小鼠孤雌胚胎干细胞系的建立及生物学特性鉴定

目的:探讨建立合适的小鼠孤雌胚胎干细胞建系方法。方法:采用氯化锶联合细胞松弛素B激活B6D2F1杂交小鼠卵母细胞,所获得的囊胚与桑椹胚分别用于孤雌胚胎干细胞的建系,观察两者的建系成功率。结果:共建立了12株小鼠孤雌胚胎干细胞系,这些细胞SSEA-1抗原阳性,SSEA-4,TRA-1-81,TRA-1-60表面抗原阴性,具有AKP活性,保持正常染色体核型,体内外分化分别形成畸胎瘤和拟胚体。结论:采用囊胚和去透明带的桑葚胚建立孤雌胚胎干细胞系获得成功。该方法为人类纯合子的胚胎干细胞建系提供基础,在自体细胞治疗领域中具有潜在的应用价值。     

定向诱导小鼠ES细胞向心肌细胞的分化

为了提高体外诱导ES细胞向心肌细胞分化的效率 ,对以往的诱导方法加以改进 ,采用直接悬浮培养和 0 8%DMSO诱导 ,建立了简便、高效的定向诱导ES细胞向心肌细胞分化的体系 .诱导第 9d起可见自发性、有节律跳动的类胚体出现 ,第 14d达到高峰 ,约有 70 %的拟胚体产生跳动 .用RT PCR的方法在跳动的拟胚体中检测到心肌细胞特异性标志物的表达 ,采用免疫荧光染色的方法在蛋白水平检测到心肌特异的α辅肌动蛋白 (α actinin)的表达 ,并可见清晰肌小节 ,表明在改进的体外诱导条件下ES细胞可分化为成熟的心肌细胞 .     

在小鼠胚胎干细胞进行基因打靶的策略

基因打靶技术是一种通过同源重组按预期方式改变生物活体的遗传信息的实验手段,与小鼠胚胎干细胞培养系统相结合,使得人们可以方便地将各种突变引入小鼠体内,得以从生物整体水平上研究高等真核生物基因的表达、调控及其生理功能.扼要介绍了近年来在小鼠胚胎干细胞进行基因打靶的研究进展.     

利用胚胎干细胞分化的拟胚体研究小鼠早期胚胎发育过程

小鼠胚胎干细胞(ES细胞)具有分化的全能性已经得到广泛共识。ES细胞在体外分化所形成的拟胚体在结构上能够模仿早期胚胎发育过程,包括在内细胞团表面形成内胚层、柱状上皮细胞的分化,以及中央空腔的形成。本文介绍利用拟胚体研究小鼠早期胚胎发育过程中各个胚胎阶段的发育、细胞程序性死亡的发生及TGF-β信号在胚胎发育过程中的作用。     

建立对未分化细胞特异性标记的小鼠胚胎干细胞系

构建pRex-1-EGFP表达载体,电穿孔转染小鼠ES细胞,用增强绿色荧光蛋白对起源于3．5d胚泡内细胞团的小鼠胚胎干细胞进行特异性标记,用荧光显微观察EGFP的表达以及RT-PCR方法检测Rex-1基因在未分化和分化中ES细胞中的表达情况。结果显示,EGFP基因成功转入小鼠ES细胞,并在未分化的ES细胞中高效表达;细胞开始分化后,EGFP的表达开始下降。由Rex-1基因启动子控制下的EGFP稳定表达的小鼠ES细胞系,对哺乳动物早期发育过程的研究以及对筛选能够调节上述过程的小分子化合物具有重要意义。     

稳定整合pTet-on载体小鼠胚胎干细胞株的建立

目的建立稳定整合Tet-on基因的ES-D3细胞系,用于人胰岛素基因(pTRE2-human-Ins)在ES-D3细胞中诱导表达调控的研究。方法通过电穿孔转染的方法,将pTet-on质粒导入ES-D3细胞中,利用G418的药物选择特性,对转染的ES-D3细胞进行压力筛选,用PCR和Southern blot方法进行DNA整合鉴定,并用瞬时转染荧光素酶报告基因(pTRE-Luc)对筛选的Tet-on阳性克隆株的功能进行鉴定。结果经400μg/mL的G418压力筛选后,获得了11个细胞克隆株,特异性核苷酸引物检测细胞基因组DNA,有9个ES-D3细胞株可以扩增出相应的核苷酸片段,部分Tet-on阳性ES-D3株Southern blot鉴定结果表明Tet-on基因片段已整合入ES-D3细胞基因组DNA,荧光素酶报告基因功能鉴定获得1株诱导表达倍率为21.31的ES-D3细胞株,且Tet-on基因阳性ES-D3细胞的形态和生长速度与正常ES-D3细胞没有差异。结论通过电穿孔转染的方法成功地获得1株诱导表达倍率为21.31的高表达ES-D3细胞株,为进行目的基因(pTRE2-human-Ins)在ES-D3细胞中转染和诱导表达打下了基础。     

