Identification and characterization of repopulating spermatogonial stem cells from the adult human testis

Hum Reprod. 2011 Jun;26(6):1296-306. doi: 10.1093/humrep/der026. Epub 2011 Feb 23.


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.

MeSH terms

  • Adult
  • Animals
  • Azoospermia / pathology
  • Biomarkers / metabolism
  • Humans
  • Integrin alpha6 / analysis
  • Male
  • Mice
  • Receptors, G-Protein-Coupled / analysis
  • Spermatogonia / cytology*
  • Stage-Specific Embryonic Antigens / analysis
  • Stem Cell Transplantation / methods
  • Stem Cells / cytology*
  • Testis / cytology*
  • Testis / pathology
  • Transplantation, Heterologous*


  • ADGRA3 protein, human
  • Biomarkers
  • Integrin alpha6
  • Receptors, G-Protein-Coupled
  • Stage-Specific Embryonic Antigens
  • stage-specific embryonic antigen-4