title: Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture.
abstract: OBJECTIVE
  Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia?
  
  
  CONCLUSIONS
  Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures.
  
  
  BACKGROUND
  The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures.
  
  
  METHODS
  Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures.
  
  
  METHODS
  Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4).
  
  
  RESULTS
  UTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected.
  
  
  CONCLUSIONS
  Different groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences.
  
  
  CONCLUSIONS
  The ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy.
  
  
  BACKGROUND
  The experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests.
  
  
  BACKGROUND
  Not applicable.
authors.affiliation: Indiana University, 800 E. Kirkwood Ave, Bloomington, IN 47405-7102, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Indiana University, 800 E. Kirkwood Ave, Bloomington, IN 47405-7102, USA.
authors.forename: A
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Indiana University, 800 E. Kirkwood Ave, Bloomington, IN 47405-7102, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Schmidt
authors.affiliation: Indiana University, 800 E. Kirkwood Ave, Bloomington, IN 47405-7102, USA ctdann@indiana.edu.
authors.forename: C T
authors.initials: CT
authors.lastname: Dann
mesh_terms: D000293:Adolescent; D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D006801:Humans; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D013091:Spermatogenesis; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D055815:Young Adult
chemical_list: D015415:Biomarkers; C101358:DAZL protein, human; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C472575:SALL4 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C484738:UTF1 protein, human
keywords: cell culture; fertility; germ cells; stage-specific embryonic antigens; testis