In vitro differentiation of male germline stem cell-like cells into mature sperm and their effect on in vivo spermatogenesis restoration

Germline stem cell (GSC) is a unique cell type that produces more stem cells through self-renewal or generating different stage/subtype stem cells during germline development and differentiates into spermatozoa and ova to produce offspring. Spermatogonial stem cells (SSCs)/GSCs obtained from testicular biopsy or primordial germ cell-like cells (PGCLCs) derived from iPSCs obtained from adult tissues can be used for autologous transplantation to restore spermatogenesis or for in vitro spermatogenesis in adult men with non-obstructive azoospermia due to genetic diseases such as Klinefelter syndrome or other causes, and in prepubertal boys diagnosed with cancer who will receive radiotherapy/chemotherapy. Patients carrying gene mutations can have their SSC or PGHBH corrected prior to transplantation using CRISPR/CAS9 technology. However, although the contribution of iPSCs to the field of in vitro gametogenesis cannot be underestimated, there are concerns regarding their safety in terms of epigenetic modifications associated with reprogramming. In this study, we showed for the first time that CD49f+ GSCs can transform into GSCLCs that show a significant increase in the expression of pluripotency markers and alkaline phosphatase activity in the presence of GDNF, EGF, bFGF, IGF2, SCF and mLIF; notably, those GSCLC colonies could be propagated for more than two months on >95% GATA4+ testicular somatic cell (TSC) feeders. AP+CD49f+ GKHBHs completed meiosis in vitro and differentiated into sperm-like cells with tail development when TGF-β/Activin/Nodal pathway inhibitor, SB431542 followed by retinoic acid were introduced. Although leptotene spermatocytes were observed after the SB431542 pre-treatment, upon the combined effect of retinoic acid, a heterogeneous population of pachytene and diplotene spermatocytes and sperm-like cells in more advanced meiosis were detected. The resulting sperm-like cells were able to induce blastocyst formation after intracytoplasmic injection. After co-transplantation of GSCLCs and Sertoli cell-rich TSCs into germ cell ablated mouse testes, DDX4+SYCP3+ cells per tubule were higher than those transplanted with TSCs alone. TSCs were able to restore endogenous spermatogenesis by reforming the impaired niche, whereas exogenous GSCLCs were able to undergo successful spermatogenesis in the presence of healthy niche cells. Overall, our approach suggests that under defined culture conditions, GSCs can be expanded in number without losing their function and viablity and our GSC-GSCLC conversion method can upregulate the expression of Oct4a, Sox2, Nanog, SSEA-1 pluripotency markers, GSCLCs can undergo stepwise differentiation into functional sperm-like cells in vitro and can also reinitiate spermatogenesis in vivo with somatic niche cell support.