胚胎干细胞分化过程中的表观遗传调控

作为一类既有自我更新能力,并具有多向分化潜能的细胞,胚胎干细胞具有非常重要的理论研究意义和临床应用前景。近期以胚胎干细胞为模型,研究有关干细胞分化的表观遗传调控已成为新的研究热点。本文就胚胎干细胞分化过程中DNA甲基化、组蛋白修饰、非编码RNA调控以及与胚胎干细胞分化密切相关的表观遗传学动态变化做一概述,对表观遗传学改变与胚胎干细胞分化关系的基础研究进行探讨。     

基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用

基因组DNA的甲基化修饰通常使基因转录失活,去甲基化或低甲基化则使基因转录活化。但是,胚胎干细胞向各种成体细胞分化过程中相关基因的转录活化与DNA甲基化修饰水平并不呈简单的正性或负性相关。因此,甲基化修饰调节基因转录是一个复杂的过程。目前,对甲基化修饰作用的研究主要集中在基因选择性活化、改变转录因子与靶基因的结合活性、与组蛋白修饰协同作用及其基因表达的阶段特异性等方面。     

表观遗传修饰与干细胞分化调控

干细胞具有自我更新和多种分化潜能的特性。干细胞向分化细胞的转变涉及到基因表达模式的改变,与自我更新有关的基因关闭．与细胞特化有关的基因激活。表观遗传调控机制,包括DNA甲基化、组蛋白修饰和微RNA（microRNA）介导的基因调控,在多个层面上控制发育过程中基因表达。近年研究表明,动态的表观遗传调控机制在干细胞自我更新和分化中起关键作用。     

胚胎干细胞分化形成囊状胚胎小体的形态学变化特征

目的观察胚胎干细胞R1(ESs)体外分化形成囊状胚胎小体(CEBs)及其血管内皮细胞和平滑肌细胞的时相规律和结构特点,探讨其在血管发育生物学研究中的作用和意义。方法体外悬浮培养ESs形成CEBs,采用HE染色观察CEBs的结构特点。用免疫荧光染色鉴定不同培养时间的CEBs中CASPASE-3的表达。加入全反式维甲酸(atRA)及联丁酰基cAMP(DBcAMP)定向诱导ESs分化,其后行平滑肌特异性标志物α-肌动蛋白(SMα-actin)及内皮细胞特异性标志物PECAM-1免疫荧光染色。结果典型的CEBs在第13d左右可以形成。第6d就可见CEBs中心部位出现调亡细胞,坏死腔开始形成,到第13d时中心坏死腔完全形成,凋亡细胞明显增多。将第6d的CEBs接种于培养皿后用atRA及DBcAMP处理,3d后可见SMα-actin染色阳性细胞。而未经处理的CEBs贴壁后,在第10d可见PECAM-1的表达。结论CEBs能够模拟体内血管发生形成的过程,可作为研究血管发育生物学的较好模型。     

组蛋白修饰与基因调控

基因表达是一个受多因素调控的复杂过程,组蛋白是染色体基本结构－核小体中的重要组成部分,其N－末端氨基酸残基可发生乙酰化、甲基化、磷酸化、泛素化、多聚ADP糖基化等多种共价修饰作用,组蛋白的修饰可通过影响组蛋白与DNA双链的亲性,从而改变染色质的疏松或凝集状态,或通过影响其它转录因子与结构基因启动子的亲和性来发挥基因调控作用,组蛋白修饰对基因表达的调控有类似DNA遗传密码的调控作用。     

组蛋白修饰调节机制的研究进展

表观遗传学涉及到DNA甲基化、组蛋白修饰、染色体重塑和非编码RNA调控等内容,其中组蛋白修饰包括组蛋白的乙酰化、磷酸化、甲基化、泛素化及ADP核糖基化等,这些多样化的修饰以及它们时间和空间上的组合与生物学功能的关系又可作为一种重要的表观标志或语言,因而被称为“组蛋白密码”．相同组蛋白残基的磷酸化与去磷酸化、乙酰化与去乙酰化、甲基化与去甲基化等,以及不同组蛋白残基的磷酸化与乙酰化、泛素化与甲基化、磷酸化与甲基化等组蛋白修 饰之间既相互协同又互相拮抗,形成了一个复杂的调节网络．对组蛋白修饰内在调节机制的研究将丰富“组蛋白密码”的内涵．     

