Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data in induced pluripotent stem cells (iPSCs) derived from a DNA repair deficient fibroblast

ABSTRACT: Cockayne syndrome (CS) is an autossomal human disorder characterized by premature aging along with other symptoms. At the molecular level, CS is characterized by a deficiency in the Transcription-couple DNA repair pathway caused by a mutation mainly in ERCC6 gene and the absence of its functional protein. It has been shown that the presence of DNA damage and the lack of some functional proteins related to DNA repair constitute a barrier for somatic cell reprogramming. Recently, it was demonstrated that one protein involved in Genome Global Repair controls the expression of an important pluripotent gene, highligting its importance for cellular reprogramming. We used microarray to confirm cellular reprogramming of CS fibroblasts at the molecular level and detail the expression of some genes invoved in cell death and aging control that might explain the unique characteristics of these iPSCs. Normal (wild type) and fibroblasts from a CS patient were reprogrammed and independent clones were isolated. After sucessive passages in culture, total RNA from donor fibroblasts, iPSCs clones and human embryonic stem cell line (HUES6) were isolated and transcriptional analysis using human genome Affimetrix Gene Chip arrays were performed. HUES6 was used to demonstrate that CS and normal iPSCS show a global gene expression similar to an embryonic stem cell, confirming that both fibroblasts were successfully reprogrammed. We also used fibroblasts from CS to point out the diiferences in global gene expression after reprogramming. We sought to use normal (control) iPSCs to compare the levels of some genes involved in cell death and aging in iPSCs from CS fibroblasts.

ORGANISM(S): Homo sapiens

SUBMITTER: Jason Nathanson

PROVIDER: E-GEOD-36648 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Evidence for premature aging due to oxidative stress in iPSCs from Cockayne syndrome.

Andrade Luciana Nogueira de Sousa LN Nathanson Jason L JL Yeo Gene W GW Menck Carlos Frederico Martins CF Muotri Alysson Renato AR

Human molecular genetics 20120601 17

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblas ...[more]

PMID: 22661500

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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.

Dataset Information

Expression data in induced pluripotent stem cells (iPSCs) derived from a DNA repair deficient fibroblast

Publications

Evidence for premature aging due to oxidative stress in iPSCs from Cockayne syndrome.

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