In-depth understanding of human spermatogenesis requires studying specific molecular signatures and interactions of spermatogonia with other testicular cell populations, for which isolation of pure populations of different cell types is crucial. Here, we describe a technique to simultaneously enrich pure, multiple testicular cell populations, including spermatogonia, endothelial (TECs), and perivascular mesenchymal stem/stromal cells (TMSCs), from testicular tissue by flow cytometry using a combination of defined markers. Immunohistochemical studies, multicolor staining, and cell sorting followed by multiplex quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that spermatogonia were highly enriched in the CD49f+CD49a-HLA-ABC-SSEA-4+ fraction of primary testicular cells. In contrast to spermatogonia, TMSCs and TECs were highly enriched in the CD49f+CD49a+HLA-ABC+CD144- and CD49f+CD49a+HLA-ABC+CD144+subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation and angiogenic capacity of the sorted populations. In this article, for the first time, we performed transcriptome profiling of highly enriched, freshly isolated human spermatogonia and compared their expression profile with that of TMSCs. Our RNA sequencing data favor the hypothesis that TMSCs are candidate niche components for spermatogonia. The composite genotype and phenotype of defined testicular cell populations combined with a robust isolation procedure from small biopsies contributes to a better understanding of cellular interactions and for the establishment of efficient culture techniques to maintain spermatogonial progenitors.
Keywords: endothelial; isolation; spermatogonia; stromal; testis.