Isolation of the Spermatogonial Stem Cell-Containing Fraction from Testes

A testis cell fraction enriched for spermatogonial stem cells (SSCs) compared with the unfractionated total testis cell population can be isolated from testes after birth and adult stages. This protocol involves dissociating the seminiferous tubules into a single-cell suspension, using enzymes and mechanical disruption. This is followed by the selection of the Thy1⁺ cell fraction that contains nearly the entire SSC population and is enriched for these cells compared with the total testis cell population. This isolated fraction can be used for molecular studies, transfer into culture to establish primary cultures, or transplantation into the testes of recipients for spermatogenesis. Typically, if the SSCs are destined for transplantation, a marker is included to visualize the clones of spermatogenesis (e.g., lacZ or green fluorescent protein [GFP] transgenes). It is important to note that the isolated Thy1⁺ cell fraction does not consist of pure SSCs and that most germ cells are non-stem-cell progenitors, and a smaller portion of the population is somatic cells.