The Study Of Culture System Optimization, Induction Into IPS And MiRNA Mediated Directional Differentiation Of Porcine BMSC

Porcine bone marrow mesenchymal stem cell(p BMSC) is one of the most widely used adult stem cells with self-renewal and multilineage differentiation capacity, which plays an important role in stem cell therapy due to clinical effectiveness and safety assessment. During the process of long term culture in vitro, the defect of p BMSC will gradually appear, such as senescence, decreasment of differentiation potential and cell proliferation ability,in addition,the unstable effect of non customize treatment of stem cell therapy is the main restrict in the clinical application of stem cells. In order to achieve the long-term culture of p BMSC in vitro, and to apply in stem cell therapy with p BMSC after directional differentiation, the study researched p BMSC in various aspects through the culture system optimization, i PS induction and directional differentiation of it, estabished the foundation for p BMSC in the area of stem cell therapy and safety assessment before clinical application.The main contents and results of the study are as follows:1. We added small molecule compounds A83-01 and RG108 to optimize the culture condition of p BMSC, the results showd that:(1) Adding 1μM A83-01 continually in medium significantly increased the expression of pluripotency genes Oct4 and Nanog, increased telomerase activity and promoted cells proliferation; A83-01 increased the amount of expression of anti-apoptosis genes, decreased the expression of apoptosis genes, and protected cells against the stimulating of apoptotic; A83-01 can also improve the efficiency of p BMSC adipogenic differentiation but there is no significant effection on osteogenic differentiation.(2) Treating p BMSC with 10μM RG108 after 48 hs could significantly increased the level of expression of pluripotency genes Oct4 and Nanog, increased telomerase activity and promoted cell proliferation; RG108 increased the expression of anti-apoptosis genes, decrease the expression of apoptosis genes, and protect cells against the stimulating of apoptotic; decreased the levels of methylation of the promoters of pluripotent gene significantly and increase the efficiency of adipogenic differentiation and osteogenic differentiation.2. The study investigated the regulation mechanism of p BMSC induced to i PS by comparing with the efficiency of i PS induction, reprogramming in pluripotency genes and the changes of DNA methylation. The results showed that:(1) Adding 1μM A83-01 in culture medium can increase the number of AP positive clones, the i PS induced efficiency increased from 0.052% to 0.0624% by statistical analysis, but there was little effect on the activation of expression of pluripotency genes during the induction;(2) p BMSC treated by 0μM RG108 after 48 hs can significantly improve the efficiency of reprogramming to 0.924%, the induction period was shortened from 10 days to 6 days and expression of pluripotency genes increased significantly during this process, p BMSC treated with RG108 can significantly decreased the level of the DNA methylation compared with the control group in the same period.3. This study investigated the role of mi R-17/21/143 during the process of adipogenic differentiation and the regulation mechanism of mi R-375 during IPC differentiation induction. The results showed that:(1) Overexpression of mimics of mi R-17, mi R-21, and mi R-143 could increase adipocyte differentiation totally; Inhibition of mi R-17 had no obvious effects on adipogenic differentiation, but the inhibition of mi R-21/143 could significantly decrease the expression of adipogenic gene and the number of oil red O-positive cells during the adipocyte differentiation; the group of combined three mi RNAs has the most effectiveness in enhancing the adipogenic differentiation.(2) Overexpression of mi R-375 by lentiviral vector could induce p BMSC to DTZ positive clones efficiently; The expression of Oct4 gene was reduced significantly during the process; the expression of SOX17 increased at D5 and then decreased after that; the expression of IPC marker genes were increased significantly; The IPC have the ability of secreting insulin and C peptide with glucose stimulation.The study, through optimized the culture system, inducted i PS and mi RNA mediated directional differentiation of p BMSC, provided theoretical and experimental basis for the superiority of p BMSC in cell reprogramming, regulation mechanism of i PS induction and mi RNA mediated directional differentiation. Estabished the foundation for p BMSC in the area of stem cell therapy and safety assessment before clinical application.