Enrichment of spermatogonial stem cells from long-term cultured human testicular cells

Objective: To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6(+), HLA(-)/ITGA6(+), GPR125(+), and HLA(-)/GPR125(+) magnetic-assisted cell sorting (MACS). Design: Experimental basic science study. Setting: Reproductive biology laboratory. Patient(s): Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. Intervention(s): Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. Main Outcome Measure(s): Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. Result(s): Compared with the nonsorted cultured testicular cells, only the ITGA6(+) and HLA(-)/GPR125(+) sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6(+). Conclusion(s): Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings. (C) 2014 by American Society for Reproductive Medicine.