Isolation and in vitro expansion of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are the only adult stem cells capable of passing genetic information to offspring through their ability to both self-renew and differentiate into mature spermatozoa. SSCs can be transplanted to establish donor-derived spermatogenesis in recipient animals, thus offering a novel reproductive tool for multiplication of elite individual animals to benefit livestock production. An optimal SSC culture in vitro can benefit various SSC-based studies and applications, such as mechanistic study of SSC biology, SSC transplantation process and SSC-based transgenesis technique. However, except for some model rodent animals, SSC culture remains an inefficient and unstable process. We here studied a workflow to isolate, purify and in vitro culture porcine SSCs from neonatal pig testes. Pig testicular cells were dissociated by two-step enzymatic digestion with collagenase type IV and trypsin. We enriched the spermatogonia from the testicular cell mix by differential plating for at least 3 times to remove firmly attached non-SSCs. We then tested the optimal culture medium formula by supplementation of different growth factors to the basic medium (DMEM/F12 + 1% FBS) and found that a combination of 20 ng/ml GDNF, 10 ng/ml LIF, 20 ng/ml FGF2 and 20 ng/ml IGF1 had the best effect on SSC growth in our defined experimental system. In the presence of 4 growth factors without specific feeders, the purified SSCs can be cultured in poly-L-lysine- and laminin-coated dishes for 28 days and remain preserving a continuous proliferation without losing the undifferentiated spermatogonial phenotype.