title: Isolation and gene expression analysis of single potential human spermatogonial stem cells.
abstract: OBJECTIVE
  It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis.
  
  
  RESULTS
  We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR.
  
  
  BACKGROUND
  The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1).
  
  
  METHODS
  We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining.
  
  
  RESULTS
  Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable.
  
  
  CONCLUSIONS
  The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design.
  
  
  CONCLUSIONS
  The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features.
  
  
  BACKGROUND
  The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests.
authors.affiliation: Department of Andrology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany k.kopylow@uke.uni-hamburg.de.
authors.forename: K
authors.initials: K
authors.lastname: von Kopylow
authors.affiliation: Department of Andrology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany MVZ Fertility Center Hamburg GmbH, amedes-group, 20095 Hamburg, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Schulze
authors.affiliation: Department of Andrology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Salzbrunn
authors.affiliation: Department of Andrology, University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: A-N
authors.initials: AN
authors.lastname: Spiess
mesh_terms: D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D002470:Cell Survival; D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D059346:Magnets; D008297:Male; D008540:Meiosis; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics* / Q000378:metabolism; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D013091:Spermatogenesis / Q000235:genetics; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D009687:Nuclear Proteins; D015534:Trans-Activators; C484738:UTF1 protein, human; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3
keywords: Dynabeads; UTF1; cell isolation; fibroblast growth factor receptor 3; human spermatogonial stem cells; immunocytochemistry; single-cell real-time RT–PCR; spermatogonia; spermatogonial stem cells