In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells

Abstract

The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation.

Bone morphogenetic protein and retinoic acid synergistically specify female germ‐cell fate in mice

“…This would also explain why early PGCs, which are specified by BMP signaling, are not directly induced into the meiotic program. In further support of this notion, we found that in the male pathway, KIT (+) spermatogonia at postnatal day 7 (P7), a precursor for meiosis in the first wave of spermatogenesis, exhibited low/no 5mC levels in the promoters of the relevant 42 genes, despite the fact that such cells re‐acquired high global 5mC levels during their development (Appendix Fig S2B) (Kubo et al , ; Ishikura et al , ). Interestingly, these genes exhibited significant demethylation during the transition from KIT (−) spermatogonia at P7 (Appendix Fig S2B) (Kubo et al , ).…”

“…E14.5 and E15.5 male and female germ cells were collected as VR (+) cells and cultured PGCLCs as SC (+) cells by FACS, and total RNAs of the samples were extracted and purified using an RNeasy Micro Kit (74004; QIAGEN) according to the manufacturer's instructions. The cDNAs synthesis, library construction, and analysis by Nextseq500 (Illumina) from 1 ng of total RNAs from each sample were performed according to the methods described previously (Nakamura et al , ; Ishikura et al , ). The cDNAs of germ cells from E9.5 to E13.5 prepared in previous studies (Kagiwada et al , ; Ohta et al , ) were also analyzed by the Nextseq500 sequencer system.…”

“…Whole‐genome bisulfite sequencing (WGBS) data of ESCs, EpiLCs, and PGCLCs were obtained from our previous studies (Shirane et al , ; Ohta et al , ), and those of KIT (−) and KIT (+) spermatogonia were obtained from a previous study (Kubo et al , ). We here defined promoters as regions between 900 bp upstream and 400 bp downstream of the transcription start sites (TSSs), and all the percent 5‐methylcytosine values (% 5mC) were calculated from the average of CpGs, in which the read depth was between 4 and 200, as previously described (Ishikura et al , ). We used the genes with promoters bearing at least one CpG site for the analysis in this study.…”

“…On the other hand, it has been shown that mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) are induced, by activin A (ACTA) and basic fibroblast growth factor (bFGF), into epiblast‐like cells (EpiLCs), which are in turn induced, essentially by BMP4, into PGC‐like cells (PGCLCs) with characteristics of migrating PGCs. Importantly, PGCLCs bear a robust capacity both for spermatogenesis and oogenesis, upon transplantation or aggregation with gonadal somatic cells followed by appropriate culture (Hayashi et al , , ; Hikabe et al , ; Ishikura et al , ; Saitou & Miyauchi, ). More recently, a system was developed which allows PGCLCs to be propagated without gonadal somatic cells for at least 1 week, creating an opportunity to reconstitute the sex‐determination pathway of germ cells under a defined condition in vitro (Ohta et al , ).…”

Male germline stem cells in non-human primates

“…Mouse induced pluripotent stem cells (iPSCs) can be reprogrammed into PGC-like cells. These in vitro generated cells can be further developed into oocyte-like or haploid male gametes with fertilization competence once an in vivo gonadal environment is provided (Zhou et al, 2016;Ishikura et al, 2016).…”