Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.
ABSTRACT The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations.
Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA).
Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity.
Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.
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Article: Human Muscle-Derived Stem Cells
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ABSTRACT: Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Commercially available human MSCs derived from bone marrow from 6 different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed by gene expression profiling using microarray technology. The phenotype of these cells did not change as reported previously, however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biological attributes that may facilitate the development of assays to test the quality of MSCs prior to clinical use.Stem Cell Research & Therapy 04/2014; 5(2):59. · 3.65 Impact Factor
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ABSTRACT: Aging is one of the major risk factors of osteoarthritis. This pathology during which chondrocytes undergo modifications of their phenotype may result from alteration of transforming growth factor β (TGFβ) signaling. This study investigates the role of TGFβ response in the process of chondrocyte dedifferentiation/redifferentiation. Dedifferentiation was induced by successive passages of human articular chondrocytes, whereas their redifferentiation was performed by three-dimensional culture in alginate. Human mesenchymal stem cells were obtained from bone marrow and differentiated into chondrocyte-like phenotype by three-dimensional culture, embedded in the same scaffold. Protein and mRNA levels were analyzed by Western blot and real-time reverse transcription PCR. Regulatory mechanism was investigated using specific inhibitors (mithramycin), mRNA silencing or decoy oligonucleotides, and expression vectors. Chondrocyte dedifferentiation interfered with TGFβ signaling by decreasing TβRII mRNA and protein levels and subsequent TGFβ response. TβRII ectopic expression in passaged chondrocytes permitted to increase the expression of several matrix genes, such as aggrecan or type II collagen. Redifferentiation of passaged chondrocytes permitted to restore, at least in part, TβRII expression and was related to differentiation of human bone marrow mesenchymal stem cells toward chondrocytes, where both specific protein 1 (Sp1) and TβRII mRNA levels were increased. Moreover, Sp1 manipulation by silencing or ectopic expression and pharmacologic inhibition revealed a link between expression levels of this transcriptional factor, which is crucial for constitutive expression of TβRII in cartilage, and TGFβ response. Therefore, these data permit us to suggest an important role of TβRII expression in the maintenance of chondrocyte phenotype, which is altered with age, and bring new insights in our understanding of chondrogenesis process.Age 08/2013; 35(4):1105-16. · 6.28 Impact Factor