CREG低表达饲养层STO细胞的建立

目的:为阐明E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)对胚胎干细胞(Embryonic stem cell,ESC)自我更新的影响及作用机制,本研究拟通过RNA干扰方法建立CREG低表达的饲养层STO细胞,为深入研究奠定基础。方法:用Western Blot方法检测饲养层细胞系STO及ESC细胞系R1中CREG基因的表达。利用Lipofectamine 2000向STO细胞中分别转染RNA干扰空对照载体,含有无意义随机序列的对照载体以及含有4种不同CREG干扰序列的载体。用1.0μg/ml嘌呤霉素筛选1 w,荧光显微镜下挑取绿色荧光蛋白表达较高的克隆进行扩增。Western Blot方法鉴定CREG基因干扰效率,获得CREG表达最低的饲养层细胞克隆。将ESC R1接种到该克隆上,不添加白血病抑制因子,连续培养3代,用碱性磷酸酶染色判断其是否分化。结果:R1几乎不表达CREG,而STO细胞高表达CREG。Western Blot结果证实筛选到的STO克隆3A干扰效果最好,达到85%。在不添加白血病抑制因子的情况下,碱性磷酸酶染色表明R1细胞在该株饲养层细胞上连续培养3代后未见明显分化。结论:成功获得CREG低表达饲养层STO细胞,为深入探讨CREG对ESC自我更新的作用及机制奠定了基础。     

饲养层细胞对绵羊胚胎干细胞体外培养的影响

家畜胚胎干细胞(embryonic stem cell,ES细胞)的研究进展缓慢,绵羊ES细胞的研究虽早有报道,但仍未建立可稳定传代的细胞系。在已建立的绵羊体外受精发育体系的基础上,摸索了饲养层(Feeder)细胞对绵羊ES细胞生长的影响,包括在一定的丝裂霉素浓度下处理Feeder的时间、细胞种类、代数、接种密度及新鲜制备和冷冻复苏后的Feeder细胞,通过试验比较研究,目的在于筛选合适的饲养层细胞,为建立绵羊ES细胞体外培养体系奠定基础。结果表明,10μg/ml丝裂霉素C处理2~2.5h获得的1~5代的SEF和1~3代的MEF及两者的1∶1混合细胞都能较好地支持绵羊ES细胞的生长。     

大鼠骨髓间充质干细胞分离与培养条件的优化研究

目的:建立和优化大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的分离培养条件,以获得群体均一、未分化状态保持良好的MSCs.方法:收集不同周龄大鼠骨髓细胞;以不同浓度Percoll密度梯度离心分离骨髓单个核细胞;以60%低糖DMEM40%MCDB201为基础培养基,培养24h去悬浮细胞;以不同接种密度传代培养;碱性磷酸酶染色和油红O染色考察MSCs向骨和脂肪组织分化的潜能.结果:采用57%Percoll液的分离效果优于70%Percoll液.6周龄(体重约180g)大鼠能在细胞分离的质和量上达到最佳效果.24h进行悬浮细胞去除、5×103/cm2接种密度传代培养,光镜和电镜显示MSCs增殖能力强,功能状态活跃,成脂成骨实验显示多向分化潜能保持良好.结论:优化大鼠周龄、分离液的密度、细胞培养条件及改进培养方法有助于获得多向分化潜能保持良好的均一的MSCs.     

长期培养小鼠胚胎干细胞拟胚体(EB)的观察

胚胎干细胞在体外培养条件下能够维持自我更新,并具有向多种细胞类型分化的能力,因此被广泛用于研究细胞分化的分子机理以及药物筛选.形成拟胚体(Embryoid body,EB)是胚胎干细胞分化常用的技术手段.为了便于今后利用EB做进一步的药物筛选及分化研究,严格规范了形成EB的条件,得到了分化状态均一性很高的EB.利用这一条件,观察到在分化条件下长期培养(长达60 d)的EB中仍有表达各项多能性指标的细胞集落.有关这一现象的进一步分析工作正在进行中.     

