Cardiomyocytes from induced pluripotent stem cells from patients and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9395354-B2
Application number	US-201213554946-A
Country	US
Kind code	B2
Filing date	Jul 20, 2012
Priority date	Jul 21, 2011
Publication date	Jul 19, 2016
Grant date	Jul 19, 2016

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.

First claim

That which is claimed is:

1. A method for cardiac disease-relevant screening of a candidate agent, the method comprising:
(a) contacting the candidate agent with an isolated population of one or more cardiomyocytes or a panel of cardiomyocytes differentiated from one or more induced human pluripotent stem cells (iPS cells) comprising at least one allele encoding a mutation associated with a cardiac disease; and
(b) determining the effect of the candidate agent on one or more phenotypes exhibited by one or more cardiomyocytes within the isolated population of cardiomyocytes or within the panel of cardiomyocytes wherein the one or more phenotypes are associated with dilated cardiomyopathy and are selected from the group consisting of: (a) relative to a cardiomyocyte produced from an induced pluripotent stem cell derived from a normal subject (“normal iPSC-CM”), an initial positive chronotropic effect in response to positive inotropic stress that later becomes negative with characteristics of failure; (b) a decreased inotropic activity compared to a normal iPSC-CM; (c) a decreased chronotropic activity compared to a normal iPSC-CM; (d) a decreased contractile force compared to a normal iPSC-CM; (e) a gene expression profile that differs from a gene expression profile of a normal iPSC-CM; (f) calcium transients that are smaller than calcium transients displayed by a normal iPSC-CM; (q) a weaker ability to resist mechanical stimulation as compared to a normal iPSC-CM; (h) cessation of spontaneous contraction in response to norepinephrine stimulation; (i) a higher frequency of punctate distribution of sarcomeric alpha-actin in comparison to a normal iPSC-CM; and (j) increased sarcomeric disorganization in response to contractile stimulation when compared to a normal iPSC-CM.

2. The method of claim 1 , wherein the mutation is in a gene selected from 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 (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); and cardiac LIM protein (CSRP3).

3. The method of claim 1 , wherein the mutation is TNNT2 R173W.

4. The method of claim 1 , wherein at least one cardiomyocyte within the isolated population of one or more cardiomyocytes or within the panel of cardiomyocytes exhibits, relative to a normal cardiomyocyte, an initially positive chronotropic effect in response to positive inotropic stress, that later become negative with characteristics of failure.

5. The method of claim 1 , wherein the contacting the candidate agent comprises contacting the candidate agent with a panel of cardiomyocytes comprising at least two cardiomyocytes having differing genotypes.

6. The method of claim 1 , wherein the contacting the candidate agent comprises contacting the candidate agent with a panel of cardiomyocytes comprising cardiomyocytes under differing environmental conditions.

7. The method of claim 6 , wherein one or more of the environmental conditions comprises stimulation with a beta-adrenergic agonist.

8. The method of claim 1 , wherein the determining the effect of the candidate agent comprises conducting a single cell analysis.

9. The method of claim 8 , wherein the single cell analysis includes one or more of atomic force microscopy, microelectrode array recordings, patch clamping, single cell PCR, and calcium imaging.

10. The method of claim 1 , wherein the candidate agent is a drug candidate.

11. The method of claim 1 , wherein the candidate agent is a genetic agent.

12. An isolated population of one or more cardiomyocytes or a panel of cardiomyocytes differentiated from one or more human induced pluripotent stem cells (iPSC) comprising at least one allele encoding a mutation associated with a cardiac disease, wherein one or more cells within the isolated population of one or more cardiomyocytes or within the panel of cardiomyocytes display a phenotype associated with the cardiac disease, wherein the cardiac disease is dilated cardiomyopathy and wherein the phenotype of the one or more cells within the isolated population of one or more cardiomyocytes or within the panel of cardiomyocytes is one or more phenotypes selected from the group consisting of: (a) relative to a cardiomyocyte produced from an induced pluripotent stem cell derived from a normal subject (“normal iPSC-CM”), an initial positive chronotropic effect in response to positive inotropic stress that later becomes negative with characteristics of failure; (b) a decreased inotropic activity compared to a normal iPSC-CM; (c) a decreased chronotropic activity compared to a normal iPSC-CM; (d) a decreased contractile force compared to a normal iPSC-CM; (e) a gene expression profile that differs from a gene expression profile of a normal iPSC-CM; (f) calcium transients that are smaller than calcium transients displayed by a normal iPSC-CM; (g) a weaker ability to resist mechanical stimulation as compared to a normal iPSC-CM; (h) cessation of spontaneous contraction in response to norepinephrine stimulation; (i) a higher frequency of punctate distribution of sarcomeric alpha-actin in comparison to a normal iPSC-CM; and (j) increased sarcomeric disorganization in response to contractile stimulation when compared to a normal iPSC-CM.

13. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein the mutation is in a gene selected from the group consisting of 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 (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); plakophilin 2 (PKP2); and cardiac LIM protein (CSRP3).

14. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein the mutation is TNNT2 R173W.

15. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein relative to a normal iPSC-CM, cells within the isolated population of one or more cardiomyocytes or within the panel of cardiomyocytes have an initially positive chronotropic effect in response to positive inotropic stress that later becomes negative with characteristics of failure.

16. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein the cardiomyocytes comprise cardiomyocytes having differing genotypes.

17. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein the isolated population of one or more cardiomyocytes or the panel of cardiomyocytes comprises cardiomyocytes under differing environmental conditions.

18. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 17 , wherein one or more of the environmental conditions comprises stimulation with a β-adrenergic agonist.

19. The isolated population of one or more cardiomyocytes or the panel of cardiomyocytes of claim 12 , wherein the isolated population of one or more cardiomyocytes is a purified population of cardiomyocytes or wherein the panel of cardiomyocytes is a panel of purified cardiomyocytes.

20. The isolated population of one or more cardiom

Assignees

• Sun Ning
• Longaker Michael T
• Robbins Robert C
• Wu Joseph
• Lan Feng
• Lee Andrew Stephen
• Burridge Paul W
• Univ Leland Stanford Junior

Inventors

• Sun Ning
• Longaker Michael T
• Robbins Robert C
• Wu Joseph
• Lan Feng
• Lee Andrew Stephen
• Burridge Paul W

Classifications

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• C12N2503/02

  Drug screening · CPC title

• G01N2800/325

  Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2501/604

  Klf-4 · CPC title