Background: Pluripotent stem cells (PSCs) are capable of differencing into various cell types in the body, providing them valuable for therapy of degenerative diseases. Patientspecific treatments using PSCs, such as mesenchymal stem cells in patient’s own body, may reduce the risk of immune rejection. Inducing the differentiation of PSCs into vascular endothelial cells (ECs) altering culture conditions or using specific growth factors is able to applied to the treatment of vascular diseases. The purpose of this study was to induce the differentiation of porcine epiblast stem cells (pEpiSCs), bone marrow-derived mesenchymal stem cells (pBM-MSCs) and adipose-derived mesenchymal stem cells (pAMSCs) into ECs and then examine the functionality of vascular ECs. Methods: Porcine pEpiSCs, pBM-MSCs and pA-MSCs were induced to differentiate into ECs on matrigel-coated plates in differentiation medium (EBM-2 + 50 ng/mL of VEGF) for 8 days. Cells differentiated from these stem cells were isolated using CD-31 positive (+) magnetic-activated cell sorting (MACS) and then proliferated in M199 medium. Evaluation of ECs differentiated from these stem cells was treated with capillary-like structure formation and three-dimensional spheroid sprouting assay. Results: Porcine pEpiSCs, pBM-MSCs and pA-MSCs showed similar expression of pluripotency-related genes (OCT-3/4. NANOG, SOX2). These stem cells were differentiated into vascular ECs, but showed different morphologies after the differentiation. Cells differentiated from pEpiSCs showed an elongated spindle-like morphology, whereas cells differentiated from pBM-MSCs showed a round pebble-like morphology. In the case of pA-MSCs, these two morphologies were mixed with each other. Additionally, vascular ECs differentiated from these stem cells showed different formation of capillary-like structure formation and three-dimensional spheroid sprouting assay. Conclusions: Cells differentiated from pEpiSCs, pBM-MSCs and pA-MSCs presented the functionality of different vascular ECs, demonstrating the potential of the excellent ECs differentiated from pEpiSCs.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Since embryonic stem cells (ESCs) were first established from explant cultures of in vivo day 3.5 mouse embryos, the establishment of ESCs from species such as primates and rat has been developed. However, this success relies on the development of culture medium suitable for human and rat cells, which has different requirements from the murine ESC. In general, the establishment of ESC from pig and cow is of great interest both the agricultural perspective and for biomedical application. Large animal models, particularly pig, are likely to provide models of human genetic diseases and transplantation research where rodent models are inappropriate. However, establishment of ESCs establishment from pigs has remained an elusive goal. In the present study, we focused on signaling transduction regulation in pig epiblast stem cells (pEpiSCs). Pig epiblasts were isolated from early tubular stage embryos collected in vivo day 10.5～12 after insemination. Epiblasts were separated from trophoblast and underlying primitive endoderm using 21G needles and fine forceps. Epiblasts were cultured on mitomycin C (10 μl/ml) treated mouse embryonic feeder cells in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% minimal essential medium (MEM) nonessential amino acids, 1% penicillin/ streptomycin, 1% glutamine, 0.007% β-mercaptoethanol, 5 ng/ml bFGF and 1 ng/ml LIF. After plating rapid differentiation of isolated epiblasts to extraembryonic cell types was visualized in most cultures but stem cells were enclosed by these differentiated cells. We have established seven pig epiblast stem cells lines (pEpiSC1-7) from Days 10.5–12 pig embryos. pEpiSC expressed the pluripotent markers including OCT4, NANOG, SOX2 and NODAL at 3-5 passage. In addition, the modification of culture condition by the inclusion of particular protein kinase inhibitor such as Akt inhibitor, PD0325091(PD), delyed rapid differentiation of pEpiSCs. These results showed that stemness of pEpiSCs can be maintained by regulation of signaling pathway. * This work was partly supported by a grant from the NPR (2011-0013703) and the Next-Generation BioGreen 21 Program (No. PJ008209), Rural Development Administration, Republic of Korea.