Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions

Objective: To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Design: Experimental basic science study. Setting: Reproductive biology laboratory. Patient(s): Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Intervention(s): Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA(-))/epithelial cell surface antigen (EPCAM(+)) in coculture with inactivated testicular feeders from the same patient. Main Outcome Measure(s): Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Result(s): Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA(-)/EPCAM(+) resulted in an enrichment of 27% VASA(+)/UTF1(+) hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm(2) after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm(2)). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. Conclusion(s): We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA(-)/EPCAM(+) sorted cells with testicular feeders improved the germ cell/somatic cell ratio. (C) 2016 American Society for Reproductive Medicine, Published by Elsevier Inc.