title: Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.
abstract: BACKGROUND
  This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes.
  
  
  METHODS
  Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers.
  
  
  RESULTS
  Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics.
  
  
  CONCLUSIONS
  This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.
authors.affiliation: PrimeGen Biotech LLC, 213 Technology Drive, Irvine, CA 92618, USA. fizadyar@primegenbiotech.com
authors.forename: Fariborz
authors.initials: F
authors.lastname: Izadyar
authors.forename: Jadelind
authors.initials: J
authors.lastname: Wong
authors.forename: Chad
authors.initials: C
authors.lastname: Maki
authors.forename: Jason
authors.initials: J
authors.lastname: Pacchiarotti
authors.forename: Thomas
authors.initials: T
authors.lastname: Ramos
authors.forename: Kyle
authors.initials: K
authors.lastname: Howerton
authors.forename: Constance
authors.initials: C
authors.lastname: Yuen
authors.forename: Scott
authors.initials: S
authors.lastname: Greilach
authors.forename: Hongyu H
authors.initials: HH
authors.lastname: Zhao
authors.forename: Michelle
authors.initials: M
authors.lastname: Chow
authors.forename: Yung-Chiong
authors.initials: YC
authors.lastname: Chow
authors.forename: Jianyu
authors.initials: J
authors.lastname: Rao
authors.forename: Jason
authors.initials: J
authors.lastname: Barritt
authors.forename: Natan
authors.initials: N
authors.lastname: Bar-Chama
authors.forename: Alan
authors.initials: A
authors.lastname: Copperman
mesh_terms: D000328:Adult; D000818:Animals; D053713:Azoospermia / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D006801:Humans; D039503:Integrin alpha6 / Q000032:analysis; D008297:Male; D051379:Mice; D043562:Receptors, G-Protein-Coupled / Q000032:analysis; D013093:Spermatogonia / Q000166:cytology*; D055746:Stage-Specific Embryonic Antigens / Q000032:analysis; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology*; D013737:Testis / Q000166:cytology* / Q000473:pathology; D014183:Transplantation, Heterologous
chemical_list: C548160:ADGRA3 protein, human; D015415:Biomarkers; D039503:Integrin alpha6; D043562:Receptors, G-Protein-Coupled; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4