PD09-11\u2003THE NEONATAL AND ADULT HUMAN TESTIS DEFINED AT THE SINGLE-CELL LEVEL

Abstract

INTRODUCTION AND OBJECTIVES: Over 100 million men worldwide suffer from infertility. In most cases, the underlying cause is unknown. Treating human infertility requires detailed knowledge of human spermatogenesis, but most of what we know about spermatogensis comes from studies in rodents. This is a significant gap in our knowledge, as human spermatogenesis is different from rodent spermatogenesis in several respects. The primary objectives of this study are to: (i) identify cell subsets in the neonatal and adult human testis, (ii) define and molecularly characterize human spermatogonial stem cells (SSCs), the only self-renewing cells in the adult testis, (iii) identify human testicular somatic cell markers, (iv) define the developmental trajectories of both germ and somatic human testicular cells, and (v) identify candidate signaling mechanisms operating between germ and somatic cells in the human testis. METHODS: We used single-cell RNA-sequencing (scRNAseq), coupled with bioinformatics approaches, to analyze neonatal and adult human testes. We validated protein markers of key cell subsets by FACS/qPCR, immunohistochemistry, and immunofluorescence analyses. RESULTS: scRNAseq analysis of adult human testes samples revealed 14 different cell types and stages, including 9 germ-cell subsets. We focused our analysis on the most primitive undifferentiated spermatogonia (SPG) subset, which are likely highly enriched for SSCs. Several novel gene and protein markers labeling this SSC-enriched subset were identified and validated. In neonatal human testes, clustering analysis identified 3 germ cell subsets, including one that resembled human primordial germ cells (PGCs) and another that had a gene expression pattern consistent with being emergent human SSCs. We identified and validated markers for these neonatal germ cell subsets. The developmental trajectory of germ cell subsets from the embryo stage, through the neonatal stage, to the adult stage, was inferred through both bioinformatics and empirical approaches. We also traced the development of somatic cells from the neonatal to adult stages. Our analysis of somatic and germ cell subsets allowed us to identify receptor-ligand signaling pairs indicative of specific cell-cell signaling mechanisms operating during the SSC genesis and maintenance phases of germ cell development in the neonatal and adult human testis, respectively. CONCLUSIONS: Our study defines cell subsets in the neonatal and adult human testis and provides a development blueprint for both the male germline and testicular somatic cell lineages. The human SSC markers we identified hold the potential to be useful for reproductive therapies to treat infertility and testicular cancer. Source of Funding: This work was supporting by National Institutes of Health grants R01 GM119128 (M.W.) and T32 HD007203 (D.B), as well as the Lalor Institute (K.T.)