Reprogramming methods influence DNA methylation and expression patterns of mouse induced pluripotent stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE92985 | GEO | 2017/06/28
SECONDARY ACCESSION(S): PRJNA359211
REPOSITORIES: GEO
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Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells through ectopic expression of transcriptional factors or chemical cocktails. Chemical reprogramming might be safe than transcriptional factors since there is no integration of exogenous genes. However, there is still little direct evidence to prove this hypothesis. In this study, we have performed whole genome profiling of the DNA methylomes of mouse chemical iPSCs (CiPSCs), transcriptional factors induced iPSCs (TF-iPSCs) and embryonic stem cells (ESCs). We find that the methylation levels of high-CpG-density promoters (HCPs) and intermediate- CpG-density promoters (ICPs) have no significant difference among them, but low-CpG-density promoters (LCP) and three retrotransposons (LINEs, LTRs and SINEs) show preference to different methylation levels. Surprisingly, CiPSCs are hypomethylated than TF-iPSCs and the major difference of methylation levels lies in the intergenic regions while two iPSCs lines are generally hypermethylated compared to ESCs. Moveover, we find the methylation states of imprinting control regions (ICRs) and the expression of imprinted genes of CiPSCs are more resemble ESCs than TF-iPSCs. Our data first provide the epigenetic states of chemical induced pluripotent stem cells and compare the difference of mouse CiPSCs, TF-iPSCs and ESCs. These observations might affect ongoing choices of reprogramming methods for disease modeling and give some guides for potential therapeutic applications.
2017-06-28 | GSE92984 | GEO
Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells through ectopic expression of transcriptional factors or chemical cocktails. Chemical reprogramming might be safe than transcriptional factors since there is no integration of exogenous genes. However, there is still little direct evidence to prove this hypothesis. In this study, we have performed whole genome profiling of the DNA methylomes of mouse chemical iPSCs (CiPSCs), transcriptional factors induced iPSCs (TF-iPSCs) and embryonic stem cells (ESCs). We find that the methylation levels of high-CpG-density promoters (HCPs) and intermediate- CpG-density promoters (ICPs) have no significant difference among them, but low-CpG-density promoters (LCP) and three retrotransposons (LINEs, LTRs and SINEs) show preference to different methylation levels. Surprisingly, CiPSCs are hypomethylated than TF-iPSCs and the major difference of methylation levels lies in the intergenic regions while two iPSCs lines are generally hypermethylated compared to ESCs. Moveover, we find the methylation states of imprinting control regions (ICRs) and the expression of imprinted genes of CiPSCs are more resemble ESCs than TF-iPSCs. Our data first provide the epigenetic states of chemical induced pluripotent stem cells and compare the difference of mouse CiPSCs, TF-iPSCs and ESCs. These observations might affect ongoing choices of reprogramming methods for disease modeling and give some guides for potential therapeutic applications.
2017-06-28 | GSE92983 | GEO
Project description:Reprogramming methods influence DNA methylation and expression patterns of mouse induced pluripotent stem cells
| PRJNA359211 | ENA
Project description:Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.
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Project description:The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.
| S-EPMC4230737 | biostudies-literature
Project description:Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.
| S-EPMC5867603 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming somatic cells through transduction with a transcription factor cocktail. However, the low efficiency of this procedure has kept iPSCs away from the study of the clinical application of stem cell biology. Our research shows that continuous passage increases the efficiency of reprogramming. Compared with conventional method of establishment of iPSCs, more embryonic stem cell (ESC)-like clones are generated by continuous passage during early reprogramming. These inchoate clones, indistinguishable from genuine ESC clones, are closer to fully reprogrammed cells compared with those derived from classical iPSC induction, which increased the expression of pluripotent gene markers and the levels of demethylation of Oct4 and Nanog. These results suggested that full reprogramming is a gradual process that does not merely end at the point of the activation of endogenous pluripotency-associated genes. Continuous passage could increase the pluripotency of induced cells and accelerate the process of reprogramming by epigenetic modification. In brief, we have provided an advanced strategy to accelerate the reprogramming and generate more nearly fully reprogrammed iPSCs efficiently and rapidly.
| S-EPMC3920923 | biostudies-literature
Project description:DNA methylation and histone methylation (H3K27me3) have been reported as major barriers to induced pluripotent stem cell (iPSC) generation using four core transcription factors (Oct4, Sox2, Klf4, and c-Myc, termed OSKM). Here, to illustrate the possibility of deriving iPSCs via demethylation, as well as the exact effects of DNA methylation and histone modification on gene expression regulation, we performed RNA sequencing to characterize the transcriptomes of ES cells and iPSCs derived by demethylation with miR-29b or shDnmt3a, and carried out integrated analyses. Results showed that OSKM + miR-29b-iPSC was more close to ES cells than the others, and up-regulated genes typically presented with methylated CpG-dense promoters and H3K27me3-enriched regions. The differentially expressed genes caused by introduction of DNA demethylation during somatic cell reprogramming mainly focus on stem cell associated GO terms and KEGG signaling pathways, which may decrease the tumorigenesis risk of iPSCs. These findings indicated that DNA methylation and histone methylation have synergetic effects on regulating gene expression during iPSC generation, and demethylation by miR-29b is better than shDnmt3a for iPSC quality. Furthermore, integrated analyses are superior for exploration of slight differences as missed by individual analysis.
| S-EPMC5290738 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.
| S-EPMC11231959 | biostudies-literature
Project description:The biomedical utility of induced pluripotent stem cells (iPSCs) will be diminished if most iPSC lines harbor deleterious genetic mutations. Recent microarray studies have shown that human iPSCs carry elevated levels of DNA copy number variation compared with those in embryonic stem cells, suggesting that these and other classes of genomic structural variation (SV), including inversions, smaller duplications and deletions, complex rearrangements, and retroelement transpositions, may frequently arise as a consequence of reprogramming. Here we employ whole-genome paired-end DNA sequencing and sensitive mapping algorithms to identify all classes of SV in three fully pluripotent mouse iPSC lines. Despite the improved scope and resolution of this study, we find few spontaneous mutations per line (one or two) and no evidence for endogenous retroelement transposition. These results show that genome stability can persist throughout reprogramming, and argue that it is possible to generate iPSCs lacking gene-disrupting mutations using current reprogramming methods.
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