Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells.
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 alpha-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.
Kubota
Hiroshi
H
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA.
Avarbock
Mary R
MR
Brinster
Ralph L
RL
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007248:Infertility, Male / Q000628:therapy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008819:Mice, Nude; D008822:Mice, Transgenic; D009414:Nerve Growth Factors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D013091:Spermatogenesis / Q000187:drug effects; D013093:Spermatogonia / Q000166:cytology* / Q000187:drug effects* / Q000637:transplantation; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects*
D016895:Culture Media, Serum-Free; C495039:GDNF protein, human; C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006133:Growth Substances; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases