PI staining), and metabolic activity (Alamar Blue assay) were evaluated at day 1, 2, 3, 4, and 7 of culture. Tissue-specific (nestin, vimentin, collagen I and IV, laminin, and fibronectin) and stem-related (BMI1, CD90, SOX2, NANOG, and OCT4) markers were evaluated at gene (qRT-PCR) and protein level (IF). After bright field imaging and by on-purpose method for automated image analysis, spheroid dimension, morphology, and compactness were quantified by their equivalent diameter (ED), sphericity (S), and border indentation (BI), respectively. After normality check by Shapiro-Wilk test, statistical significance was assessed by t-test or Wilcoxon test (p < 0.05).

Conclusions
The human DPSC spheroids can be easily and quickly created by a low-cost procedure in 24 h and they may efficiently enhance the therapeutic action of DPSCs. Moreover, automated image analysis here proves as a valuable tool for the finest analysis of DPSC spheroid morphology in future preclinical setting applications.

Objective
Mesenchymal Stem/stromal Cells, and, in particular, adipose-derived ones (ASCs), show great therapeutic potential in counteracting orthopaedic conditions. Since a large part of ASC action is exerted through paracrine signalling, in the last years, we focused on the study of their conditioned medium (CM) in terms of molecular composition and biological action on experimental models of osteoarthritis (OA).

Materials and Methods
The CM was obtained from confluent ASCs cultured for 72 h in the absence of FBS, then concentrated through filter devices with a 3 kDa cut off. Its components were investigated through Raman Spectroscopy, NTA, -omic approaches, and ELISA. Articular chondrocytes (CHs) and osteochondral explants (OEs) were harvested from patients undergoing arthroplasty at IRCCS Istituto Ortopedico Galeazzi prior to approval of the ethics committee. OA phenotype was induced by stimulation with 10 ng/mL TNFα, and specimens were treated with the CM derived from 0.5-1 × 10 6 ASCs. The levels of inflammatory, hypertrophic, and catabolic factors were monitored through time by immunological or enzymatic assays. Results ASC-CM peculiar Raman fingerprint and its characteristic vesicular profile were depicted. The analysis of ASC-CM composition showed the presence of stable levels of bioactive factors that can be putative players in counteracting the OA process. Among other potential effectors, the abundance of the chondroprotective factors Dkk-1 and TIMP-1/2 was particularly intriguing. Accordingly, in both experimental OA models, i.e., articular chondrocytes (CHs) and osteochondral explants (OEs), ASC-CM efficiently buffered the TNFα-induced aberrant activity of matrix metalloproteinases. Furthermore, ASC-CM reverted TNFαinduced expression of PGE2 and COL10A1 in CHs, while it lowered the catabolic release of glycosaminoglycans on OEs. Conclusions These in vitro and ex vivo experiments confirm ASC-CM beneficial potential in dampening OA-related hallmarks, encouraging deeper investigations of this product in the perspective of its future clinical translation as a cell-free orthobiologic.

P-13. LATTICE-BASED SCAFFOLDS FOR THE BULK REINFORCEMENT OF SOFT MATERIALS FOR CARTILAGE REGENERATION
the metastatic volume by magnetic resonance. Secondly, the MSCs viability after treatment with nPTX, as well as drug uptake and release, were investigated in vitro. The biodistribution of luciferase (LUC)-transduced MSCs intraportally injected in tumourbearing mice was investigated by in vivo imaging systems (IVIS). Finally, the effect on the metastatic reduction of nPTX-loaded MSCs was evaluated on tumour-bearing mice after intraportal injection. Results NPTX significantly reduced the metastatic volume of tumour-bearing mice, demonstrating the responsiveness of our model to this drug. In vitro, after 24 h, we could appreciate a high uptake of nPTX by MSCs without a reduction of cell viability. NPTX release by MSCs was higher after 24 h but sustained until 72 h, as reported by mass-spectrometry analysis. Biodistribution studies revealed a high and prolonged accumulation of MSCs in the liver after intraportal injection. Results obtained in vivo on the antitumour efficacy of this system reported a significantly higher metastatic reduction in mice treated with nPTXloaded MSCs with respect to control mice treated with not loaded MSCs or free nPTX.

Conclusions
The ability of MSCs to incorporate nPTX to a high extent and without reporting toxicity is promising. This system allowed to lower the drug dose, thus reducing nPTX toxicity and specifically targeting the tumour site reporting an effective reduction of the metastatic burden. Obtained results open new insights into the use of MSCs for delivering nPTX as a suitable and promising therapeutic option for PDAC.

