Transcriptomics

Dataset Information

6

Emergence of a developmental stage-dependent human liver disease signature demonstrated by directed differentiation of alpha-1 antitrypsin deficient iPS cells [HumanMethylation450]


ABSTRACT: We monitored 9 pluripotent stem cell lines across three time points of hepatic directed differentiation, representing 3 developmental stages: undifferentiated (T0), definitive endoderm (T5), and early hepatocyte (T24). ESCs (n=3) and patient-derived normal (n=3) or PiZZ (n=3) iPSCs were analyzed in the undifferentiated state (T0), after differentiation to definitive endoderm (T5), and upon reaching hepatic stage (T24) for a total of 27 samples. We sought to test the hypothesis that a single transgene-free iPSC clone from each donor could be used to detect disease-specific differences between the normal cohort and the PiZZ cohort, anticipating that this difference would emerge only at a developmental stage in which the mutant AAT gene is expressed. Cells were sorted before analysis at T0 and T5 after antibody staining for TRA1-80+/SSEA3+ (T0) or C-kit+/CXCR4+ (T5) cells. ESCs (n=3) and patient-derived normal (n=3) or PiZZ (n=3) iPSCs were analyzed in the undifferentiated state (T0), after differentiation to definitive endoderm (T5), and upon reaching hepatic stage (T24) for a total of 27 samples. Cells were sorted before analysis at T0 and T5 after antibody staining for TRA1-80+/SSEA3+ (T0) or C-kit+/CXCR4+ (T5) cells. RNA was extracted at each stage, biotin labeled, and hybridized to either Affymetrix GeneChip Human Gene 1.0 ST or miRNA 2.0 arrays. For methylation analysis, gDNA was sorted from the same 27 samples before undergoing bisulfite conversion. Bisulfite converted DNA was then amplified and purified prior to overnight hybridization to Illumina’s Infinium HumanMethylation 450 BeadChips. Next day staining of hybridized arrays produced methylation-dependent differential fluorescence that was detected via an Illumina iScan array scanner.

ORGANISM(S): Homo sapiens  

SUBMITTER: Darrell N Kotton   Andrew A Wilson  Adam C Gower 

PROVIDER: E-GEOD-66077 | ArrayExpress | 2015-04-15

SECONDARY ACCESSION(S): GSE66077PRJNA275835

REPOSITORIES: GEO, ArrayExpress

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Publications


Induced pluripotent stem cells (iPSCs) provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ) in the gene responsible for alpha-1 antitrypsin (AAT) deficiency, we find that the global transcriptomes of Pi  ...[more]

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