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1. (WO2018098597) GENERATING ATRIAL AND VENTRICULAR CARDIOMYOCYTE LINEAGES FROM HUMAN PLURIPOTENT STEM CELLS
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Claims:

1 . A population of cardiomyocytes enriched for ventricular cardiomyocytes, wherein said population is essentially free of pacemaker cells.

2. The population of claim 1 wherein said population is devoid of pacemaker cells.

3. A pharmaceutical composition for treating heart failure or myocardial infarction in a patient, the pharmaceutical composition comprising the population of cardiomyocytes of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. A method of producing a population of cardiomyocytes enriched for ventricular cardiomyocytes, the method comprising:

incubating pluripotent stem cells in mesoderm induction medium, said

mesoderm induction medium comprising a BMP component and an effective amount of an activin component sufficient to generate ventricular mesoderm; and thereafter

culturing said incubated cells in suitable medium(s) to generate a population of cardiomyocytes enriched for ventricular cardiomyocytes.

5. The method of claim 4, wherein the concentration of the activin component is greater than the concentration of the BMP component.

6. The method of claim 4 or 5 wherein the ratio of the BMP component to the activin component is about 0.3-1 : 1 , about 0.5: 1 , or about 0.8:1 .

7. The method of any one of claims 4 to 6, wherein the concentration of the activin component is determined by measuring the level of CD235a expressing mesoderm cells and comparing this to the level of RALDH2 expressing meso- derm cells.

8. The method of any one of claims 4 to 7, wherein the concentration of the activin component is chosen by determining a concentration which preferentially results in more CD235a expressing mesoderm cells as compared with RALDH2 expressing mesoderm cells.

9. The method of any one of claims 4 to 8 wherein the activin component is added in an amount of from about 4 ng/ml to about 20 ng/ml.

10. The method of any one of claims 4 to 9 wherein the concentration of the activin component is between 6 - 20ng/ml

1 1 . The method of any one of claims 4 to 10 wherein the concentration of the BMP component is between about 3ng/ml to about 20ng/ml.

12. The method of any one of claims 4 to 1 1 , wherein the concentration of the BMP component is 5ng/ml or 10ng/ml.

13. The method of any one of claims 4 to 12, wherein the concentration of the activin component is 12ng/ml.

14. The method of any one of claims 4 to 13, wherein said BMP component is

BMP4.

15. The method of any one of claims 4 to 14 wherein said activin component is activin A.

16. The method of any one of claims 4 to 15, wherein at least a portion of said population of cardiomyocytes generated is used to treat a subject in need of cardiac repair.

17. The method of claim 16, wherein the subject in need of cardiac repair is at risk of heart failure, suffering heart failure and/or suffering a myocardial infarction event.

18. The method of claim 17 wherein said treatment is before, during or after a myocardial infarction event.

19. A method of producing a population of cardiomyocytes enriched for atrial cardiomyocytes, the method comprising:

incubating pluripotent stem cells in mesoderm induction medium, said

mesoderm induction medium comprising a BMP component and an effective amount of an activin component sufficient to generate atrial mesoderm; and thereafter

adding a retinoic acid component to the cells, wherein said addition of the retinoic acid component occurs during or after the incubation in mesoderm induction medium; and

culturing the incubated cells so that a population of cardiomyocytes enriched for atrial cardiomyocytes is generated.

20. The method of claim 19 wherein said retinoic acid component is added when said cells are RALDH2 positive and CD235a negative.

21 . The method of claim 19 or 20 wherein the ratio of the BMP component to the activin component is about 1 .5 to 1 or greater.

22. The method of any one of claims 19 to 21 wherein the ratio of the BMP com-ponent to the activin component is 3:2.

23. The method of any one of claims 19 to 22 wherein the BMP component is present at a concentration of from about 3 ng/ml to about 100 ng/ml.

24. The method of any one of claims 19 to 23 wherein the BMP component is present in an amount of about 3ng/ml.

25. The method of any one of claims 19 to 24 wherein the activin component is present in an amount of from about 0.01 ng/ml to about 6 ng/ml.

26. The method of any one of claims 19 to 25 wherein the activin component is present in an amount of about 2ng/ml.

27. The method of claim any one of claims 19 to 26 wherein the retinoic acid component is trans retinoic acid or retinol.

