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Human testis development in prenatal life involves complex changes in germline and somatic cell identity. To better understand, we profiled and analyzed ∼32,500 single-cell transcriptomes of testicular cells from embryonic, fetal, and infant stages. Our data show that at 6-7 weeks postfertilization, as the testicular cords are established, the Sertoli and interstitial cells originate from a common heterogeneous progenitor pool, which then resolves into fetal Sertoli cells (expressing tube-forming genes) or interstitial cells (including Leydig-lineage cells expressing steroidogenesis genes). Almost 10 weeks later, beginning at 14-16 weeks postfertilization, the male primordial germ cells exit mitosis, downregulate pluripotent transcription factors, and transition into cells that strongly resemble the state 0 spermatogonia originally defined in the infant and adult testes. Therefore, we called these fetal spermatogonia "state f0." Overall, we reveal multiple insights into the coordinated and temporal development of the embryonic, fetal, and postnatal male germline together with the somatic niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.

The rhesus macaque induced pluripotent stem cell (riPSC) line, UCLAi090-A (riPSC90), was generated from rhesus embryonic fibroblast (REF) cells called REF90. REF90 cells and the riPSC90 line were authenticated by short tandem repeat analysis and had a normal male (42, XY) karyotype. The riPSC90 line expressed markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA4, and generated teratomas after transplantation into immunocompromised mice. riPSC90 could be used in parallel with riPSC89, which was derived from REFs cultured from a different rhesus macaque embryo (Sosa et al. 2016).

We generated a rhesus macaque induced pluripotent stem cell (riPSC) line, riPSC89, from rhesus embryonic fibroblasts (REFs). Fibroblasts were expanded from the skin of a rhesus macaque embryo at embryonic day 47. REFs and riPSCs had a normal male (42, XY) karyotype. The riPSC89 line was positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA4. Pluripotency was demonstrated through the generation of teratomas using transplantation into immunocompromised mice. The riPSC89 line may be a useful non-human primate resource to uncover developmental origins of disease, or used as a basic model to understand lineage specification in the primate embryo.