Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation

Objectives:  The key to fertility in adult males is production of mature spermatogenic cells. Spermatogonial stem cells (SSC) have the dual capacity of self-renewal and of differentiation into mature sperm. SSC transplantation may provide potential treatment for specific male infertilities. However, until now, there has been no evidence of offspring produced by transplantation of adult SSC line cells in humans or other mammals.
Materials and methods:  A new line of SSCs from adult C57BL/6 mouse was established by using magnetic-activated cell sorting. The cell line was characterized by immunocytochemistry, karyotype analysis and telomeric repeat amplification protocol (TRAP) telomerase activity assay. Spermatogenic function was examined by allograft into germ cell-ablated recipient mice.
Results:  For more than 14 months with more than 65 maintenance passages, the cell line showed a normal karyotype (40, XY) and high telomerase activity. It represented a Thy-1+, Oct4+, SSEA-1-, c-kit- (99 ± 1%) cell subpopulation. We cryopreserved these SSCs and successfully produced normal offspring after transplanting them into testes of busulphan-sterilized mice.
Conclusions:  We established and long-term maintained an adult SSC line with normal spermatogenic function, without the need of genetic modification; thus, this study provides a model system for basic research and clinical application.