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• W3092822976 abstract "Identify the protein markers of human and monkey spermatogonial stem cells (SSCs) and the signaling pathways regulating their self-renewal and proliferation, to facilitate the development of SSC-based therapies for the treatment of male infertility. We performed high throughput, unbiased, single-cell RNA-sequencing of normal adult primate testicular tissue. Clustering analysis coupled with the evaluation of expression patterns identified potential marker genes of primate stem/progenitor spermatogonia. The expression of these candidate markers on human testicular tissue was evaluated by immunostaining techniques. SSC transplantation was used to determine the candidate markers that may be utilized to sort and enrich human SSCs for downstream clinical applications. We performed drop-seq single cell RNA sequencing of 5 human and 4 rhesus macaque healthy adult donor testicular tissue, generating ∼13,560 and 20,242 single cell transcriptomes respectively. Principal component analysis (PCA) dimensionality reduction and T-distributed Stochastic Neighbor Embedding (tsne)/ Uniform Manifold Approximation and Projection(UMAP) unsupervised clustering analysis partitioned the primate cells into transcriptionally distinct populations, representing the known primate testicular cell types. Differential expression analysis identified marker genes of the undifferentiated spermatogonia cell populations, that were validated by immunoflourescence techniques. Fluorescence activated cell sorting (FACS) and human to nude mouse xenotransplantation was used to evaluate SSC colonization activity. Our transcriptome data identified CDK17, MAGEB2, MORC1, TCF3, PIWIL4, GPX1,DNAJB6, FMR1 and TSPAN33 as candidate markers genes of primate stem/progenitor spermatogonia. These genes exhibited unambiguous staining of cells on the basement membrane of human seminiferous tubules and demonstrated overlapping expression with human undifferentiated spermatogonia UCHL1, and more limited overlap with the differentiation marker cKIT. Colometric staining revealed that expression of these markers is mainly restricted to Adark and Apale spermatogonia which are thought to represent the SSC population in primate testes. Flow cytometry analysis of cell surface proteins TSPAN33 and PLPPR3 found that ∼3% and ∼1.5% of human testicular cells express these proteins respectively. When FACS sorted TSPAN33+, TSPAN33- or PLPPR3+,PLPPR3- and unsorted control fractions were xenotransplantated into infertile recipient mice, we observed significant enrichment of SSC colonization activity in the TSPAN33+ and PLPPR3+ fractions. We have identified putative marker genes of primate stem/progenitor spermatogonia. These genes are implicated in WNT, Hedgehog, FGF, BMP, MAP2K/AKT signaling as well as metabolism that may be important in regulating primate SSC growth dynamics in vivo and/or in vitro. Our transcriptome data may reveal markers/niche signaling pathways that can be exploited to isolate and enrich primate SSCs and expand them in vitro." @default.
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• W3092822976 date "2020-09-01" @default.
• W3092822976 modified "2023-10-18" @default.
• W3092822976 title "SINGLE CELL TRANSCRIPTOME ANALYSIS IDENTIFIES PUTATIVE CELL SURFACE MARKERS OF HUMAN SPERMATOGONIAL STEM CELLS" @default.
• W3092822976 doi "https://doi.org/10.1016/j.fertnstert.2020.08.305" @default.
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