66 ISOLATION, CHARACTERIZATION, AND NUCLEAR REPROGRAMMING OF STEM CELLS DERIVED FROM ADULT PORCINE LIVER AND FAT

Abstract

Genetic manipulation of the porcine genome to produce genetically modified pigs for various biomedical and agriculture applications has been hampered by the lack of an ideal cell type that can grow in culture for a long period of time and is amenable to various genetic manipulations with high efficiency. The cell type currently used for various genetic manipulations is fetal fibroblast. These cells have very limited life span in culture and the efficiency of gene targeting is very low. There is no report of isolation of functional embryonic stem cells (ESC) from pig that would have been used to produce transgenic pigs with high efficiency as has been possible in mice. Recently, porcine induced pluripotent stem cells (iPSC) have been reported by 3 groups. However, they have yet to be tested for genetic manipulations and production of transgenic pigs. In this study, we developed a simple but novel strategy to recover stem cells from adult porcine liver and adipose tissues. Small colonies with few cells became visible as early as 2 to 3 days under reduced oxygen conditions on collagen-coated plates, and a full-grown colony with a fibroblast-like morphology took 10 to 14 days to form. Ten single colonies per tissue were isolated, subcultured, and monitored for growth and gene expression. Both of these cell types maintained steady growth through 70 population doublings (at the time of writing) and are still growing without any change in their morphology. Reverse transcription PCR was used to monitor gene expression. Both cell types show strong expression of c-Myc and KLF4. Moreover, low expression of Oct-4 and Lin28, 2 important genes related to pluripotency, was also detected in cells derived from adipose tissues, indicating that these cells could be easily reprogrammed to pluripotent state. These cells maintained a normal karyotype after long-term culture. Cell lines with stable genetic modifications and extended expression of transgene were obtained when these cells were transfected with a plasmid containing the neomycin resistance gene and selected under G418. Further, these 2 cell types, liver stem cells (LSC) and fat stem cells (FSC), and fetal fibroblasts (FF) as a control were used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos. The average fusion rates were 87, 81, and 89% for LSC, FSC, and FF, respectively. Of 2 recipients receiving nuclear transfer embryos produced with each cell type, one established pregnancy at Day 30 (50%). Efficiencies were 5% (11 fetuses/223 embryos transferred), 1.8% (4 fetuses/228 embryos transferred),and 5% (11 fetuses/219 embryos transferred) for LSC, FSC, and FF, respectively. Thus, these adult liver and fat stem cells are attractive cell types for cloning valuable adult animals with high efficiency and for SCNT transgenesis.