METHODS OF GENERATING HUMAN CARDIAC CELLS AND TISSUES AND USES THEREOF

• US 20100196910A1
• Filed: 02/08/2010
• Published: 08/05/2010
• Est. Priority Date: 07/20/2001
• Status: Abandoned Application

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: GEPSTEIN, Lior, Kehat, Izhak, Itskovitz-Eldor, Joseph, Amit, Michal
• Application Number:
  US12/701,648
• Publication Number:
  US 20100196910A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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: 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 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

A method of generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype is disclosed. The method comprises (a) partially dispersing a confluent cultured population of human stem cells, thereby generating a cell population including cell aggregates; (b) subjecting the cell aggregates to culturing conditions suitable for generating embryoid bodies; (c) subjecting the embryoid bodies to culturing conditions suitable for inducing cardiac lineage differentiation in at least a portion of the cells of the embryoid bodies, the culturing conditions suitable for inducing cardiac lineage differentiation including adherence of the embryoid bodies to a surface, and culture, medium supplemented with serum, thereby generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype.

Claims

1. A method of generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype, the method comprising:

(a) partially dispersing a confluent cultured population of human stem cells, thereby generating a cell population including cell aggregates;

(b) subjecting said cell aggregates to culturing conditions suitable for generating embryoid bodies; and

(c) subjecting said embryoid bodies to culturing conditions suitable for inducing cardiac lineage differentiation in at least a portion of the cells of said embryoid bodies, said culturing conditions suitable for inducing cardiac lineage differentiation including adherence of said embryoid bodies to a surface, thereby generating cells predominantly displaying at least one characteristic associated with the cardiac phenotype.

2. The method of claim 1, further comprising isolating said cell aggregates from said cell population prior to step (b).

3. The method of claim 1, further comprising isolating said embryoid bodies prior to step (c).

4. The method of claim 1, wherein said culturing conditions suitable for inducing cardiac lineage differentiation further include culture medium supplemented with serum.

5. The method of claim 1, further comprising screening and optionally isolating cells predominantly displaying at least one characteristic associated with a cardiac phenotype, said screening effected by at least one method selected from the group consisting of detection of mechanical contraction, detection of a cardiac specific structure, detection of a cardiac specific protein, detection of a cardiac specific RNA, detection of cardiac specific electrical activity, detection of cardiac specific changes in the intracellular concentration of a physiological ion.

6. The method of claim 5, wherein said detection of cardiac specific electrical activity is effected using a microelectrode array.

7. The method of claim 6, wherein said multielectrode array comprises electrodes positioned 100 μ

m or less apart.

8. The method of claim 6, wherein said multielectrode array comprises at least 60 electrodes.

9. The method of claim 6, wherein said multielectrode array is configured to obtain data characterizing said cardiac specific electrical activity with a frequency greater than a range selected from 1-25 kHz.

10. The method of claim 5, further comprising screening and optionally isolating cells substantially displaying proliferation.

11. The method of claim 1, wherein said human stem cells are embryonic stem cells.

12. The method of claim 1, wherein said human stem cells are clonal.

13. The method of claim 1, wherein said human stem cells are H9.2 cells.

14. The method of claim 1, wherein said partially dispersing a confluent cultured population of human stem cells is effected via a non-trypsin based method.

15. The method of claim 1, wherein said partially dispersing a confluent cultured population of human stem cells is effected via treatment with collagenase.

16. The method of claim 1, wherein said culturing in step (b) is effected for a time period selected from the range of 7 to 10 days.

17. The method of claim 1, wherein said culturing conditions in step (b) include inhibiting adherence of said cell aggregates to a surface.

18. The method of claim 1, wherein said culturing conditions in step (b) include culture medium supplemented with serum.

19. The method of claim 1, wherein said culturing in step (c) is effected for at least as long as a time period selected from the range of 1-60 days.

20. The method of claim 1, wherein said culturing in step (c) is effected in the presence of dimethyl sulfoxide.

21. The method of claim 1, wherein said culturing conditions in step (c) include exposing said embryoid bodies to a surface coated with gelatin.

22. The method of claim 1, wherein said at least one characteristic associated with a cardiac phenotype is selected from the group consisting of cardiac specific mechanical contraction, a cardiac specific structure, expression of a cardiac specific RNA, expression of a cardiac specific protein, cardiac specific changes in the intracellular concentration of a physiological ion, cardiac specific electrical activity.

23. The method of claim 22, wherein said cardiac specific mechanical contraction is selected from the group consisting of spontaneous mechanical contraction, rhythmic mechanical contraction, synchronous mechanical contraction, and propagative mechanical contraction.

24. The method of claim 22, wherein said cardiac specific structure is selected from the group consisting of a sarcomere, a Z-band, an intercalated disc, a gap junction, a desmosome, a fibrillar bundle, a fibrillar bundle striation, and a myocytic syncytium.

25. The method of claim 22, wherein said cardiac specific RNA encodes a protein selected from the group consisting of cardiac α

-myosin heavy chain, cardiac β

-myosin heavy chain, α

-actinin, cardiac troponin I, cardiac troponin T, GATA-4, Nkx×

2.5, MLC-2A, MLC-2V, atrial myosin light chain, ventricular myosin light chain, and connexin-43.

26. The method of claim 22, wherein said cardiac specific protein is selected from the group consisting of cardiac α

-myosin heavy chain, cardiac β

-myosin heavy chain, atrial natriuretic peptide, cardiac troponin I, desmin and connexin-43.

27. The method of claim 22, wherein said cardiac specific electrical activity is selected from the group consisting of spontaneous electrical activity, rhythmic electrical activity, synchronized electrical activity, and propagative electrical activity.

28. The method of claim 27, wherein said propagative electrical activity is characterized by slow conduction.