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Title: Transcription profiling of mouse embryonic stem cells to identify novel cardiac genes through differentiation      
dateReleased:
03-15-2009
description:
Cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world. However, progress toward improved clinical treatments is hampered by an incomplete understanding of the genetic programs controlling early cardiogenesis. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) differentiating along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. Approximately 24% (43/176) of the transcripts enriched in the CPC population have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a subset 31/133 (23%) of the remaining candidate genes by in situ hybridization and report that all were expressed in key cardiac structures during cardiogenesis (embryonic day, E7.5 - 9.5), many of which were previously uncharacterized. These data demonstrate the power of mESC differentiation to model specific developmental processes and provide a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis. Experiment Overall Design: Transgenic mouse embryonic stem cell line (Nkx2-5-GFP, PMID:9834187) contains an Nkx2-5 cardiac-specific transcription factor driving expression of green fluorescent protein (GFP). Cells were differentiated through embryoid body formation (hanging droplet technique) as described in PMID:8155574. Following primary mouse embryonic fibroblasts (PMEF) feeder subtraction, mESCs were dissociated and resuspended in differentiation medium. Cells at this time point were designated as day 0. Following incubation for two days, the EBs were transferred, in suspension, to poly-HEMA coated tissue culture dishes to prevent cell attachment and grown in medium containing ascorbic acid PMID:12668514) which was replenished every 3 days thereafter. Experiment Overall Design: Cells were harvested on days 0, 4 and 6 in three biological replicates Experiment Overall Design: each (1a, 2a and 3a), washed in PBS for 30 minutes at 37ºC then resuspended in 0.25% trypsin for 7 minutes. Experiment Overall Design: Cells from day 6 were then sorted using the BD Biosciences FACSAria™ Cell sorting system into GFP+ and GFP- fractions into RLT buffer (RNeasy, Qiagen). This sorting separates cells into two groups reflecting their differentiation into those fated to a non-cardiac lineage (GFP negative) and to a cardiac lineage (GFP positive).
privacy:
not applicable
aggregation:
instance of dataset
ID:
E-GEOD-10970
refinement:
raw
alternateIdentifiers:
10970
dateSubmitted:
03-27-2008
keywords:
functional genomics
dateModified:
06-10-2011
creators:
John D. Gearhart
availability:
available
types:
gene expression
name:
Mus musculus
name:
unknown experiment type
ID:
A-AFFY-45
name:
Affymetrix GeneChip Mouse Genome 430 2.0 [Mouse430_2]
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-10970/E-GEOD-10970.raw.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ebi.ac.uk/arrayexpress/files/E-GEOD-10970/E-GEOD-10970.processed.1.zip
storedIn:
ArrayExpress
qualifier:
gzip compressed
format:
TXT
accessType:
download
authentication:
none
authorization:
none
accessURL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE10970
storedIn:
Gene Expression Omnibus
qualifier:
not compressed
format:
HTML
accessType:
landing page
primary:
true
authentication:
none
authorization:
none
abbreviation:
EBI
homePage: http://www.ebi.ac.uk/
ID:
SCR:004727
name:
European Bioinformatics Institute
homePage: https://www.ebi.ac.uk/arrayexpress/
ID:
SCR:002964
name:
ArrayExpress