Diverse Epigenetic Profile of Novel Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) are a promising model for studying mechanisms ofregulation of early development and differentiation. OCT4, NANOG, OCT4-related genes andsome others were recently described to be important in pluripotency maintenance. Lesser isknown about molecular mechanisms involved in their regulation. Apart from genetic regulationof gene expression epigenetic events, particularly methylation, play an important role in earlydevelopment. Using RT-PCR we studied the expression of pluripotency-related genes OCT4,NANOG, DPPA3, and DPPA5 during hESCs differentiation to embryoid bodies. Analysis ofmethylation profiles of promoter or putative regulatory regions of the indicated genesdemonstrated that expression of the pluripotency-maintaining genes correlated with theirmethylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. Wepropose that DNA methylation underlies the developmental stage-specific mechanisms ofpluripotency-related genes expression and reactivation and may have an impact ondifferentiation potential of hESC lines.     

Epigenetic regulation of gene expression in porcine epiblast, hypoblast, trophectoderm and epiblast-derived neural progenitor cells

After fertilization, lineage specification is governed by a complicated molecular network in which permissiveness and repression of expression of pluripotency- and differentiation-associated genes are regulated by epigenetic modifications. DNA methylation operates as a very stable repressive mark in this process. In this study, we investigated the relationship between DNA methylation and expression of pluripotency-associated genes (OCT4, NANOG and SOX2), a trophectoderm (TE)-specific gene (ELF5), and genes associated with neural differentiation (SOX2 and VIMENTIN) in porcine Day 10 (E10) epiblast, hypoblast, and TE as well as in epiblast-derived neural progenitor cells (NPCs). We found that OCT4, NANOG, and SOX2 were highly expressed in the epiblast and hypoblast, while VIMENTIN was only highly expressed in the epiblast. Moreover, low expression of OCT4, NANOG, SOX2 and VIMENTIN was noted in the TE. Most CpG sites of OCT4, NANOG, SOX2 and VIMENTIN displayed low methylation levels in the epiblast and hypoblast and, strikingly, also in the TE. Hence, the expression patterns of these genes were not directly related to levels of DNA methylation in the TE in contrast to the situation in the mouse. In contrast, ELF5 was exclusively expressed in the TE and was correspondingly hypomethylated in this tissue. In NPCs, we observed down-regulation of NANOG and OCT4 expression, which correlated with hypermethylation of their promoters, whereas VIMENTIN displayed up-regulation in accordance with hypomethylation of its promoter. In conclusion, DNA methylation is an inconsistently operating epigenetic mechanism in porcine E10 blastocysts, whereas in porcine epiblast-derived NPCs, expression of pluripotency-associated and differentiation genes appear to be regulated by this modification.     

Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells

Nanog, Oct4, and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated, the mechanistic functions are not well defined. Here, we identify general and cell-line-specific requirements for NANOG, OCT4, and SOX2 in hESCs. We show that OCT4 regulates, and interacts with, the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages, whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs.     

1号染色体长臂扩增削弱人胚胎干细胞神经分化潜能

目的探讨1号染色体长臂（1q）的扩增对人胚胎干细胞（hESCs）神经分化的影响。 方法通过对H9 hESCs克隆化培养的方法获得1q扩增的hESCs系。中期染色体计数的方法明确细胞内的染色体数目,核型分析鉴定染色体变异的情况,全基因组测序（WGS）分析基因组片段拷贝数变异的情况。使用碱性磷酸酶（AP）染色法检测细胞干性维持的情况,RT-qPCR和免疫荧光染色等方法检测胚胎干细胞（ESCs）标志物OCT4、SOX2、NANOG、REX1和SSEA4等的表达。拟胚体（EB）形成实验进行hESCs不定向分化、全反式视黄酸（RA）诱导hESCs向外胚层分化、使用STEMdiff? SMADi Neural Induction Kit诱导hESCs向神经祖细胞（NPCs）定向分化,并通过RT-qPCR、AP染色和免疫荧光染色等方法检测其分化能力。两组间比较采用独立样本t检验。 结果分离获得一株1q发生2个拷贝扩增的细胞,核型分析发现额外获得的2个1q是等臂染色体,核型为[47,XX,+i （1q）],将其命名为Amp （1q）。Amp （1q）AP染色呈阳性,且表达ESCs标志物OCT4、SOX2、NANOG、REX1和SSEA4,具备干细胞自我更新的特征。EB分化过程中,与H9细胞相比,Amp （1q）向外胚层的分化能力下降,MAP2 （29.67±1.53比66.67±1.15）和PAX6 （8001±567.09比28308.00±1692.50）的表达降低（P均< 0.05）;RA诱导分化实验进一步证明,与H9细胞相比,Amp （1q）存在向外胚层分化的缺陷,MAP2 （22.50±3.54比42.50±2.12）和PAX6 （5403.00±569.93比38756.00±1068.44）的表达降低（P均< 0.05）。当定向诱导向神经谱系分化时,Amp （1q）形成NPCs的能力降低,NPCs标志物PAX6的表达水平低于H9细胞（13.83±3.75比88.33±1.53） （P均< 0.05）。 结论Amp （1q）具有ESCs自我更新的能力,但1q的扩增会削弱hESCs神经分化的能力。     

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.     

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.     

LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells

We identify LSD1 (lysine-specific demethylase 1; also known as KDM1A and AOF2) as a key histone modifier that participates in the maintenance of pluripotency through the regulation of bivalent domains, a chromatin environment present at the regulatory regions of developmental genes that contains both H3K4 di/trimethylation and H3K27 trimethylation marks. LSD1 occupies the promoters of a subset of developmental genes that contain bivalent domains and are co-occupied by OCT4 and NANOG in human embryonic stem cells, where it controls the levels of H3K4 methylation through its demethylase activity. Thus, LSD1 has a role in maintaining the silencing of several developmental genes in human embryonic stem cells by regulating the critical balance between H3K4 and H3K27 methylation at their regulatory regions.     

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.