Objective
Environmental microplastic (MPs, 1 µm-5 mm) degradation has become a problem for human health due to the possible production of toxic metabolites, contaminating water, air, food, and several daily used products. Bisphenol A (BPA) is the most representative chemical component of MPs debris, and it is reported to alter cellular functions by acting as an endocrine disruptor. Endocrine disrupting chemicals (EDC) are able to interfere with endogenous hormone biosynthesis, metabolism, and functions through the binding with typical and peculiar oestrogen receptors, triggering a dysregulation in cellular physiological processes such as oxidative stress. Placental tissues are supposed to be susceptible to EDC for the abundance of hormone receptor expression. Thus, it has been hypothesised that exposure of the mother to MPs-derived chemicals, such as BPA, can lead to an imbalance of the physiological processes that contribute to a successful pregnancy, increasing the risk of gestation-related complications. Based on this evidence, our purpose is to evaluate the effects of BPA on mesenchymal stromal cells isolated from the amniotic membrane of the human term placenta (hAMSC).

Materials and Methods
Our preliminary experiments aimed to evaluate the impact of BPA on hAMSC properties and functions in vitro. Cellular viability, metabolism, and apoptotic rate were analysed after a 24 h exposure to increasing concentrations of BPA (0.1; 0.2; 0.3; 0.4 µM) by MTT assay, ATP lite assay, and PI/Annexin kit, respectively. Concomitantly, the loss of cellular oxidative balance has been assessed by flow cytometry 3 h and 24 h after BPA exposure. In addition, dysregulation in the expression of cell cycle mediators was evaluated by RT-PCR.

Results
We first observed a dose-dependent reduction in hAMSC viability and metabolic capacity as well as an enhancement in cellular apoptotic rate after the treatment with increasing concentrations of BPA. At the highest concentration of BPA used in our study, hAMSC-intracellular oxidative stress and the gene expression of typical cell cycle regulators both increased.

Conclusions
Our preliminary data suggest that BPA may affect hAMSC functions. In light of these observations, additional studies will be performed to evaluate intracellular pathways through which BPA can act, supposing that adverse consequences on placenta resident MSC may be related to a negative outcome of gestation and riskiness for the baby.

Objective
In the last decade, the scientific interest in the secretome of Mesenchymal Stem/stromal Cells (MSCs) has increased tremendously due to its promising potential as an alternative to cell therapy. Mid-term serum starvation represents a convenient strategy for secretome production since it stimulates cell secretion while reducing the drawbacks associated with the use of animal-derived supplements. Nevertheless, the impact of this procedure on the metabolic status of donor cells still needs to be defined. Here, we investigate this aspect through metabolomics by comparing MSCs cultured with 10% foetal bovine serum (FBS) and serum-starved ones.

Materials and Methods
Primary human adipose-derived MSCs were grown in complete culture medium until confluence. Then, cells were either collected and analysed or rinsed and cultured for three days in the absence of FBS. Samples were screened for polar and apolar molecules by untargeted metabolomics at the Proteomics and Metabolomics Facility of IRCCS Ospedale San Raffaele. The differences revealed by this analysis were further validated by ad hoc biochemical and functional assays.

Results
Differential metabolomics shows a clear clustering between samples grown in standard conditions and under serum deprivation. Metabolite set enrichment analysis reveals several processes affected by serum withdrawal, most of them occurring at the mitochondrial level such as Mitochondrial Electron Transport Chain, Oxidation of Branched Chain Fatty Acids, and Citric Acid Cycle. The impairment of mitochondrial metabolism is further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. At last, cells exposed to serum starvation show higher expression levels of mitochondrial superoxide dismutase.

Conclusions
Mid-term serum deprivation affects cell metabolomes by impairing mitochondrial activity and inducing oxidative stress. We hypothesise that the metabolic stress occurring during serum starvation may trigger the release by donor cells of multiple bioactive factors mediating the pro-angiogenic, trophic, and antioxidant effects of their secretome.

Conclusions
Type I collagenase injection induced Achilles tendinopathy since structural changes and cellular alterations were clearly visible. As regards the inflammation and the recruitment of immune cells, the data obtained suggest their partial involvement, which should be further investigated at other timing of the tendinopathy to understand possible interaction between TSPCs and macrophages.

