387 DERIVATION OF PUTATIVE EMBRYONIC STEM CELLS FROM PORCINE PARTHENOGENETIC BLASTOCYSTS AND THE LOSS OF PARENTAL-SPECIFIC EXPRESSION PATTERNS IN Igf2/H19 GENES

Abstract

Porcine embryonic stem cells (ESC) can be a useful tool for the production of a transgenic animal and the study of developmental gene regulation. The study of porcine parthenogenetic ESC might also provide advantages in the understanding of changes in human parthenogenetic embryonic stem cells in the culture environment. Because human embryonic stem cells must be maintained stably for therapeutic uses, parthenogenetic porcine embryonic stem cells can give us precious information to help understand human parthenogenetic embryonic stem cells. Three putative porcine embryonic stem cell lines were derived from 99 parthenogenetic embryos. Cumulus-oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro. Diploid parthenogenetic zygotes were produced by electrical activation followed by cytochalasin B treatment to suppress second polar body extrusion. Embryos were cultured to the blastocyst stage. Hatched blastocysts were directly cultured on mitomycin C-inactivated murine embryonic fibroblasts as feeder layers. Primary colonies were formed after 7 days of culture, and the colonies were transferred to new culture dishes 7 days after. They were passsaged every 5 days by physical dissociation, with one colony divided into small clumps and maintained for over 30 passages. These cells morphologically resembled human embryonic stem cells and consistently expressed the markers of pluripotent cells such as alkaline phosphatase, NANOG, OCT-4, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. They could be maintained holding the previous characteristics after cryopreservation. Furthermore, we conducted experiments to confirm the expression patterns of the imprinted genes Igf2 and H19 in these ESC and IVF/parthenogenetic blastocysts using quantitative real-time PCR. At the blastocyst stage, the 2 genes were expressed in a parental-specific manner according to their origins in normal fertilized embryos and uniparental embryos. The putative parthenogenetic ESC, on the other hand, showed a high expression of Igf2, the paternally expressed gene, when compared with their blastocyst counterparts. Current work aims to confirm the authenticity of these ESC via teratoma formation in severe combined immunodeficiency mice following injection with these putative parthenogenetic ESC.

This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).