Cardiac differentiation of embryonic stem cells recapitulates embryonic cardiac development.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE5671 | GEO | 2007/08/30
SECONDARY ACCESSION(S): PRJNA96951
REPOSITORIES: GEO
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Project description:Human cardiomyocytes can be generated from human embryonic stem cells (hESCs) in vitro by a variety of methods, including co-culture with visceral endoderm-like cell lines and growth factor directed differentiation as monolayers or three-dimensional embryonic bodies. To enable the identification, purification and characterisation of human embryonic stem cell derived cardiomyocytes (CMs) and cardiac progenitor cells (CPCs), we introduced sequences encoding GFP into the NKX2-5 locus by homologous recombination. We found that NKX2-5GFP hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells and cardiomyocytes and the standardization of differentiation protocols.
2011-11-16 | E-MEXP-3371 | biostudies-arrayexpress
Project 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).
2009-03-15 | E-GEOD-10970 | biostudies-arrayexpress
Project 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. Keywords: time course, differentiated cardiac cell fate
2009-03-05 | GSE10970 | GEO
Project description:Rationale: Cardiogenesis is regulated by a complex interplay between transcription factors and chromatin-modifying enzymes. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective: To identify novel regulators of mesodermal cardiac lineage commitment. Methods and Results: We performed a bioinformatic-based transcription factor-binding site analysis on upstream promoter regions of genes that are enriched in ES cell-derived CPCs. From 32 candidate transcription factors screened, we found that YY1, a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1 kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays and in vivo chromatin immunoprecipitation (ChIP) and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with GATA4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by ES cell-based assays where we show that the over-expression of YY1 enhanced the cardiogenic differentiation ES cells into CPCs in a cell autonomous manner. Conclusion: These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes. We report the identification of putative YY1 target genes in cardiac progenitor cells (CPCs). Two samples of independently FACS-purified eGFP+ CPCs were examined against the input.
2013-06-01 | E-GEOD-35370 | biostudies-arrayexpress
Project description:Mouse embryonic stem cells can differentiate in vitro into spontaneously contracting cardiomyocytes. The main objective of this study was to investigate cardiogenesis in cultures of differentiating embryonic stem cells (ESCs) and to determine how closely it mimics in vivo cardiac development. We identified and isolated a population of cardiac progenitor cells (CPCs) through the use of a reporter DNA construct that allowed the expression of a selectable marker under the control of the Nkx2.5 enhancer. We proceeded to characterize these CPCs by examining their capacity to differentiate into cardiomyocytes and to proliferate. We then performed a large-scale temporal microarray expression analysis in order to identify genes that are uniquely upregulated or downregulated in the CPC population. We determined that the transcriptional profile of the mESC derived CPCs was consistent with pathways known to be active during embryonic cardiac development. We conclude that in vitro differentiation of mESCs recapitulates the early steps of mouse cardiac development. Experiment Overall Design: Cell culture and cell differentiation Experiment Overall Design: Mouse embryonic stem cells (mESCs) were maintained undifferentiated by culturing on a feeder layer of mitomycin C pretreated primary mouse embryonic fibroblasts (PMEFs, Specialty Media, cat.# PMEF-H) at 37oC, 90% humidity and 5% CO2. The cell line used was the mESC D3 (passage #12-18). The basic culture medium was comprised of DMEM (Invitrogen, cat.# 11965-092), heat inactivated (56oC for 30 minutes) fetal bovine serum (Hyclone, cat#. S11550, 15%), Glutamax (Invitrogen, cat.# 35050-065, 1%), non-essential amino acids (Invitrogen, cat.# 11140-050, 1%), sodium pyruvate (Invitrogen, cat.# 11360-070, 1%), ?-mercaptoethanol (Sigma, cat.# M7522, 0.1mM), gentamicin (Cambrex, cat.# 17-518, 25?g/ml) and ESGRO leukemia inhibitory factor (Chemicon, cat.# ESG1106, 1000units/ml). Prior to initiation of differentiation The PMEF feeder layer was subtracted and the mESCs were maintained on a 0.01% gelatin pretreated plate for 24-48 hours. Differentiation through embryoid body (EB) formation was initiated by dissociating the mESCs using trypsin (Invitrogen, cat#. 