title: Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.
abstract: OBJECTIVE
  To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions.
  
  
  METHODS
  Experimental basic science study.
  
  
  METHODS
  Reproductive biology laboratory.
  
  
  METHODS
  Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment.
  
  
  METHODS
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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/cm2after 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Reproductive Medicine Unit, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain; Fundación Instituto Valenciano de Infertilidad (FIVI), INCLIVA, Department of Pediatrics, Obstetrics and Gynecology, Valencia University, Valencia, Spain.
authors.forename: Jose V
authors.initials: JV
authors.lastname: Medrano
authors.affiliation: Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Rombaut
authors.affiliation: Fundación Instituto Valenciano de Infertilidad (FIVI), INCLIVA, Department of Pediatrics, Obstetrics and Gynecology, Valencia University, Valencia, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Simon
authors.affiliation: Reproductive Medicine Unit, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pellicer
authors.affiliation: Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium. Electronic address: ellen.goossens@uzbrussel.be.
authors.forename: Ellen
authors.initials: E
authors.lastname: Goossens
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D000071858:Epithelial Cell Adhesion Molecule / Q000276:immunology; D061252:Feeder Cells; D005786:Gene Expression Regulation; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D010641:Phenotype; D013093:Spermatogonia / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors
chemical_list: D015415:Biomarkers; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D006680:HLA Antigens
keywords: Human spermatogonial stem cells; in vitro propagation; male fertility preservation