RESUMO
Male germ-cell development comprises primordial germ-cell (PGC) development, spermatogonium differentiation, and ensuing spermatogenesis. We present a step-by-step protocol for differentiation of mouse pluripotent stem cells (PSCs) into germline stem-cell-like cells (GSCLCs) via PGC-like cell and spermatogonium-like cell intermediates. The differentiation protocol has higher fidelity than our previous protocol. Upon transplantation into testes in vivo or culture for testis transplants, GSCLCs robustly contribute to spermatogenesis, providing a paradigm for PSC-based reconstitution of mammalian male germ-cell development. For complete details on the use and execution of this protocol, please refer to Ishikura et al. (2021).
Assuntos
Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Masculino , Mamíferos , Camundongos , Espermatogênese , Espermatogônias , TestículoRESUMO
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.