Objective
The purpose of the present study was a comparative analysis of the biological characteristics of human Mesenchymal Stromal Cells (MSCs) derived from Adipose Tissue (ADSCs) and Dental Pulp (DPSCs) in order to evaluate their use in regenerative medicine.

Materials and Methods
ADSCs and DPSCs were obtained from healthy patients. ADSCs were isolated by two methods known as Stromal Vascular Fraction (SVF) and Mechanical Fragmentation (MF), while DPSCs were obtained from open apex third molars by MF after separation of the radicular (RPSCs) and the coronal region (CPSCs). These populations were compared based on their morphological features by TEM and IF, and their capacity to proliferate by WST-1 assay. Expression of surface markers was measured by flow cytometry, and the ability to undergo trilineage differentiation was also studied. Conditioned medium (CM) was collected after 3 days of culture and the Bio-Plex Pro Human Cytokine 27-plex Assay (Bio-Rad) was performed. Extracellular vesicles were separated by ExoQuick-TC and suspensions were examined by flow cytometry. In DPSCs, more than 90% were Nestin + and only 10% αSMA + compared with <1% and 30% in ADSCs, respectively. Multivariate analysis by PCA for the expression of 16 surface markers measured by flow cytometry showed that organ source (dental pulp or adipose tissue) was the most critical factor that discriminates cell phenotypes. The Bio-Plex assay showed that, whereas the analyte composition of CMs from the two DPSCs appeared very similar, the isolation method was responsible for large differences among CMs from ADSCs in terms of cytokines, chemokines, and growth factors secretion. Finally, ADSCs released a significantly higher number of the smaller EVs named exosomes, ≤100 nm in diameter, than DPSCs.

Conclusions
These results indicate that the four MSC populations show different phenotypic and functional signatures depending on both the tissue source and the extraction method. These differences may be crucial for cell-free therapies using CMs from such cells.

Materials and Methods
From the same platelet pool, we obtained the standard HPL-E and the new HPL-S. We investigated if the treatment with Ca-Gluconate could interfere with the chemical characteristics and the growth factor amounts. Moreover, we compared the cellular growth, immunophenotype, and multipotent capacity of MSCs isolated and expanded in HPL-E and -S. We also isolated lyo-secretome from cell supernatant after serum starvation, ultrafiltration, and freeze-drying from MSCs. Lyo-secretome was analysed for lipid, protein, and growth factor contents, extra-vesicle size and concentrations, immunophenotype, anti-elastase activity, and immunomodulant properties. Results HPL-S did not contain PLTs and fibrinogen; total protein and growth factor amounts were comparable with HPL-E. The number of colony-forming unit fibroblasts showed no significant differences between the two groups. MSCs with HPL-E showed a cumulative Population Doubling higher in the earlier passages with an inversion of the growth trend in passage 4. Stem cell markers were maintained during expansion. Immunophenotypic analysis showed a significantly different expression of HLA-DR (1.30% with HPL-S, 14.10% with HPL-E) and of CD146 (>50% with HPL-S, <30% with HPL-E). Lyo-secretome obtained from MSCs with HPL-E or HPL-S did not show significant chemical/biological differences.

Conclusions
The use of HPL-S is an effective alternative for MSC production in GMP conditions and the obtained lyo-secretome could replace MSC-cellular therapy as a cell-free surrogate.

Objective
The growth in vitro of medicinal stem cells depends on the culture method, including the attachment substrate required for adhering to cell culture. Strategies that involve the testing of new biocompatible substrates for stem cell modulation or expansion, for example, nature-inspired biomaterials, may improve the phenotype and the functionality of these cells. The chicken eggshells and honey bee pollen were usually considered waste-like materials. We aim to evaluate these two organic sources obtainable by the food industry for supporting mesenchymal stem culture.

Materials and Methods
Human Dental Pulp Stem Cells (DP) were selected as a mesenchymal stem cell culture model. Resazurin reduction assay was used to assess the adhesion to the eggshell membrane by living cells and, in parallel, to evaluate cytotoxicity in presence of bee pollen solution. Immunofluorescence staining was performed to display morphology of cells attached to the eggshell membrane. Monochlorobimane probe was used to evaluate glutathione levels after exposure to bee pollen.

Results
The viability signal of DP stem cells plated on the eggshell membrane increased according to seeded cell density. The cell attachment to the membrane was also confirmed by immunofluorescence performed on fixed cultured membranes. Bee pollen solution was able to increase metabolism-based signal of viability, as well as to increase antioxidant cellular glutathione content.

