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The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air–liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming. This protocol describes how to differentiate human induced pluripotent stem cells into oogonia in vitro. It is suitable for investigating the mechanisms of human primordial germ cell specification and epigenetic reprogramming.

Human pluripotent stem cells (hPSCs) have been induced into human primordial germ cell–like cells (hPGCLCs) in vitro, the first step toward human in vitro gametogenesis. Yamashiro et al. went a step closer to generating mature gametes by culturing hPSCs with mouse embryonic ovarian somatic cells in xenogeneic reconstituted ovaries (see the Perspective by Gill and Peters). Over a period of 4 months, hPGCLCs underwent hallmark epigenetic reprogramming and differentiated progressively into cells closely resembling human oogonia, an immediate embryonic precursor for human oocytes. This study creates opportunities for human germ cell research and provides a foundation for human in vitro gametogenesis. Science , this issue p. 356 ; see also p. 291 Human primordial germ cell–like cells differentiate into oogonia in xenogeneic reconstituted ovaries in vitro. Human in vitro gametogenesis may transform reproductive medicine. Human pluripotent stem cells (hPSCs) have been induced into primordial germ cell–like cells (hPGCLCs); however, further differentiation to a mature germ cell has not been achieved. Here, we show that hPGCLCs differentiate progressively into oogonia-like cells during a long-term in vitro culture (approximately 4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. The hPGCLC-derived oogonia display hallmarks of epigenetic reprogramming—genome-wide DNA demethylation, imprint erasure, and extinguishment of aberrant DNA methylation in hPSCs—and acquire an immediate precursory state for meiotic recombination. Furthermore, the inactive X chromosome shows a progressive demethylation and reactivation, albeit partially. These findings establish the germline competence of hPSCs and provide a critical step toward human in vitro gametogenesis.

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency 1 . In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >10 10 -fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells 2 , 3 , differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine. A new strategy that involves signalling-molecule-driven differentiation can induce epigenetic reprogramming of human pluripotent stem cell-derived primordial germ cell-like cells to pro-spermatogonia and oogonia-like cells with massive propagation and high efficiency.

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17 , TFAP2C , and BLIMP1 , which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2 , immediate BMP effectors, combined with SOX17 and TFAP2C , generated hPGCLCs. GATA3 / GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3 / GATA2 expression remained unaffected in SOX17 , TFAP2C , or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3 , SOX17 , and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis.

An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) to a mouse ear caused a transient skin swelling, and the repetition of the challenge enlarged the contact dermatitis. The repeated challenge with DNFB also induced eosinophil infiltration on the application site. Administration of a chymase inhibitor significantly inhibited the ear swelling as well as eosinophil accumulation. An intradermal injection of human chymase to the mouse ear also elicited transient skin swelling and eosinophil infiltration, both of which were augmented in proportion to the number of injections. Human serum albumin and heat-inactivated chymase failed to induce such skin reactions, suggesting the participation of proteolytic activity of the enzyme. In addition, chymase stimulated eosinophil migration in vitro in a concentration-dependent manner. Taken together, these observations suggest that mast cell chymase may contribute to development of the DNFB-induced dermatitis, probably by promoting eosinophil infiltration. It is therefore possible that chymase plays a role in pathogenesis of chronic dermatitis such as atopic dermatitis.