Abstract

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.