Long‐Term Culture and Transplantation of Murine Testicular Germ Cells

Abstract

The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.

In-Vitro Culture and Characterization of Spermatogonial Stem Cells (SSCs) in Pre-Pubertal Mice

“…Though the cells exhibited different morphology (viz., round, stellate, polyhedral, and triangular) the stellate shaped cells were found to be more. A similar observation has been made by Jeong et al, (2003) in mice and Wang et al, (2015) in pig SSCs.…”

Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat

“…Because the cell population was highly enriched for stem cells and a defined medium was used, it was possible to identify the growth factor requirements for continuous self-renewal as glial cell line-derived neurotrophic factor (GDNF), GDNF-family receptor ␣-1 (GFR␣1) and basic fibroblast growth factor (bFGF). Other workers have obtained in vitro proliferation of mouse SSC by using less defined media with FBS and a mixture of growth factors, with or without GDNF (10,11). Because the SSCs of many species replicate in the seminiferous tubules of the mouse, we hypothesized that GDNF, GFR␣1, and bFGF, as identified in mice, also would support rat SSC self-renewal in vitro.…”

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

“…Longand short-term culture of SSCs have been attempted in several laboratories (12)(13)(14), and in our previous studies, GDNF appeared to have a beneficial effect on maintenance during a 7-day culture period (6,14). In a recent report, SSCs derived from DBA͞2 background mouse gonocytes, precursors of SSCs in neonate testis, proliferated in culture using a complex, undefined medium containing leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), GDNF, and 1% FBS (13).…”