Generation of male germ cells in vitro from the stem cells

Infertility has become a serious disease since it affects 10%–15%of couples worldwide,and male infertility contributes to about 50%of the cases.Notably,a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China.As such,it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility.Stem cells,including embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs),spermatogonial stem cells(SSCs),and mesenchymal stem cells(MSCs),possess the abilities of both self-renewal and differentiation into germ cells.Significantly,much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions,e.g.,the coculturing with Sertoli cells,three-dimensional culture system,the addition of growth factors and cytokines,and/or the overexpression of germ cell-related genes.In this review,we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us,and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis

Spermatogonial stem cells(SSCs)have great applications in both reproductive and regenerative medicine.Primates including monkeys are very similar to humans with regard to physiology and pathology.Nevertheless,little is known about the isolation,the characteristics,and the culture of primate SSCs.This study was designed to identify,isolate,and culture monkey SSCs.Immunocytochemistry was used to identify markers for monkey SSCs.Glial cell line-derived neurotrophic factor family receptor alpha-1(GFRAl)-enriched spermatogonia were isolated from monkeys,namely Macaca fascicularis(M.fascicularis),by two-step enzymatic digestion and magnetic-activated cell sorting,and they were cultured on precoated plates in the conditioned medium.Reverse transcription-polymerase chain reaction(RT-PCR),immunocytochemistry,and RNA sequencing were used to compare phenotype and transcriptomes in GFRAl-enriched spermatogonia between 0 day and 14 days of culture,and xenotransplantation was performed to evaluate the function of GFRAl-enriched spermatogonia.SSCs shared some phenotypes with rodent and human SSCs.GFRAl-enriched spermatogonia with high purity and viability were isolated from M.fascicularis testes.The freshly isolated cells expressed numerous markers for rodent SSCs,and they were cultured for 14 days.The expression of numerous SSC markers was maintained during the cultivation of GFRAl-enriched spermatogonia.RNA sequencing reflected a 97.3%similarity in global gene profiles between 0 day and 14 days of culture.The xenotransplantation assay indicated that the GFRAl-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kitw/w(W)mutant mice.Collectively,GFRAl-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo.This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.