Volume 106, Issue 6, Pages 1539-1549
Jose V. Medrano, Ph.D., Charlotte Rombaut, M.Sc., Carlos Simon, M.D., Antonio Pellicer, M.D., Ellen Goossens, Ph.D.
To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cells (mSSCs) culture conditions.
Experimental basic science study.
Reproductive biology laboratory.
Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment.
Testicular cells used to create in vitro cell cultures corresponding to the following groups:  unsorted human testicular cells,  differentially plated human testicular cells, and  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.
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/cm2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm2) and differentially plated cells (49 hSSCS/cm2). 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.
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.