Abstract
The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment.
Keywords:
Calcium imaging; Cardiomyocytes; Disease modeling; Human induced pluripotent stem cells.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Antigens, Surface / genetics
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Antigens, Surface / metabolism
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Biomarkers / metabolism
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Calcium / metabolism
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Cardiomyopathy, Dilated / genetics
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Cardiomyopathy, Dilated / metabolism
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Cardiomyopathy, Dilated / pathology*
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Cell Differentiation
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Cellular Reprogramming*
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Cryopreservation
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Dermis / cytology
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Dermis / metabolism
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Fibroblasts / cytology*
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Fibroblasts / metabolism
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Gene Expression
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Humans
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Induced Pluripotent Stem Cells / cytology*
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Induced Pluripotent Stem Cells / drug effects
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Induced Pluripotent Stem Cells / metabolism
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Intercellular Signaling Peptides and Proteins / pharmacology
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Models, Biological*
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Molecular Imaging
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Mutation
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Myocytes, Cardiac / cytology*
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Myocytes, Cardiac / drug effects
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Myocytes, Cardiac / metabolism
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Plasmids / chemistry
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Plasmids / metabolism
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Primary Cell Culture
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Proteoglycans / genetics
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Proteoglycans / metabolism
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SOXB1 Transcription Factors / genetics
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SOXB1 Transcription Factors / metabolism
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Serial Passage
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Troponin T / genetics
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Troponin T / metabolism
Substances
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Antigens, Surface
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Biomarkers
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Intercellular Signaling Peptides and Proteins
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Proteoglycans
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SOX2 protein, human
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SOXB1 Transcription Factors
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TNNT2 protein, human
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TRA-1-60 antigen, human
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Troponin T
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Calcium