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。     

潮霉素抗性的小鼠胚胎干细胞滋养层的制备

目的：获得潮霉素抗性的小鼠胚胎干细胞滋养层。方法：利用基因组整合了潮霉素B磷酸转移酶基因的小鼠品系Smad4hygro＋/-,从受精14d的小鼠胚胎中制备原代胚胎成纤维细胞;鉴定基因型后,挑选一株潮霉素B磷酸转移酶基因杂合型胚胎成纤维细胞,用丝裂霉素C处理,以获得潮霉素抗性的滋养层细胞。结果：经潮霉素B加压处理后,杂合型滋养层细胞可存活2周左右,并可维持胚胎干细胞的生长。结论：制备的滋养层细胞可用于潮霉素抗性胚胎干细胞的筛选。     

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.     

小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础     

Using embryonic stem cells to form a biological pacemaker via tissue engineering technology

Biological pacemakers can be achieved by various gene‐based and cell‐based approaches. Embryonic stem cells (ESCs)‐derived pacemaker cells might be the most promising way to form biological pacemakers, but there are challenges as to how to control the differentiation of ESCs and to overcome the neoplasia, proarrhythmia, or immunogenicity resulting from the use of ESCs. As a potential approach to solve these difficult problems, tissue‐engineering techniques may provide a precise control on the different cell components of multicellular aggregates and the forming of a construct with‐defined architectures and functional properties. The combined interactions between ESC‐derived pacemaker cells, supporting cells, and matrices may completely reproduce pacemaker properties and result in a steady functional unit to induce rhythmic electrical and contractile activities. As ESCs have a high capability for self‐renewal, proliferation, and potential differentiation, we hypothesize that ESCs can be used as a source of pacemaker cells for tissue‐engineering applications and the ambitious goal of biological cardiac pacemakers may ultimately be achieved with ESCs via tissue‐engineering technology.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立

建立一种精确定量人胚胎干细胞线粒体DNA拷贝数的方法。构建包含线粒体DNANDl和核单拷贝基β-globin基因序列的重组质粒作为标准品;收集无饲养层培养体系下人胚胎干细胞DNA样本,结合2个单独的Taqman探针实时荧光定量PCR对待测样本中线粒体NDl和核β-globin基因分别进行定量,从而对人胚胎干细胞线粒体DNA的含量进行了精确定量。结果提示,人胚胎干细胞线粒体DNA的平均拷贝数／细胞为1321±228。研究表明,该技术可对人胚胎干细胞线粒体DNA拷贝数进行准确的测定,为研究培养条件对人胚胎干细胞线粒体DNA拷贝数的影响及优化体外培养条件奠定了基础。     

Metabolic flux analysis of embryonic stem cells using three distinct differentiation protocols and comparison to gene expression patterns

Metabolic flux analysis (MFA) was performed on mouse embryonic stem cells cultured under three distinct differentiation conditions: classical embryoid body formation (EB), and on surfaces coated with either gelatin (GEL) or matrigel (MAT). MFA was based on 15 metabolic reactions and eight transport steps, and was carried out based on measurements of four substrates and/or metabolites: glucose, lactate, glutamine, and glutamate. Fluxes representing biomass production remained fairly constant for all three culture conditions with at most a 40% variation. In contrast, major temporal variations, up to 500%, were observed for all other major central metabolic fluxes across all culture conditions. Fluxes were compared to gene-expression patterns measured by microarray analysis. Particularly interesting is the correlation between the metabolic fluxes with expression patterns of the corresponding genes of the pyruvate to lactate reaction, whereby the genes for several isoforms of the lactate dehydrogenase enzyme were examined. The patterns of this flux were notably different in the EB cultures compared to the GEL and MAT cultures and reflected differences in oxygen and nutrient transport in EB vs. the GEL and MAT cultures. Another novel finding of this study is an event occurring between Days 4 and 5 of differentiation, which was identified by a notable change in both the metabolic fluxes and gene-expression patterns. This suggests that metabolic patterns can be used as effective beacons of changes in differentiating stem cells. Overall, and qualitatively, core metabolic fluxes, under the three culture conditions examined, correlated well with gene-expression patterns.