Conclusions
The biocompatibility of both eggshell membranes and bee pollen treatment in DP stem cells was confirmed. The experiments with both the organic materials showed positive results in terms of cell metabolism stimulation and no significant alteration of classical morphology of mesenchymal stem cells in vitro. We conclude that these data can be promising for continuing this study and starting new analyses concerning specific stem cell properties changes, in comparison to standard tissue culture plates.

Objective
The periodontal ligament (PDL) plays a key role in providing mechanical stability and absorbing the high forces associated with mastication. These capacities deteriorate if PDL is affected by periodontitis, a degenerating disease that leads to loss of the PDL and the supporting alveolar bone. In view of PDL engineering and PDL regeneration, we investigated the hPDLSCs and adipose-derived stem cells (ASCs) behaviour under controlled uniaxial stretch stimuli, exploiting a previously developed bioreactor and customized flexible substrates.

Materials and Methods
ASCs (ASC52 -telo hTERT, ATCC) and primary hPDLSCs were characterised for the expression of mesenchymal markers by means of a FACS analysis. The same cell types were seeded on customised flexible substrates and exposed to a controlled uniaxial stretch stimulus (15% of strain, 1 Hz, for 90 s every 6 h for 3 days, n = 3) using a previously in-house developed bioreactor. Cells cultured in static conditions were used as control (n = 3). For both cell types and both conditions, DMEM was used as a culture medium. At the end of the culture period, cells were collected and the expression of stemness markers (NANOG, SOX2, and OCT3/4) and osteogenic markers (ALP, OCN, and RUNX2) were assessed through Real-Time PCR. Results FACS analysis highlighted that the phenotypes of ASCs and hPDLSCs are comparable, both expressed CD73, CD90, CD105, and CD44, while they were negative for CD45 and HLADR. The basal stemness marker expression seems to be higher for hPDLSCs than ASCs, nonetheless, both cell types showed an increasing trend in their expression after stretch stimulation. After the stretch stimulus, the expression of the osteogenic genes was increased, especially the OCN (p < 0.05), in the hPDLSCs, but not in the ASCs.

Conclusions
The results obtained by FACS analysis demonstrated that both cell types expressed the typical mesenchymal markers, and the expression of the stemness genes showed an increasing trend after stretch stimulation. Osteogenic genes were upregulated in hPDLSCs when cultured under controlled uniaxial stretch conditions. According to these results, hPDLSCs showed more osteo differentiating ability than ASCs. Further studies are ongoing to define the response of hPDLSCs and ASCs to combined stimuli, such as mechanical (stretch) and chemical (tenogenic medium) ones, to obtain an effective system for PDL regeneration.

P-24. 3D BRAIN ORGANOID MODELS AS A TOOL TO SCREEN FOR NUTRACEUTICALS
Arianna Minoia 1 , Luca Dalle Carbonare 1 , Jens C. Schwamborn 2 , Silvia Bolognin 2 and Maria Teresa Valenti 3 scription was done using High-Capacity cDNA Transcription Kit. Real-time data: TaqMan probes and TaqMan Universal Master Mix to analyse the gene expression. Flow cytometry: Flow cytometry was performed using BD LSR-Fortessa. Sectioning organoids. Immunofluorescence staining. Confocal imaging

Results
We investigated the gene expression of PARK2, NR2F1, CTNNB1, and LRP5 for untreated 3D organoids and treated 3D organoids with DMSO, lipoic acid, and JH-II in the two cell lines. Comparing the expression of PARK2 between the organoids deriving from the Bil-WT line towards the treated organoids with mutation LRRK2-G2019S, we note a higher expression of PARK2 in the samples treated with the molecules of interest. We also found this trend of a significant increase in expression for the NR2F1 gene. As regards the LRP5 gene, we note an identification in the samples treated with lipoic acid and an increase in expression trend for the organoids treated with the JH-II inhibitor. As regards the last gene analysed, CTNNB1, there is a higher expression for the samples treated with the inhibitor of the LRRK2-G2019S mutation.

Conclusions
The molecules impact on the expression of the genes associated with the Wnt/β-catenin pathway upstream and downstream appear to have a beneficial impact on the rise in the expression of the target genes. Considering an improvement in the conditions of PD patients, the treatment with the molecules we examined would result in an increase in the expression of the gene of interest.

