Abstract: PD08-03
Sources of Funding: This work was supported by HHMI.

Introduction

Male fertility relies on the proper regulation of human adult spermatogonial stem cells (SSCs), which either self-renew or commit to unipotent differentiation (spermatogenesis). The future of male fertility lies in culturing human SSCs, and is currently the core limitation of the field. Mouse SSCs can be cultured, manipulated, and transplanted back into the testis. However, the conditions used to culture mouse SSCs fail to support the growth and germline identity of human SSCs. To better understand human SSC self-renewal and differentiation, and to enable long-term culturing, we aimed to conduct molecular/genomic profiling of human SSCs.

Methods

First, we derived methods to purify the self-renewing human SSC (which bear the surface marker SSEA4), and differentiating human spermatogonia (which bear KIT). We profiled RNA/transcription (via bulk and single-cell RNA sequencing), DNA methylation (via whole-genome bisulfite sequencing) and determined the promoters and enhancers that have open chromatin (via ATAC-seq) in SSEA4-enriched SSCs. We also conducted RNA/transcription profiling of differentiating KIT+ spermatogonia to provide comparisons to SSEA4+ SSCs.

Results

Here, we successfully profiled RNA, chromatin and DNA methylation in human SSEA4+ SSCs, and RNA in KIT+ spermatogonia. We identified transcription factors and signaling pathway components that are unique to either SSEA4+ SSCs or KIT+ spermatogonia. Importantly, several factors including receptor/ligand systems and transcription factors appear unique to humans (not utilized in the mouse), results of which inform the biology of human SSC self-renewal and differentiation, and should inform and guide human SSC cell culturing. Remarkably, we find the core pluripotentcy genes (e.g. OCT4, SOX2, NANOG) repressed in SSCs, with portion apparently poised for activation in SSCs by open chromatin, creating a condition of ‘latent’ pluripotency likely to impact their developmental potential.

Conclusions

Human SSCs have been extensively profiled, revealing signaling and transcription factor pathways of likely importance. Latent pluripotency in SSCs may enable rapid activation of pluripotency following fertilization, but may also create susceptibiliy to forming germ cell tumors – enabling their plasticity to form tumors with all three germ layers. Overall, our findings provide insights into understanding unipotency and pluripotency in human SSCs, possible mechanisms for germ cell tumor formation, and guidance for in vitro culturing of human SSCs.

Funding

This work was supported by HHMI.