Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming (original) (raw)
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Nature genetics, 2010
The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome 1-5 , resulting in altered patterns of gene expression 2,6-9 . Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) 10,11 that occurs upon derivation of human iPSCs, and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined 10 lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells (ESCs), suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription
Single-Cell RNA-Seq Reveals Dynamic Early Embryonic-like Programs during Chemical Reprogramming
Cell stem cell, 2018
Chemical reprogramming provides a powerful platform for exploring the molecular dynamics that lead to pluripotency. Although previous studies have uncovered an intermediate extraembryonic endoderm (XEN)-like state during this process, the molecular underpinnings of pluripotency acquisition remain largely undefined. Here, we profile 36,199 single-cell transcriptomes at multiple time points throughout a highly efficient chemical reprogramming system using RNA-sequencing and reconstruct their progression trajectories. Through identifying sequential molecular events, we reveal that the dynamic early embryonic-like programs are key aspects of successful reprogramming from XEN-like state to pluripotency, including the concomitant transcriptomic signatures of two-cell (2C) embryonic-like and early pluripotency programs and the epigenetic signature of notable genome-wide DNA demethylation. Moreover, via enhancing the 2C-like program by fine-tuning chemical treatment, the reprogramming proce...
Cell, 2012
During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
2017
Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy, as well as for expanding our understanding of the general principles of gene regulation. MYOD-mediated myogenic reprogramming, which converts many cell types into contractile myotubes, remains one of the best characterized model system for direct conversion by defined factors. However, why MYOD can efficiently convert some cell types into myotubes but not others remains poorly understood. Here, we analyze MYOD-mediated reprogramming of human fibroblasts at pseudotemporal resolution using single-cell RNA-Seq. Successfully reprogrammed cells navigate a trajectory with two branches that correspond to two barriers to reprogramming, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Differential analysis of the major branch points alongside alignment of the successful reprogramming path ...
2019
To unravel the mechanism of human cellular reprogramming process at single-cell resolution, we performed parallel scRNA-Seq and scATAC-Seq analysis. Our analysis reveals that the cells undergoing reprogramming proceed in an asynchronous trajectory and diversify into heterogeneous sub-populations. BDD2-C8 fluorescent probe staining and negative staining for CD13, CD44 and CD201 markers, could enrich for the GDF3+ early reprogrammed cells. Combinatory usage of the surface markers enables the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 or a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Altogether, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrativ...
Transcriptome Signature and Regulation in Human Somatic Cell Reprogramming
Stem Cell Reports, 2015
Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming.
Global Epigenetic State Network Governs Cellular Pluripotent Reprogramming and Transdifferentiation
arXiv: Molecular Networks, 2012
How do mammalian cells that share the same genome exist in notably distinct phenotypes, exhibiting differences in morphology, gene expression patterns, and epigenetic chromatin statuses? Furthermore how do cells of different phenotypes differentiate reproducibly from a single fertilized egg? These fundamental questions are closely related to a deeply rooted paradigm in developmental biology that cell differentiation is irreversible. Yet, recently a growing body of research suggests the possibility of cell reprogramming, which offers the potential for us to convert one type of cell into another. Despite the significance of quantitative understandings of cell reprogramming, theoretical efforts often suffer from the complexity of large circuits maintaining cell phenotypes coupled at many different epigenetic and gene regulation levels. To capture the global architecture of cell phenotypes, we propose an "epigenetic state network" approach that translates the classical concept...
Cell Cycle, 2015
I dentification of functionally relevant differences between induced pluripotent stem cells (iPSC) and reference embryonic stem cells (ESC) remains a central question for therapeutic applications. Differences in gene expression between iPSC and ESC have been examined by microarray and more recently with RNA-SEQ technologies. We here report an in depth analyses of nuclear and cytoplasmic transcriptomes, using the CAGE (cap analysis of gene expression) technology, for 5 iPSC clones derived from mouse lymphocytes B and 3 ESC lines. This approach reveals nuclear transcriptomes significantly more complex in ESC than in iPSC. Hundreds of yet not annotated putative non-coding RNAs and enhancer-associated transcripts specifically transcribed in ESC have been detected and supported with epigenetic and chromatin-chromatin interactions data. We identified superenhancers transcriptionally active specifically in ESC and associated with genes implicated in the maintenance of pluripotency. Similarly, we detected non-coding transcripts of yet unknown function being regulated by ESC specific superenhancers. Taken together, these results demonstrate that current protocols of iPSC reprogramming do not trigger activation of numerous cis-regulatory regions. It thus reinforces the need for already suggested deeper monitoring of the non-coding transcriptome when characterizing iPSC clones. Such differences in regulatory transcript expression may indeed impact their potential for clinical applications.