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Differentiation of Human Umbilical Cord Wharton’s Jelly-Derived Mesenchymal Stem Cells into Nucleus Pulposus Cells in Vitro

Author: ZhangYan
Tutor: RuanDiKe
School: Second Military Medical University
Course: Surgery
Keywords: Umbilical cord Huatong gum mesenchymal stem cells Mesenchymal stem cells Flow cytometry Telomerase activity Nucleus pulposus cells Co-culture Differentiation Disc degeneration
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 58
Quote: 0
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Abstract

Objective: umbilical China through plastic mesenchymal stem cells (Wharton's jelly-derived mesenchymal stem cells, WJMSCs) is derived from the umbilical cord blood around the tube Huatong gum tissue of mesenchymal stem cells, stem cells the basic characteristics that the cells have self-renewal and the ability to differentiate to different tissues or cells. Isolated from human umbilical cord the extracted the Huatong gum mesenchymal stem cells, to observe the cell morphology, detect proliferation, cell phenotype and telomerase activity, the identified isolation cells with stem cell properties for the next step through the cell co-culture method is to lay the foundation for nucleus pulposus cells induced to differentiate. Methods: Take a full-term healthy newborns umbilical cord separation the Huatong gum organizations, and cut it into pieces with collagenase and trypsin digestion, DMEM/F12 medium and fetal bovine serum extracted from the cells, cell fusion, 80-90 subcultured%. Cell morphology was observed, and the determination of different generations of cell proliferation. 3rd generation cell line telomerase activity analysis and flow cytometry to detect cell immune phenotype the the (CD34/CD45/CD105/CD73/CD90/HLA-DR and HLA-ABC). RESULTS: The primary cultured for 3-5 days in some cells adherent growth morphology of spindle and polygonal form colonies after 1 week, 2 weeks, 90% cell fusion. After passage, cells were spindle morphology consistent cells were swirling growth, cell proliferation ability, 5-6 days to passage, no decline in proliferative capacity passaged for 16 generations. Telomerase activity was measured as positive, flow cytometric analysis of cell phenotype results show that CD90/CD105/CD73/HLA-ABC the positive, CD34/CD45/HLA-DR negative. Conclusion: human umbilical cord is rich in mesenchymal stem cells can be extracted from the umbilical cord Huatong gum by enzymatic digestion of mesenchymal stem cells, the cells were fusiform, whorled growth, proliferation ability. Flow cytometry to detect cell phenotype cells express mesenchymal stem cell immune phenotype, do not express the hematopoietic stem cell phenotype, telomerase activity was positive. The Huatong gum mesenchymal stem cells due to stronger proliferative activity, better amplification ability and easier to obtain expected to become the ideal seed cells for tissue engineering and cell therapy technology. Background and Purpose: degenerative disc disease (degenerative disc disease, DDD) is common and frequently-occurring disease, low back pain caused serious impact on people's work and life. The current treatment is conservative treatment and surgical treatment, temporary relief of clinical symptoms, but the long-term effect is not ideal, and complications may occur. In recent years, for the purpose of tissue engineering to repair the disc degeneration restore disc height and cell therapy technology is increasingly considered to be a promising treatment method. However, both tissue engineering or cell therapy technology in the treatment of degenerative disc disease is still a lack of an ideal seed cells. Investigated the effects of human umbilical cord the Huatong gum mesenchymal stem cells (Wharton's jelly-derived mesenchymal stem cells, WJMSCs) to the differentiation potential of nucleus pulposus cells (nucleus pulposus cells, NPCs) provide an ideal for the treatment of degenerative disc disease source of seed cells. Methods: normal full-term infant umbilical cord separated digestion the umbilical cord huatong gum tissue, collected cultured of Huatong gum mesenchymal stem cells; take normal non degenerated intervertebral disc (T12-L1) nucleus pulposus cells were isolated and cultured, enzyme digestion. . Take stable proliferation 3rd generation Huatong gum mesenchymal stem cells and nucleus pulposus cells were co-cultured. CFSE-labeled the Huatong gum mesenchymal stem cells. Transwell culture plates with inserted layer non-contact co-culture, the insertion layer has a 0.4μm high density Aperture; the lower inoculated with Huatong gum mesenchymal stem cells, the nucleus pulposus cells of the upper inoculated. Ordinary six-well plates-contact co-culture. According to the total proportion of cultured cells were divided into 3 groups (25:75 WJMSCs / hNPCs, 50:50 WJMSCs / hNPCs, 75:25 WJMSCs / hNPCs), one week after co-culture MoFlo high-speed flow cytometry sorter points Huatong gum selected collection-contact co-culture of mesenchymal stem cells. Extraction of the Huatong gum mesenchymal stem cell total RNA was reverse transcribed to to get cDNA, using Real-Time PCR method for detection of proteoglycan and collagen type I, type II collagen VI collagen type, SOX-9 and pluripotent glycan genes expression of the housekeeping gene GAPDH was used as an internal reference, the Huatong gum cultured alone mesenchymal stem cells as a control, using the 2-ΔΔCt method relative gene expression changes. Results: cell sorting bitmap display the CFSE labeled WJMSCs and the no fluorescent nucleus pulposus cells labeled complete separation and sorting. Sorting cells Real-Time PCR to detect the Huatong gum mesenchymal stem cells relative gene expression changes. After 7 days of co-culture, the results show that contactless co-cultured mesenchymal stem cell groups Huatong gum SOX-9, collagen type II and proteoglycan expression has improved significantly (P lt; 0.05) relatively 25:75 WJMSCs / hNPCs group raised the magnitude of the largest (SOX-9 raised 2429 times collagen type Ⅱ raised 9463 times, proteoglycan raised 5974 times); non-contact total training group groups Huatong glue mesenchymal stem cells of SOX-9, Ⅱ relative expression of type I collagen and proteoglycan also significantly improve (P lt; 0.05), but the gene rate increases than-contact co-culture group of small (P lt; 0.05) which the 25:75 WJMSCs / hNPCs set of genes express the maximum increase rate (SOX -9 increased 114-fold increased 57-fold, collagen type II, the proteoglycan raised 67 times). Co-culture groups of type I collagen VI collagen type and multi-glycans gene gene expression change was not statistically significant (P gt; 0.05). CONCLUSION: Human umbilical cord the Huatong gum mesenchymal stem cell differentiation potential of nucleus pulposus cells. Cells were cultured for 7 days able to induce the Huatong gum mesenchymal stem cells to differentiate into nucleus pulposus cells optimal cell ratio of 25:75 WJMSCs / and of NPCs. -Contact co-culture is more conducive to the differentiation of mesenchymal stem cells, can be inferred Huatong gum mesenchymal stem cells transplanted into the nucleus pulposus, nucleus pulposus cells capable of inducing the implanted cells to differentiate into nucleus pulposus cells and the secretion of the nucleus pulposus matrix, in order to restore the disc height. Therefore, the of Huatong gum mesenchymal stem cells because of its greater proliferative activity, better amplification capabilities and easier access is expected to become the ideal seed cells for the treatment of degenerative disc disease.

Differentiation of Human Umbilical Cord Wharton’s Jelly-Derived Mesenchymal Stem Cells into Nucleus Pulposus Cells in Vitro

Abstract

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