Metabolic properties of chicken embryonic stem cells

Cellular energy metabolism correlates with cell fate,but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood.Using a previously established chES cell model and electron microscopy (EM),we found that undifferentiated chES cells stored glycogen.Additionally,undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells,suggesting that chES cells direct glucose flux towards the glycogenic pathway.Moreover,we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway,as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs.Additionally,cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining,but it could be rescued by exogenous G6P.However,we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining.Moreover,differentiated chES cells expressed higher levels of GLUT1,HK1 and PFK mRNAs,while the level of GYS mRNA remained similar in control CEF cells.These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P,while differentiated chES cells have a decreased glycogen reserve,which suggests that the amount of glycogen is indicative of the chES cell state.

Effect of Down-regulated Expression of cPouV and cNanog on Pluripotency of Chicken(Gallus gallus) Embryonic Stem Cells(cESCs)

To explore the pluripotency maintenance and update the functional influence of pluripotency genes cNanog and cPouV in chicken （ C,a/lus gallus） embry- onic stem cells （ cESCs）, the stable RNAi vectors pSuper-cNanog and pSuper-cPouV constructed previously were used to transfect cESCs. The mRNA levels of two target genes were detected with real- time PCR. These two genes were down-regulated since the 48^th and the down-reg-lation continued with the extension of time, the interference efficiency reached 65% at 96^th hour （P 〈0.05）. With the down-regulation of cNanog or cPouV gene, cESCs showed differentiation and prolifera- tion rate of these cells slowed down, the domed colony of these cells disappeared gradually when the edge of colony became irregular. At 96^th hour after transfection, the alkline phosphatase （AKP） and stage-specific embryonic antigen-1 （ SSEA-1 ） were not be detected in cNanog gene-knecked out eESCs, but it was done in that with cPouV gene -knocked out. The cPouV-suppressing cESCs were again transfected with pSuper-cNanog, the pluripotency markers AKP and SSEA-1 were both not found expressing at the 48^th hour. The results showed that cPouV and cNartog genes played an important role in maintaining pluripotency and self- renewal in cESCs, and cNanog gene was dominant. To sum up, our results may provide insights into the molecular regulation mechanism of avian during development.