31 PORCINE SKIN-DERIVED STEM CELLS MAY BE A SUPERIOR SOURCE OF DONOR NUCLEI FOR EFFICIENT GENETIC MODIFICATION OF CLONED EMBRYOS

Abstract

Somatic cell nuclear transfer (SCNT) in pigs relies primarily on the utilization of fetal-derived fibroblast cells, and the resultant clones tend to exhibit a significant level of phenotypic instability, which may be due to epigenetic reprogramming and/or genomic damage in the donor cells. In addition to the compromised phenotypic stability, production of transgenic clones through SCNT is inefficient, because the restricted lifespan of somatic donor cells in culture can be limiting when the genetic modification requires selection. In contrast, stem cells proliferate rapidly and do not undergo senescence at a high rate, so the selection process can be extended. Since there is no report of an embryonic stem cell line derived in the pig that could contribute to the germ line, we decided to investigate the utility of porcine skin-derived stem cells (SSCs). Porcine SSCs were isolated from the skin on the back of day 35 to 50 Yorkshire fetuses. The SSCs were cultured continually in SSCs medium (DMEM/F12 containing B-27, 20 ng mL^–1 of epidermal growth factor, and 40 ng mL^–1 of basic fibroblast growth factor) at 37.8°C, 5% CO₂, 95% air. The SSCs expressed the neural progenitor marker nestin, as well as genes that are critical for pluripotency, such as Oct4 and Stat3. The SSCs proliferated actively in vitro and retained a normal karyotype after long-term culture. Electron microscopy revealed 2 distinct cell types within the spheres; elongated cells at the sphere periphery had invaginated nuclear envelopes and prominent nucleoli, and these cells displayed few, but large elongated mitochondria with transversal cristae as well as large cisternae of rough endoplasmic reticulum. In contrast, the cells in the center of the spheres were predominantly round-shaped, with a large round nucleus or cuboidal. The SSCs can be genetically modified with long-term positive selection, and 50 μg mL^–1 G418 appeared to be an appropriate dose of G418 for selection of the transfected SSCs. Finally, NT embryos reconstructed with SSCs showed high rates of pre- and post-implantation development.The cell number in the blastocyst stage embryos derived from cloning with the SSC was significantly higher than those of the blastocysts derived from IVF (28.5 ± 1.9, 16.8 ± 4.0, respectively, P < 0.05), although there was no significant difference in blastocyst formation rates between these groups (21 to 25%). Three of the animals became pregnant in 4 surrogate gilts which received cloned embryos and reached to term. Two healthy male cloned piglets and 1 healthy female cloned piglet are genetically identical to the SSCs.

Funding for this study was provided by the National Institutes of Health.