Isolation and in vitro expansion of porcine spermatogonial stem cells

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.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture

“…This regulation of stem cell fate is mediated by mechanical, adhesive, and topological cues interacting with dynamic soluble factors [80][81][82] where the biophysical cues can initiate mechanotransduction inducing intracellular biochemical and functional responses by stem cells [83,84]. Feeder-free systems have recapitulated this structural support by using hydrogels including Matrigel [19], agarose [85], poly-L-lysine and laminin [86,87]. Spermatogonia cultured in transwells were not provided with similar structural support, which may have in turn modulated their response to soluble factors, reducing their proliferation.…”

Optimized Recovery of Immature Germ Cells after Prepubertal Testicular Tissue Digestion and Multi-Step Differential Plating: A Step towards Fertility Restoration with Cancer-Cell-Contaminated Tissue

“…Furthermore, recovery of undifferentiated germ cells is also challenging because these cells are known to be highly vulnerable to exposure to cell stress-inducing conditions [ 23 ], including high concentrations of digestion enzymes [ 24 ], and do not properly adhere to uncoated plastic dishes [ 25 ].…”

“…So far, the majority of studies aimed at germ cell recovery or enrichment rely on the adherence property of testicular somatic cells to uncoated plastic dishes for their elimination and do not consider the fraction of germ cells that adhere to such dishes and are thus discarded [ 25 , 26 ]. Germ cell-enriched populations thus only originate from the second plating as reported in experiments using human adult testicular tissue [ 27 , 28 ].…”