Successful human spermatogonial stem cell (hSSC) culture could enable cell therapy for male infertility. Mammalian extracellular matrix (ECM) promotes mitogenesis, migration, and/or differentiation of various stem/progenitor cells, and can plausibly facilitate hSSC survival in culture. Hydrogel forms of human testicular ECM (htECM), porcine testicular ECM (ptECM), porcine small intestinal submucosa ECM (SIS), and porcine urinary bladder ECM (UBM) were used to coat tissue culture plates for hSSC culture. In addition, hSSCs were cultured on Sandos inbred mice (SIM) 6-thioguanine-resistance, ouabain-resistant (STO) mouse embryonic fibroblast feeder cells (control), murine laminin, or human laminin. Undifferentiated embryonic cell transcription factor 1-positive (UTF1+) human spermatogonia were quantified at days 0, 7, and 14 of culture. htECM was the only condition that retained a significantly higher number of UTF1+ cells than control STO feeder cell cultures (22% vs. 3%). Overall, the number of hSSCs declined during the 14 day culture period under all conditions. A multiparameter flow cytometry analysis of cells cultured on htECM and ptECM revealed that stage-specific embryonic antigen 4+ undifferentiated spermatogonia may be lost to differentiation (cKIT+ spermatogonia) and apoptosis (annexin V+ spermatogonia). Proliferation of undifferentiated human spermatogonia (Ki67+) was limited, suggesting that hSSCs may have different growth factor requirements than mouse SSCs. ECM from the homologous species (human) and homologous tissue (testis) was the most effective substrate for hSSCs, and establishes a foundational feeder-free, serum-free condition for future iterative testing of culture conditions toward the long-term goal of stable hSSC cultures.
Impact Statement
This study developed and characterized human testis extracellular matrix (htECM) and porcine testis ECM (ptECM) for testing in human spermatogonial stem cell (hSSC) culture. Results confirmed the hypothesis that ECM from the homologous species (human) and homologous tissue (testis) is optimal for maintaining hSSCs. We describe a simplified feeder-free, serum-free condition for future iterative testing to achieve the long-term goal of stable hSSC cultures. To facilitate analysis and understand the fate of hSSCs in culture, we describe a multiparameter, high-throughput, quantitative flow cytometry approach to rapidly count undifferentiated spermatogonia, differentiated spermatogonia, apoptotic spermatogonia, and proliferative spermatogonia in hSSC cultures.
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