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 DBA͞2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor ␣-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNFinduced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clumpforming 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.

Efficient Enhancement of Lentiviral Transduction Efficiency in Murine Spermatogonial Stem Cells

“…Therefore lentiviral vector could be useful vector for transduction of cells that have a slow cell cycle, such as SSCs (Kim et al, 2010;Kubota et al, 2004;Nagano et al, 2002). In previous studies, transgenic animals were also produced by lentiviral transduction and transplantation of SSCs (Hamra et al, 2002;Ryu et al, 2007).…”

“…For the in vitro culture of stem cells including SSCs, feeder layer with supplements that promote cell growth, self-renewal and signal transduction are important elements. In the case of SSCs, commonly used feeder cells are STO and mouse embryonic fibroblast (MEF) (Kanatsu-Shinohara et al, 2003;Kubota et al, 2004;Ryu et al, 2005). However, the effect of STO and MEF on the transduction and survival rate of stem cells including SSCs has not been studied.…”

“…The donor cells were cultured based on the method presented by Kubota et al (2004) with minor modifications. Donor cells were extracted from ROSA and C57 mice 6-8 days after birth.…”

“…For most of these studies, the transgenic animals were produced by lentiviral infection of highly purified SSCs and transplanting them right into testes of the recipient animals. With the recent developments of the in vitro culture technique of rodent SSCs, the possibility of germ-line modification for cultured cells has been proposed (Kanatsu-Shinohara et al, 2003;Kubota et al, 2004). If genetic modification and culture techniques of SSC are used simultaneously with a drug selection method, improvements in purity and mass propagation of trans-duced cells are possible.…”

In vitro production of fertile sperm from murine spermatogonial stem cell lines

“…On the other hand, the in vitro proliferation of murine SSCs has become possible by adding glial cell line-derived neurotrophic factor to the SSCs culture media 8,9 . The cultured SSCs, termed germline stem (GS) cells or SSC lines, proliferated in a self-renewing manner for up to or beyond 2 years, while remaining stable genetically, epigenetically and karyotypically 10 .…”