小鼠胚胎干细胞分化扩增期连续低密度传代对原始细胞中Oct-4阳性细胞比例及神经分化能力的影响

目的：探讨在分化扩增期采用连续低密度传代的方法是否能降低小鼠胚胎干细胞向神经细胞分化的前体细胞中Oct-4阳性细胞的比例,以及对神经分化能力的影响。方法：采用“五步法”将小鼠胚胎干细胞向神经元分化,进入扩增期后采用连续低密度传代的方法连续传10代。然后应用细胞免疫组化鉴定Oct-4阳性细胞、神经元与胶质细胞、流式细胞仪检测Oct-4阳性细胞比例、凋亡试剂盒测定细胞凋亡。结果：流式细胞仪检测出扩增期连续低密度传代得到的前体细胞中Oct-4阳性细胞的比例由16.17±4.8％降至4.33±1.63％,扩增期低密度传代细胞和正常高密度传代细胞的细胞凋亡率鉴定分别为15.16±3.64% 和11.88±2.63%,步骤5诱导分化后的细胞GFAP和Tuj-1免疫组化染色呈阴性。结论：低密度传代培养可以减少分化后Oct-4阳性细胞的比例,且该比例下降不是由Oct-4阳性细胞的凋亡引起的。同时可能是因为低密度传代培养和高密度相比引起了细胞的质变、或者改变了前体细胞向神经元分化的某种微环境,导致了前体细胞不能分化为神经细胞。提示高密度培养在前体细胞向神经元分化过程中具有重要作用,高密度和低密度培养的比较,提供了研究ES细胞向神经元分化机制的新平台和研究方向。     

Lefty1对小鼠胚胎干细胞分化过程的影响

目的:研究Lefty1在小鼠胚胎干细胞分化过程中的作用。方法:根据染色质免疫沉淀测序结果,在临近Lefty1转录起始位点以及与之相距10 kb的上游区域有TGF-β信号通路Smad2/3蛋白的四个结合区域,通过CRISPR/Cas9方法获得四个区域敲除的单克隆细胞,利用荧光实时定量PCR(qRT-PCR)检测各细胞中Lefty1的转录水平,并用TGF-β信号通路的激活剂AC和抑制剂SB分别处理敲除的细胞,检测其对TGF-β信号的响应,最后通过胚状体形成实验,检测敲除细胞系在中内胚层分化过程中的标志分子Gsc和Mixl1的表达。结果:利用CRISPR/Cas9方法成功获得不同区域敲除的单克隆细胞,与野生型E14细胞相比,四种区域敲除的细胞中Lefty1 RNA含量明显降低,并且在干细胞状态和分化状态下,敲除细胞系对TGF-β信号的响应减弱。在胚状体形成的实验中,与野生型E14细胞相比,敲除细胞系在分化过程中Lefty1表达的基础水平明显降低,中内胚层分化的标志分子Gsc和Mixl1转录水平也明显下降。结论:在胚胎干细胞中,Lefty1转录起始位点附近以及上游10 kb的这四段区域通过TGF-β信号通路对Lefty1的转录发挥调控作用,从而影响中内胚层分化过程中的标志分子Gsc和Mixl1的表达。     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

组蛋白修饰酶对基因转录的调控

基因在转录过程中,需招募多种组蛋白修饰酶来对组蛋白进行化学修饰,这些化学修饰包括:组蛋白的甲基化、乙酰化、磷酸化、泛素化和SUMO化等.大多数组蛋白修饰酶能与不同的转录因子形成复合物,并引起组蛋白和DNA之间相互作用的改变,从而调控基因的转录.本文总结了各种组蛋白修饰酶复合物的组成、结构及功能方面的研究进展.     

Nuclear and chromatin reorganization in the MHC-Oct3/4 locus at developmental phases of embryonic stem cell differentiation

Epigenetic gene control is involved in mechanisms of development. Little is known about the cooperation of nuclear and chromatin events in programmed differentiation from mouse embryonic stem cells (ESC). To address this, Oct3/4-positive ESC and differentiated progenies, Sox1-positive neural precursor cells (NPC) and post-mitotic neurons (PMN), were isolated using a stage-selected culture system. We first investigated global nuclear organization at the each stage. Chromocenter preexists in ESC, disperses in NPC and becomes integrated into large heterochromatic foci in PMN, while the formation of PML bodies markedly decreases in neural differentiation. We next focused on the gene-dense MHC-Oct3/4 region. Oct3/4 gene is expressed preferentially adjacent to PML bodies in ESC and are repressed in the absence of chromocenter association in NPC and PMN. Histone deacetylation in NPC, demethylation of lysine 4 of histone H3 (H3K4), tri-methylation of H3K27, and CpG methylation in PMN are targeted for the Oct3/4 promoter within the region. Interestingly, di-methyl H3K4 mark is present in Oct3/4 promoter in NPC as well as ESC. These findings provide insights into the molecular basis of global nuclear reorganization and euchromatic gene silencing in differentiation through the spatiotemporal order of epigenetic controls.     

DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells

Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

Artificial niche substrates for embryonic and induced pluripotent stem cell cultures

Stem cells possess the ability to self-renew and differentiate into other cell types. In vivo, stem cells reside in their own anatomic niches in a defined physiological environment, from which they are released to differentiate into a required cell type when deemed appropriate. While a resident within the niche, the stem cell receives signals that in turn maintain the cell in a pluripotent state. In addition, the niche also provides nourishment to the cell. Physically, the niche also serves to anchor the cell via various ECM components and cell-adhesion molecules. Therefore, in vitro models that replicate the in vivo niche will lead to a better understanding of stem cell fate and turnover. In turn, this will help inform attempts to culture stem cells in vitro on artificial niche-like substrates. In this review, we have highlighted recent studies describing artificial niche-like substrates used to culture embryonic and induced pluripotent stem cells in vitro.     

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

The embryonic midbrain directs neuronal specification of embryonic stem cells at early stages of differentiation

Specific neuronal differentiation of Embryonic Stem Cells (ESCs) depends on their capacity to interpret environmental cues. At present, it is not clear at which stage of differentiation ESCs become competent to produce multiple neuronal lineages in response to the niche of the embryonic brain. To unfold the developmental potential of ESC-derived precursors, we transplanted these cells into the embryonic midbrain explants, where neurogenesis occurs as in normal midbrain development. Using this experimental design, we show that the transition from ESCs to Embryoid Body (EB) precursors is necessary to differentiate into Lmx1a⁺/Ptx3⁺/TH⁺ dopaminergic neurons around the ventral midline of the midbrain. In addition, EB cells placed at other dorsal-ventral levels of the midbrain give rise to Nkx6.1⁺ red nucleus (RN) neurons, Nkx2.2⁺ ventral interneurons and Pax7⁺ dorsal neurons at the correct positions. Notably, differentiation of ESCs into Neural Precursor Cells (NPCs) prior to transplantation markedly reduces specification at the Lmx1a, Nkx6.1 and Pax7 expression domains, without affecting neuronal differentiation. Finally, exposure to Fgf8 and Shh in vitro promotes commitment of some ESC-derived NPCs to differentiate into putative Lmx1a⁺ dopaminergic neurons in the midbrain. Our data demonstrate intrinsic developmental potential differences among ESC-derived precursor populations.     

Ghrelin promotes the differentiation of human embryonic stem cells in infarcted cardiac microenvironment

Ghrelin is broadly expressed in myocardial tissues, where it exerts different functions. It also has been found to have a wide variety of biological functions on cell differentiation and tissue development. The aim of this study was to investigate the effect of ghrelin on human embryonic stem cell (hESC) differentiation in infarcted cardiac microenvironment. The hESCs grown on feeder layers expressed several pluripotential markers including alkaline phosphatase (AKP). Four weeks after transplantation into rat infarcted hearts, the hESCs and their progeny cells survived and formed intracardiac grafts were 54.7% and 19.6% respectively in ghrelin- and phosphate-buffered saline (PBS)-treated groups. Double immunostaining with anti-human Sox9 and anti-HNA or anti-human fetal liver kinase-1 (Flk1) and anti β-tubulin showed that the human grafts were in development. However, double positive stains were only found in the ghrelin-treated group. In addition, the hESC injection protocol was insufficient to restore heart function of the acute myocardial infarction model. Our study, therefore, provides a new insight of ghrelin on promoting hESC survival and differentiation in rat infarcted cardiac microenvironment. This may give a clue for therapy for myocardial infarction by hESCs or progeny cells.     

In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells

Embryonic stem (ES) cells are omnipotent; they can differentiate into every cell type of the body. The development of culture conditions that allow their differentiation has made it conceivable to produce large numbers of cells with lineage-specific characteristics in vitro. Here, we describe a method by which murine ES cells can be differentiated into cells with characteristics of epidermal keratinocytes. Keratinocyte-like cells were isolated from embryoid bodies and grown in culture. Potential applications of this method are the in vitro differentiation of cells of interest from ES cells of mice with lethal phenotypes during embryonic development and the production of genetically modified epidermal keratinocytes that could be used as temporary wound dressing or as carriers of genes of interest in gene therapeutic treatments.