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| ZHANG Yan,RUAN Dike,ZHANG Chao.Differentiation of human umbilical cord Wharton′s jelly-derived mesenchymal stem cells into nucleus pulposus-like cells by coculture with cell-cell contact in vitro[J].Chinese Journal of Spine and Spinal Cord,2012,(10):936-942. |
| Differentiation of human umbilical cord Wharton′s jelly-derived mesenchymal stem cells into nucleus pulposus-like cells by coculture with cell-cell contact in vitro |
| Received:June 26, 2012 Revised:August 13, 2012 |
| English Keywords:Wharton′s jelly-derived mesenchymal stem cells Coculture Differentiation Nucleus pulposus cells |
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| 【Abstract】 Objectives: To investigate the differentiation of human Wharton′s jelly-derived mesenhymal stem cells(WJMSCs) into nucleus pulposus-like cells by coculture with cell-cell contact in vitro. Methods: Umbilical cord was obtained from normal birth, and the mesenchyamal stem cells were isolated from the umbilical cord Wharton′s jelly. Human nucleus pulposus cells(NPCs) were isolated from human intervertebral nucleus pulposus. WJMSCs were taken for flow cytometric analysis(CD73/CD90/CD105/CD34/CD45/HLA-ABC/HLA-DR). Fluorescent labeled WJMSCs and no-labled NPCs were cocultured for 7 days by cell-cell contact method. Cells were seeded at ratio of 1∶1. WJMSCs cultured alone served as controls. MoFlo high-speed cell sorting was conducted to separate fluorescent labeled WJMSCs and NPCs after coculture. Total RNA was extracted from cells using Trizol reagent. After the cDNA was obtained by reverse transcription, relative gene expressions of type Ⅰ collagen, type Ⅱ collagen, type Ⅵ collagen, aggrecan, SOX-9 and versican were determined by Real-Time PCR and normalized to the GAPDH housekeeping gene. WJMSCs cultured alone served as controls. The 2-ΔΔCt value was then used to calculate relative expression of each target gene. Results: WJMSCs were isolated from human umbilical cord and expanded as primary cultures. After attachment, the cells gradually spread out and were shown as fibroblast-like morphology. According to the immunophenotypic analysis of flow cytometry, the WJMSCs expressed CD73, CD90, CD105, and HLA-ABC, but did not express CD34, CD45 and HLA-DR. While WJMSCs and NPCs were cocultured with cell-cell contact, SOX-9, type Ⅱ collagen and aggrecan mRNA increased significantly in WJMSCs(P<0.01). There were no significant changes in type Ⅰ collagen, type Ⅵ collagen and versican mRNA expression(P>0.05). Conclusions: WJMSCs display the characteristics of mesenchymal stem cells. Coculture of NPCs and WJMSCs with cell-cell contact can induce WJMSCs differentiating into a NPCs phenotype within 7 days, which indicates that WJMSCs may be a promising seed cell for tissue engineering therapies or cell-based therapies for degenerated intervertebral discs. |
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