Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate
Undifferentiated spermatogonia (uSPG), including spermatogonial stem cells, are essential for long-term spermatogenesis and hold therapeutic potential for treating male infertility. While rodent uSPG are well characterized, human uSPG remain poorly understood. Here, we screened antibodies against 21 proteins encoded by genes enriched in human uSPG subsets identified from single-cell RNA sequencing analysis. Among them, we identified FSD1 as a pan-uSPG cell-surface marker suitable for purifying the entire uSPG population. Transcriptomic profiling of FSD1+ uSPG uncovered candidate signaling pathways and transcriptional networks involved in uSPG development. PIWIL4 and EGR4 label primitive, quiescent uSPG, whereas PPP1R36 and NANOS3 mark proliferative uSPG subsets. PIWIL4+ and NANOS3+ cells do not overlap, highlighting distinct uSPG developmental states. We provide empirical evidence that RAS signaling regulates the transition between these states. Together, our study provides valuable tools for studying uSPG development and facilitating future translational advances. Overall design: FSD1+ cells were purified from testis biopsies from four individuals by FACS and performed bulk RNAseq. For comparison, bulk RNAseq analysis was performed in parallel on unfractionated human testicular cells from the same donors.