Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

Serum-free culture of rhesus monkey embryonic stem cells

Previous studies have shown that the maintenance and proliferation of undifferentiated rhesus monkey embryonic stem (rES) cells requires medium supplemented with fetal bovine serum (FBS). Due to the uncharacterized composition and variation in serum nature, the present study aimed to replace the serum-containing medium with a serum-free medium in the rES cell culture. The results showed that after the initial 48-h culture in the routinely used serum-containing medium, rES cells can grow and proliferate for a prolonged period in the serum-free medium composed of DMEM supplemented with a cocktail of BSA, IGF-1, TGF-alpha, bFGF, aFGF, estradiol, and progesterone. rES cells cultured in the serum-free medium maintained high level of alkaline phosphatase activity and OCT4 level. There was no indication of differentiation as judged by the marker gene expression of all three embryonic germ layers and trophoblast. In addition, serum-free culture would not affect the passage capacity and differentiation potential of rES cells. This work will facilitate the future study of induced differentiation of rES cells and other applications.

Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells.

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Morphine induces Beclin 1-and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus

Chronic exposure to morphine can induce drug addiction and neural injury, but the exact mechanism is not fully understood. Here we show that morphine induces autophagy in neuroblastoma SH-SY5Y cells and in the rat hippocampus. Pharmacological approach shows that this effect appears to be mediated by PTX-sensitive G protein-coupled receptors signaling cascade. Morphine increases Beclin 1 expression and reduces the interaction between Beclin 1 and Bcl-2, thus releasing Beclin 1 for its pro-autophagic activity. Bcl-2 overexpression inhibits morphine-induced autophagy, whereas knockdown of Beclin 1 or knockout of ATG5 prevents morphine-induced autophagy. In addition, chronic treatment with morphine induces cell death, which is increased by autophagy inhibition through Beclin 1 RNAi. Our data are the first to reveal that Beclin 1 and ATG5 play key roles in morphine-induced autophagy, which may contribute to morphine-induced neuronal injury.