Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling by array of fibroblasts from eight individuals to evaluate variability in the establishment of induced pluripotent stem cell lines and to develop future cellular models of disease.

ABSTRACT: Fibroblasts from eight individuals were chosen to evaluate variability in the establishment of iPSC lines and to develop future cellular models of disease. From the eight patients four were healthy controls (Cont) and four were schizophrenic (Sz). All the patients were males with ages ranging from 21-51 and both groups (Cont vs Sz), were age matched. Primary fibroblasts were reprogrammed using lentivirus expressing Oct4, Sox2, Klf4 and cMyc

ORGANISM(S): Homo sapiens

SUBMITTER: Nicholas Matigian

PROVIDER: E-MTAB-1040 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Variability in the generation of induced pluripotent stem cells: importance for disease modeling.

Vitale Alejandra M AM Matigian Nicholas A NA Ravishankar Sugandha S Bellette Bernadette B Wood Stephen A SA Wolvetang Ernst J EJ Mackay-Sim Alan A

Stem cells translational medicine 20120907 9

In the field of disease modeling, induced pluripotent stem cells (iPSCs) have become an appealing choice, especially for diseases that do not have an animal model. They can be generated from patients with known clinical features and compared with cells from healthy controls to identify the biological bases of disease. This study was undertaken to determine the variability in iPSC lines derived from different individuals, with the aim of determining criteria for selecting iPSC lines for disease m ...[more]

PMID: 23197870

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Project description:Epigenetics describes mechanisms via which the environment can act on the genome, including DNA methylation of promoter regions of genes. This could be a way that environmental risk factors could affect neurodevelopment in individuals at risk for schizophrenia. Patient-derived cells provide a means whereby epigenetics and gene-environment interactions might be investigated. Induced pluripotent stem cells (iPS cells) present an attractive patient-derived cellular model because they can be differentiated into cell types of interest in schizophrenia. In this study the influence of the reprogramming process on schizophrenia-associated DNA methylation was investigated through genome-wide DNA methylation profiling and gene expression of patient-derived iPS cells and the fibroblasts from which they were derived. Olfactory neurosphere-derived cells (ONS cells) from the same patients were also profiled to provide a window on epigenetic regulation in three distinct cell types. Patient-derived cell were compared to cells derived from healthy controls to identify differences in DNA methylation and gene expression associated with schizophrenia in three cell types. The main finding is that iPS cells, prior to neuronal differentiation, demonstrated significant schizophrenia-associated differences in DNA methylation. This is in contrast to the fibroblasts from which they were derived, which show lesser schizophrenia-associated differences in DNA methylation. Like iPS cells, ONS cells showed robust significant schizophrenia-associated differences in DNA methylation. Notably, the schizophrenia-associated differences in DNA methylation were unique for each cell type, with little overlap between them, and only 5 genes commonly methylated in all cell types. Gene expression differences among the cell types mirrored the DNA methylation differences in magnitude, again with little overlap in specific genes affected. Gene Ontology analysis of the affected genes identified common cellular process affected in all three cell types without the same genes contributing. These data suggest that most DNA methylation differences are driven by the demands imposed by each cell type with schizophrenia-associated differences also being cell-type specific. Thus DNA methylation may not be a cause of schizophrenia-associated differences in cell functions in these cells but rather a downstream consequence of some unidentified causative mechanisms. This dataset represents the DNA methylation part of the study. The gene expression data is deposited under accession E-MTAB-5016.

