| Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotentstem cells (iPSCs) have a great potential for use in cell-based therapies. However,their clinical applications are limited because of ethical issues and safety. Therefore,new approaches need to be developed for pluripotent cell generation. Previous studieshave demonstrated that spermatogonial stem cells (SSCs) from newborn, adult mouseand human testis tissue can be converted into embryonic stem cell (ESC)-like cellsunder specific in vitro conditions without genetic manipulations. This methodovercomes the problems associated with the therapeutic use of ESCs and iPSCs byproviding cells that are non-embryonic, patient-specific, and free from geneticmodifications.Our previous studies have shown that female germline stem cells (FGSCs) exist inpostnatal (neonatal or adult) mouse ovarian tissues. The isolation from postnatal miceand long-term culture has gained a great deal of interest in stem cell biology andreproduction biology. Recently, Tilly’s group from Massachusetts General Hospitalhave followed our work using fluorescence-activated cell sorting (FACS) to isolateand culture similar mouse FGSCs, and then extended our work by getting humanFGSCs from20to30years old woman ovarian tissues. Based on previous researchand the experience during our experiment, we found that the shape, growth pattern, and function of FGSCs during gametogenesis are similar to those of SSCs. Althoughthe pluripotent stem cells converted from SSCs has gained interest in stem cellresearch, whether FGSCs can be converted into pluripotent stem cells similar to thatof spermatogonial stem cells is unknown. Thus, we hypothesized that FGSCs can alsobe converted into pluripotent stem cells using the method for SSCs conversion.In the present study, the newly isolated and purified FGSCs from neonatal andprepubertal mouse ovaries have similar characteristic such as shape, growth pattern,and gene expression profile in vitro culture. The female embryonic stem-like cells(fESLCs) were generated from neonatal and prepubertal mouse FGSCs with stablyproliferating ability under ESC culture conditions. Valproic acid and vitamine Cshowed a synergistic effect on the fESLCs colony formation and growth undersuitable initial number of seeded FGSCs.The Nanog promoter regions were changed from methylation to unmethylationduring the fESLCs generation. fESLCs exhibited a strong alkaline phosphataseactivity than FGSCs, and have properties similar to those of ESCs in terms of marker(Oct4、Nanog、Sox2、Utf1、Esg1、Rex1and SSEA-1) expression. Both FGSCs andfESLCs had a normal karyotype. During the FGSCs conversion into fESLCs, therewere no obvious changes in Peg10and Igf2r methylation pattern, while paternallyimprinted regions (H19and Meg3IG) showed partial methylation in fESLCs.Embryoid bodies from fESLCs were formed under differentiation culture condition,and pluripotency-associated gene expression decreased during the differentiationperiod. They are also able to differentiate into derivatives of all three embryonic germ layers in vitro, have teratoma formation ability in immunodeficient mouse. Wheninjected into the3.5dpc blastocyst, fESLCs contribute to the development of variouscells, tissues or organs and have germline chimeric ability.Thus, our findings suggest that FGSCs converted into pluripotent stem cells in vitrousing the method for SSCs conversion is feasible, and this research will provide afoundation for personalized regenerative applications based on pluripotent stem cellsin future. Moreover, FGSCs conversion can be used as research models for theconversion involved in pluripotency acquisition. This will help us to determine theunderlying mechanisms of pluripotency and why germline cells can be converted to apluripotent state by culture conditions alone, whereas somatic cells require exogenousexpression of pluripotency-related factors. |
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