Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of day 31 hESC-cardiomyocytes treated with DMSO (control) or retinoic acid (RA) during differentiation

ABSTRACT: Human embryonic stem cell-reporter line hESC-NKX2.5(eGFP/w) were differentiated to cardiomyocytes (CMs) by utilizing the spin embryoid body method. During differentiation the cells were treated with DMSO or retinoic acid (RA) from day 4-7. At day 31, cells were sorted based on GFP prior to RNA isolation. The results of this microarray demonstrate that CMs treated with RA during differentiation exhibit atrial-like gene expression profile, while DMSO-treated cells show ventricular-like gene profile. CMs treated with DMSO or RA during differentiation were sorted for GFP and analyzed for differential gene expression.

ORGANISM(S): Homo sapiens

SUBMITTER: Harsha Devalla

PROVIDER: E-GEOD-61154 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recent establishment of induced pluripotent stem (iPS) cells opened new avenues for generating human patient-specific stem cell derivatives that can be used for in vitro modeling of human disease, drug development and cell replacement therapy. In this study we analyzed the molecular and functional properties of cardiomyocytes (CM) differentiated from human iPS cells. Clusters of synchronously beating cells were first observed at day 11 of iPS cell differentiation. Beating clusters that were microdissected at day 18 of differentiation expressed high levels of cardiospecific transcripts NKx2.5, alpha-MHC, MLC2v, alpha-actinin and troponin T. Immunocytochemical stainings for alpha-actinin and troponin T revealed cross-striations typical of CM. Functional assessment of iPS cell-derived CM showed that these cells possess intact calcium transients and respond to beta-adrenergic and muscarinic stimulation. Molecular, structural and electrophysiological properties of iPS cell-derived CMs were highly comparable to their human ES cell-derived counterparts at the same differentiation stage. Comparison of global gene expression profiles of human ES and iPS cells and the corresponding microdissected beating areas further confirmed their similarity. We conclude that human iPS cells can differentiate into functional CM, which are indistinguishable from human ES cell-derived CMs and may fulfill the basic requirement for their use in disease modeling, drug screening and future therapeutic applications. Six different experimental groups were included into analysis: undifferentiated human ES cells (1) and undifferentiated human iPS cells (2), human ES cell-derived cardiomyocytes (3) and human iPS cell-derived cardiomyocytes (4) enriched by microdissection of spontaneously contracting embryoid body outgrowths, and human fetal (5) and adult (6) heart tissue. Total RNA samples were prepared from three independent biological replicates in groups 1-4. In groups 5 and 6, single RNA probes were analyzed as three technical replicates.

Project description:Cardiovascular diseases are a major cause of life-threatening burden around the world. The heart has a very low regeneration capacity and donor organs for transplantation are scarce. Therefore regeneration of lost myocardium with stem cell-derived cardiomyocytes (CMs) provides an attractive strategy for heart repair. Human pluripotent stem cells (hPSCs) can be efficiently differentiated in vitro into CMs but the molecular mechanisms behind this process are still not fully understood. In particular identification of secreted autocrine and/or paracrine factors that function as important extrinsic signals remained elusive because the mass spectrometry (MS)-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. Thus we established an albumin-free cardiomyogenic differentiation medium and performed secretomics at seven different time points during in vitro differentiation. This analysis led to the identification of 4832 proteins with 1802 being significantly altered during differentiation and 431 of these were annotated as secreted according to gene ontology. Bioinformatics revealed enrichment of extrinsic Wnt pathway-related proteins 3 days upon induction of differentiation and of extracellular matrix proteins in the resulting CMs. Numerous extrinsic components of Wnt, Activin A, Nodal, TGFβ, BMP or FGF signaling pathways were quantitatively assessed during differentiation. Notably, the abundance of pathway agonists was generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. We hypothesize that Activin A, Nodal and TGFβ signaling are turned down shortly upon initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation and interestingly, Activin A and TGFβ signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.

Project description:The aim of the study was to define the specific gene expression signature of pancreatic endoderm cells (PECs), by designing a strategy for generating putative PECs (PDX1+/NKX6-1+, and posterior foregut endoderm (PFG) cells (PDX1+/NKX6-1-). Cell line: PDX1-eGFP reporter cell line (PDXeG clone 170-3) generated using the HUES-4 embryonic stem cell line. Human embryonic stem cells (hESCs) were maintained on irradiated mouse embryonic fibroblasts (MEFs). Generation of a hESC-derived PDX1-eGFP reporter cell line: A PDX1-targeting vector was constructed by inserting an eGFP-pSV40-NeoR reporter cassette into the 5â² untranslated region of the PDX1 gene upstream of the PDX1 start codon (ATG), resulting in an GFP-tagged PDX1 allele. The GFP cassette was flanked by a 12.5kb homologous arm on the 5â direction and by a 3,5kb homologous arm on the 3 direction. The final targeting construct was verified by PCR, restriction analysis and sequencing. After approximately 2 weeks emerging clones were picked, expanded and analyzed by PCR. The Neomycin cassette was deleted by using a CRE expression vector (NLS-CRE-IRES-Puro) that was transiently co-transfected with a DsRED or GFP plasmid into selected targeted cloned. 24 hours post electroporation, GFP/DsRED positive single cells were FACS sorted and plated into 96-well plates. Clones were expanded and characterized for Neo excision by PCR. Differentiation protocol: hESCs cultured on MEFs were grown until 90% confluency and differentiated into definitive endoderm (DE) by using a modified version of the DâAmour protocol (D'Amour et al., 2005). The first day of differentiation, hESCs were cultured in the presence of 100ng/ml human Activin A (Peprotech) and 25ng/ml Wnt3a (R&D systems) in RPMI medium (Life Technologies). The four following days 100ng/ml human Activin A was added together with 1x B27-insulin in RPMI medium (Life Technologies). To generate posterior foregut cells, DE cells were treated with 64 ng/ml bFGF for additional 12 days. To generate pancreatic endoderm cells, DE cells were first treated with 2uM retinoic acid (RA, Sigma Aldrich) in DMEM/F12 medium containing 1x B27 +insulin (Life Technologies) for three days and then finally treated with 64 ng/ml FGF2 in combination with 100 ng/ml Noggin for the remaining 9 days.

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Genome-wide analysis of day 31 hESC-cardiomyocytes treated with DMSO (control) or retinoic acid (RA) during differentiation

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