Objective
Periodontitis is one of the most common diseases worldwide, causing a progressive destruction of the tooth supporting tissues. Thus, different regenerative approaches are under investigation, and this study aims to evaluate the biological properties of polycaprolactone (PCL) 3D-Bioprinted scaffolds w/or w/o alumina toughened zirconia (ATZ) filler, as suitable materials for alveolar bone and periodontal ligament (PDL) regeneration.

Materials and Methods
Three different types of blends were compared to each other, respectively: pure PCL, 80/20 w/w PCL/ATZ, and 60/40 w/w PCL/ATZ. The ATZ was incorporated in the PCL matrix through dissolution in chloroform by solvent casting method, then the bioink was 3D bioprinted to generate scaffolds with a standardised porous and circular geometry. ASC52 hTert cells (ASCs) were seeded on these scaffolds for 24 h to test their adhesion on the material surface. To evaluate the biocompatibility of the scaffolds, ASCs were cultured for 3, 7, and 14 days to allow scaffold colonisation, and registered their growth through the quantification of the ATP release in culture by viable cells (CellTiter-Glo kit). To investigate whether the scaffold had osteoinductive properties, ASCs were cultured in an osteogenic medium for 2 months, then RNA was extracted and the expression of osteogenic genes (ALP, COLL1, OCN, and RUNX2) were quantified with Real-Time PCR. SEM and EDX analysis were performed to evaluate the newly formed bone and to quantify the presence of calcium deposition, respectively.

Conclusions
In conclusion, we developed a lyophilised HA-based/PRP device that improves and preserves the PRP activity over time. HA/PRP allows the development of promising products for topical, intradermic, and intra-articular applications. Objective Preeclampsia (PE), the most severe human pregnancy-related syndrome, is a leading cause of foetal-maternal mortality and morbidity and lack of an effective therapy. The main hallmarks of PE are severe maternal hypertension and proteinuria, expression of generalised endothelial damage, and inflammation that could lead to Foetal Growth Restriction (FGR). Human Placenta-Derived Mesenchymal Stromal Cells (hPDMSCs) are well renowned for their pro-angiogenic and anti-inflammatory effects exerted via paracrine interactions. Herein, we tested the effects of hPDMSCs-CM (Conditioned-Media) on a mouse model of preeclampsia.

Materials and Methods
PDMSCs were isolated from control placentae and plated (1 × 10 5 cells/mL) in DMEM without FBS at passage 5. After 48 h, CM was collected. Preeclampsia was induced in pregnant C57BL/6NCrl mice by intravenous bacterial Lipopolysaccharide (LPS) injection. Starting from d9, maternal blood pressure and proteinuria were monitored until d19. At d11 of pregnancy, dams were injected with E.Coli LPS (1 µg/Kg). At d12, mice were randomly divided into two groups (n = 7 each) and treated intravenously as follows: plain vehicle (300 µL, placebo) and hPDMSCs-CM (300 µL, treated). At d19, mice were sacrificed. A number of foetuses, FGR, foetal reabsorption, and placental weight were evaluated. Next, placentae were processed for mRNA and protein isolation. sFlt-1, IL-6, and TNF-α gene and protein expression were evaluated by Real-Time PCR and Enzyme-Linked Immunosorbent Assay (ELISA).

Results
Injection of hPDMSCs-CM on d12 significantly decreased maternal systolic blood pressure and proteinuria by day 13 until term relative to placebo group. No FGR and/or reabsorbed foetuses were delivered by hPDMSCs-CM treated PE mice, while 5 FGR foetuses were found in the placebo group. No differences were found in placental weight between groups. hPDMSCs-CM injection significantly decreased sFlt-1, IL-6, and TNF-α levels in PE mice.

Conclusions
Our data indicate that hPDMSCs-derived trophic mediators can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia. Supported by Corion Biotech s.r.l. (CAT) and Superoxide Dismutase1 (SOD1) and of OxS-triggered cell death modulators PARP1, Caspase3, and LDOC1 in normal and PE-PDMSCs. Finally, we tested the hypothesis that PDMSCs-CM (Conditioned-Media) could restore CAT and SOD1 in H2O2-treated villous explants.