从早期胚胎多能干细胞生成的嵌合鼠

本文利用囊胚注射法将小鼠胚胎多能干细胞－ＣＣＥ细胞注射到发育３天半的昆明和Ｃ３７ＢＬ／６Ｊ小鼠受体囊胚腔内,经假孕鼠借腹怀胎,获３只ＣＣＥ细胞毛色嵌合鼠。实验共注射胚胎６５４个,经培养其恢复成活率７３．８％,胚胎移植后,假母受孕率及产仔率分别为３２．９％和５３％。在所获３只嵌合鼠中,２只为ＣＣＥ－昆明毛色嵌合鼠,１只为ＣＣＥ－Ｃ３７ＢＬ／６Ｊ毛色嵌合鼠,这是国内首次利用胚胎多能干细胞获得嵌合鼠。为     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

分离和克隆小鼠ES细胞集落的主要影响因素

目的　研究不同培养条件分离和克隆小鼠ES细胞集落的效率。方法　以PMEF饲养层、NIH3T3细胞饲养层或培养液中加入LIF为培养条件 ,分离和克隆昆明小鼠ES细胞集落 ,比较其效率。结果　饲养层的培养条件明显优于培养液中加入LIF的培养条件 ;有饲养层的培养条件下 ,桑椹胚的ES细胞集落出现率显著低于囊胚 ;两种饲养层培养囊胚 ,其ES细胞集落的出现率差异无显著性。结论　以PMEF或NIH3T3细胞作饲养层 ,培养昆明小鼠的囊胚 ,适时离散ICM ,是比较理想的分离ES细胞集落的方法。     

Pluripotency and Epigenetic Factors in Mouse Embryonic Stem Cell Fate Regulation

Embryonic stem cells (ESCs) are characterized by their ability to self-renew and to differentiate into all cell types of a given organism. Understanding the molecular mechanisms that govern the ESC state is of great interest not only for basic research—for instance, ESCs represent a perfect system to study cellular differentiation in vitro—but also for their potential implications in human health, as these mechanisms are likewise involved in cancer progression and could be exploited in regenerative medicine. In this minireview, we focus on the latest insights into the molecular mechanisms mediated by the pluripotency factors as well as their roles during differentiation. We also discuss recent advances in understanding the function of the epigenetic regulators, Polycomb and MLL complexes, in ESC biology.     

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum‐free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.Download video file.^{(69M, mov)}     

小鼠胚胎干细胞的培养

目的:建立小鼠胚胎干细胞(embryonic stem cells,ES)的培养方法。方法:制备G418抗性的原代小鼠胚胎成纤维细胞,经丝裂霉素C处理后成滋养层细胞,将小鼠胚胎干细胞复苏后,应用含白血病抑制因子的ES细胞培养液,培养小鼠ES细胞,观察集落的生长情况,并在光镜下观察细胞形态。结果:小鼠胚胎成纤维细胞生长良好,ES细胞呈克隆状生长,且保持未分化状态。结论:建立了小鼠胚胎干细胞培养的有效方法,为下一步基因打靶奠定基础。     

Cell Cycle-Dependent Turnover of 5-Hydroxymethyl Cytosine in Mouse Embryonic Stem Cells

Hydroxymethylcytosine in the genome is reported to be an intermediate of demethylation. In the present study, we demonstrated that maintenance methyltransferase Dnmt1 scarcely catalyzed hemi-hydroxymethylated DNA and that the hemi-hydroxymethylated DNA was not selectively recognized by the SRA domain of Uhrf1, indicating that hydroxymethylcytosine is diluted in a replication-dependent manner. A high level of 5-hydroxymethylcytosine in mouse embryonic stem cells was produced from the methylcytosine supplied mainly by de novo-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the HoxA gene cluster showed a high hydroxymethylation level whilst the methylcytosine level was quite low, suggesting that methylated CpG is actively hydroxylated during proliferation. All the results indicate that removal and production of hydroxymethylcytosine are regulated in replication-dependent manners in mouse embryonic stem cells.     

小鼠胚胎干细胞向生殖细胞分化的研究

小鼠胚胎干细胞(ESC)在体外可以分化为多种细胞类型,其中包括各阶段的生殖细胞,甚至精细胞和成熟卵母细胞。ESC向生殖细胞分化的效率受到包括生长因子、激素和体细胞等多种因素的影响,在体外形成的是雌性配子还是雄性配子与ESC是XX型还是XY型没有必然联系。简要综述了小鼠生殖细胞在体内外的分化发育、性别决定和增殖等,并总结和展望了ESC向生殖细胞分化研究面临的问题和应用前景。     

