Study On The Induced Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Chondrocytes

Objective:1.To investigate the ability of Multilineage differentiation of human umbilicalcord-MSCs by subculturing to23passages.2. To investigate a better method of inducinghUC-MSCs into chondrocytes in non-scaffold culture in vitro.Methods: Mesenchymal stem cells were isolated from umbilical cord by collagenaseⅡdigestion and were digested by trypsin for continuous passage culture. A series of differentgeneration cultured-cells were collected for following experiment. The cells surface antigens andcell cycle were measured by flow cytometry; The cell growth curve was analyzed by MTT assay;Adipogenic、osteogenic and chondrogenetic ability were evaluated by specially inducing culture;OCT-4、SOX-2and Nanog mRNA expression level were assayed by real-time fluorescencequantitative PCR. Third generation hUC-MSCs were induced into chondrocytes in two kinds ofnon-scaffold culture.The histological structure of neocartilage was observed by HE staining inpellet culture. Type Ⅱ collagen expression was observed by immunohistochemistry. GAGexpression was observed by Alcian blue and DMMB staining. The content GAG and type Ⅱcollagen were estimated from the determination of hydroxyproline content and Alcian Bluemethod. Expressions of GAG, type Ⅱ collagen and Sox-9were assayed by real-time fluorescencequantitative PCR.Result: By subculturing to23passages in vitro, the expression rate of surface marker in cell wasnot significant changed; the shape of cell growth curves were similarity; there was also no obviousdifference in cell cycle; cells can be induced into osteoblasts, chondrocytes and adipocytes; Oct-4、Sox-2and Nanog mRNA expression level were no obvious difference. HE staining results displayedthat hUC-MSCs incubated in pellet culture to induce into chondrocytes,the tissue with the uniquestructure of the cartilage tissue.Immunohistochemistry, Alcian blue and DMMB staining resultsshowed that chondrogenic induction cells were expressed GAG,type Ⅱ collagen in two kinds ofnon-scaffold culture. The determination of hydroxyproline content and Alcian Blue method displaythat the content of GAG and type Ⅱ collagen in experiment groups were more than that in controlgroups in two kinds of non-scaffold culture,in which the cells were incubated in pellet culture withhigher expression. Real-time PCR results demonstrated that chondrogenic induction cells were expressed GAG,type Ⅱ collagen and Sox-9, and the cells were incubated in pellet culture withhigher expression.Conclusion:1.Subculturing to23generation in vitro, the ability of mutilineagedifferentiation of hUC-MSCs was not influenced with increase in numbers of subculture.2.hUC-MSCs incubated in pellet culture can strongly express GAG and type Ⅱ collagen afterchondgenic induction, so it’s more conducive to inducing hUC-MSCs into chondrocytes.