Generation of Mesenchymal Cell Lines Derived from Aged Donors.

Background: Mesenchymal stromal cells (MSCs) have the capacity for self-renewal and multi-differentiation, and for this reason they are considered a potential cellular source in regenerative medicine of cartilage and bone. However, research on this field is impaired by the predisposition of primary MSCs to senescence during culture expansion. Therefore, the aim of this study was to generate and characterize immortalized MSC (iMSC) lines from aged donors. Methods: Primary MSCs were immortalized by transduction of simian virus 40 large T antigen (SV40LT) and human telomerase reverse transcriptase (hTERT). Proliferation, senescence, phenotype and multi-differentiation potential of the resulting iMSC lines were analyzed. Results: MSCs proliferate faster than primary MSCs, overcome senescence and are phenotypically similar to primary MSCs. Nevertheless, their multi-differentiation potential is unbalanced towards the osteogenic lineage. There are no clear differences between osteoarthritis (OA) and non-OA iMSCs in terms of proliferation, senescence, phenotype or differentiation potential. Conclusions: Primary MSCs obtained from elderly patients can be immortalized by transduction of SV40LT and hTERT. The high osteogenic potential of iMSCs converts them into an excellent cellular source to take part in in vitro models to study bone tissue engineering.

Proteomic Analysis of Synovial Fibroblasts and Articular Chondrocytes Co-Cultures Reveals Valuable VIP-Modulated Inflammatory and Degradative Proteins in Osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast-derived ADAMTS-7 and -12.

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS-7 and -12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK-Runx2 axis and Wnt/ß-catenin signalling in ADAMTS-12 and ADAMTS-7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL-1ß or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS-12 expression in OA-SF, also reducing Fn-fs-induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS-7 and COMP degradation in OA-SF as well. In addition, Wnt7B expression was induced by IL-1ß and by itself, also increasing ADAMTS-7. Our results could contribute to the development of disease-modifying OA drugs targeting ADAMTS-7 and -12 for the prevention of extracellular matrix components degradation like COMP.

Human Cartilage Engineering in an In Vitro Repair Model Using Collagen Scaffolds and Mesenchymal Stromal Cells.

The purpose of this study was to investigate cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (hBMSCs) with different collagen (Col) scaffolds. Lesions were made in human cartilage biopsies. Injured samples were pre-treated with interleukin 1ß (IL1ß) for 24 h; also, samples were not pre-treated. hBMSCs were seeded on different types of collagen scaffolds. The resulting constructs were placed into the lesions, and the biopsies were cultured for 2 months in chondrogenic medium. Using the modified ICRSII scale, neotissues from the different scaffolds showed ICRS II overall assessment scores ranging from 50% (fibrocartilage) to 100% (hyaline cartilage), except for the Col I +Col II +HS constructs (fibrocartilage/hyaline cartilage, 73%). Data showed that hBMSCs cultured only on Col I +Col II +HS scaffolds displayed a chondrocyte-like morphology and cartilage-like matrix close to native cartilage. Furthermore, IL1ß pre-treated biopsies decreased capacity for repair by hBMSCs and decreased levels of chondrogenic phenotype of human cartilage lesions.

Ovine Mesenchymal Stromal Cells: Morphologic, Phenotypic and Functional Characterization for Osteochondral Tissue Engineering.

INTRODUCTION: Knowledge of ovine mesenchymal stromal cells (oMSCs) is currently expanding. Tissue engineering combining scaffolding with oMSCs provides promising therapies for the treatment of osteochondral diseases. PURPOSE: The aim was to isolate and characterize oMSCs from bone marrow aspirates (oBMSCs) and to assess their usefulness for osteochondral repair using ß-tricalcium phosphate (bTCP) and type I collagen (Col I) scaffolds. METHODS: Cells isolated from ovine bone marrow were characterized morphologically, phenotypically, and functionally. oBMSCs were cultured with osteogenic medium on bTCP and Col I scaffolds. The resulting constructs were evaluated by histology, immunohistochemistry and electron microscopy studies. Furthermore, oBMSCs were cultured on Col I scaffolds to develop an in vitro cartilage repair model that was assessed using a modified International Cartilage Research Society (ICRS) II scale. RESULTS: oBMSCs presented morphology, surface marker pattern and multipotent capacities similar to those of human BMSCs. oBMSCs seeded on Col I gave rise to osteogenic neotissue. Assessment by the modified ICRS II scale revealed that fibrocartilage/hyaline cartilage was obtained in the in vitro repair model. CONCLUSIONS: The isolated ovine cells were demonstrated to be oBMSCs. oBMSCs cultured on Col I sponges successfully synthesized osteochondral tissue. The data suggest that oBMSCs have potential for use in preclinical models prior to human clinical studies.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

INTRODUCTION: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. MATERIAL AND METHODS: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. RESULTS: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. CONCLUSION: HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.

Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines.

INTRODUCTION: The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. METHODOLOGY: Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. RESULTS: The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower. CONCLUSION: Among the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation.

Differentiation of human mesenchymal stromal cells cultured on collagen sponges for cartilage repair.

AIM: The aim of this study was to evaluate proliferation and chondrogenic differentiation of human bone-marrow mesenchymal stromal cells (hBMSCs) cultured on collagen biomaterials. MATERIALS AND METHODS: hBMSCs were seeded on five different collagen (Col) sponges: C1C2 (types I and II Col), C1C2HS (types I and II Col plus heparan sulphate (HS)), C1C2CHS (types I and II Col plus chondroitin sulphate (CHS)), C1-OLH3 (type I Col plus low molecular weight heparin) and C1CHS (type I Col plus CHS). The resulting constructs were analyzed by histological and immunohistochemical staining, molecular biology and electron microscopy. Col released into culture media was measured by a dye-binding method Results: hBMSCs on biomaterials C1C2, C1C2HS and C1C2CHS had more capacity to attach, proliferate and synthesize Col II and proteoglycans in the extracellular matrix (ECM) than on C1-OLH3 and C1CHS. The presence of aggrecan was detected only at the gene level. Total Col liberated by the cells in the supernatants in all scaffold cultures was detected. The level of Col I in the ECM was lower in C1-OLH3 and that of Col II was highest in C1C2 and C1C2HS. Electron microscopy showed differently shaped cells, from rounded to flattened, in all constructs. Col fibers in bundles were observed in C1C2CHS by transmission electron microscopy. CONCLUSIONS: The results show that Col I and Col II (C1C2, C1C2HS and C1C2CHS) biomaterials allowed cell proliferation and chondrogenic-like differentiation of hBMSCs at an early stage. Constructs cultured on C1C2HS and C1C2CHS showed better cartilage-like phenotype than the other ones.

Alternative protocols to induce chondrogenic differentiation: transforming growth factor-ß superfamily.

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 (TGFß) 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 TGFß 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 TGFß-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 TGFß-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.

Effects of severe hypoxia on bone marrow mesenchymal stem cells differentiation potential.

Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed.

Cryopreservation effect on proliferative and chondrogenic potential of human chondrocytes isolated from superficial and deep cartilage.

OBJECTIVES: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. MATERIALS AND METHODOLOGY: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. RESULTS: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). CONCLUSIONS: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.

Human amniotic membrane as an alternative source of stem cells for regenerative medicine.

The human amniotic membrane (HAM) is a highly abundant and readily available tissue. This amniotic tissue has considerable advantageous characteristics to be considered as an attractive material in the field of regenerative medicine. It has low immunogenicity, anti-inflammatory properties and their cells can be isolated without the sacrifice of human embryos. Since it is discarded post-partum it may be useful for regenerative medicine and cell therapy. Amniotic membranes have already been used extensively as biologic dressings in ophthalmic, abdominal and plastic surgery. HAM contains two cell types, from different embryological origins, which display some characteristic properties of stem cells. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. Both populations have similar immunophenotype and multipotential for in vitro differentiation into the major mesodermal lineages, however they differ in cell yield. Therefore, HAM has been proposed as a good candidate to be used in cell therapy or regenerative medicine to treat damaged or diseased tissues.

Quantification of cells expressing mesenchymal stem cell markers in healthy and osteoarthritic synovial membranes.

OBJECTIVE: To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints. METHODS: Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. RESULTS: Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. CONCLUSION: Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

Bone marrow cells immunomagnetically selected for CD271+ antigen promote in vitro the repair of articular cartilage defects.

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.

Isolation and characterization of mesenchymal stem cells from human amniotic membrane.

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 multipotentiality, 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.

Molecular profile and cellular characterization of human bone marrow mesenchymal stem cells: donor influence on chondrogenesis.

