Chromatin and Single-Cell RNA-Seq Profiling Reveal Dynamic Signaling and Metabolic Transitions during Human Spermatogonial Stem Cell Development

Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4⁺ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4⁺ hSSCs and differentiating c-KIT⁺ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic “poising” in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation. Cairns and colleagues show that human spermatogonial stem cells (hSSCs) bear unique DNA methylation and open chromatin landscapes, which may enable proper development, niche responsiveness, and “poised” pluripotency. Interestingly, single-cell transcriptome and immunofluorescence analyses reveal four cellular states, spanning from quiescent hSSCs to proliferating, metabolically active, differentiating spermatogonia.