Abstract
To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs’ gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs’ epigenetic memory.
Highlights
Embryonic stem cells (ESCs) hold great promise for therapeutic and regenerative medicine applications due to their inherent ability to produce tissue from all three germ layers
To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells, we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors
G4 mouse Embryonic stem cells were pruchased from the Lunenfeld-Tanenbaum Research Institute [28], mouse fibroblast induced pluripotent stem cell line (FiPSC) was obtained from System Biosciences [29] and mouse granulosa cell derived-iPSCs were generated in our laboratory
Summary
Embryonic stem cells (ESCs) hold great promise for therapeutic and regenerative medicine applications due to their inherent ability to produce tissue from all three germ layers. A significant scientific hurdle to using ESCs or iPSCs in regenerative medicine is the paucity of information on the precise molecular signals necessary to direct differentiation into specific tissues Despite their overall similarity to ESCs, accumulating evidence suggests that iPSC lines vary in their capacity to produce certain tissue types upon spontaneous differentiation [5,6,7,8]. This restriction is in part related to reprogrammed cells’ epigenetic signature, which may be defined as a mechanism by which cells retain a functional memory of their originating identity throughout cell divisions [9,10,11,12]. With these techniques we test our hypothesis that tissue-specific iPSCs favor homotypic differentiation, reverting preferentially to their originating cell type
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