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[Abstract] Mesenchymal stem cells (MSCs) are an accepted candidate for cell-based therapy of multiple diseases. The interest in MSCs and their possible application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently, like aggregation and transforming growth factor beta (TGFβ) delivery, hypoxia has been indicated as crucial for complete chondrogenesis. The aim of this study was to test different culture conditions for directing stem cell differentiation into the chondrogenic lineage in vitro by testing different TGFβ superfamily members into the culture media under normoxic conditions. All chondrogenic culture conditions used allowed the differentiation of bone marrow-MSCs (BM-MSCs) into chondrogenic lineage. Chondrogenic induction capacity depended on the growth factor added to the culture media. In particular, the chondrogenic culture condition that better induced chondrogenesis was the medium that included the combination of three growth factors: bone morphogenetic protein-2 (BMP-2), BMP-7 and TGFβ-3. In this culture media, differentiated cells showed the highest levels expression of two markers of chondrogenesis, SOX9 and COL2A1, compared to the control points (p < 0.05, two-tailed t test). In our experimental conditions, the combination of BMP-2, BMP-7 and TGFβ-3 was the most effective in promoting chondrogenesis of BM-MSCs. These results underline the importance of determining in each experimental design the best protocol for in vitro directing stem cell differentiation into the chondrogenic lineage. Galicia. Consellería de Sanidade; PS07/84 Ministerio de Ciencia e Innovacion; PLE2009-0144 Instituto de Salud Carlos III; PI 08/2028

presentado en el 2014 World Congress on Osteoarthritis: Promoting Clinical and Basic Research in Osteoarthritis

[Abstract] Objective: The purposes of this project were to quantify the cells expressing the mesenchymal stem cell (MSC) marker CD271 in synovial membranes from human osteoarthritic (OA) and healthy joints, and to determine if those CD271 cells were involved in spontaneous human cartilage repair and were beneficial for the repair of human articular cartilage defects. Methods: The coexpression of CD44/CD271, CD90/CD271, and CD105/CD271 antigens was determined by immunofluorescence in OA and healthy synovial membranes and during spontaneous cartilage repair. Isolated MSCs from the bone marrow of four OA patients (mean age: 64 years) were magnetically separated into MSC CD271+ and MSC CD271 subsets. The separated cell subsets were then implanted into 2 mm focal defects of articular cartilage. These implants were cultured in chondrogenic differentiation medium supplemented with recombinant human transforming growth factor-beta3 for 8 weeks. The repair tissues were analyzed by histochemistry (hematoxylin–eosin and safranin O) and immunohistochemistry for collage types I and II. Results: Cells expressing the CD271 antigen were diffusely distributed in OA synovial membranes and localized in the subintimal zone in healthy synovial membranes. The number of cells expressing MSC markers was higher in OA synovial membranes than in synovia from healthy joints, corresponding to the highest level of coexpression of CD90/CD271 antigens (9.8% vs. 2.6%). Spontaneous repair tissue showed more cells expressing the CD271 antigen (9.9% ± 4.0%). The highest levels of expression were found to be associated with CD44; 64% of positive CD271 cells coexpressed the CD44 antigen. In both implant cell types, the repair tissue morphology resembled articular cartilage, having an extracellular matrix with a hyaline aspect and numerous lacunae containing cells, and was immunopositive for collagen types I and II. Statistical analyses of the repair tissue demonstrated that the implantation of MSC CD271+ provided such benefits as a greater filling of the chondral defect and better integration between the repair tissue and native cartilage. Safranin O staining of repair tissue was negative in implants of MSC CD271- but more positive in implants with MSC CD271+. The overall histologic score for CD271 implants was 9.5 ± 0.89 and 12.19 ± 1.01 for CD271+ implants. Conclusions: Synovial membranes from OA patients contain more cells expressing CD271 antigen than those from healthy joints, and spontaneous cartilage repair tissue contains cells positive for CD271 antigen. These data suggest the involvement of CD271 antigen in spontaneous cartilage repair and indicate that the cell subset MSC CD271+ provides higher quality chondral repair than the CD271- subset. Galicia. Consellería de Economía, Industria e Comercio; PGIDIT05SAN08PR Galicia. Consellería de Economía, Industria e Comercio; PGIDIT04PXIC91602PN Galicia. Consellería de Economía, Industria e Comercio; PGIDIT04PXIC91601PN Instituto de Salud Carlos III; FISCP03/00127 Instituto de Salud Carlos III; FIS05/2495

[Abstract] Introduction: The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. Aim: To compare two previously published protocols for the isolation of human amnion mesenchymal stromal cells (hAMSCs), including their phenotypic characterization and in vitro potential for differentiation toward osteogenic, adipogenic, and chondrogenic mesodermal lineages. Materials and Methods: Human placentas were obtained from selected caesarean-sectioned births. Two different protocols (Alviano et al.1 and Soncini et al.2) for the isolation of hAMSCs were performed. After monolayer expansion of adherent cells from both protocols, the cells were characterized by flow cytometry and for multi- potentiality, as assessed by their capability to differentiate toward adipocyte-, osteoblast-, and chondrocyte-like cells. Results: Both protocols yielded hAMSCs that showed plastic adherence, fibroblast-like growth, and well-defined human MSC markers. The cell yield and mesodermal differentiation capability of hAMSCs were higher in cells isolated using the Soncini protocol. Conclusions: Our data demonstrated the successful isolation of hAMSCs from full-term placentas using two published protocols. Differences between the two protocols in cell yield and in vitro differentiation potential are shown. Servizo Galego de Saúde; PS07/84 Instituto de Salud Carlos III; CIBER BBN CB06-01-0040

(score decline -64%) and biochemical analysis of GAG (-75%) and total collagen (-30%) content. Gene expression of catabolic enzymes (MMP13, MMP1, MMP2), and genes involved in inflammatory response (IL8, CCL2, CCL4 CXCL2) was significantly increased (>4-fold) and genes related to ECM formation (COL2A1, COMP, Aggrecan, COL8A1, COL11A1) were decreased (

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normal tissue. OA-derived stem cell clones had highly variable life-spans as measured by population doublings. When cultured long term, we observed that some of the OA-derived stem cells lines expanded significantly more than others (>50PD) whilst remaining cell lines became senescent at approximately 30PD. Quantitative assays for senescence and proliferative index support the conclusion that the stem cell population in OA cartilage is heterogeneous in nature. Average telomere length of OA derived stem cell lines was more variable than normal stem cell lines (Fig.1) and corresponded to their individual growth curves, where cell lines with low average telomere length became senescent at approximately 30PD. The chondrogenic potency of OA-derived stem cell lines as assessed by pellet culture was also variable. Conclusions: These data demonstrate the presence of stem cells within OA cartilage and, interestingly, describe the heterogeneity of life-span within OA derived stem cell lines. This variability in stem cell characteristics will have a major negative impact on clinical applications in relation to stem cell potency and the capacity of cells to repair or regenerate articular cartilage lesions. These findings have allowed us to focus on developing methods of isolating highly proliferative stem cells from OA cartilage. The future aim of which is to improve their potency to that of normal stem cells in order that OA-derived stem cells will be of benefit in autologous tissue engineering procedures for cartilage defects.