| Description |
Human spermatogonial stem cells (SSCs) are the only stem cells in the male germline, which can either self-renew to replenish themselves, or differentiate to ensure continuous and life-long spermatogenesis. Studying human spermatogonial stem cell development and spermatogenesis is of great significance to understand the basis of male infertility and germline biology. However, due to the limitation of sample access and lack of genetic tools, the study of the human germline and SSCs is far behind that of the mouse. Here, we utilized multiple molecular approaches to study human SSCs and germline. First, we enriched for self-renewing SSCs (SSEA4+) and differentiating spermatogonia (KIT+) by cell sorting, and performed genomic profiling of their global DNA methylation status (via whole genome bisulfite sequencing), open chromatin status (via ATAC-seq), and transcriptome (via bulk and single-cell RNA sequencing). We found certain regions in the genome of SSCs devoid of chromatin and DNAme to allow quick pluripotency activation after fertilization, as well as hormone receptor and transcription factor binding. By analyzing the 172 single cells profiled, we identified four continuous cell states during SSC differentiation, accompanied by transcriptional, signaling, and metabolic transitions. Next, to further investigate additional stages of germline development and germline-niche interaction, we performed additional largescale single-cell RNA sequencing from unsorted testicular cells, which includes different iv types of germ cells and niche cells. With around 6,500 single cells profiled, we successfully reconstructed the whole process of spermatogenesis. We discovered key gene and non-coding RNA expression dynamics during spermatogenesis. We also identified different types of niche cells in the testis, including macrophages, endothelial, myoid, Leydig, and Sertoli cells. Moreover, using this unbiased profiling approach, we identified a novel state of SSCs. Notably, epigenetic/DNA methylation and nascent RNA analyses revealed considerable plasticity within SSC development. Lastly, we verified all our genomic findings using immunofluorescence as well as sequential RNA fluorescence in situ hybridization. Taken together, our studies provide the first chromatin and transcription landscape of human SSC, and cell atlas of human testis, which will shed light on the study of male infertility and speed the development of human SSC in vitro culture. |
