The Research Of Differentiation Of Rat IPS Cells And Es Cells Into Granulosa Cell-like Cells In Vitro

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles before40years of age, representing one major cause of female infertility. The women with POF will have Premature Menopause, high level gonadotrophic hormone and hypoestrogenemia. The etiology of POF is still unclear, but some factor maybe induce the development of POF, including heredity, enzyme deficiency, iatrogenic and immunity. For this reason, the therapy of POF is direction therapy, including hormone replacement therapy, assisted reproductive technology and cell therapy of stem cell. The stem cell therapy bring us the useful strategy to treat POF and decrease the Clinical symptoms of descend of hormone. Stem cell therapy is still not used in clinic, because of the technical disturbance of ethics and immunologic rejection. The iPS cells is a new method to obtain the stem cell from somatic cell of patient, and it can overcome the disturbance of embryonic stem cell.This research is divided into two parts:First, the establishment of rat embryonic stem cell from inner cell mass of blastaea and rat Induced pluripotent stem cells; Second, Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitro and the expression level of candidate gene of POF in these two induced granulosa cell-like cells. Experimental methods and results were as follows: Part1The establishment of rat embryonic stem cells and rat Induced pluripotent stem cellsObjective:To establish rat embryonic stem cells and rat Induced pluripotent stem cells.Methods:The method of culture of inner cell mass of blastaea is used to establish rat embryonic stem cells; The introduction of six transcription factor s into somatic cell with lentivirus vctor is used to establish rat Induced pluripotent stem cells; Identification of the characteristics of stem cell, including alkaline phosphatase activities, SSEA-1marker expression, Nanog marker expression, chromosome karyotype analysis, teratoma and expression of undifferentiation marker gene (Oct4、Sox2、Nanog、Nodal、Fgf4、Gal、Leftyb、Lin28、Gabra).Results:1. The rat embryonic stem cells is successfully established; The positive result is observed; The embryonic stem cells express the stem cell marker SSEA-1and Nanog; The normal rat karyotype is maintained in the procedure of passage of embryonic stem cells; The embryonic stem cells have the potency of differentiation to ectoderm, mesoderm and endoderm; The undifferentiation marker genes (Oct4、Sox2、Nanog、 Nodal、Fgf4、Gal、Leftyb、Lin28、Gabra) are significantly high expressed in this embryonic stem cell line. 2. The induced pluripotent stem cells is successfully established; The positive result is observed; The iPS cells express the stem cell marker SSEA-land Nanog; The normal rat karyotype is maintained in the procedure of passage of iPS cells; The iPS cells have the potency of differentiation to ectoderm, mesoderm and endoderm; The undifferentiation marker genes (Oct4、Sox2、Nanog、Nodal、Fgf4、 Gal、Leftyb、Lin28、Gabra) are significantly high expressed in this embryonic stem cell line.Conclusions:The establishment of rat embryonic stem cells and rat Induced pluripotent stem cells can be supply the fine material for the research. Part2Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitroObjective:To induce rat embryonic stem cells and rat Induced pluripotent stem cells to differentiate into granulosa cell-like cells and investigate the function of induced cells.Methods:The method of co-culture with granulosa cell induce differentiation of rat embryonic stem cells and rat Induced pluripotent stem cells into granulosa cell-like cells; E2Hormone assay is performed to analysis the secretion function of induced granulosa cell-like cells; Immunostaining is used to analysis the expression of FSHR; Real-time PCR is applied to analysis the expression level of candidate genes of POF, including BMP15, FMR1, FSHR, INHA, AMH, NOBOX, FOXO3, EIF2B, FIGLA and GDF9.Results:1. The induced cells from rat embryonic stem cells are like granulosa cell in morphology; Immunostaining results show the expression of FSHR which is the marker of granulosa cell on membrane of induced cells; E2Hormone assay certify that induced cells from rat embryonic stem cells can secrete E2.2. The induced cells from rat induced pluripotent stem cells are like granulosa cell in morphology; Immunostaining results show the expression of FSHR which is the marker of granulosa cell on membrane of induced cells; E2Hormone assay certify that induced cells from rat induced pluripotent stem cells can secrete E2.3. Real-time PCR was performed to analyze the expression levels of the BMP15, FMR1, INHA, FSHR, AMH, NOBOX, FOXO3, EIF2B, FIGLA, and GDF9genes in iPSCs and ESCs after induction. The BMP15, INHA, FSHR, AMH, NOBOX, and GDF9genes were significantly up-regulated in both rat iPSCs and ESCs following co-culture with GCs in comparison to cells prior to co-culture. The FMR1gene was up-regulated in co-cultured iPSCs, but not changed in co-cultured ESCs. Additionally, the EIF2B and FOXO3genes were significantly up-regulated in co-cultured iPSCs and weakly up-regulated in co-cultured ESCs. The FIGLA gene was up-regulated in co-cultured ESCs but down-regulated in co-cultured iPSCs.Conclusions:1. Rat embryonic stem cells are successfully induced into granulosa cell-like cells and the induced cells have the function of granulosa cell.2. Rat induced pluripotent stem cells are successfully induced into granulosa cell-like cells and the induced cells have the function of granulosa cell.3. In the candidate genes of POF, BMP15, INHA, FSHR, AMH, NOBOX and GDF9genes have the same high expression level trend in both rat embryonic stem cells and rat Induced pluripotent stem cells. The results indicate these genes maybe play the more important role in the procedure of POF. It can provide the useful clue for the research.