615小鼠ES细胞集落的分离及初步鉴定

目的　分离和培养 6 15小鼠的ES细胞集落 ,为建系打下基础。方法　以PMEF为饲养层分离 6 15小鼠的ES细胞集落 ,进行无饲养层培养 ,并对其进行初步鉴定。结果　ES细胞集落的出现率和传代成功率为 2 2 6 %和 0 94 % ,其ALP染色阳性 ,具有稳定的二倍体核型 ,可自发分化为多种类型的细胞。结论　成功分离和培养了6 15小鼠的ES细胞集落     

Targeted Proteomics of the Secretory Pathway Reveals the Secretome of Mouse Embryonic Fibroblasts and Human Embryonic Stem Cells

Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach for interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs, respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study in MEFs and hESCs, respectively, have not been reported in previous MS analyses.The identification of low abundance secreted proteins via MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 h. In contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology: obtaining a temporal snapshot of proteins secreted in response to a differentiation trigger, and identifying proteins secreted by cells that are isolated from a heterogeneous population.Human embryonic stem cells (hESCs)¹ are pluripotent cells isolated from the inner cell mass of a pre-implantation blastocyst stage embryo (1). They have potential applications in regenerative medicine, are an attractive source of human cells for drug evaluation, and are useful models for understanding human development. The self-renewal or differentiation of hESCs is controlled by endogenous proteins secreted by hESCs and by exogenous factors present in cell culture medium (2, 3). For instance, hESCs are routinely cultured on feeder layers of mouse embryonic fibroblasts (MEFs) or on Matrigel-coated plates in mouse embryonic fibroblast–conditioned medium (MEF-CM). In these cases, cytokines secreted by MEFs and present in MEF-CM, together with cytokines and extracellular matrix (ECM) proteins secreted by hESCs, form a localized microenvironment that regulates hESC fate.The comprehensive identification of proteins secreted by MEFs and hESCs—their cellular secretome—can help unravel the molecular mechanisms that regulate hESC fate. Yet the use of MS-based approaches for secretome analysis remains challenging. In general, secretome studies of various cell types have relied on MS analysis of cell culture supernatants (reviewed in Ref. 4). However, such an approach typically results in the identification of small numbers of extracellular proteins. This was indeed the case with MS analysis of conditioned medium (CM) from MEFs or other feeder cells that support the maintenance of undifferentiated hESCs (5–8). A low abundance of secreted proteins of interest and a high concentration of serum proteins in cell culture media significantly impede MS analysis. To overcome these limitations, Bendall et al. implemented an iterative-exclusion MS (IE-MS) strategy, in conjunction with the use of medium without serum or serum replacer, for the identification of proteins secreted by MEFs and hESCs (2). Using this approach, large numbers of previously unreported proteins secreted by MEFs and hESCs could be identified, showing that IE-MS is a powerful strategy for the identification of low abundance proteins. However, the use of medium without serum or serum replacer for secretomic analysis can be problematic. Specifically, the use of a “blank” or serum-free medium might alter cellular physiology and, consequently, the profile of secreted proteins. Indeed, we observe that hESCs are highly prone to apoptosis under such growth conditions. Moreover, an analysis of the cell culture supernatant is not specifically targeted toward endogenously secreted ECM proteins, which are also an important component of the cellular microenvironment. ECM proteins form a matrix that associates with the cell and might not be present in the cell culture supernatant. Moreover, many growth factors are known to be sequestered by ECM proteins and might not be released into the culture medium (9). Here we present a rigorous evaluation of an alternate strategy to interrogate the entire cellular secretome, including cytokines and ECM proteins. Notably, our approach does not require the use of customized media lacking serum and serum replacers, and it is compatible with cell culture systems utilizing media of unknown or poorly defined composition, such as CM from MEFs.To identify the secretome of MEFs and hESCs, we carried out an MS analysis of their subcellular fractions that were enriched in secretory pathway organelles. The secretory pathway comprises the endoplasmic reticulum (ER), the Golgi apparatus, and the associated transport vesicles. Detailed MS analysis of these organelles identifies the secretory cargo (i.e. proteins destined to be secreted) in addition to the secretory pathway proteome (10). Indeed, we have previously identified several secreted proteins in hESCs as a result of contamination by the ER and Golgi (11) in our subcellular fractions. In light of these reports, we hypothesized that targeted proteomic analysis of the secretory pathway is a viable approach for comprehensive characterization of the cellular secretome. Accordingly, we developed protocols to isolate subcellular fractions enriched in the ER and Golgi compartments from MEFs and hESCs, and we subsequently carried out MS analysis on these samples. Several proteins secreted by MEFs and hESCs could be identified in this manner. Strikingly, the numbers of proteins identified were comparable to those obtained with the highly efficient IE-MS approach. Furthermore, we also show that short-term changes in medium composition affect the profile and quantitative levels of several proteins that transit through the secretory pathway, including secreted and membrane proteins. Taken together, our results validate the use of targeted secretory pathway proteomics as a powerful alternate approach to interrogate the cellular secretome.