title: A combined approach facilitates the reliable detection of human spermatogonia in vitro.
abstract: OBJECTIVE
  Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro?
  
  
  CONCLUSIONS
  Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures.
  
  
  BACKGROUND
  The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies.
  
  
  METHODS
  Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3).
  
  
  METHODS
  Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels.
  
  
  RESULTS
  Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions.
  
  
  CONCLUSIONS
  There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models.
  
  
  CONCLUSIONS
  While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future.
  
  
  BACKGROUND
  This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest.
  
  
  BACKGROUND
  Not applicable.
authors.affiliation: Institute for Reproductive and Regenerative Biology, Centre of Reproductive Medicine and Andrology, University Hospital Münster, Albert-Schweitzer-Campus 1 (D11), Münster 48149, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Kossack
authors.forename: N
authors.initials: N
authors.lastname: Terwort
authors.forename: J
authors.initials: J
authors.lastname: Wistuba
authors.forename: J
authors.initials: J
authors.lastname: Ehmcke
authors.forename: S
authors.initials: S
authors.lastname: Schlatt
authors.forename: H
authors.initials: H
authors.lastname: Schöler
authors.forename: S
authors.initials: S
authors.lastname: Kliesch
authors.forename: J
authors.initials: J
authors.lastname: Gromoll
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D001706:Biopsy; D018929:Cell Culture Techniques; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005819:Genetic Markers; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000473:pathology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D005819:Genetic Markers; C095204:MAGEA4 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015534:Trans-Activators; C484738:UTF1 protein, human; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases
keywords: combined approach; germ cell-specific markers; human germ cell culture; spermatogonia; testicular biopsies