title: Separation of somatic and germ cells is required to establish primate spermatogonial cultures.
abstract: OBJECTIVE
  Can primate spermatogonial cultures be optimized by application of separation steps and well defined culture conditions?
  
  
  CONCLUSIONS
  We identified the cell fraction which provides the best source for primate spermatogonia when prolonged culture is desired.
  
  
  BACKGROUND
  Man and marmoset show similar characteristics in regard to germ cell development and function. Several protocols for isolation and culture of human testis-derived germline stem cells have been described. Subsequent analysis revealed doubts on the germline origin of these cells and characterized them as mesenchymal stem cells or fibroblasts. Studies using marmosets as preclinical model confirmed that the published isolation protocols did not lead to propagation of germline cells.
  
  
  METHODS
  Testicular cells derived from nine adult marmoset monkeys (Callithrix jacchus) were cultured for 1, 3, 6 and 11 days and consecutively analyzed for the presence of spermatogonia, differentiating germ cells and testicular somatic cells.
  
  
  METHODS
  Testicular tissue of nine adult marmoset monkeys was enzymatically dissociated and subjected to two different cell culture approaches. In the first approach all cells were kept in the same dish (non-separate culture, n = 5). In the second approach the supernatant cells were transferred into a new dish 24 h after seeding and subsequently supernatant and attached cells were cultured separately (separate culture, n = 4). Real-time quantitative PCR and immunofluorescence were used to analyze the expression of reliable germ cell and somatic markers throughout the culture period. Germ cell transplantation assays and subsequent wholemount analyses were performed to functionally evaluate the colonization of spermatogonial cells.
  
  
  RESULTS
  This is the first report revealing an efficient isolation and culture of putative marmoset spermatogonial stem cells with colonization ability. Our results indicate that a separation of spermatogonia from testicular somatic cells is a crucial step during cell preparation. We identified the overgrowth of more rapidly expanding somatic cells to be a major problem when establishing spermatogonial cultures. Initiating germ cell cultures from the supernatant and maintaining germ cells in suspension cultures minimized the somatic cell contamination and provided enriched germ cell fractions which displayed after 11 days of culture a significantly higher expression of germ cell markers genes (DDX-4, MAGE A-4; P < 0.05) compared with separately cultured attached cells. Additionally, germ cell transplantation experiments demonstrated a significantly higher absolute number of cells with colonization ability (P < 0.001) in supernatant cells after 11 days of separate culture.
  
  
  CONCLUSIONS
  This study presents a relevant aspect for the successful setup of spermatogonial cultures but provides limited data regarding the question of whether the long-term maintenance of spermatogonia can be achieved. Transfer of these preclinical data to man may require modifications of the protocol.
  
  
  CONCLUSIONS
  Spermatogonial cultures from rodents have become important and innovative tools for basic and applied research in reproductive biology and veterinary medicine. It is expected that spermatogonia-based strategies will be transformed into clinical applications for the treatment of male infertility. Our data in the marmoset monkey may be highly relevant to establish spermatogonial cultures of human testes.
  
  
  BACKGROUND
  Funding was provided by the DFG-Research Unit FOR 1041 Germ Cell Potential (SCHL394/11-2) and by the Graduate Program Cell Dynamics and Disease (CEDAD) together with the International Max Planck Research School - Molecular Biomedicine (IMPRS-MBM). The authors declare that there is no conflict of interest.
  
  
  BACKGROUND
  Not applicable.
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany.
authors: Daniel
authors: D
authors: Langenstroth
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany.
authors: Nina
authors: N
authors: Kossack
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany.
authors: Birgit
authors: B
authors: Westernströer
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany.
authors: Joachim
authors: J
authors: Wistuba
authors: Stem Cell Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany.
authors: Rüdiger
authors: R
authors: Behr
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany.
authors: Jörg
authors: J
authors: Gromoll
authors: Institute of Reproduction and Regenerative Biology, Centre of Reproductive Medicine and Andrology, Albert-Schweitzer-Campus 1, Building D11, 48149 Münster, Germany stefan.schlatt@ukmuenster.de.
authors: Stefan
authors: S
authors: Schlatt
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002144:Callithrix / Q000502:physiology*; D002454:Cell Differentiation; D002469:Cell Separation / Q000662:veterinary; D008297:Male; D008819:Mice, Nude; D061251:Primary Cell Culture / Q000379:methods / Q000662:veterinary*; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000378:metabolism; D013997:Time Factors
chemical_list: D015415:Biomarkers
keywords: germ cell; marmoset monkey; spermatogonial stem cell; testicular cell culture; xenotransplantation