Whole-Genome Methylation Profile of Fibroblast-Derived Isogenic Induced Pluripotent Stem Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE105093 | GEO | 2017/10/19
SECONDARY ACCESSION(S): PRJNA414786
REPOSITORIES: GEO
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Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates
2014-09-12 | E-GEOD-55107 | biostudies-arrayexpress
Project description:Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hyper-methylated regions compared with embryonic stem cells. These random aberrant hyper-methylations might be a cause of the differences in the properties of each of the iPSC lines.
2018-07-12 | GSE116924 | GEO
Project description:Whole-genome expression analysis of non-integrating Sendai virus (SeV) vector and retroviral vector-generated human iPS cells.
2010-09-22 | GSE24240 | GEO
Project description:Epigenetic memory in induced pluripotent stem cells (iPSCs), with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, iPSCs from human CD34+ hematopoietic stem cells (HSCs) might be more suitable for hematopoietic differentiation than commonly used fibroblast-derived iPSCs. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). Although genome-wide DNA methylation profiling at all promoter regions demonstrates an epigenetic memory of iPSCs with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. All human iPSC lines showed terminal maturation into normoblasts and enucleated RBCs, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the CD34+ HSC-derived iPSCs. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+ iPSCs and NSC iPSCs, which confirms their comparable erythroid differentiation potential. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300ng of total RNA per sample was used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating the biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. 8 samples were analyzed: CD34 1, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 1, 1 replicate CD34 2, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 2, 1 replicate CD34 OSiPS 1, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSKMiPS 1, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSiPS 2, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate CD34 OSKMiPS 2, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate H1, Human H1 embryonic stem cell (ESC), 1 replicate H9, Human H9 embryonic stem cell (ESC), 1 replicate
2015-02-20 | E-GEOD-55109 | biostudies-arrayexpress
Project description:Whole-genome expression analysis of non-integrating Sendai virus (SeV) vector and retroviral vector-generated human iPS cells. Parental fibroblast and induced pluripotent stem (iPS) cells.
2010-09-22 | E-GEOD-24240 | biostudies-arrayexpress
Project description:We examined genome-wide DNA methylation with 20 human iPSC lines derived from five different cell types,and 7 human ESC lines using Illumina’s Infinium HumanMethylation450K and compared among iPSCs by retrovirus, Sendai virus or episomal vectors.
2017-11-06 | GSE73938 | GEO
Project description:The inclusion of oocyte factors together with Yamanaka’s previously identified reprogramming factors (OCT4, SOX2, KLF4 with or without cMYC; OSK(M)) may facilitate the reprogramming process that leads to induced pluripotent stem cells (iPSCs). We previously applied label-free LC-MS/MS analysis to search for such facilitators of reprogramming (reprogrammome), resulting in a catalog of 28 candidates that are (i) able to robustly access the cell nucleus, and (ii) shared between mature mouse oocytes and pluripotent embryonic stem (ES) cells. In the present study we hypothesized that our 28 reprogrammome candidates would also be (iii) abundant in mature mouse oocytes, (iv) depleted after oocyte-to-embryo transition, and (v) able to potentiate or replace the OSKM factors during iPSC reprogramming. Using LC-MS/MS and isotopic labeling methods we found that the abundance profiles of the 28 proteins was below that of known oocyte-specific and housekeeping proteins. Out of the 28 proteins only arginine methyltransferase 7 (PRMT7) presented a substantially changing profile during mouse embryogenesis and impacted on the conversion of mouse fibroblasts into iPSCs. PRMT7 indeed could very efficiently replace SOX2 in a factor-substitution assay yielding iPSCs. These findings show that proteomics can be used to prioritize the functional analysis of reprogrammome candidates.
2016-05-31 | PXD003093 | Pride
Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.
2014-03-11 | E-GEOD-55725 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) are a valuable resource in veterinary regenerative medicine and cellular therapy, particularly for advancing species-specific applications such as feline medicine. This study employs RNA sequencing (RNA-seq) to investigate the transcriptomic profiles of feline iPSCs generated using the Sendai virus method and mesenchymal stem cells (MSCs) derived from these iPSCs. The comparative analysis reveals unique expression patterns linked to the Sendai virus reprogramming approach, identifying key regulatory pathways and gene networks characteristic of Sendai virus-derived iPSCs. Furthermore, the distinct transcriptome of iPSC-derived MSCs showcases markers associated with mesenchymal lineage commitment and MSC functionality. These findings provide valuable insights into the impact of Sendai virus reprogramming on feline iPSC properties and contribute to advancing stem cell-based therapies tailored to feline-specific needs.
2024-11-27 | GSE282965 | GEO
Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture. 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived.
2015-06-08 | E-GEOD-69632 | biostudies-arrayexpress