Differentiation protocol for making human cardiomyocytes

• US 20050164382A1
• Filed: 01/21/2005
• Published: 07/28/2005
• Est. Priority Date: 07/12/2001
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Chunhui Xu
• Inventors: Xu, Chunhui
• Granted Patent: US 7,763,464 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/62 DMSO
• CPC Class Codes: C12N 2501/06 Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

This invention provides populations human cells of the cardiomyocyte lineage. The cells are obtained by causing cultures of pluripotent stem cells to differentiate in vitro, and then harvesting cells with certain phenotypic features. Differentiated cells bear cell surface and morphologic markers characteristic of cardiomyocytes, and a proportion of them undergo spontaneous periodic contraction. Highly enriched populations of cardiomyocytes and their replicating precursors can be obtained, suitable for use in a variety of applications, such as drug screening and therapy for cardiac disease.

Claims

1. A method of differentiating primate pluripotent stem (pPS) cells into cardiomyocyte lineage cells, comprising:

a) obtaining pPS cells that were produced from a human blastocyst;

b) culturing the cells with a first factor selected from activin, transforming growth factor (TGF), and insulin-like growth factor (IGF), and with a second factor selected from bone morphogenic protein (BMP), fibroblast growth factor (FGF), insulin, and platelet-derived growth factor (PDGF); and

then

c) collecting a population of cells from the culture that have differentiated into cardiomyocyte lineage cells, characterized in that at least about 5% of the cells express cardiac troponin I (cTnI), cardiac troponin T (cTnT), or atrial natriuretic factor (AN F) from an endogenous gene.

2. The method of claim 1, wherein the first factor is selected from Activin A, TGF β

1, and IGF II.

3. The method of claim 1, wherein the cells are cultured with Activin A, TGF β

1, and IGF II.

4. The method of claim 1, wherein the second factor is selected from BMP-4, FGF-4, Insulin, basic FGF, and PDGF-BB.

5. The method of claim 1, wherein the first factor is an activin, and the second factor is a bone morphogenic protein.

6. The method of claim 5, comprising culturing the cells in a medium containing an activin, at least two growth factors, and a bone morphogenic protein

7. The method of claim 5, wherein the cells are cultured with Activin A.

8. The method of claim 5, wherein the cells are cultured with BMP-4.

9. The method of claim 5, wherein the cells are cultured with both Activin A and BMP-4.

10. The method of claim 1, wherein the pPS cells are from an established line of human embryonic stem cells.

11. The method of claim 1, comprising culturing pPS cells in an environment essentially free of feeder cells;

and then culturing the cells with said factors so as to differentiate into cardiomyocyte lineage cells.

12. The method of claim 1, comprising initiating differentiation of the pPS cells by forming embryoid bodies before culturing with said factors.

13. The method of claim 1, comprising differentiating the cells on a surface coated with gelatin or with an extracellular matrix comprising laminin.

14. The method of claim 1, further comprising culturing the cells with a nucleotide analog that affects DNA methylation;

such as 5-aza-deoxy-cytidine.

15. The method of claim 1, further comprising separating the cells after culturing with said factors by density centrifugation.

17. The method of claim 1, further comprising culturing the collected cells for at least 1 week in a medium containing creatine, carnitine, or taurine.

18. The method of claim 1, wherein at least about 20% of the collected cells express at least three markers selected from:

cTnI, cTNT, sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, P1-adrenoceptor (β

1-AR), ANF, MEF-2A, MEF-2B MEF-2C, MEF-2D creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (AN F).

19. The method of claim 1, wherein at least about 60% of the collected cells express cardiac-specific myosin heavy chain.

20. The method of claim 1, wherein at least about 5% of the collected cells have spontaneous contractile activity.

16. The method of claim 16, wherein the separating comprises distributing cells in the population according to their density, and collecting cells at a density between about 1.05 and about 1.075 g/mL.