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| Status |
Public on Apr 15, 2015 |
| Title |
Emergence of a developmental stage-dependent human liver disease signature demonstrated by directed differentiation of alpha-1 antitrypsin deficient iPS cells [miRNA-2_0] |
| Platform organism |
synthetic construct |
| Sample organism |
Homo sapiens |
| Experiment type |
Non-coding RNA profiling by array
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| Summary |
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.
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| Overall design |
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.
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| Contributor(s) |
Wilson AA, Kotton DN |
| Citation(s) |
25843048 |
| |
| Submission date |
Feb 19, 2015 |
| Last update date |
May 02, 2017 |
| Contact name |
Adam C Gower |
| E-mail(s) |
[email protected]
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| Phone |
617-358-7138
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| Organization name |
Boston University School of Medicine
|
| Department |
Department of Medicine
|
| Lab |
Division of Computational Biomedicine
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| Street address |
72 East Concord Street, E632
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
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| Platforms (1) |
| GPL14613 |
[miRNA-2] Affymetrix Multispecies miRNA-2 Array |
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| Samples (27)
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| This SubSeries is part of SuperSeries: |
| GSE66078 |
Emergence of a developmental stage-dependent human liver disease signature demonstrated by directed differentiation of alpha-1 antitrypsin deficient iPS cells |
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| Relations |
| BioProject |
PRJNA275832 |
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