BACKGROUND: The use of autologous or allogenic stem cells has recently been suggested as an alternative therapeutic approach for treatment of cartilage defects. Bone marrow mesenchymal stem cells (BM-MSCs) are well-characterized multipotent cells that can differentiate into different cell types. Understanding the potential of these cells and the molecular mechanisms underlying their differentiation should lead to innovative protocols for clinical applications. The aim of this study was to evaluate the usefulness of surface antigen selection of BM-MSCs and to understand the mechanisms underlying their differentiation. METHODS: MSCs were isolated from BM stroma and expanded. CD105+ subpopulation was isolated using a magnetic separator. We compared culture-expanded selected cells with non-selected cells. We analyzed the phenotypic profiles, the expression of the stem cell marker genes Nanog, Oct3/4, and Sox2 and the multi-lineage differentiation potential (adipogenic, osteogenic, and chondrogenic). The multi-lineage differentiation was confirmed using histochemistry, immunohistochemistry and/or real-time polymerase chain reaction (qPCR) techniques. RESULTS: The selected and non-selected cells displayed similar phenotypes and multi-lineage differentiation potentials. Analyzing each cell source individually, we could divide the six donors into two groups: one with a high percentage of CD29 (ß1-integrin) expression (HL); one with a low percentage of CD29 (LL). These two groups had different chondrogenic capacities and different expression levels of the stem cell marker genes. CONCLUSIONS: This study showed that phenotypic profiles of donors were related to the chondrogenic potential of human BM-MSCs. The chondrogenic potential of donors was related to CD29 expression levels. The high expression of CD29 antigen seemed necessary for chondrogenic differentiation. Further investigation into the mechanisms responsible for these differences in BM-MSCs chondrogenesis is therefore warranted. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous human BM-MSCs for articular cartilage repair.

Multilineage differentiation potential of cells isolated from the human amniotic membrane.

The human amniotic membrane (HAM) contains two cell types from different embryological origins. Human amnion epithelial cells (hAECs) are derived from the embryonic ectoderm, while human amnion mesenchymal stromal cells (hAMSCs) are derived from the embryonic mesoderm. In this study, we localized, isolated, quantified and phenotypically characterized HAM-derived cells and analysed their in vitro differentiation potential towards mesodermal cell lineages. Human amnion-derived cells were isolated and characterized by flow cytometry. Immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction studies were performed for the analysis of multipotentiality. Immunophenotypic characterization of both cell types demonstrated the presence of the common, well-defined human mesenchymal stem cell (MSC) markers (CD90, CD44, CD73, CD166, CD105, CD29), as well as the embryonic stem-cell markers SSEA-4 and STRO-1. Phenotypes of both cell populations were maintained from passages P0 to P9. The assessment of multilineage potential demonstrated that the hAMSCs showed greater adipogenic and chondrogenic potential. Both populations had the ability to retain their capacity for differentiation during culture passages from P0 to P4. Our data demonstrate the successful localization and isolation of hAMSCs and hAECs from the HAM. Both cell populations possessed similar immunophenotype. However, they differed in cell yield and multipotential for differentiation into the major mesodermal lineages. Our functional differentiation studies demonstrated that hAMSCs possess a much greater mesodermal differentiation capacity than hAECs. These considerations will be important for use of these cells for cell therapy.

[Treatment of joint cartilage lesions with cell therapy]. / Tratamiento de lesiones del cartílago articular con terapia celular.

Articular cartilage lesions which do not affect the integrity of subchondral bone, they are not able to repair it expontaneously. The asymptomatic nature of these lesions induces articular cartilage degeneration and development of an arthrosic process. To avoid the necessity to receive joint replacement surgery, it has been developed different treatments of cellular therapy which are focused to create new tissues whose structure, biochemistry composition and function will be the same than native articular cartilage. Approaches used to access the stream produce a fibrocartilaginose tissue which is not an articular cartilage. Implantation of autologous chondrocytes and autologous mosaicplasties induces a quality better articular cartilage. Furthermore both techniques involve damage in the sane cartilage; because of trying to get a big amount of chondrocytes or because of extraction osteochondral cylinder which will be implanted in the injured joint. The stem cells are a promising toll to repair articular cartilage, however they are in a previous experimentation step yet. Although the present studies using cellular therapy improves clinically and functionally, it is not able to regenerate an articular cartilage which offer resistance the degeneration process.