hESC Transcriptome Research Articles

NAT10-mediated N4-acetylcytidine mRNA modification regulates self-renewal in human embryonic stem cells.

NAT10-catalyzed N4-acetylcytidine (ac4C) has emerged as a vital post-transcriptional modulator on the coding transcriptome by promoting mRNA stability. However, its role in mammalian development remains unclear. Here, we found that NAT10 expression positively correlates with pluripotency in vivo and in vitro. High throughput ac4C-targeted RNA immunoprecipitation sequencing (ac4C-RIP-seq), NaCNBH3-based chemical ac4C sequencing (ac4C-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays revealed noticeable ac4C modifications in transcriptome of hESCs, among which transcripts encoding core pluripotency transcription factors are favorable targets of ac4C modification. Further validation assays demonstrate that genetic inactivation of NAT10, the ac4C writer enzyme, led to ac4C level decrease on target genes, promoted the core pluripotency regulator OCT4 (POU5F1) transcript decay, and finally impaired self-renewal and promoted early differentiation in hESCs. Together, our work presented here elucidates a previously unrecognized interconnectivity between the core pluripotent transcriptional network for the maintenance of human ESC self-renewal and NAT10-catalyzed ac4C RNA epigenetic modification.

Network based meta-analysis prediction of microenvironmental relays involved in stemness of human embryonic stem cells.

Background. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of in vitro fertilised blastocysts, which can either be maintained in an undifferentiated state or committed into lineages under determined culture conditions. These cells offer great potential for regenerative medicine, but at present, little is known about the mechanisms that regulate hESC stemness; in particular, the role of cell–cell and cell-extracellular matrix interactions remain relatively unexplored.Methods and Results. In this study we have performed an in silico analysis of cell-microenvironment interactions to identify novel proteins that may be responsible for the maintenance of hESC stemness. A hESC transcriptome of 8,934 mRNAs was assembled using a meta-analysis approach combining the analysis of microarrays and the use of databases for annotation. The STRING database was utilised to construct a protein–protein interaction network focused on extracellular and transcription factor components contained within the assembled transcriptome. This interactome was structurally studied and filtered to identify a short list of 92 candidate proteins, which may regulate hESC stemness.Conclusion. We hypothesise that this list of proteins, either connecting extracellular components with transcriptional networks, or with hub or bottleneck properties, may contain proteins likely to be involved in determining stemness.

The abbreviated pluripotent cell cycle

Human embryonic stem cells (hESCs) and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory, and structural. The primary temporal context that the pluripotent self-renewal cell cycle of hESCs is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the embryonic stem cell (ESC) cell cycle. This supports the requirements of pluripotent cells to self-propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated ESC cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle.

Ascorbate Promotes Epigenetic Activation of CD30 in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions.

Abstract LB-250: Smoking reprograms human airway basal cells in vivo toward an embryonic stem cell-like molecular phenotype similar to that of lung adenocarcinoma

Abstract Activation of the human embryonic stem cell (hESC)-like transcriptional program has been observed in various epithelial cancers, including lung adenocarcinomas, in association with tumor aggressiveness. We hypothesized that initial changes related to the acquisition of this “cancerous” hESC-like phenotype occur in the stem/progenitor cell compartment of healthy epithelia under the influence of a chronic carcinogenic stress as a step toward malignant tissue derangement. Given that cigarette smoking is capable of inducing molecular changes in the airway epithelium leading to lung cancer, we analyzed expression of hESC-specific genes in the freshly isolated large airway epithelium of healthy nonsmokers (LAE-NS; n=21) and healthy smokers (LAE-S; n=31) and in the pure basal cell (BC) populations obtained from the LAE-NS (BC-NS; n=4) and LAE-S (BC-S; n=4). Expression patterns were then compared to those in primary human lung adenocarcinoma samples (n=193) and pure human lung adenocarcinoma cells propagated as xenografts in the NOD/SCID/IL2Rg-null immunodeficient mice (n=4) using a list of 40 hESC-specific genes based on published meta-analysis of the hESC transcriptome. The microarray analysis revealed low-level constitutive expression of a subset of hESC-specific genes in the LAE-NS, with higher expression in the BC population. Smoking dramatically and selectively up-regulated hESC-specific gene expression in BC, an observation confirmed by massive parallel sequencing (RNA-Seq). Remarkably, 35% of known hESC-specific genes were found up-regulated in BC-S by both microarrays and RNA-Seq. The majority of these genes (71%) were not detected in nonsmokers’ BC indicative of their de novo induction by smoking. Strikingly, 82% of hESC-specific genes induced in BC-S were also significantly up-regulated in both primary human lung adenocarcinomas and xenograft-derived human lung adenocarcinoma cells. Clustering analysis identified a subgroup of lung adenocarcinoma samples (n=32; 6%) among 193 primary lung adenocarcinoma samples based on the high expression of hESC-specific signature found up-regulated in BC of healthy smokers. This subset of adenocarcinoma subjects exhibited distinct clinical/pathological characteristics, such as longer smoking history (p<0.0002), lower lung function parameters such as FEV1 and DLCO (both p<0.001), larger tumor size (p<0.0002), more advanced tumor stage, poorer differentiation grade, and higher frequency of TP53 gene mutations (50% vs 23% in other adenocarcinoma subjects). Thus, prior to any clinical manifestations of lung cancer, cigarette smoking reprograms airway basal stem/progenitor cell population in vivo toward a lung cancer-associated hESC-like molecular state. The subset of lung adenocarcinomas highly expressing smoking-induced basal cell hESC-like gene signature have a distinct, aggressive clinical phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-250.

A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas.

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.