25300-054, 0.05%) and resuspending them in basic culture medium (without the leukemia inhibitory factor) at a final concentration of 5x104 cells/ml. The hanging droplet (HD) technique was used for EB formation. HDs were plated on the lid of H2O containing tissue culture dishes at a volume of 20?L/droplet (approximately 1000 cells/ droplet). Two days post initiation of differentiation the EBs were transferred in suspension on poly-HEMA (Sigma, cat#. P3932) treated tissue culture dishes. The plates were pretreated with poly-HEMA in order to avoid any cell attachment on the plastic. Basic culture medium was supplemented with ascorbic acid(Takahashi et al., 2003) (Sigma, cat#. A4403, 0.1mg/ml) and it was exchanged with fresh medium every 2 days during differentiation. Experiment Overall Design: Transfection of mouse embryonic stem cells Experiment Overall Design: The mouse Nkx2.5 enhancer fragment #5(Lien et al., 1999) and the hsp68 minimal promoter was excised from the provided vector (XhoI/ NcoI) and inserted in front of the EGFP gene in the Bluescript vector. A vector containing the hygromycin resistance gene (hygromycin phosphotransferase) under the control of the mouse polymerase II promoter was also used in order to select for the transfected mESCs. Prior to transfection the two vectors were linearized. Undifferentiated mESCs were grown to confluency on a layer of primary embryonic fibroblasts and dissociated with trypsin. The cells were combined with the linearized DNA and electroporated (Biorad Gene Pulser Xcell, 240V/ 500?F). The cells were then replated on a fresh layer of PMEFs. Hygromycin B (Invitrogen, cat.# 10687-010, 50?g/ml) was used for 7 days after transfection for selection. Resistant colonies were picked and grown on layers of PMEFs in the presence of antibiotic. Once sufficient cells were present the clones were differentiated in order to check for the correct expression of the GFP in the cardiac areas (spontaneously contracting) of the differentiating cultures. Successful clones were further amplified and used for characterization of the GFP expressing cardiac progenitor cells. Experiment Overall Design: In order to select mESC derived cardiomyocytes for the microarray analysis experiment, a stable transgenic clone of mESCs was made that allowed the expression of the neomycin resistance gene under the control of the alpha myosin heavy chain promoter (U71441) as described by Klug et al., (Klug et al., 1996). The transgenic clone was prepared as described above. Experiment Overall Design: FACS Analysis Experiment Overall Design: For determination of the CPC yield in the differentiating cells, EBs were harvested at the indicated timepoints (between days 6 and 10 of differentiation). Each sample contained approximately 100 hanging droplets on day 0 of differentiation. The EBs were washed in PBS and resuspended in 0.05% trypsin at 4oC (1/2 hour) and 37oC (10 minutes). The percentage of GFP+ cells was determined in a BD FACS Calibur (Cell Quest Pro software). Age matched EBs from untransfected mESCs were used as negative controls. Cells from the same samples were also counted. Experiment Overall Design: FACS sorting Experiment Overall Design: The BD Biosciences FACSAria Cell sorting system was used to sort the GFP+ and GFP- cells between days 5 and 10 of differentiation. EBs were harvested and dissociated as described above. The cells were suspended in sorting medium (96% DMEM without phenol red, 1% fetal bovine serum, 1% Glutamax, 1% non essential amino acids, 1% sodium pyruvate, 25?g/ml gentamicin) and kept at 4oC. The cells were sorted into a fetal bovine serum rich medium (20%) in order to increase their survival. For further culture the cells were resuspended in mESC culture medium at a concentration of 5x105 cells/ml and cultured in suspension for 48 hours. The cells were then transferred on tissue culture plates for another 48 hours. For RNA isolation the cells were spun down and washed in PBS before the RNA isolation procedure. Experiment Overall Design: To assay their proliferation capacity, GFP+ CPCs were FACS sorted on day 5 of differentiation. Cells were reaggregated in suspension (5x105 cells/ ml) in the presence of mouse recombinant Igf1 (Chemicon, Cat.# GF121, 100ng/ml) for 14 days. Cell aggregates were fixed and stained for the colocalization of the antigens: Nkx2-5 and Ki67 or Pcna. The same ICC reagents were used as described above. Experiment Overall Design: GeneChip microarray hybridization Experiment Overall Design: Total RNA for the microarray expression analysis was isolated from FACS sorted cells (GFP+ and GFP-) on days 5, 6, 7, and 8 of differentiation as described above. Two separate differentiation batches were prepared for the sake of data reproducibility. Total RNA was also isolated from terminally differentiated mESC derived genetically selected cardiomyocytes 3 weeks post initiation of differentiation. The GeneChip® two cycle target labeling kit (Affymetrix, cat.# 900494) was used to convert 100ng of total RNA to biotinylated labeled cRNA. This was then hybridized to the Affymetrix murine MOE430 2.0 genome GeneChip® array (Affymetrix, cat.# 900496). Fluorescence was detected using the Affymetrix GS3000 GeneArray scanner and image analysis for each chip was done through the GeneChip® operating system software from Affymetrix (GCOS1.3) using the standard default settings. Experiment Overall Design: Microarray data analysis Experiment Overall Design: To estimate the gene expression signals, data analysis was conducted on the chipsâ CEL file probe signal values at the Affymetrix probe pair (perfect match (PM) probe and mismatch (MM) probe) level, using the statistical technique RMA (Robust Multiarray Analysis) (Irizarry et al., 2003) with the bioconductor package Affy. The data normalization procedure utilized the quantile normalization method (Bolstad et al., 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. Experiment Overall Design: The Spotfire DecisionSite software package was used for the identification of uniquely upregulated or downregulated (at least 1.5 times increase or decrease in the expression