Project description:Cell-based models of many neurological and psychiatric diseases, established by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), have now been reported. While numerous reports have demonstrated that neuronal cells differentiated from hiPSCs are electrophysiologically active mature neurons, the âageâ of these cells relative to cells in the human brain remains unresolved. Comparisons of gene expression profiles of hiPSC-derived neural progenitor cells (NPCs) and neurons to the Allen BrainSpan Atlas indicate that hiPSC neural cells most resemble first trimester neural tissue. Consequently, we posit that hiPSC-derived neural cells may most accurately be used to model the early developmental defects that contribute to disease predisposition rather than the late features of the disease. Though the characteristic symptoms of schizophrenia SZ generally appear late in adolescence, it is now thought to be a neurodevelopmental condition, often predated by a prodromal period that can appear in early childhood. Postmortem studies of SZ brain tissue typically describe defects in mature neurons, such as reduced neuronal size and spine density in the prefrontal cortex and hippocampus, but abnormalities of neuronal organization, particularly in the cortex, have also been reported. We postulated that defects in cortical organization in SZ might result from abnormal migration of neural cells. To test this hypothesis, we directly reprogrammed fibroblasts from SZ patients into hiPSCs and subsequently differentiated these disorder-specific hiPSCs into NPCs. SZ hiPSC differentiated into forebrain NPCs have altered expression of a number of cellular adhesion genes and WNT signaling. Methods: We compared global transcription of forebrain NPCs from six control and four SZ patients by RNAseq. Results: Multi-dimensional scaling (MDS) resolved most SZ and control hiPSC NPC samples; 848 genes were significantly differentially expressed (FDR<0.01) Conclusions: The WNT signaling pathway was enriched 2-fold (fisher exact test p-value = 0.031). 1-2 independent differentiations (biological replicates) for each of four control and four schizophrenia patients were analyzed; samples were generated in parallel to neuron RNAseq data.

Project description:To follow-up findings that miR-9 was abundantly expressed in control NPCs, significantly down-regulated in a subset of SZ NPCs, and that miR-9 levels/activity, neural migration and diagnosis were strongly correlated, we tested the effect of manipulating miR-9 at cellular, proteomic and transcriptomic levels. Unexpectedly, proteomic- and RNAseq-based analysis revealed that these effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes. Together these data indicate that aberrant levels and activity of miR-9 may be one of the many factors that contribute to SZ risk, at least in a subset of patients. Methods: We compared global transcription of forebrain NPCs from two control and two SZ patients with manipulated miR-9 levels by RNAseq. Results: Although RNAseq analysis revealed large inter-individual heterogeneity, we were able to resolve several functional consistencies in the effects of our miR-9 perturbations: i) the change in miR-9 activity was consistent with the inhibitory role of miR-9, ii) the gene expression fold-change of miR-9 target genes (between each perturbation and its corresponding control, summarized by the first principal component) was correlated (r=0.95, p=3.92e-04) with miR-9 fold change and iii) the differentially expressed (DE; p <0.01) gene list resulting from miR-9 perturbation (paired t-test) was enriched for miR-9 targets (1.53-fold, p=1.2e-5). Conclusions: We integrated the miR-9 perturbation RNAseq data with our existing RNAseq datasets contrasting control and SZ hiPSC NPC expression from our cohort 1 (six controls, four patients), to ask whether there was any relationship between the âSZ NPC signatureâ and âmiR-9 perturbationâ datasets; we observed that the DE (p-value <0.01) in âSZ NPC signatureâ is enriched for DE (fdr<0.01) in âmiR-9 perturbationâ (the overall enrichment is 2.31-fold (p=9.39e-09)); there is significant correlation between DE fold-change in these two datasets (overall genes r=0.188; p<10e-50). Effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes Biological duplicates of passage-matched NPCs from 1 control (female) and 1 SZ patient (female) were transduced with either RV-GFP or RV-miR-9-GFP; GFP-positive NPCs were purified by fluorescent activated cell sorting (FACS) and expanded for two passages. In parallel, passage-matched NPCs from 2 controls (1 male, 1 female) and 2 SZ patients (1 male, 1 female) were transiently transfected with either scrambled or miR-9 LNA probes. In both instances, miR-9 perturbation was confirmed by qPCR.

Dataset Information

Transcription profiling by array of fibroblasts from eight individuals to evaluate variability in the establishment of induced pluripotent stem cell lines and to develop future cellular models of disease.

Publications

Variability in the generation of induced pluripotent stem cells: importance for disease modeling.

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