Cardiomyocytes From Induced Pluripotent Stem Cells From Patients and Methods of Use Thereof

Abstract

Human somatic cells obtained from individuals with a genetic heart condition are reprogrammed to become induced pluripotent stem cells (iPS cells), and differentiated into cardiomyocytes for use in analysis, screening programs, and the like.

1 - 32 . (canceled) 
     
     
         33 . An in vitro-generated cardiomyocyte wherein:
 a. the in-vitro-generated cardiomyocyte is generated from a pluripotent stem cell or a reprogrammed cell in vitro;   b. the in-vitro-generated cardiomyocyte comprises at least one mutation in a gene encoding a sarcomeric protein; and   c. the in-vitro-generated cardiomyocyte displays a phenotype associated with hypertrophic cardiomyopathy.   
     
     
         34 . The in vitro-generated cardiomyocyte of  claim 33 , wherein the in-vitro-generated cardiomyocyte exhibits, relative to a normal cardiomyocyte, one or more phenotypes selected from the group consisting of: an electrophysiological phenotype, contractile arrhythmia, an increased intracellular calcium level, and an increased ratio of β-myosin expression to α-myosin expression. 
     
     
         35 . The in-vitro-generated cardiomyocyte of  claim 33 , wherein the in-vitro-generated cardiomyocyte is generated from the pluripotent stem cell, and the pluripotent stem cell is an induced pluripotent stem cell. 
     
     
         36 . The in-vitro-generated cardiomyocyte of  claim 35 , wherein the induced pluripotent stem cell is derived from a subject with hypertrophic cardiomyopathy, arrhythmia, or both. 
     
     
         37 . The in-vitro-generated cardiomyocyte of  claim 33 , wherein the gene encoding a sarcomeric protein is cardiac troponin T (TNNT2), myosin heavy chain (MYH7), tropomyosin 1 (TPM1), myosin binding protein C (MYBPC3), 5′-AMP-activated protein kinase subunit gamma-2 (PRKAG2), troponin I type 3 (TNNI3), titin (UN), myosin light chain 2 (MYL2), actin alpha cardiac muscle 1 (ACTC1), or cardiac LIM protein (CSRP3). 
     
     
         38 . The in-vitro-generated cardiomyocyte of  claim 33 , further comprising at least one mutation in caveolin 3 (CAV3), galactosidase alpha (GLA), lysosomal-associated membrane protein 2 (LAMP2), mitochondrial transfer RNA glycine (MTTG), mitochondrial transfer RNA isoleucine (MTTI), mitochondrial transfer RNA lysine (MTTK), mitochondrial transfer RNA glutamine (MTTQ), myosin light chain 3 (MYL3), troponin C (TNNC1), Transthyretin (TTR), GATA4, or a combination thereof. 
     
     
         39 . The in-vitro-generated cardiomyocyte of  claim 33 , wherein the at least one mutation is in MYH7. 
     
     
         40 . The in-vitro-generated cardiomyocyte of  claim 33 , wherein the at least one mutation is a MYH7 R663H mutation 
     
     
         41 . The in-vitro-generated cardiomyocyte of  claim 33 , wherein the phenotype associated with hypertrophic cardiomyopathy comprises, relative to a normal cardiomyocyte, electrophysiological arrhythmia, contractile arrhythmia, an increased intracellular calcium level, an increased ratio of β-myosin expression to α-myosin expression, an increased cell size, irregular calcium transient, an increased intracellular calcium level, calcineurin activation, nuclear translocation of nuclear factor of activated T-cells (NFAT), or an increased hypertrophic response to a positive inotropic stress. 
     
     
         42 . A population or panel of cells, wherein the population or panel of cells comprises the in-vitro-generated cardiomyocyte of  claim 33 . 
     
     
         43 . The population or panel of cells of  claim 42 , wherein the population or panel of cells has a cardiomyocyte purity of greater than 60%. 
     
     
         44 . A cell culture comprising:
 a) the in-vitro-generated cardiomyocyte of  claim 33 ; and   b) a candidate agent selected from the group consisting of: a calcium channel blocker, a sodium channel blocker, a potassium channel blocker, a beta blocker, and a combination thereof.   
     
     
         45 . A method for screening a candidate agent, the method comprising:
 a) contacting the candidate agent with a cardiomyocyte in vitro, wherein the cardiomyocyte
 i) comprises at least one mutation in a gene encoding a sarcomeric protein; and 
 ii) displays a phenotype associated with hypertrophic cardiomyopathy; and 
   b) using an in vitro assay to detect an effect of the candidate agent on the phenotype associated with hypertrophic cardiomyopathy.   
     
     
         46 . The method of  claim 45 , wherein the candidate agent comprises a drug candidate. 
     
     
         47 . The method of  claim 45 , wherein the in vitro assay comprises atomic force microscopy, microelectrode array recordings, patch clamping, single cell PCR, or calcium imaging. 
     
     
         48 . The method of  claim 45 , wherein the cardiomyocyte is generated from a pluripotent stem cell in vitro. 
     
     
         49 . The method of  claim 45 , wherein the gene encoding a sarcomeric protein is TNNT2, MYH7, TPM1, MYBPC3, PRKAG2, TNNI3, UN, MYL2, ACTC1, CSRP3, CAV3, GLA, LAMP2, MTTG, MTTI, MTTK, MTTQ, MYL3, TNNC1, TTR, or GATA4. 
     
     
         50 . The method of  claim 45 , wherein the phenotype associated with hypertrophic cardiomyopathy comprises, relative to a normal cardiomyocyte, electrophysiological arrhythmia, contractile arrhythmia, an increased intracellular calcium level, an increased ratio of β-myosin expression to α-myosin expression, an increased cell size, irregular calcium transient, an increased intracellular calcium level, calcineurin activation, nuclear translocation of NFAT, or an increased hypertrophic response to a positive inotropic stress. 
     
     
         51 . The method of  claim 45 , further comprising subjecting the cardiomyocyte to electrical stimulation. 
     
     
         52 . The method of  claim 45 , further comprising subjecting the cardiomyocyte to drug stimulation. 
     
     
         53 . The method of  claim 45 , further comprising, prior to the contacting the cardiomyocyte with the candidate agent, generating the cardiomyocyte from a pluripotent stem cell derived from a subject with hypertrophic cardiomyopathy. 
     
     
         54 . The method of  claim 53 , further comprising administering the candidate agent to the subject with hypertrophic cardiomyopathy based on the effect of the candidate agent on the phenotype. 
     
     
         55 . The method of  claim 45 , wherein the candidate agent is a calcium channel blocker or a sodium channel blocker.