title: Comparison of colony formation of human spermatogonial stem cells (SSCs) with and without collagen.
abstract: OBJECTIVE
  To investigate the effects of collagen and growth factors on in vitro proliferation of human spermatogonial stem cells obtained from patients with non-obstructive azoospermia.
  
  
  METHODS
  The experimental cross-sectional study was conducted from February 2013 to April 2015 after obtaining approval from the ethics committee of Ahvaz Jundishapur University of Medical Sciences, Iran. Testicular sperm extractions of non-obstructive azoospermic patients were obtained from the Clinical Urology and Embryology, In Vitro Fertilization Department of Imam Khomeini Hospital. Spermatogonial stem cells and Sertoli cells, obtained from human testis biopsies by a two-step enzymatic digestion method, were purified using fluorescence- activated cell-sorting and daturastramonium-lectin, and were cultured separately. To investigate a more direct influential factor on colony formation, one control and two experimental groups were formed. Group 1 acted as the control in which spermatogonial stem cells were co-cultured with Sertoli cells alone. In group 2 they were co-cultured with Sertoli cells and growth factors such as leukaemia inhibitory factor, epidermal growth factor and glial cell-derived neurotrophic factor, and in group 3 with Sertoli cells along with growth factors in the presence of collagen-coated dishes. Number and diameter of the colonies were evaluated after 7 weeks.
  
  
  RESULTS
  Specimens obtained related to 21 patients. Number and diameter of the colonies in group 3 (18±2.6 and 276.6±45.5) were significantly more than both groups 1 (3.5±1 and D1:81.6±12) and group 2(11±2.2 and 165.2±32.5) (p<0.05 each). Also, the number and diameter of colony in group 2 were significantly better than the control group (p<0.05).Expression profile of the VASA, promyelocytic leukaemia zinc-finger (PLZF), Octamer-binding transcription factor 4 (OCT4) and integrin a6 (INTGa6) were detected in all groups. Based on cytochemical findings, OCT4 was expressed in the colonies of all three groups.
  
  
  CONCLUSIONS
  According to positive effects of collagen and growth factors on the colonisation of spermatogonial stem cells, it seems that using the cells may lead to better colonisation of this type of stem cells.
authors.affiliation: PhD Student, Anatomical Sciences, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Razi
authors.initials: R
authors.lastname: Shiva
authors.affiliation: Physiological Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Saki
authors.initials: S
authors.lastname: Ghasem
authors.affiliation: Fertility and Infertility Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Hemadi
authors.initials: H
authors.lastname: Masoud
authors.affiliation: Department of Anatomical Sciences, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Khorsandi Laya
authors.initials: KL
authors.lastname: Sadat
authors.affiliation: Department of Immunology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Khodadadi
authors.initials: K
authors.lastname: Ali
authors.affiliation: Department of Urology, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
authors.forename: Dadfar Mohammad
authors.initials: DM
authors.lastname: Reza
mesh_terms: D000072956:Adult Germline Stem Cells / Q000166:cytology / Q000187:drug effects*; D053713:Azoospermia; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D018920:Coculture Techniques; D003094:Collagen / Q000494:pharmacology*; D003430:Cross-Sectional Studies; D053487:DEAD-box RNA Helicases / Q000187:drug effects / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005434:Flow Cytometry; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D006801:Humans; D039503:Integrin alpha6 / Q000187:drug effects / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000187:drug effects / Q000378:metabolism; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000187:drug effects / Q000378:metabolism; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D012708:Sertoli Cells
chemical_list: D051100:Glial Cell Line-Derived Neurotrophic Factor; D039503:Integrin alpha6; D051741:Kruppel-Like Transcription Factors; D053605:Leukemia Inhibitory Factor; D050814:Octamer Transcription Factor-3; D000075722:Promyelocytic Leukemia Zinc Finger Protein; C092037:ZBTB16 protein, human; D004815:Epidermal Growth Factor; D003094:Collagen; C498288:DDX4 protein, human; D053487:DEAD-box RNA Helicases
keywords: Spermatogonial stem cells, SSCs, Growth factor, Collagen, Co-culture.