Growth Factors Essential for Self-Renewal and Expansion of Mouse Spermatogonial Stem Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 101; no. 47; pp. 16489 - 16494
• Main Authors: Kubota, Hiroshi, Avarbock, Mary R., Brinster, Ralph L.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 23.11.2004; National Acad Sciences
• Subjects: Animals; Biological Sciences; Cell Differentiation - drug effects; Cell growth; Cells, Cultured; Cellular biology; Culture Media, Serum-Free; Cultured cells; Feeder cells; Fibroblast Growth Factor 2 - pharmacology; Germ cells; Glial Cell Line-Derived Neurotrophic Factor; Glial Cell Line-Derived Neurotrophic Factor Receptors; Growth Substances - pharmacology; Humans; Infertility, Male - therapy; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Nude; Mice, Transgenic; Nerve Growth Factors - pharmacology; Neurons; Pluripotent stem cells; Proto-Oncogene Proteins - pharmacology; Proto-Oncogene Proteins c-ret; Rats; Receptor Protein-Tyrosine Kinases - pharmacology; Receptors; Recombinant Proteins - pharmacology; Rodents; Signal Transduction; Spermatogenesis - drug effects; Spermatogonia - cytology; Spermatogonia - drug effects; Spermatogonia - transplantation; Stem Cell Transplantation; Stem cells; Stem Cells - cytology; Stem Cells - drug effects; Testes; Transplantation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0407063101

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA/2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor α-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNF-induced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clump-forming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.