Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells

Abstract

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.

Fig. 1.

Expansion of DBA × ROSA SSCs in serum-free medium supplemented with GDNF. (A) DBA × ROSA pup testis cells formed clumps with tight intercellular contacts in culture. (B) Cultured cells in clumps express GCNA1 (dark blue), a marker for germ cells. (C) Expression of β-gal was detected only in germ cell clumps. β-gal expressing cells stain blue with X-gal. (D) Macroscopic appearance of recipient testis 2 months after transplantation with DBA × ROSA MACS Thy-1 cells cultured for 10 weeks in the presence of GDNF. Each blue-stained area indicates donor-derived spermatogenesis. Testis was stained with X-gal. (E) Freshly isolated MACS Thy-1 cells and cultured cells were transplanted into recipient testes. The number of donor-derived spermatogenic colonies per 10⁵ MACS Thy-1 cells originally seeded in culture is shown. The transplantation assay demonstrated expansion of DBA × ROSA SSCs in culture with GDNF. DBA × ROSA SSCs cultured without GDNF and C57 × ROSA SSCs cultured with or without GDNF were not maintained. Data are shown as means ± SEM for six recipient testes per time point. Most error bars do not extend beyond the symbol. (Scale bar, 100 μmin A–C and 2 mm in D.)

Fig. 2.

Expansion of SSCs in serum-free medium supplemented with GDNF, soluble GFRα1, and bFGF. (A) MACS Thy-1 cells from C57 × ROSA were cultured in the conditions indicated. Fresh MACS Thy-1 cells and cultured cells were transplanted into recipient testes. The number of donor-derived spermatogenic colonies per 10⁵ MACS Thy-1 cells originally seeded in culture is shown. The transplantation assay demonstrated a synergistic effect of soluble GFRα1 and bFGF on expansion of C57 × ROSA SSCs cultured with GDNF. Data are shown as means ± SEM for six recipient testes per time point. Most error bars do not extend beyond symbol. (B–E) Development and growth of germ cell clumps from 129/SvCP MACS Thy-1 pup testis cells. (Scale bar, 50 μm.) MACS Thy-1 cells on STO feeders after 5 h in culture (B), initiation of cell clump formation at 2 days (C), growth of germ cell clumps at 5 days (D), and continuous expansion of germ cell clumps at 5 months (E) are shown.

Fig. 3.

Phenotypic characteristics of cultured 129/SvCP SSCs. (A) Immunocytochemistry of c-Ret receptor tyrosine kinase. All cells in germ cell clumps express the c-Ret receptor (green). (B) FACS analyses for GFRα1 and NCAM expression on clump-forming germ cells. Filled histograms represent stained germ cells with the antibodies indicated. Open histograms indicate isotype control antibody-stained cells. Cultured SSCs expressed GFRα1 and NCAM. (x axis, Log fluorescence intensity.) (C) AP activity on SSCs and ES cells. Cultured germ cell clumps have lower AP activity (red) than ES cells. Staining time and protocol were identical for the two cell types. (D) Immunocytochemistry of Oct-4 on SSCs and ES cells. Germ cell clumps and ES cells express a high level of Oct-4 (green). (Scale bar, 50 μm in A and 100 μmin C and D.)

Fig. 4.

Biological characteristics of cultured SSCs. (A) Effect of FBS on proliferation of SSCs. C57 × ROSA SSCs were exposed to FBS at the concentration indicated for 2 weeks. Cells were transplanted after 7 and 14 days of culture. At each time point, the number of spermatogenic colonies formed per 10⁵ cells placed in culture is shown. All values are means ± SEM, and five to six recipient testes were analyzed per time point. Proliferation of SSCs at 14 days was decreased significantly in all concentrations of FBS compared to serum-free medium (Bonferroni-adjusted P value < 0.001). (B) Effect of soluble factors on proliferation of SSCs. Soluble factors indicated were added individually in the culture of C57 × ROSA SSCs. Control culture contained GDNF, soluble GFRα1, and bFGF with no additional factors. After 6 weeks of culture with additional factors, cultured SSCs were transplanted into recipient testes to evaluate stem cell activity. The data are shown as relative colonization activity, the number of colonies per 10⁵ donor cells originally placed in culture relative to that obtained with the control culture (means ± SEM, n = 10–12). A significant effect (asterisk) was observed in culture with IGF-1 (2.77 ± 0.68-fold increase, Bonferroni-adjusted P value < 0.001). (C) Restoration of fertility in infertile recipients by transplantation of cultured SSCs. Progeny from W⁵⁴/W^v mice transplanted with C57GFP × ROSA-derived germ cell clumps that were cultured for 11 weeks in vitro. Because the transplanted SSCs are haploid for the GFP transgene, 50% of progeny should express GFP. EGF, epidermal growth factor.