Comment[ArrayExpressAccession] E-GEOD-43197 MAGE-TAB Version 1.1 Public Release Date 2013-09-23 Investigation Title Natural cardiogenesis-based template predicts cardiogenic potential of induced pluripotent stem cell lines Comment[Submitted Name] Natural cardiogenesis-based template predicts cardiogenic potential of induced pluripotent stem cell lines Experiment Description Rationale: Cardiac development is a complex process that results in the first integrated, multi-lineage embryonic tissue. Imperfect developmental progression leads to congenital heart disease, the most common birth defect with developmental corruption affecting more than 1% of all live births. Interrogation of individual genes has provided the backbone for cardiac developmental biology, yet a comprehensive transcriptome derived from natural cardiogenesis is required to establish an unbiased roadmap to gauge innate developmental milestones necessary for stem cell-based differentiation and in vitro disease modeling. Objective: Establish a contextual expression database of spatial-temporal cardiac structures, and validate a predictive tool to diagnose and predict cardiogenic outcomes from individual pluripotent stem cell lines. Methods and Results: Stage-specific cardiac structures were dissected from eight distinctive embryonic time points to produce a genome-wide expressome analysis across the spectrum of early to late cardiogenesis. Hierarchical clustering of the time course dataset demonstrated discrete gene expression profiles during natural embryonic development. In reference to the native cardiogenic expression roadmap, disruptive iPSC-derived cardiac expression profiles were revealed from pro-cardiogenic 3-factor (SOX2, OCT4, KLF4) compared to non-cardiogenic 4-factor (addition of c-MYC) reprogramming regimens upon stage-specific differentiation. Expression of cardiac-related genes from 3F-iPSC differentiated in vitro at day 0, 5, and 11 recapitulated expression of natural embryos at days 0, E7.5-E8.5, and E14.5-E18.5, respectively. In contrast, 4F-iPSC demonstrated variable gastrulation gene expression profiles beginning at day 5 of differentiation. Differential gene expression within the pluripotent ground state between the archetypical high cardiogenic potential of embryonic stem cells recapitulated in 3F-iPSC vs. the low cardiogenic potential of 4F-iPSC revealed 23 distinguishing candidate genes. Upon subsequent differentiation, cell line-specific gene expression differences were magnified to 399 genes at day 5 and 726 genes at day 11. A confirmed panel of 20 genes, differentially expressed between high and low cardiogenic cell lines, was transformed into a predictive score that was sufficient to correctly rank independent iPSC lines according to cardiogenic potential. Conclusions: Transcriptome analysis attuned to the embryonic developing heart provides a robust platform to probe coordinated cardiac specification and maturation from stem cell-based cardiogenesis model systems. Based on this genome-wide expressome roadmap, a panel of pre-cardiac genes was extracted that allowed differential prognosis of cardiogenic competency from individual reprogrammed cell lines at the pluripotent state. The overall experimental design includes 3 time points (Day0, Day5, Day11) and 3 different stem cell lines: R1 embryonic stem cells (ESCs), H9 induced pluripotent stem cells (H9-iPSCs) generated/reprogrammed by 3 transcription factors (called 3F-iPSCs), and 19BL induced pluripotent stem cells (19BL-iPSCs) generated/reprogrammed by 4 transcription factors (called 4F-iPSC). At each time point, each cell line has 3 biological replicates. In total, there are 27 samples. R1-embryonic stem cells (R1-ESCs): Day0 undifferentiated ESCs - 3 biological replicates, Differentiated for 5 days (Day5) - 3 biological replicates, Differentiated for 11 days (Day11) - 3 biological replicates. 3F-iPSC (H9 iPSCs): Day 0 undifferentiated - 3 biological replicates, Differentiated for 5 days (Day5) - 3 biological replicates, Differentiated for 11 days (Day11) - 3 biological replicates. 4F-iPSC (19BL-iPSCs): Day0 undifferentiated - 3 biological replicates, Differentiated for 5 days (Day5) - 3 biological replicates, Differentiated for 11 days (Day11) - 3 biological replicates. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name LI Martinez-Fernandez Li Hartjes Terzic Nelson Person First Name XING Almudena Xing Katherine Andre Timothy Person Mid Initials A J Person Email li.xing@mayo.edu Person Affiliation Mayo Clinic Person Address Health Sciences Research, Mayo Clinic, 200 1st street SW, Rochester, MN, USA Person Roles submitter Protocol Name P-GSE43197-1 P-GSE43197-5 P-GSE43197-6 P-GSE43197-2 P-GSE43197-3 P-GSE43197-4 P-GSE43197-7 Protocol Description The data were analyzed with R/Bioconductor using RMA. ID_REF = VALUE = Log2 RMA signal intensity Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA. Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome 430 2.0 Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400. Pluripotent cells were spontaneously differentiated following an embryoid body (EB) formation protocol in differentiation medium with 20% FBS in the absence of LIF. Cells were grown onto mitomycin-inactivated feeders and cultured in mouse embryonic stem cell medium containing leukemia inhibitory factor (LIF). Qiagen RNeasy kit. GeneChips were scanned using the GeneChip Scanner 3000. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name cell line growth condition time Experimental Factor Type cell line growth condition time Publication Title Natural Cardiogenesis-Based Template Predicts Cardiogenic Potential of Induced Pluripotent Stem Cell Lines. Publication Author List Martinez-Fernandez A, Li X, Hartjes KA, Terzic A, Nelson TJ PubMed ID 24036272 Publication DOI 10.1161/CIRCGENETICS.113.000045 Comment[SecondaryAccession] GSE43197 Comment[GEOReleaseDate] 2013-09-23 Comment[ArrayExpressSubmissionDate] 2012-12-28 Comment[GEOLastUpdateDate] 2013-09-26 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-43197.sdrf.txt