Reconstitution of sex determination and the testicular niche using mouse pluripotent stem cells.

Proper differentiation of gonadal somatic cells is crucial for sex determination and the production of sex hormones and gametes, and reconstituting this process in culture would both deepen our understanding of this process and enable the generation of gametes in vitro. Here, we report the reconstitution of testicular somatic cells using mouse pluripotent stem cells. The reconstitution recapitulated the sex-determination process, yielding cell types that formed seminiferous tubules and adjacent interstitial tissues. The reconstituted testicular tissue incorporated pluripotent stem cell–derived primordial germ cells and supported their differentiation into spermatogonial stem cells. These spermatogonial stem cells differentiated into functional spermatozoa upon transplantation into testis. This study contributes to a deeper understanding of the sex-determination process and to the creation of an alternative source for the male germ line in culture. Editor's summary: In vitro gametogenesis, the production of viable gametes from stem cells outside the body, could be beneficial in various settings ranging from human reproduction to helping with recovery of endangered species. Achieving this goal has been difficult, however, because it is not sufficient to generate primordial germ cells alone. Those germ cells need to be either placed in the appropriate reproductive organ in vivo or further cultured in the correct milieu in vitro to give rise to fully functional gametes. Yoshino et al. achieved this aim by reconstituting the appropriate combination of testicular somatic cells, yielding the appropriate conditions for sperm maturation in vitro. The authors confirmed that these sperm were fully functional by producing two generations of viable mouse pups (see the Perspective by Mitchell). —Yevgeniya Nusinovich INTRODUCTION: Sex determination in mammals depends on the differentiation of gonadal somatic cells, which create a sex-specific environment that directs germ cell fate and ensures fertility. Although genetic sex is fixed at fertilization, germ cells initially remain sexually undifferentiated and acquire male or female identity after interacting with the gonadal somatic cells. Reconstitution of this process in vitro would provide a powerful platform to study the mechanisms underlying sex determination and enable in vitro gametogenesis. However, despite advances in creating primordial germ cell–like cells (PGCLCs) from pluripotent stem cells, reconstruction of a functional gonadal somatic environment, particularly the testicular niche, has remained a major challenge. RATIONALE: In vivo, the bipotential gonad undergoes sex determination through a tightly regulated gene network initiated by the expression of Sry in XY embryos, leading to Sertoli cell differentiation and testis formation. This process is stabilized by antagonistic signaling pathways and paracrine interactions that suppress the female fate. We hypothesized that faithful reconstitution of testicular development in culture requires (i) generation of a bipotential gonadal state, (ii) delivery of appropriate sex-determining signals, and (iii) coordinated interactions between testicular somatic cells and primordial germ cells. To test this, we developed a pluripotent stem cell–based system that reconstructs gonadal sex determination and supports male germ cell differentiation in vitro. RESULTS: Using mouse embryonic stem cells harboring sex-specific reporters, Sox9-GFP (testis) and Foxl2-tdTomato (ovary), we established culture conditions that drive cells through a bipotential gonadal state into testicular somatic cell–like cells (TesLCs) in a Y chromosome–dependent manner. Modulation of bone morphogenetic protein (BMP) and WNT signaling induced SOX9-positive supporting cells exclusively from XY cells, accompanied by the formation of seminiferous tubule–like structures containing Sertoli- and interstitial-like cells. Single-cell transcriptome analyses revealed that the in vitro differentiation trajectory was closely reminiscent of the sex-determination process in vivo, with emergence of a male-specific population enriched for Sry, Sox9, Amh, and Dmrt1, genes that are important for Sertoli cell differentiation and/or function. When combined with PGCLCs, TesLCs formed testicular organoids that supported male germ cell differentiation. In these organoids, PGCLCs sequentially differentiated into prospermatogonia and spermatogonia and further progressed to spermatocytes that initiated meiosis. Germline stem cell–like cells (GSCLCs) were established from the organoids and propagated indefinitely in vitro. Upon transplantation into infertile recipient mice, GSCLCs reconstituted spermatogenesis and produced mature sperm capable of fertilization, leading to healthy and fertile offspring. The system also revealed intrinsic features of sex determination. Whereas differentiation of SOX9-positive cells strictly depended on the presence of a Y chromosome, FOXL2-positive cells could be generated from XY cells and supported oogenesis, underscoring a fundamental asymmetry between male and female sex-determination pathways. CONCLUSION: This study establishes a fully pluripotent stem cell–derived model of testicular development that reconstructs the sex-determination process and generates functional spermatogonial stem cells without the use of embryonic tissue. This platform enables mechanistic analyses of gonadal development, sex determination, and germ-somatic interactions and provides a foundation for extending germline reconstruction to other mammalian species, with broad implications for reproductive biology and medicine. Generation of testicular organoids.: Testicular cells (TesLCs) can be induced from pluripotent stem cells through the sex-determination process and assembled with pluripotent stem cell–derived PGCLCs to form testicular organoids, in which PGCLCs differentiate into functional spermatogonial stem cells capable of giving rise to mature sperm upon transplantation into recipient testes. At the bottom right is a fluorescence image of a testicular organoid. Y, Y chromosome. [ABSTRACT FROM AUTHOR]