Unknown,Transcriptomics,Genomics,Proteomics

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Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

ABSTRACT: In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Examination of differential methylation between iPSC and ESCs through differentiated states. Examination of differential methylation between iPSC and somatic cell source and iPSC and ESCs.

ORGANISM(S): Homo sapiens

SUBMITTER: Dinh Diep

PROVIDER: E-GEOD-40372 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells.

Ruiz Sergio S Diep Dinh D Gore Athurva A Panopoulos Athanasia D AD Montserrat Nuria N Plongthongkum Nongluk N Kumar Sachin S Fung Ho-Lim HL Giorgetti Alessandra A Giorgetti Alessandra A Bilic Josipa J Batchelder Erika M EM Zaehres Holm H Kan Natalia G NG Schöler Hans Robert HR Mercola Mark M Zhang Kun K Izpisua Belmonte Juan Carlos JC

Proceedings of the National Academy of Sciences of the United States of America 20120918 40

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, ...[more]

PMID: 22991473

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Project description:Here we describe a method for whole genome bisulfite sequencing in very small cell populations (µWGBS) and in single cells (scWGBS). Our method is optimized for profiling DNA methylation in many samples at low coverage, and we developed a bioinformatic method that uses collections of single-cell methylomes to infer cell state dynamics. Using these technological advances, we studied epigenetic cell state dynamics in three in vitro models of cellular differentiation and pluripotency and identified patterns of epigenetic remodeling and cell-to-cell heterogeneity. 1-cell or 4-cell samples were collected from a variety of human and mouse cell lines (KBM7, CCE, K562, 32D, HL60). We also included negative controls (no cells), and cross controls (cells of one species aligned to the other). K562 cells were treated with 5-azacytidine in a final concentration of 1µM to induce demethylation. HL60 cells were treated with 1α,25-dihydroxyvitamin D3 in a final concentration of 10nM to induce differentiation. Naïve pluripotency of CCE ES cells was induced by culturing the cells in ESGRO-2i medium with the addition of 1% FCS. Differentiation of CCE ES cells was induced by treating the cells with all-trans retinoic acid (ATRA) in a final concentration of 0,3µM every third day along with complete LIF-free medium exchange. Embryoid body formation from CCE ES cells was induced by hanging-drop culture for 5 days, followed by 9 days on gelatinized dishes (see treatment annotation). Each experiment, with treated and untreated cells, was performed in multiple biological replicates. We then measured DNA methylation in the individual 1-cell or 4-cell experiments with bisulfite treatment and high-throughput sequencing. For sequencing, most experiments pooled a 4-cell experiment from one species with a 1-cell experiment from the other, but some replicates were sequenced as a single cell (indicated with SC in the replicate annotation). Two samples (marked as “deep”) were sequenced much deeper than the others. See Farlik et al. (2015) Cell Reports for complete study details.

Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation. Examination of CpG methylation profiles in Knock-out and wild type mice We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. CpG Islands (samples labeled as CpG) and trans gene (samples labeled as LacZ) were amplified after Bisulfite treatment. Please note that the same gDNA was used to amplify CpG Island (Gene_CpG_KO ) and LacZ (Gene_LacZ_KO) reporter for the same gene. PCR product of amplification was gel separated, isolated and pooled. After libraries were prepared and sequenced, the alignment was performed. CpG island and LacZ alignments were done separately resulting in three different Processed Data files per gene investigated: Gene_CpG_KO, Gene_LacZ_KO and Gene_CpG_WT. LacZ reference is included in the submission, but is also available for download from KOMP Phenotype website (www.kompphenotype.org). Also please note that the libraries were prepared using Illumina TruSeq RNA Sample Prep Kit starting from adapter binding step as samples were double stranded Bisulfite treated DNA amplicons. So the library preparation was done as with RNASeq, but samples investigated were bisulfite treated.

Dataset Information

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

Publications

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells.

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