Materials and Methods
PDMSCs were isolated from control (n = 10) and PE (n = 10) placentae. At passage 5, cells were plated (1 × 10 5 cells/mL) in DMEM without FBS. After 48 h, CM was collected, and total antioxidant capacity was tested by Cayman's Antioxidant Assay. Control (n = 24) villous explants were treated for 24 h by H2O2 and next by control PDMSCs-CM for 48 h. CAT, SOD1, PARP1, Caspase3, and LDOC1 gene expression were evaluated by Real Time PCR.

Results
We reported a lower total antioxidant capacity (1.42 Fold Decrease) in PE-PDMSCs relative to control. We reported decreased CAT and SOD1 and increased PARP1 (p = 0.03), Caspase3 (p = 0.04), and LDOC1 (p = 0.05) gene levels in PE relative to control PDMSCs. After 24 h with H2O2, normal PDMSCs-CM treatment increased CAT and SOD1 and significantly decreased PARP1 (p = 0.02), Caspase3, and LDOC1 mRNA levels relative to controls.

Conclusions
Herein, we demonstrated that pathological PE-PDMSCs are characterised by aberrant antioxidant properties followed by increased production of OxS-related cell death effectors. Moreover, PDMSCs-CM promotes the expression of antioxidant enzymes, thus inhibiting OxS-mediated cell death. Indeed, our data suggest that PDMSCs-CM could be used to neutralise the exacerbated OxS typical of PE placentae, thus opening to novel PDMSCsbased therapeutic options.

Objective
To assess the role of mesenchymal stem cells (MSCs) on skin wound healing both in vitro and in vivo after dermatological surgical procedures performed for oncological purposes.

Materials and Methods
MSCs were obtained from discarded adipose tissue during dermato-surgical procedures. After isolation, MSCs were cultured in ascorbic-acid enriched medium in order to obtain MSC-based dermal scaffolds. Organotypic cultures were also performed to assess the pro-epithelizing properties of MSCs. MSCs were also seeded on commercially available acellular dermal matrices (ADMs) to assess whether they could exert a synergistic action. Finally, we also looked at whether MSCs were spontaneously recruited in vivo at wound sites in the presence of ADMs. Classical histology, immunofluorescence, and ELISA tests have been employed in the aforementioned experimental settings.

Results
MSCs can efficiently be used to produce dermal equivalents. MSCs secrete all the main components (collagen and fibronectin) of the extracellular matrix upon stimulation. MSCs guide wound re-epithelialization in vitro in the presence of keratinocytes, mainly through a paracrine action on epidermal basal stem cells. When seeded on acellular dermal collagenic substitutes, MSCs significantly increase extracellular-matrix production, therefore, confirming the potential effectiveness of combination treatments. CD90+ STRO-1+ cells were detected in the neodermis after ADM positioning, therefore suggesting efficient ADM-mediated MSC recruitment.

Conclusions
MSCs represent a promising tool in the regenerative setting, especially for the treatment of large surgical wounds after dermatological surgical procedures. However, further studies are needed to confirm their safety in oncological patients since a potential tumourpromoting role has recently been postulated for MSCs.

Objective
Mesenchymal stromal cells (MSCs) derived extracellular vesicles (EVs) have been studied for the treatment of Osteoarthritis (OA), the most common chronic disease of the joint cartilage. A large-scale expansion of MSCs is required to meet clinical demand and this could affect the effectiveness of cells and cell products. Therefore, MSC generated from induced pluripotent stem cells (iMSC) represent a promising cellular source for the manufacture of EV therapeutics. In this study, we isolated and tested the efficacy of EV secreted by MSCs and iMSC in the treatment of OA in vitro.

Methods
MSCs and iMSCs were cultured in vitro in serum-free clinical grade conditions. Cells were characterised during long-term in vitro expansion for surface expression pattern, proliferation ability, senescence rate, and differentiation capacity. EVs were isolated using an FPLC-anion exchange chromatography (AEX) approach and their biological effect on IL-1α treated human chondrocytes was examined.

Results
The use of a serum-free, chemically defined medium for isolation and culture of hMSCs allowed us to expand a population with a stable phenotype from early to late passages. It is already well known that MSC proliferation, differentiation, and function decline with passaging. After three passages, we indeed observed a drastic impact on cell growth and differentiation. The paracrine activity of hMSCs during long-term expansion was also evaluated. The number and size of vesicles released by hMSCs increased proportionally with their age. The anti-inflammatory activity of MSC-EVs was investigated in an in vitro model on osteoarthritic chondrocytes and the expression of inflammatory cytokines such as IL-6 and IL-8 were measured. Administration of hMSC-EVs showed relevant antiinflammatory effects only for early passages-derived vesicles (until passage three). iMSCs were also expanded for the long term to define the best culture conditions and the best time window for the isolation of EVs with maximum biological activity.

