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In vitro expansion of human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived PGC model, has proved challenging due to rapid loss of primordial germ cell (PGC)-like identity and limited cell survival/proliferation. Here, we describe long-term culture hPGCLCs (LTC-hPGCLCs), which actively proliferate in a serum-free, feeder-free condition without apparent limit as highly homogeneous diploid cell populations maintaining transcriptomic and epigenomic characteristics of hPGCLCs. Histone proteomics confirmed reduced H3K9me2 and increased H3K27me3 marks in LTC-hPGCLCs compared with induced pluripotent stem cells (iPSCs). LTC-hPGCLCs established from multiple human iPSC clones of both sexes were telomerase positive, senescence-free cells readily passaged with minimal cell death or deviation from the PGC-like identity. LTC-hPGCLCs are capable of differentiating to DAZL-positive M-spermatogonia-like cells in the xenogeneic reconstituted testis (xrTestis) organ culture milieu as well as efficiently producing fully pluripotent embryonic germ cell-like cells in the presence of stem cell factor and fibroblast growth factor 2. Thus, LTC-hPGCLCs provide convenient access to unlimited amounts of high-quality and homogeneous hPGCLCs., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We recently reported a robust and defined culture system for the specification of human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs), both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro (Irie et al. Cell 160: 253-268, 2015). Similar attempts previously produced hPGCLCs from hPSCs at a very low efficiency, and the resulting cells were not fully characterized. A key step, which facilitated efficient hPGCLC specification from hPSCs, was the induction of a "competent" state for PGC fate via the medium containing a cocktail of four inhibitors. The competency of hPSCs can be maintained indefinitely and interchangeably with the conventional/low-competent hPSCs. Specification of hPGCLC occurs following sequential expression of key germ cell fate regulators, notably SOX17 and BLIMP1, as well as initiation of epigenetic resetting over 5 days. The hPGCLCs can be isolated using specific cell surface markers without the need for generating germ cell-specific reporter hPSC lines. This powerful method for the induction and isolation of hPGCLCs can be applied to both hESCs and iPSCs, which can be used for advances in human germ line biology.

Abstract In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms.

In vitro gametogenesis (IVG) has been a topic of great interest in recent years not only because it allows for further exploration of mechanisms of germ cell development, but also because of its prospect for innovative medical applications especially for the treatment of infertility. Elucidation of the mechanisms underlying gamete development in vivo has inspired scientists to attempt to recapitulate the entire process of gametogenesis in vitro. While earlier studies have established IVG methods largely using pluripotent stem cells of embryonic origin, the scarcity of sources for these cells and the ethical issues involved in their use are serious limitations to the progress of IVG research especially in humans. However, with the emergence of induced pluripotent stem cells (iPSCs) due to the revolutionary discovery of dedifferentiation and reprogramming factors, IVG research has progressed remarkably in the last decade. This paper extensively reviews developments in IVG using iPSCs. First, the paper presents key concepts from groundwork studies on IVG including earlier researches demonstrating that IVG methods using embryonic stem cells (ESCs) also apply when using iPSCs. Techniques for the derivation of iPSCs are briefly discussed, highlighting the importance of generating transgene-free iPSCs with a high capacity for germline transmission to improve efficacy when used for IVG. The main part of the paper discusses recent advances in IVG research using iPSCs in various stages of gametogenesis. In addition, current clinical applications of IVG are presented, and potential future applications are discussed. Although IVG is still faced with many challenges in terms of technical issues, as well as efficacy and safety, novel IVG methodologies are emerging, and IVG using iPSCs may usher in the next era of reproductive medicine sooner than expected. This raises both ethical and social concerns and calls for the scientific community to cautiously develop IVG technology to ensure it is not only efficacious but also safe and adheres to social and ethical norms. [ABSTRACT FROM AUTHOR]