Ovarian granulosa cells are a viable source of iPSCs with the capacity for epigenetically-biased differentiation into steroidogenic tissue of the ovary

ObjectiveTo test the hypothesis that induced pluripotent stem cells (iPSCs) derived from mouse ovarian granulosa cells (GCs) retain an ovarian epigenetic memory that favors differentiation into homotypic ovarian cell types.DesignBasic Science.Materials and MethodsMouse ovarian GCs were reprogrammed into iPSCs (mGriPSCs) using the retrovirally introduced pluripotency genes Sox2, cMyc, Klf4 and Oct4. Putative iPSC colonies were characterized to confirm stem cell identity using immunohistochemistry (IHC), alkaline phosphatase staining and in vitro differentiation assays. mGriPSCs and mouse embryonic stem cells (mESCs) were differentiated into embryoid bodies (EBs) to generate steroidogenic cells. EBs underwent IHC, chromosome and gene expression microarray, and micro-RNA analysis. Hormone synthesis was assessed by radioimmunoassay.ResultsOvarian GCs are successfully reprogrammed into iPSCs. mGriPSC-derived EBs preferentially produce estradiol compared to mESC-derived EBs (359 vs 55 pg/mL; p<0.02) while mESC-derived EBs produce more progesterone (783 vs. 30 pg/mL; p<0.01). IHC shows mGriPSCs are 1.3-2.8X more likely than mESCs to differentiate into cells expressing the oocyte antigens GDF9, BOULE, DAZL, and MVH (p<0.001-0.06). Gene expression microarrays reveal concomitant upregulation in steroidogenic genes paralleling functional differentiation. Micro-RNA profiling shows close association of mGriPSCs with the originating GCs. As typically observed with iPSC lines, chromosome arrays demonstrate some genomic instability, likely associated with retroviral integration.ConclusionMouse ovarian GCs are a viable source for generating iPSCs. Our results confirm that tissue-specific iPSCs retain an epigenetic memory favoring spontaneous differentiation into homotypic cell types. Analysis of epigenetic signatures may prove advantageous in directed differentiation of iPSCs into functional cell types for regenerative therapies. ObjectiveTo test the hypothesis that induced pluripotent stem cells (iPSCs) derived from mouse ovarian granulosa cells (GCs) retain an ovarian epigenetic memory that favors differentiation into homotypic ovarian cell types. To test the hypothesis that induced pluripotent stem cells (iPSCs) derived from mouse ovarian granulosa cells (GCs) retain an ovarian epigenetic memory that favors differentiation into homotypic ovarian cell types. DesignBasic Science. Basic Science. Materials and MethodsMouse ovarian GCs were reprogrammed into iPSCs (mGriPSCs) using the retrovirally introduced pluripotency genes Sox2, cMyc, Klf4 and Oct4. Putative iPSC colonies were characterized to confirm stem cell identity using immunohistochemistry (IHC), alkaline phosphatase staining and in vitro differentiation assays. mGriPSCs and mouse embryonic stem cells (mESCs) were differentiated into embryoid bodies (EBs) to generate steroidogenic cells. EBs underwent IHC, chromosome and gene expression microarray, and micro-RNA analysis. Hormone synthesis was assessed by radioimmunoassay. Mouse ovarian GCs were reprogrammed into iPSCs (mGriPSCs) using the retrovirally introduced pluripotency genes Sox2, cMyc, Klf4 and Oct4. Putative iPSC colonies were characterized to confirm stem cell identity using immunohistochemistry (IHC), alkaline phosphatase staining and in vitro differentiation assays. mGriPSCs and mouse embryonic stem cells (mESCs) were differentiated into embryoid bodies (EBs) to generate steroidogenic cells. EBs underwent IHC, chromosome and gene expression microarray, and micro-RNA analysis. Hormone synthesis was assessed by radioimmunoassay. ResultsOvarian GCs are successfully reprogrammed into iPSCs. mGriPSC-derived EBs preferentially produce estradiol compared to mESC-derived EBs (359 vs 55 pg/mL; p<0.02) while mESC-derived EBs produce more progesterone (783 vs. 30 pg/mL; p<0.01). IHC shows mGriPSCs are 1.3-2.8X more likely than mESCs to differentiate into cells expressing the oocyte antigens GDF9, BOULE, DAZL, and MVH (p<0.001-0.06). Gene expression microarrays reveal concomitant upregulation in steroidogenic genes paralleling functional differentiation. Micro-RNA profiling shows close association of mGriPSCs with the originating GCs. As typically observed with iPSC lines, chromosome arrays demonstrate some genomic instability, likely associated with retroviral integration. Ovarian GCs are successfully reprogrammed into iPSCs. mGriPSC-derived EBs preferentially produce estradiol compared to mESC-derived EBs (359 vs 55 pg/mL; p<0.02) while mESC-derived EBs produce more progesterone (783 vs. 30 pg/mL; p<0.01). IHC shows mGriPSCs are 1.3-2.8X more likely than mESCs to differentiate into cells expressing the oocyte antigens GDF9, BOULE, DAZL, and MVH (p<0.001-0.06). Gene expression microarrays reveal concomitant upregulation in steroidogenic genes paralleling functional differentiation. Micro-RNA profiling shows close association of mGriPSCs with the originating GCs. As typically observed with iPSC lines, chromosome arrays demonstrate some genomic instability, likely associated with retroviral integration. ConclusionMouse ovarian GCs are a viable source for generating iPSCs. Our results confirm that tissue-specific iPSCs retain an epigenetic memory favoring spontaneous differentiation into homotypic cell types. Analysis of epigenetic signatures may prove advantageous in directed differentiation of iPSCs into functional cell types for regenerative therapies. Mouse ovarian GCs are a viable source for generating iPSCs. Our results confirm that tissue-specific iPSCs retain an epigenetic memory favoring spontaneous differentiation into homotypic cell types. Analysis of epigenetic signatures may prove advantageous in directed differentiation of iPSCs into functional cell types for regenerative therapies.