Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Keywords: in vitro, primordial germ cells, embryonic stem cells, stella
Project description:In this study, we developed a feeder-, serum-, and animal product-free culture condition, and successfully induced high percentage of SLCs from human PSCs. Genome-wide expression analyses demonstrated a significant upregulation of germ cell specific genes in SLCs derived from PSCs compared to in vivo isolated human CD90+ SSCs. And furthermore, under this optimized feeder-free condition SLCs have went through meiosis and formed putative round spermatids. Our data demonstrated a robust feeder-, serum- and xeno-free protocol to induce differentiation of PSCs to SLCs from which putative haploid spermatids may develop.
Project description:Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.
Project description:Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.
Project description:Sus domesticus (pig) are an excellent large mammalian model for comparative studiesdue to their relatively comparable physiology and organ size to humans. The derivation of transgene-free porcine pluripotent stem cells (PiPSCs) will therefore benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. Established PiPSCs can robustly differentiate into derivates representing the primary germ layers in vitro and in vivo. Furthermore, the transgene- free PiPSCs preserve intrinsic species-specific developmental timing in culture. This capacity is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ~ 3.7 hours, a time scale recapitulating in vivo porcine somitogenesis. We therefore propose that these transgene-free PiPSCs can serve as a powerful tool for modeling development and disease, informing both conserved and unique features of mammalian pluripotency and developmental timing mechanisms.