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.
LI JiaZHANG BaoLuHAN HongBingCAO ZhiChengLIAN ZhengXingLI Ning
The chicken embryo is a classic model used to investigate embryonic development, gene expression, and tissue differentiation, and is also an important research tool in studying the animal functional genomics. The whole blastoderms of fresh unincubated eggs from White Leg-horn chickens were collected with a paper ring, mechanically broken into small pieces and cul-tured in medium. Then the small pieces would develop into blastocyte-like structures (BLS), which could be facilitated by an addition of fetal bovine serum (FBS) to the primary culture and their diameter was nearly doubled from 12 to 24 h. The additional yolk had no positive effect on the development in the first 12 h but encouraged the BLSs attaching and inner cells differentiat-ing instead in 24 h. The inner cells of the BLS showing a high alkaline-phosphatase activity similar to those in mouse embryonic stem (ES) cells and also expressing a large amount of the specific stage embryonic antigen-1 (SSEA-1) on the surface, which was known to be the char-acteristic of non-differentiated mouse and avian ES cells, could finally differentiate into nerve-like cells, fibroblast cells and so on in the medium. Leukemia inhibitory factor (LIF) facilitated the cells’ proliferation and prevented differentiation in the suspended culture of the BLSs. So we drew the conclusion that the BLS obtained from broken blastoderm can be used to amplify avian ES cells so as to initiate a new method of producing transgenic chickens.
LI Jia1, PAN Qiuzhen1, LI Junying1, HAN Hongbing1, SUN Shufeng1, YANG Jun1, XU Shuguang, TIAN Liang, LIAN Zhengxing1,2, YANG Ning1,2 & LI Ning2,3 1. College of Animal Science and Technology, China Agricultural University, Beijing 100094, China
由鸡Ⅹ期胚盘获得的类囊胚包含禽类胚胎干细胞,且具有较强的增殖与分化能力。体外培养12、24、36 h的类囊胚细胞S tathm in基因的表达高于Ⅹ期胚盘细胞,证实增殖的细胞为禽类胚胎干细胞。超微结构观察表明,类囊胚细胞在早期增殖过程中以卵黄蛋白为能量的主要来源,当卵黄蛋白消耗殆尽,则细胞出现大量死亡。胚胎干细胞培养液(ESM)能够促进类囊胚细胞的增殖却不能减少死亡。在ESM基础上添加5 mm o l/L 6-磷酸葡萄糖(G 6P),不仅各期细胞增殖显著,且24 h细胞死亡数的比例(D/T)由23%下降到5.6 7%(P<0.0 5),3 6 h细胞D/T由3 0%下降到1 5.6 3%(P<0.0 5)。结果表明,在ESM基础上添加5 mm o l/L G 6P可促进类囊胚细胞的增殖,并明显改善能量匮乏导致的细胞死亡。
