Nuclear transcriptome profiling of induced pluripotent stem cells and embryonic stem cells identify non-coding loci resistant to reprogramming (original) (raw)
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Global Transcription in Pluripotent Embryonic Stem Cells
Cell Stem Cell, 2008
The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem (ES) cells are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage specific genes or by selective silencing of genome regions during differentiation. Here we show that the ES cell genome is transcriptionally globally hyperactive and undergoes global silencing as cells differentiate. Normally silent repeat regions are active in ES cells and tissue-specific genes are sporadically expressed at low levels. Whole genome tiling arrays demonstrate widespread transcription in both coding and non-coding regions in pluripotent ES cells whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ES cells is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery, but not histone modifying activities. Interference with several chromatin remodeling activities in ES cells affects their proliferation and differentiation behavior. We propose that global transcriptional activity is a hallmark of pluripotent ES cells that contributes to their plasticity and that lineage specification is strongly driven by reduction of the actively transcribed portion of the genome. *Correspondence: meshorer@cc.huji.ac.il (E.M.), mistelit@mail.nih.gov (T.M.). 7 Current address :
Induced Pluripotent Stem Cells-Emphasis on Transcriptomics and Recent Advances in Therapeutic Potential, 2016
In our previous review we reviewed the history of embryonic stem cells, advantages and disadvantages of ESC, parthenogenetic ESC and their therapeutic applications, cloning along with merits of SCNT, in this shot commentary we have just concentrated on induced pluripotent stem cells, mainly their transcriptomics, along with special emphasis on global transcriptomics in candidate oocyte factors and various advances in therapeutic applications.
Stem cell reports, 2016
The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from t...
Stem Cells, 2005
Global gene expression profiling was performed on murine embryonic stem cells (ESCs) induced to differentiate by removal of leukemia inhibitory factor (LIF) to identify genes whose change in expression correlates with loss of pluripotency. To identify appropriate time points for the gene expression analysis, the dynamics of loss of pluripotency were investigated using three functional assays: chimeric mouse formation, embryoid body generation, and colony-forming ability. A rapid loss of pluripotency was detected within 24 hours, with very low residual activity in all assays by 72 hours. Gene expression profiles of undifferentiated ESCs and ESCs cultured for 18 and 72 hours in the absence of LIF were determined using the Affymetrix GeneChip U74v2. In total, 473 genes were identified as significantly differentially expressed, with approximately one third having unknown biological function. Among the 275 genes whose expression decreased with ESC differentiation were several factors previously identified as important for, or markers of, ESC pluripotency, including Stat3, Rex1, Sox2, Gbx2, and Bmp4. A significant number of the decreased genes also overlap with previously published mouse and human ESC data. Furthermore, several membrane proteins were among the 48 decreased genes correlating most closely with the functional assays, including the stem cell factor receptor c-Kit. Through identification of genes whose expression closely follows functional properties of ESCs during early differentiation, this study lays the foundation for further elucidating the molecular mechanisms regulating the maintenance of ESC pluripotency and facilitates the identification of more reliable molecular markers of the undifferentiated state.
Regulatory Non-Coding RNAs in Pluripotent Stem Cells
International Journal of Molecular Sciences, 2013
The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation.
Nucleic Acids Research, 2013
The transcriptional landscape in embryonic stem cells (ESCs) and during ESC differentiation has received considerable attention, albeit mostly confined to the polyadenylated fraction of RNA, whereas the non-polyadenylated (NPA) fraction remained largely unexplored. Notwithstanding, the NPA RNA super-family has every potential to participate in the regulation of pluripotency and stem cell fate. We conducted a comprehensive analysis of NPA RNA in ESCs using a combination of wholegenome tiling arrays and deep sequencing technologies. In addition to identifying previously characterized and new non-coding RNA members, we describe a group of novel conserved RNAs (snacRNAs: small NPA conserved), some of which are differentially expressed between ESC and neuronal progenitor cells, providing the first evidence of a novel group of potentially functional NPA RNA involved in the regulation of pluripotency and stem cell fate. We further show that minor spliceosomal small nuclear RNAs, which are NPA, are almost completely absent in ESCs and are upregulated in differentiation. Finally, we show differential processing of the minor intron of the polycomb group gene Eed. Our data suggest that NPA RNA, both known and novel, play important roles in ESCs.
Insight into the key genes of pluripotency in human and their interrelationships is necessary for understanding the underlying mechanism of pluripotency and hence their successful application in regenerative medicine. The recent advances in transcriptomics technologies have created new opportunities to decipher the genes involved in pluripotency, genetic network that governs the unique properties of embryonic stem cells and lineage differentiation mechanisms in a deeper scale. There are a large number of experimental studies on human embryonic stem cells (hESCs) being routinely conducted for unfolding the underlying biology of embryogenesis and their clinical prospects. However, the outcome of these studies often lacks consensus due to differences in samples, experimental techniques and/or analysis protocols. A universal stemness gene list is still lacking. In this quest, we compared transcriptomic profiles of pluripotent and non-pluripotent samples from diverse cell lines/types gen...
DNA and Chromatin Modification Networks Distinguish Stem Cell Pluripotent Ground States
Molecular & Cellular Proteomics, 2012
Pluripotent stem cells are capable of differentiating into all cell types of the body and therefore hold tremendous promise for regenerative medicine. Despite their widespread use in laboratories across the world, a detailed understanding of the molecular mechanisms that regulate the pluripotent state is currently lacking. Mouse embryonic (mESC) and epiblast (mEpiSC) stem cells are two closely related classes of pluripotent stem cells, derived from distinct embryonic tissues. Although both mESC and mEpiSC are pluripotent, these cell types show important differences in their properties suggesting distinct pluripotent ground states. To understand the molecular basis of pluripotency, we analyzed the nuclear proteomes of mESCs and mEpiSCs to identify protein networks that regulate their respective pluripotent states. Our study used label-free LC-MS/MS to identify and quantify 1597 proteins in embryonic and epiblast stem cell nuclei. Immunoblotting of a selected protein subset was used to confirm that key components of chromatin regulatory networks are differentially expressed in mESCs and mEpiSCs. Specifically, we identify differential expression of DNA methylation, ATP-dependent chromatin remodeling and nucleosome remodeling networks in mESC and mEpiSC nuclei. This study is the first comparative study of protein networks in cells representing the two distinct, pluripotent states, and points to the importance of DNA and chromatin modification processes in regulating pluripotency. In addition, by integrating our data with existing pluripotency networks, we provide detailed maps of protein networks that regulate pluripotency that will further both the fundamental understanding of pluripotency as well as efforts to reliably control the differentiation of these cells into functional cell fates.