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

Cell Stem Cell. 2017 Oct 5;21(4):533-546.e6. doi: 10.1016/j.stem.2017.09.003.


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.

Keywords: DNA methylation; hormone receptors; human spermatogonial stem cells; metabolism; open chromatin; pluripotency; single-cell RNA-seq; spermatogenesis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Chromatin / metabolism*
  • Cluster Analysis
  • DNA / metabolism
  • DNA Methylation / genetics
  • Genomics
  • Humans
  • Male
  • Meiosis
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism
  • Receptors, Cell Surface / metabolism
  • Repetitive Sequences, Nucleic Acid / genetics
  • Reproducibility of Results
  • Seminiferous Tubules / cytology
  • Sequence Analysis, RNA / methods*
  • Signal Transduction*
  • Single-Cell Analysis / methods*
  • Spermatogonia / cytology*
  • Stage-Specific Embryonic Antigens / metabolism
  • Stem Cells / cytology*
  • Stem Cells / metabolism*
  • Transcription, Genetic
  • Transcriptome / genetics


  • Chromatin
  • Receptors, Cell Surface
  • Stage-Specific Embryonic Antigens
  • stage-specific embryonic antigen-4
  • DNA