Conclusions
Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. The focus of our study was directed on the determination of the optimal range of time in which MSCs and iMSC are biologically functionally in a serum-free culture system. This paracrine application may represent a novel therapeutic approach for the treatment of OA.

Background and Objectives
The tumour microenvironment (TME) is a complex milieu that contains cancer cells and non-malignant cellular and non-cellular components which together orchestrate a complex dialog. In the last decades, several papers have proposed that mesenchymal stem cells (MSCs) play a critical role in TME formation and function. In our work, we evaluated the influence of MSCs in combination with endothelial cells on Osteosarcoma (OS) cells using multicellular spheroids. Our hypothesis is that MSCs may have a pro-tumorigenic action.

Materials and Methods
The OS cell lines MG-63, U-2 OS, SaOS-2, and 143B were engineered to express GFP, Endothelial Cells (HUVEC) were purchased, while MSCs were isolated at the Rizzoli. Spheroids were made by self assembly in ultra-low attachment 96 well plates. Based on previous experience in generating multicellular spheroids, we combined OS/HUVEC/MSCs with a ratio of 5:3:2.

Results
Commercially available OS cell lines show a different ability to grow as stable spheroids; only some of them grow rapidly as spheroids but develop necrotic cores over time. Our results revealed that the metabolic activity of cells in OS cell-only spheroids decreased with time, however, increased in hybrid spheroids with MSCs/HUVEC. OS spheroids composed of U-2 OS and SaOS-2 cells formed irregular spheroids while in the hybrid configuration, with MSCs and HUVEC, they assembled forming regular spheroids. OS spheroids composed by 143B and MG-63 cells only had a rounded and compact morphology. The hybrid spheroids of 143B cells and MSCs/HUVEC had an extremely regular and smooth surface, and over a long time, they began to form buds. The hybrid spheroids composed of MG-63 and MSCs/HUVEC had a smooth and regular surface; interestingly, after 96 h MG-63 cells started to leave the spheroid's core and after 7 and 12 days, they formed buds. Bud formation and OS cell migration suggest an increase in OS cell motility and migration when they are cultured with MSCs/HUVEC. Several studies have shown that spheroids' roughness is indicative of cellular invasiveness. Thus, we compared the roughness between simple and hybrid spheroids demonstrating that all hybrid spheroids exhibit greater roughness than spheroids composed of OS cells only.

Conclusions
Data obtained shows that combined MSCs and HUVEC support OS growth and may influence the spheroid morphology and invasiveness, thus sustaining the hypothesis of the pro-tumorigenic effect of these cells.

Objective
Conventional therapies for pancreatic ductal adenocarcinoma (PDAC) present limits due to drug toxicity and the chemoresistance of PDAC cells. That can be due to the PDAC stroma, which constitutes a barrier for the transport of drugs to tumour cells and a drugdelivery system might improve the treatment. The aims of this study were firstly to develop a preclinical metastatic model of PDAC and secondly to set up a model of treatment with mesenchymal stem cells (MSCs) for future applications as a drug-delivery tool.

Materials and Methods
To generate the liver metastatic model, three different cell lines derived from KPC mice were used: K8484 (from PdxCre/LSL-KrasG12D-Trp53R172H), DT6606 (from PdxCre/LSL-KrasG12D), and DT6606lm (from liver metastases after intraportal injection of DT6606). Cells were injected into the portal vein of immunocompetent eight weeks C57BL/6N male mice in a dose range of 1 × 10 3 -5 × 10 5 . On day 20, after tumour induction, the metastatic growth was assessed by seven-tesla magnetic resonance (MR). On day 21, the first group of tumour-bearing mice underwent intravenous (i.v.) injection of luciferase-transduced MSCs (LUC + MSCs) to evaluate the MSCs biodistribution using in vivo imaging system (IVIS). A second group of tumour-bearing mice underwent an intraportal injection of LUC + MSCs and the LUC signal intensity of the two groups was compared.

