Comment[ArrayExpressAccession]	E-GEOD-24487
Public Release Date	2011-03-14															
Investigation Title	Recapitulation of human premature aging by using iPSCs from Hutchinson-Gilford progeria syndrome  															
Comment[Submitted Name]	Recapitulation of human premature aging by using iPSCs from Hutchinson-Gilford progeria syndrome  
Experiment Description	Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature aging disease1-5, characterized by premature atherosclerosis and degeneration of vascular smooth muscle cells (SMCs)6-8. HGPS is caused by a single-point mutation in the LMNA gene, resulting in the generation of progerin, a truncated mutant of lamin A. Accumulation of progerin leads to various aging-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin9-12. Here, we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature aging. Upon differentiation of HGPS-iPSCs, progerin and its associated aging consequences are restored. In particular, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescent SMC phenotypes associated with vascular aging. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs) as a component of the progerin-containing protein complex. The absence of nuclear DNAPKcs correlates with premature as well as physiological aging. Since progerin also accumulates during physiological aging6,12,13, our results provide an in vitro iPSC-based model with an acceleration progerin accumulation to study the pathogenesis of human premature and physiological vascular aging. Microarray gene expression profiling was done to: (1) Compare differences between WT fibroblasts and fibroblasts from patients suffering of the Hutchinson-Gilford progeria syndrome (2) Check that iPSC originating from WT and patients are in fact similar to ESC															
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Term Source Name	EFO
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Term Source File	http://www.ebi.ac.uk/efo/efo.owl															
Person Last Name	Boue	Liu	Barkho	Ruiz	Diep	Yang	Qu	Kurian	Walsh	Panopoulos	Thompson	Boue	Fung	Zhang	Yates III	Izpisua-Belmonte
Person First Name	Stephanie	Guang-Hui	Basam	Sergio	Dinh	Sheng-Lian	Jing	Leo	Christopher	Athanasia	James	Stephanie	Ho-Lim	Kun	John	Juan-Carlos
Person Mid Initials										D						
Person Email	geo@ncbi.nlm.nih.gov															
Person Affiliation	CMRB															
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Person Address	CMRB, Dr Aiguader 88, Barcelona, Spain															
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Protocol Name	P-GSE24487-1	P-GSE24487-5	P-GSE24487-2	P-GSE24487-9	P-GSE24487-7	P-GSE24487-6	P-GSE24487-8	P-GSE24487-3	P-GSE24487-4							
Protocol Description	ID_REF = <br>VALUE = RMA normalized autosome probes-only	The GeneChip microarray processing was performed by the Functional Genomica Core in the Institute for Research in Biomedicine (Barcelona, Spain) according to the manufacturerM-bM-^@M-^Ys protocols (Affymetrix, Santa Clara, CA). The amplification and labeling were processed as indicated in Nugen protocol with 25ng starting RNA. For each sample, 3.75ug ssDNA were labeled and hybridized to the Affymetrix HG-U133 Plus 2.0 chips.	All fibroblasts were cultured in 5% CO2 and at 37M-0C in DMEM containing 15% FBS, 2 mM L-glutamine, 0.1 mM non-essential aminoacids, sodium Pyruvate, and 55 M-NM-<M b-mercaptoethanol.	H9 hESCs (WiCell Research) and iPSCs were maintained on a layer of mitotically inactivated MEFs in DMEM/F12 (Invitrogen) supplemented with 0.1 mM non-essential amino acids (Invitrogen), 1 mM glutamax (Invitrogen), 20% Knockout Serum Replacement (Invitrogen), 55 M-NM-<M b-mercaptoethanol (Invitrogen) and 10 ng/ml bFGF (Joint Protein Central). hESCs and iPSCs were also cultured in Matrigel (BD Biosciences) with mTeSR medium (Stem Cell Technologies).	The statistical analysis of the data was performed using ArrayStar 3. Briefly, raw CEL files were imported together with gene annotation from NetAffix (from 11/13/2009) and normalized using RMA algorithm and quantile normalization. As both H9 ESC and HGPS-iPSC originate from female samples, and in order to check for any possible bias introduced by the X and Y chromosome-coded genes, we performed the same analysis with only autosome genes.	Expression signals were scanned on an Affymetrix GeneChip Scanner (7G upgrade). The data extraction was done by the Affymetrix GCOS software v.1.4.	iPSCs Generation. For the generation of human iPSCs, human fibroblasts were seeded in a 6-well plate and spin-infected with a mix of high-quality retroviruses encoding OCT4, SOX2, KLF4, c-MYC, and GFP in the presence of 4 M-oM-^AM--g/ml polybrene. Three infections on consecutive days were performed. 6 days after the first spin-infection, fibroblasts were gently individualized with TrypLE (invitrogen) and seeded onto fresh MEFs in the fibroblast culture medium. After 24 h, the medium was switched to hESC medium. The media was changed every 1M-bM-^@M-^S2 days depending on cell density. To establish the iPSC lines, colonies were manually picked and transferred onto MEF feeder cells for several passages before further transfer to Matrigel/mTesR conditions.	Total RNA was isolated using Trizol (Invitrogen) followed by cDNA synthesis using High capability RNA-to-cDNA Mater Mix (Invitrogen).	The GeneChip microarray processing was performed by the Functional Genomica Core in the Institute for Research in Biomedicine (Barcelona, Spain) according to the manufacturerM-bM-^@M-^Ys protocols (Affymetrix, Santa Clara, CA). The amplification and labeling were processed as indicated in Nugen protocol with 25ng starting RNA. For each sample, 3.75ug ssDNA were labeled and hybridized to the Affymetrix HG-U133 Plus 2.0 chips.							
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Protocol Type	bioassay_data_transformation	hybridization	grow	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling							
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Experimental Factor Type	cell line	BioSourceProvider														
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Publication Title	Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.															
Publication Author List	Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J 3rd, Izpisua Belmonte JC															
PubMed ID	21346760															
Publication DOI	10.1038/nature09879															
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Comment[SecondaryAccession]	GSE24487															
Comment[GEOLastUpdateDate]	2012-04-11
Comment[AEExperimentType]	transcription profiling by array															
Comment[GEOReleaseDate]	2011-03-14
Comment[ArrayExpressSubmissionDate]	2010-09-30
SDRF File	E-GEOD-24487.sdrf.txt