TRANSCRIPTOME ANALYSIS REVEALS CONDITIONS FOR CULTURING HUMAN SPERMATOGONIAL STEM-LIKE CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (MP38)1 Apr 2020MP38-11 TRANSCRIPTOME ANALYSIS REVEALS CONDITIONS FOR CULTURING HUMAN SPERMATOGONIAL STEM-LIKE CELLS Kun Tan, Hye-Won Song, Merlin Thompson, Sarah Munyoki, Meena Sukhwani, Tung-Chin Hsieh, Kyle Orwig, and Miles Wilkinson* Kun TanKun Tan More articles by this author , Hye-Won SongHye-Won Song More articles by this author , Merlin ThompsonMerlin Thompson More articles by this author , Sarah MunyokiSarah Munyoki More articles by this author , Meena SukhwaniMeena Sukhwani More articles by this author , Tung-Chin HsiehTung-Chin Hsieh More articles by this author , Kyle OrwigKyle Orwig More articles by this author , and Miles Wilkinson*Miles Wilkinson* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000887.011AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Spermatogonial stem cells (SSCs) have potential therapeutic value for male infertility, a condition that afflicts >100 million men world-wide. A major limitation is the lack of reproducible methods to culture human SSCs, which is the goal of this study. METHODS: Immunofluorescence (IF) analysis and the xenotransplantation assay were used to characterize PLPPR3+ human spermatogonia (SPG). RNAseq was used to define the transcriptomes of PLPPR3+ and KIT+ human SPG purified by FACS. Integrin-alpha6+ human testicular cells were purified using magnetic-activated cell sorting and cultured with standard media containing FGF2 and the factors described below. Quantitative PCR (qPCR) was used to determine the expression of marker genes. RESULTS: As a novel approach to elucidate conditions for culturing human SSCs, we elected to define the transcriptome of human undifferentiated (undiff) SPG. To achieve this, it was critical to identify a cell-surface protein that specifically marks these cells. As a candidate, we chose PLPPR3, as we found this surface protein is encoded by a gene selectively expressed in the human undiff-SPG subset. Follow-up IF co-staining experiments showed that the PLPPR3 protein also specifically marks undiff-SPG, consistent with its RNA expression. To assess whether PLPPR3 labels SSCs within the undiff-SPG population, we performed xenotransplantation analysis and found PLPPR3+ SPG were ∼20-fold enriched for SSC activity compared with unfractionated cells. Satisfied that PLPPR3 specifically marks human undiff-SPG and enriches for SSCs, we performed RNAseq analysis on purified PLPPR3+ cells by FACS from human testes biopsies. Comparison with differentiating (KIT+) SPG from the same biopsies revealed a surprisingly large number of differentially expressed genes between differentiating- and undiff-SPG, including genes encoding many signaling pathway components. This led us to hypothesize that these differentially activated signaling pathways could be leveraged to selectively culture human SSCs. In support, we found that inhibition of the AKT pathway in human testicular cultures selectively supported undiff-SPG over differentiating SPG. In contrast, GDNF and BMP8B were not selective, as they support both differentiating and undiff-SPG. CONCLUSIONS: The finding that AKT pathway inhibition specifically favors undifferentiated SPG suggests this approach can be used to culture and expand human SSCs for therapeutic use in the future. Source of Funding: This work was supported by NIH grants R01 GM119128 (M.F.W.) and the Lalor Institute (K.T.). © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e573-e573 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kun Tan More articles by this author Hye-Won Song More articles by this author Merlin Thompson More articles by this author Sarah Munyoki More articles by this author Meena Sukhwani More articles by this author Tung-Chin Hsieh More articles by this author Kyle Orwig More articles by this author Miles Wilkinson* More articles by this author Expand All Advertisement PDF downloadLoading ...