Generation of Mouse Spermatogonial Stem-Cell-Colonies
in A Non-Adherent Culture
The properties of self-renewal and division in spermatogonial stem cells (SSCs)
support spermatogenesis. There is a number of reported methods for
Materials and Methods
In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco’s modified Eagle’s medium
(DMEM) medium (CTRL group), 10% fetal bovine serum (FBS)+DMEM (10% group), and
growth factor (G group) that contained 2% FBS, glial cell-derived neurotrophic factor (GDNF),
epidermal growth factor (EGF), and fibroblast growth factor (FGF). Mouse spermatogonial
stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue.
After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed
by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and
flow cytometry against the germ cell markers
We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression (P<0.05) of germ cell markers in the G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermato- gonial stem-like colonies were partially positive.
A non-adherent culture system could provide a favorable method for