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Establishment and Characterization of Pig iPS and Female Germ Line Stem Cells
Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. To overcome difficulties in preparing mice oocyte extract, a pig GV oocyte extract (pGV extract) was developed to investigate the epigenetic reprogramming events in treated somatic cell nuclei. The pGV extract promoted colony formation concomitant with the expression of stem cell markers and repression of differentiated cell markers in treated cells. Using fibroblasts transfected with human Oct‐4 promoter‐ driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4‐EGFP cells by 10 days post‐treatment. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Using donor nuclei treated with pGV extract, increase the number of high‐quality blastocysts that expressed Me‐H3‐K9, Oct4 and Nanog at levels comparable to in vitro fertilized embryos. The pGV extracttreated fibroblast cells can differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig. Next, we identified germ line stem cells that supported oogenesis. female germ line stem cells (FGSC) from neonatal pig was established and cultured for more than 6 months. After long‐term culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. To further study developmental potential of oocyte‐like cells generated from FGSCs, these cells were aggregated with granulosa cells collected from neonatal pig ovaries. Interestingly after overnight culture in hanging drops, oocyte‐like cells aggregated with granulosa cells and formed structures very similar to primordial follicles containing the oocyte‐like cell in the middle and a layer of granulosa cells around it. Our results demonstrate the presence of a population of germ line stem cells in postnatal pig ovary with the ability to self‐renew and differentiate to oocyte‐like cells that might be useful for follicle engineering and assisted reproductive technologies. However, the functionality of FGSC‐derived oocytes us-ing in vitro maturation, fertilization and embryo development as well as ovarian transplantation is currently under investigation. In conclusion, gene manipulation of FGSCs or iPS cells is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.
