Optimization Of The Culture System For The Mesenchymal Stem Cell From The Placenta Chorion In Vitro And The Preliminary Analysis Of Its Biological Characteristic

Mesenchymal stem cells (MSCs) are specialized cells capable of self-renewal as well as multilineage differentiation, and with great perspective of clinical application. Previous research mainly concentrated on bone marrow-derived MSCs, however, it can only be obtained by invasion method which distribute sparsely, therefore, many new sources of MSCs were founed, for example, peripheral blood, cord blood, umbilical cord, deciduous teeth and placenta. Placenta draws great interests not only because it is rich in MSCs with immunogenicity, but also it is readily avilable and easily procured, and its use does not elicit ethical debate, which may render these cells as good potential sources for future therapectic applications. In 2007, the first international workshop on placenta derived stem cells was held in Italy, which provided the preliminary indications of the possibility of using these cells in the future clinical applications. But till now the accurate distrabution and how to expand the placenta derived MSCs effectivly in vitro is still a problem needed to be solved.In our research, we set up a system to isolate and expand the placenta chorion derived MSC (hCMSCs) in vitro, on the basis of these, the preliminary analysis of the amplification conditions and the biological characteristic were carried out, which laid a foundation to the clinical application of these cells as the seed cells.Part I Isolation and Identification of hCMSCs in vitroObjective: To isolate the MSC within the chorion of human placenta in vitro after determining its localization, then the biological characteristic of the hCMSCs was explored by inducing these cells to the adipocytes and the neuron like cells as well, which offered the experimental data for the optimizing the culture system of hCMSCs. Methods: Immunohistochemistry was applied to show the localization of the Mesenchymal Stem Cell within the placenta chorion, then the corresponding part of chorion was digested by collagenase IV to get the cell suspension for primary culture and subculture; Flow Cytometry was used to detect the surface marker of hCMSCs; the morphology of hCMSCs was observed under inverted microscope; then under different induction conditions, hCMSCs was induced to the adipocytes and the neuron like cells, respectively, to determine its differentiation potential.Results: (1) The result of immunohistochemistry showed CD105, CD90 positive hCMSCs was much easier to be found around the blood vessels within the villous of chorion;(2) The hCMSCs then could be obtained by mechanical dissociation and collagenase digestion effectively. (3) The hCMSCs showed plastic adherence and typical fibroblastic morphology by light microscopy, and the Flow Cytometry analysis showed that hCMSCs highly expressed CD73,CD90,CD105, negative for CD14,CD34,CD45,HLA-DR; (4) To induce neural differentiation, in addition to the morphological changes, the differentiated cells was detected by immunofluorescence staining for the expression of NSE; (5) Futhermore, hCMSCs were treated with adipogentic medium for 2 weeks. The differentiated adipocytes were indicated by accumulation of neutral oil droplet that positively stained using oil-red O staining.Conclusion: Human placenta chorion was proved to be rich in MSCs, and the hCMSCs could be obtained from the chorion by mechanical dissociation and collagenase digestion effectively. The results of the Flow Cytometry and induction indicated that the cultured hCMSCs well maintained the surface markers together with the differentiation potential as MSCs.Part II The Effect of Cytokines on the Proliferation and Characteristic of hCMSCsObjiect: To optimize the culture to expand the hCMSCs in vitro efficiently, and to analysis the characteristic of the expaned hCMSCs as well, so as to provide the hCMSCs as the seed cells in stem cell based therapies in clinical trials fast and effectively. Methods: The corresponding part of chorion was digested by 0.1% collagenase IV to get the cell suspension for primary culture and subculture; after two passages the hCMSCs were used for the cell proliferation assay. MTT method was adopted to show the different proliferation promoting effects of different cytokines, such as the interleukin-6 (IL-6), glycoprotein130 (GP130), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), flt3 ligand (FL), and basic fibroblast growth factor (bFGF). Thereafer, under the optimized culture condition, the morphology of the expanded hCMSCs were observed under the microscope, the cell phenotype was detected by the Flow Cytometry analysis, and oil-red O staining together with the Immunofluorescence staining were applied to identified the differentiation potential of the hCMSCs to adipocytes and neuron like cells, respectively. Finally PT-PCR was used to detect the expression of flt3.Results: (1) The results of MTT and cell counting indicated that through comparation both bFGF and FL showed well proliferation promoting effects on the hCMSCs, but the effect of FL was more obvious. (2) hCMSCs showed plastic adherence and typical fibroblastic morphology by light microscopy, both under the optimized culture conditions with bFGF or FL added.(3) The Flow Cytometry analysis showed that under the optimized culture conditions containing bFGF or FL, hCMSCs still highly expressed CD73,CD90,CD105, negative for CD14,CD34,CD45,HLA-DR. (4) Under the optimized culture conditions containing bFGF or FL, and after induction, the differentiated neuron like cells were detected NSE positive by immunofluorescence staining, and within the differentiated adipocytes oil droplets were observed instead. (5) The result of RT-PCR showed that flt3 was expressed in the cultured hCMSCs.Conclusion: Both bFGF and FL showed well proliferation promoting effects on the hCMSCs among all selected cytokines, but the effect of FL was more obvious and at the same time FL could well maintain the cell morphology, cell phenotype and the differentiation potential of the expanded hCMSCs as well, therefore, FL could be adopted as a good cytokine for the optimization of the culture system for the hCMSCs.In summary, this research identified the location of the MSC within the placenta chorion. Effective culture system was estabilished sucesssfully to isolated and expanded hCMSCs in vitro. And basis on these, FL was proved having good proliferation promoting effects on the hCMSCs, and the expression of flt3, the ligand of FL, was also detected, which provide basis for the further study of the mechanisms of the proliferation promoting effects of FL on the hCMSCs.