Results
Among the three investigated cell lines, only the K8484 cell line was chosen to generate the metastatic model. K8484 cell-derived liver metastases, indeed, exhibited a largely glandular architecture, more similar to human metastases. Moreover, the use of the intraportal model allowed a homogeneous and synchronous metastatic growth in mice 20 days after the injection. Studies on biodistribution of i.v. injected MSCs revealed a cell accumulation in the lung after injection with a low-intensity signal and several animals died after cell administration. The mice subjected to intraportal injection of MSCs reported a high cell accumulation in the liver with a prolonged signal up to six days after injection, making the intraportal injection preferable to allow MSCs to reach the tumour site.

Conclusions
The development of this metastatic model of PDAC in the liver allowed us to evaluate MSCs as a promising therapeutic option for PDAC, especially using intraportal injection to deliver them directly to the tumour site. Objective Tumour-associated stromal cells, also known as carcinoma-associated fibroblasts (CAFs), play a pivotal role in favouring tumour growth by their interactions with both tumour cells and cells present in the tumour microenvironment (TME). Recent studies demonstrate that CAFs can favour metastasis and are responsible for chemo-resistance mechanisms. Human amniotic mesenchymal stromal cells (hAMSC) are known to exert immune-modulatory and anti-fibrotic effects, targeting not only immune cells but also stromal cells in damaged tissues. The aim of our study was to determine if hAMSC could also exert effects on stromal cells within the tumour microenvironment, namely CAFs. To this end, we in vitro stimulated the differentiation of normal adult fibroblasts towards CAFs with exogenous TGF-β. The acquisition of CAF features was assessed by immunofluorescence for αSMA expression and for cytoskeleton organisation. In addition, we used Real-Time PCR and flow cytometry to assess the expression of the most relevant CAF markers. Finally, we evaluated the ability of hAMSC conditioned medium (CM-hAMSC) to counteract the acquisition of CAF-like gene expression and functionality.

Materials and Methods
Conditioned medium from hAMSC was obtained by culturing hAMSC at passage 2 in DMEM-F12 without serum for 5 days. Human dermal fibroblasts were treated with TGF-β ± CM-hAMSC for 3, 7, and 11 days. At each timepoint, flow cytometry, immunofluorescence, and RT-PCR were performed to assess CAF-like phenotype.

Results
Flow cytometry showed a high percentage of αSMA + cells after TGF-β treatment, which decreased after CM-hAMSC treatment. Concurrently TGF-β-treated fibroblasts displayed well-organised cytoskeletal αSMA and presented extracellular MFAP5 deposition, whereas CM-hAMSC treatment interfered with these processes. These data are supported by RT-PCR analysis which showed αSMA and MFAP5 downregulation when dermal fibroblasts were treated with CM-hAMSC. On the other hand, we observed an increase of PDPN gene expression and positive cells after CM-hAMSC treatment.

Conclusions
Our preliminary results suggest that hAMSC-secreted factors are able to inhibit CAF activation. Further studies will be aimed at confirming these results and understanding if hAMSC-secreted factors inhibit CAF migration and ultimately clarify the use of hAMSC as a potential therapeutic strategy able to not only target tumour cells, but also stromal cells within the tumour microenvironment.

Objective
In recent years, mesenchymal stromal cells (MSC) have received a strong boost in veterinary medicine due to their pro-regenerative properties. MSCs are virtually present in all the organs possessing a vascular stroma. The aim of this study was to evaluate the potential therapeutic use of MSC from adipose tissue in dogs with complete cranial cruciate ligament (CCL) ruptures that had not undergone surgical treatment. CCL rupture is the most common cause of lameness in dogs and can be treated conservatively, but surgical therapy is the treatment of choice to restore stability and functionality of the joint. However, surgery does not prevent the development of osteoarthritis, which over time leads to relapse of lameness and pain. It is worth mentioning that an increasing number of public and private veterinary hospitals all over the world treat partial CCL rupture with MSC as a second-line or even first-line therapy. There is no evidence of MSC use in complete ruptures.

Material and Methods
Tyson, an American Staffordshire Terrier, male, 4 years old, received a diagnosis of complete CCL rupture followed by 4 intra-articular infiltrations of autologous MSC. Post-infiltration monitoring was performed by orthopaedic, ultrasound, radiography examination, and nuclear magnetic resonance (NMR). A 20-month follow up was performed.

Results
MSC did not trigger adverse effects in the short to medium term, nor they cause regeneration of the CCL. However, they displayed a strong anti-inflammatory activity responsible for symptom remission. The therapeutic effectiveness of MSC seems to be attributable to their chondroprotective effect which slows down the development and progression of osteoarthritis.