Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.