value) probe sets in the CPC population when compared to the rest of the cells in the differentiating EBs. Probe sets that were considered unique for the CPC population were found to be commonly upregulated or downregulated during all four days of analysis. Probe sets that exhibited an increasing or decreasing pattern of expression with at least 1.5 times increase or decrease in the expression values of day 5 CPCs and day 8 CPCs were also reported. Probe sets of the CPC population that exhibited upregulation or downregulation by at least 1.5 times when compared to the mESC derived cardiomyocytes were also reported. The final analysis included probe sets that exhibited a different temporal pattern of expression in the CPC population when compared to the rest of the cells along the four days of differentiation. Specifically in order to identify these probe sets gene expression curve over time was modeled flexibly
2008-06-14 | E-GEOD-5671 | biostudies-arrayexpress
Project description:Adult cardioyocytes undergo remarkable dedifferentiation and cell cycle reprogramming/reentery when cultured in mitogen-rich medium, continuously, and give rise to cardiac progenitor cells (CPCs). Using microfluidic device for single-cell capture and whole-transcriptomic amplification, we analyze the whole-genome transcriptomic profile in adult mouse cardiomyocytes (Ctl) and in their derived CPCs, and validate the gene expression by single-cell PCR and PCR array. Using two platforms for DNA methylation profiling: NimbleGen DNA methylation array and CHARM array, the whole-genome DNA methylation profile in myocytes (Ctl) and CPCs were compared and their regulations in relationship to the transcriptomics profile were analyzed. The results demonstrated remarkable molecular reprogramming pertaining to dedifferentiation, loss of cardiac contractile, structure, and function molecules, and reactivation of cell cycle and proliferation genes, significant changes in metabolisms. The reduction of cardiac function and structure genes are highly related to their hypermethylation of the promoter regions. GO and Pathway enrichment analysis revealed distinct coordianted transcriptomic and epigenetic regulation in the CPCs derived from cardiomyocytes. Mouse single cardiomyocytes (Ctl) and their derievd single CPCs were captured using microfluidic deviced and cDNA synthesized and amplified and labelled using NuGENE kits, and regular Affymetrix hybridization and wash protocols were used to process the mouse whole-genome array GeneChip 430 2.0.
2016-07-24 | E-GEOD-49448 | biostudies-arrayexpress
Project description:The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs. YFP+ cells marking precardiac mesoderm (Isl1-cre domain) were FACS-sorted (w/wo stabilized Beta-catenin). Their total RNA was amplified with the WT-Ovation Pico RNA Amplification System, fragmented and labeled with the FL-Ovation cDNA Biotin Module V2 (Nugen). The hybridization, staining and scanning of the Affymetrix GeneChips were performed in the Gladstone Genomics Core Lab. Raw data generated from at least three independent experiments were further analyzed by the group of Dr. Ru-Fang Yeh at the Center for Informatics and Molecular Biostatistics, UCSF.
2009-05-13 | E-GEOD-15232 | biostudies-arrayexpress
Project description:Adult cardioyocytes undergo remarkable dedifferentiation and cell cycle reprogramming/reentery when cultured in mitogen-rich medium, continuously, and give rise to cardiac progenitor cells (CPCs). Using microfluidic device for single-cell capture and whole-transcriptomic amplification, we analyze the whole-genome transcriptomic profile in adult mouse cardiomyocytes (Ctl) and in their derived CPCs, and validate the gene expression by single-cell PCR and PCR array. Using two platforms for DNA methylation profiling: NimbleGen DNA methylation array and CHARM array, the whole-genome DNA methylation profile in myocytes (Ctl) and CPCs were compared and their regulations in relationship to the transcriptomics profile were analyzed. The results demonstrated remarkable molecular reprogramming pertaining to dedifferentiation, loss of cardiac contractile, structure, and function molecules, and reactivation of cell cycle and proliferation genes, significant changes in metabolisms. The reduction of cardiac function and structure genes are highly related to their hypermethylation of the promoter regions. GO and Pathway enrichment analysis revealed distinct coordianted transcriptomic and epigenetic regulation in the CPCs derived from cardiomyocytes. Genomic DNA isolated from adult mouse cardiomyocytes (Ctl) or cardiomyocyte-derived progenitor cells (CPCs) was subjected to digestion by methylcytosine-sensitive enzyme McrBC, and subsequently purification and amplification, labeling and hybridization according to NimbleGen protocol. Percentage methylation, hypermethylation or hypomethylation, and peak enrichment were assessed using CHARM protocol.
2016-07-24 | E-GEOD-49676 | biostudies-arrayexpress
Project description:The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.
2009-05-13 | GSE15232 | GEO
Project description:Baseline gene expression of mouse Cardiac Progenitor Cells (CPC) We used microarrays to analyze the global gene expression of mouse CPCs. Compare the gloable gene expression of mouse CPCs and Cardiomyocytes. Although the chip is two color, we only used Cy5 as the data channel.
2012-12-01 | E-GEOD-41175 | biostudies-arrayexpress