Establishment of porcine embryonic stem cells from parthenogenetically activated blastocysts

Porcine has been known to have a great impact on the studies of organ transplantation, biomaterial production andspecific biomodel development such as transgenic animals. To achieve such therapeutic purposes, establishment ofporcine embryonic stem cells (pESCs) will be needed. Especially, in vitro differentiation toward neural cells from pESCscan be a useful tool for the study of early neural development and neurodegenerative disorders. In addition, these cellscan also be used in cell replacement therapies and drug development for neuroprotective and/or neurotoxic reagents.Although several studies reported the successful isolation of pES-like cells, it has been a big challenge to determineoptimal conditions to generate pESCs without loss of pluripotency for a long time. The present study was performedfor generation and characterization of putative pESCs, and differentiation into neurons and astrocytes. In this study,porcine blastocysts were produced by parthenogenetically activated oocytes. The putative pESCs were cultured in pESCgrowth media supplemented with a growth factor and cytokines (bFGF, LIF and SCF). Subculture of pESCs wasconducted by mechanical dissociation using syringe needles after 4~5 days of incubation. As results, six putative pESClines were maintained over thirty passages. The putative pESCs were compact, round, flat, and single layered, whichwere similar to human embryonic stem cell morphologically. Six pES-like cells were positive for alkaline phosphataseactivity at every three passages. Furthermore, Oct-3/4, Sox-2, Nanog and SSEA-4 were shown to be expressed in thosecells. Also, normal karyotypes of pESCs were observed by Giemsa-staining. Differentiation potential into the three germlayers of the putative pESCs was demonstrated by the formation of embryoid bodies (EB). Besides, the study of ESCis very important in aspect of its application to not only the cell-based replacement therapies but also cellular differentiationresearch. Our results also showed that RA and N2 supplements activated the neural differentiation in pESCs.Neurofilament-160 were expressed in neural precursor cells. The expression of markers for specific neural lineages, suchas Microtubule-associated protein-2 expressed in matured neuron, was also induced from embryonic neural progenitors.In summary, the pESCs were generated from the parthenogenetically activated blastocysts and the typical characteristics of the cells were maintained for the long term culture. Furthermore, it was successful to differentiate the pESCs intovarious neural lineages through in vitro neurogenesis system. Eventually, pESCs will be excellent biomedicine inincurable and/or zoonotic diseases by regenerating the damaged tissue.