Transcriptome analysis reveals conditions for culturing human primitive undifferentiated spermatogonia

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for male infertility, which afflicts >100 million men world-wide. To devise SSC therapy approaches, it is critical to first develop methods to culture human SSCs in vitro. Here, we report on a transcriptome approach to address this question. Using single-cell RNA-seq (scRNAseq), immunofluorescence, and germ-cell xenograft transplantation analyses, we identified a cell-surface protein, PLPPR3, that purifies human primitive undifferentiated spermatogonia (uSPG) enriched for SSCs. Comparative RNAseq analysis of PLPPR3+ cells (primitive uSPG) with KIT+ cells (enriched for differentiating [d] SPG) revealed that these two stages differentially express a remarkably large number of genes, including genes encoding key components in the TGF, GDNF, AKT, and JAK-STAT signaling pathways. Using scRNAseq analysis and conventional approaches, we tested the effect of manipulating these signaling pathways on cultured human SPG. This revealed that GDNF and BMP8B broadly support the culture of SPG, Activin A supports more advanced SPG, and one condition—AKT pathway inhibition—had the unique ability to selectively support primitive uSPG. These findings have implications for methods to culture and expand human SSCs for therapeutic uses in the future. Bulk RNAseq was performed on PLPPR3+ and KIT+ cells FACS purified from 4 independent human testicular biopsy samples (aged from 30 to 41). For single cell RNA-Seq, testicular cells from biopsies obtained from two fertile men aged 32- and 37-years, respectively, were cultured under different conditions (FGF2 alone vs AKT-I+FGF2) for two weeks and then collected for sequencing.