28. The method of any one of claims 19 to 27 wherein the retinoic acid component is added in a concentration of between 50nm and 5μΜ.

29. The method of any one of claims 19 to 28 wherein the retinoic acid component is added at a concentration of 500nM.

30. The method of any one of claims 19 to 29 wherein the BMP component is

BMP4.

31 . The method of anyone of claims 19 to 30, wherein the activin component is

Activin A.

32. The method of any one of claims 19 to 31 , wherein the BMP component is added to the mesoderm induction medium after one day..

33. The method of any one of claims 19 to 31 wherein the activin component is added to the mesoderm induction medium after one day .

34. The method of any one of claims 19 to 33 wherein the retinoic acid component is added at about day 3 to day 5 of the method.

35. The method of any one of claims 19 to 34 wherein additional BMP component is not added to the mesoderm induction medium at day 3 of the method.

36. The method of any one of claims 19 to 35, wherein an FGF inhibitor is excluded from the mesoderm induction medium at day 3 of the method.

37. The method of any one of claims 4 to 36, further comprising:

incubating the pluripotent stem cells in a medium suitable for aggregate and/or embryoid body formation, prior to incubating the pluripotent stem cells in the mesoderm induction medium.

38. The method of any one of claims 4 to 37, wherein the cells produced by the method are utilized in an in vitro assay to screen for potential therapeutic compounds.

39. An isolated population of cardiomyocytes enriched for atrial cardiomyo-cytes comprising at least or about 50% of atrial cardiomyocytes, at least or about 60% of atrial cardiomyocytes, at least or about 70% of atrial cardiomyocytes, at least or about 80% of atrial cardiomyocytes, or at least or about 90% of atrial cardiomyocytes.

40. The population of cardiomyocytes of claim 39 wherein said population is obtained according to the method of any one of claims 18 to 38.

41 . An isolated population of cardiomyocytes enriched for ventricular cardiomyocytes comprising at least or about 50% of ventricular cardiomyocytes, at least or about 60% of ventricular cardiomyocytes, at least or about 70% of ventricular cardiomyocytes, at least or about 80% of ventricular cardiomyocytes, or at least or about 90% of ventricular cardiomyocytes.

42. The population of claim 41 , wherein said population is essentially free of pacemaker cells or devoid of pacemaker cells.

43. The population of claim 41 or 42, wherein said population is obtained according to the method of any one of claims 4 to 17.

44. A method of treating a subject in need of cardiac repair, comprising administering to the subject the population of cardiomyocytes according to any one of claims 1 , 2, or 41 -43.

45. The method of claim 44, wherein said subject is at risk for heart failure, is suffering heart failure and/or has experienced a myocardial infarction event.

46. The method of claim45, wherein the myocardial infarction is in the ventricle of the patient.

47. The population of cardiomyocytes of any one of claims 1 , 2, or 40 to 42, for use in the treatment of a subject in need of cardiac repair.

48. Use of the population of cardiomyocytes of any of one of claims 1 , 2, or 41 to 43, in the preparation of a medicament for the treatment of a subject in need of cardiac repair.

49. A process for detecting atrial mesoderm in a population of mesoderm cells, comprising detecting RALDH2, wherein a presence of RALDH2 is indicative of atrial mesoderm.

50. A process for detecting ventricular mesoderm in a population of mesoderm cells, comprising detecting CD235a, wherein a presence of CD235a is indicative of ventricular mesoderm.

51 . A method for producing a population of cardiomyocytes enriched for sinoatrial nodal pacemaker cells or epicardial cells, the method comprising: incubating pluripotent stem cells in mesoderm induction medium, said mesoderm induction medium further comprising a BMP component and an ac- tivin component in amounts sufficient to generate ALDH+/CD235- mesoderm; and thereafter

culturing said incubated cells in suitable medium(s) with one or more of WNT, FGFi and BMP to generate a population of cardiomyocytes enriched for sinoatrial nodal pacemaker cells or epicardial cells.

52. A population of cardiomyocytes produced by the method of claim 51 .

53. A method of screening or evaluating the potential cardiac toxicity of a test compound or agent, comprising the steps of exposing a population of cardiomyocytes according to any of the foregoing cell population claims to the test compound and evaluating the viability, contractility, changes in electric potentials and/or other functionalities of the cells.