Differentiation Of Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Into Nucleus Pulposus Cells In Vitro

Background and Purpose Wharton's jelly-derived mesenchymal stem cells (WJMSCs) was a primitive stromal population isolated from umbilical cord extracellular matrix, and they displayed the characteristics of mesenchymal stem cells (MSCs). They growed as adherent cells with mesenchymal morphology, were self-renewing, expressed cell surface markers displayed by MSCs and could be differentiated into other cells. This study was conducted to isolate MSCs from human umbilical cord Wharton's jelly, observe the cell morphology, identify the cell phenotype and detect the activity of telomerase.Methods: Human umbilical cord was obtained after normal birth, arteries, veins and epithelium were stripped and discarded from umbilical cord tissue. The Wharton's jelly was treated with collagenase and trypsin digestion, and the cells were cultured in DMEM/F12 medium supplemented with fetal bovine serum in 5% CO2 in 37oC incubator. The ability of proliferation of WJMSCs was observed by means of growth curve. WJMSCs at passage 3 were taken for telomerase activities detection and flow cytometric analysis (CD34/CD45/CD105/CD73/ CD90/HLA-DR and HLA-ABC).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 following treatment with FBS-DMEM/F12. The ability of proliferation of WJMSCs didn't decline from passage 2 to passage 16. According to the immunophenotypic analysis of flow cytometry, the WJMSCs expressed CD73\CD105\CD90 and HLA-ABC, but did not express CD34\CD45 and HLA-DR. WJMSCs were positive in telomerase activity assay.Conclusions: In this study, we isolated mesenchymal stem cells from human umbilical cord wharton's jelly. We have observed that the WJMSCs expressed the surface markers of MSCs, didn't express the hematopoietic lineage markers and showed positive in telomerase activity assay. WJMSCs displayed the characteristics of mesenchymal stem cells. WJMSCs might be an ideal seed cells for tissue engineeering or as cell-based therapy because of faster proliferation, greater expansion capabilities and easier obtained. Background and Purpose Nowadays, the degenerative disc disease (DDD) was commonly to be seen in low back pain patients, seriously affecting their daily life and work. The conventional treatment of DDD consisted of therapies, both conservative and invasive, that were aimed at symptomatic relief, often had short-term solutions and lead to complications. New therapeutic strategies which were based on tissue engineering or cell-based therapies approaches had been proposed as promising treatments for DDD, aiming at restoring the disc extracellular matrix and ultimately disc height. However, the lack of ideal seed cells remained a serious problem among these methods. The objective of this study was to investigate for the first time in differentiation of human Wharton's jelly-derived mesenhymal stem cells towards nucleus pulposus cells.Methods: WJMSCs were isolated from human umbilical cord after normal birth and NPCs were isolated from human spine (T12–L1). Fluorescent labeled WJMSCs and NPCs were cocultured for 7 days. Transwell six-well plates with 0.4μm pore size, high pore density, polyethylene terephthalate (PET) track-etched tissue culture inserts were used for coculture without contact. MoFlo high-speed cell sorting was conducted to separate fluorescent labeled WJMSCs and NPCs after coculture with cell–cell contact, which allowed examination of individual cell populations. Cells were seeded at ratios of 25%:75%WJMSCs/NPCs, 50%:50% WJMSCs/NPCs, and 75%:25% WJMSCs/NPCs. Total RNA was extracted from cells using Trizol reagent. After the cDNA were obtained by reverse transcription, relative gene expressions of aggrecan, type I collagen, type II collagen, type VI collagen, versican and SOX-9 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: While WJMSCs and NPCs were cocultured with contact, SOX-9,type II collagen and aggrecan mRNA were significantly (p<0.05) increased in WJMSCs in all three cell ratios, with the largest increase being seen at a ratio of 25%:75% WJMSCs/NPCs (2429-fold at SOX-9, 9463-fold at type II collagen and 5974-fold at aggrecan). The coculture without contact gene expression levels of SOX-9,type II collagen and aggrecan also had significantly increase in all ratios(p<0.05), but the increase was more lightly than that of coculture with cell-cell contact. The highest increase in coculture group without contact could be seen at a ratio of 25%:75% WJCs/NPCs(114-fold at SOX-9, 57-fold at type II collagen and 67-fold at aggrecan). There were no significant changes in type I collagen, type VI collagen and versican mRNA expression at all groups (P>0.05).Conclusions: Coculture of NPCs with WJMSCs causes WJMSCs differentiating into an NP phenotype and that the optimum cell ratio for differentiation is 25:75 WJMSCs/NPCs. It is shown that this differentiation can be achieved within 7 days, and that direct cell-cell contact is more important for WJMSCs differentiating into NPCs. Furthermore, the effects of NP cells on WJMSCs suggest that if WJMSCs were implanted into the intervertebral disc, the WJMSCs would be induced by the surrounding NPCs to differentiate into NPCs phenotype and product nucleus pulposus matrix. The present results indicate that WJMSCs might be a promising seed cells for tissue engineering or as a cell-based therapy for DDD because of its faster proliferation, greater expansion capability and easier obtained.