Derivation and propagation of spermatogonial stem cells from human pluripotent cells

This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs).

hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes.

SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125⁺ spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules.

SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125⁺ spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.