Neural stem cells (NSCs) are self-renewing tripotent cell populations and have capacity of neuronal (neurons) and glial (astrocytes and oligodendrocytes) differentiation. Many researchers have reported that NSCs have therapeutic effects in neurological disease by transplantation. However, it is not easy to obtain NSCs in vitro. Recently, Yamanaka and colleagues showed that somatic cells could be reprogrammed into pluripotent state by enforcing reprogramming factors. Induced pluripotent stem (iPS) cells undergo unlimited self-renewal and have differentiation potential into various types of cells like embryonic stem cells. Direct differentiation into a specialized cell types from iPS cells hold considerable promise for regenerative medicine as well as basic research. Here, we induced differentiation of iPS cells into NSCs in vitro and in vivo, which were compared with embryonic stem (ES) cell-derived NSCs and brain derived NSCs. NSCs from ES and iPS cells were morphologically indistinguishable from brain derived NSCs and stained positive for NSCs markers Nestin and Sox2. ES cells derived NSCs were transcriptionally distinguishable from brain derived NSCs. However, global gene expression pattern were similar but distinct between iPS derived NSCs and brain derived NSCs. Moreover, iPS derived NSCs were spontaneously aggregated upon passaging, formed ES cell like colonies, and finally reactivated Oct4-GFP. The spontaneously reverted GFP-positive cells (iPS-NSC-iPS) expressed similar levels of pluripotency markers (Oct4,Nanog) to ES and iPS cells, and could form germ line chimera. One possible explanation for this phenomenon is that spontaneously re-reprogramming was associated with transgene re-activation when iPS cells were differentiated into NSCs. However, NSCs from dox-inducible iPScells could not be reprogrammed into pluripotent state without doxycycline. Taken together, iPS derived NSCs were morphologically and similar to brain derived NSCs, but differ in gene expression pattern and maintenance. * This work was supported by the Next Generation Bio-Green21 Program funded by the Rural Development Administration (Grant PJ008009).

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.