Comparation Of Two Lines Of Different Originated Human 45,XO-induced Pluripotent Stem Cells And Their Neural Differentiation Study

Objective Turner Syndrome(TS)is a syndrome of sex chromosome's abnormality caused by the complete or partial deletion of the X chromosome.The pathogenesis of Turner Syndrome is unclear up to now.The iPS cells by the artificial mean over express embryonic stem cell,s main transcription factor genes,which is a kind of " embryoid stem cells" obtained by reprogramming adult cells,providing a new method for people to study the disease's pathogenesis.The objective of the research is to generate 45,XO induced pluripotent stem cell lines derived from two kinds of cells(villous cells and peripheral blood mononuclear cells),at the same time to research the 45,XO induced pluripotent stem cells' differentiation from totipotent cells to neural cells and to provide better cell models for the investigation of the pathogenesis of Turner syndrome and X chromosome's functions.Method1.The 45,XO villus cells and peripheral blood mononuclear cells were reprogrammed into induced pluripotent stem cells(iPS cells)by the Sendai virus,which were encoding human Oct4,Sox2,c-Myc and Klf4.2.Analyze the karyotype of induced pluripotent stem cells and identify the pluripotency and differentiation ability in vivo and in vitro by alkaline phosphatase staining,immunofluorescence staining,teratoma experiment and so on.3.Compare the generation efficiency of ips cells derived from two kinds of cells by comparing the appearance time of stem cell colonies and the number of colonies after reprogramming two kinds of cells.4.Induce ips cells' differentiation to neural stem cells in vitro,compare the positive rate and perform immunofluorescence staining.Add specific cytokines to neural stem cells to further induce neuronal differentiation and identify neurons by immunofluorescence staining.Results1.45,XO villus cells,morphology changes from the fifth day after infection and stem cell colonies appear on the fifteenth day.Stem cell colonies derived from 45,XO peripheral blood mononuclear cells appear on the nineth day after infection.Picked mechanically,these cells can be passaged in vitro stably for a long time and keep the 45,XO karyotype and self-renewal.2.The AKP staining of the two lines of 45,XO-i PSCs is positive.Immunofluorescence staining shows that SSEA-3,SSEA-4,Oct4 are positive.Induced pluripotent stem cells can form three germ layer-derived cells in the differentiation in vitro.The tissues and cells derived from three germ layers appear in teratoma,which forms by injecting subcutaneously clone cells into SCID mice.3.1X10~5 infected cells of the two types are respectively inoculated on the feeder cells.On the twenty-fifth day stain colonies by alkaline phosphatase.300 positive colonies are found in the ips cells derived from peripheral blood mononuclear cells and the efficiency is 0.300%.25 positive colonies are found in the ips cells derived from villus cells,with an efficiency of 0.025%.X2 test shows that P<0.05 and the efficiency's difference is statistically significant.4.Induce neural differentiation of 45,XO-i PSCs.The immunofluorescence staining of neural stem cells differentiated in vitro from 45,XO-i,PSCs and normal people iPSCs shows that Nestin is positive and there are no significant differences in the induction efficiency.The neural stem cells are continually induced to neuronal cell lines,and immunofluorescence staining shows that the expression of the surface markers of the neuronal cells(Tuj1 and Neuro)is positive.Conclusion1.We have generated two iPS cell lines derived from villus cells and peripheral blood mononuclear cells.The two iPS cell lines can maintain abnormal karyotype and stem cell's pluripotency in the long-time passage in vitro,which could be used as cell models of Turner syndrome;2.Comparing the efficiency of generating the two 45,XO-i PSCs,we find the peripheral blood cells,s efficiency is higher than that of villus cells.Peripheral blood cells may be the ideal cell type of generating 45,XO-i PSCs.3.The two lines of 45,XO-i PSCs have no significant differences with the normal line of i PSCs in the differentiation to neural stem cells.