104. PROLIFERATION ABILITY, TELOMERASE ACTIVITY AND  MOLECULAR CHARACTERIZATION OF PLURIPOTENT CELL LINES FROM IVF AND PARTHENOGENTIC PIG EMBRYOS

Abstract

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.