Tissue modeling in embryonic stem (ES) cell system

• US 9,321,997 B2
• Filed: 06/21/2004
• Issued: 04/26/2016
• Est. Priority Date: 06/20/2003
• Status: Active Grant

• Current Assignee: Evotec International GmbH (Evotec SE)
• Original Assignee: Axiogenesis AG
• Inventors: Kolossov, Eugen, Bohlen, Heribert, Fleischmann, Bernd, Ehlich, Andreas, Hescheler, Jrgen, Rll, Wilhelm, Knigsmann, Jessica
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US10/561,780
• Publication Number:
  US 20070258948A1
• Time in Patent Office: 4,327 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• 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 2502/081 neurons
• CPC Class Codes: C12N 2502/086 glial cells
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/4836 using multielectrode arrays
• CPC Class Codes: G01N 33/5088 of vertebrates

Abstract

Provided are embryonic stem (ES) cell-derived tissue modeling systems. In particular, systems for the de novo generation of tissue by parallel drug selection of cell types constituting the tissue of interest in one culture of differentiating ES cells is described as well as the use of such systems in transplantation and drug development.

Claims

1. A method of modeling or producing a cardiac tissue comprising:

(a) obtaining cardiomyocytes, wherein a first population of pluripotent cells is differentiated into said cardiomyocytes, wherein said differentiation includes forming embryoid bodies and isolating cardiomyocytes from said embryoid bodies;

(b) obtaining fibroblasts, wherein a second population of pluripotent cells is differentiated into said fibroblasts;

(c) co-culturing the cardiomyocytes obtained in step (a) with the fibroblasts obtained in step (b);

(d) allowing integration and alignment of the cardiomyocytes and fibroblasts into a viable cardiac tissue;

wherein the cardiomyocytes acquire longitudinal morphology upon integration and alignment with the fibroblasts; and

wherein said viable cardiac tissue exhibits contractions for at least 2 weeks.

2. The method of claim 1, wherein the first population of pluripotent cells comprises a selectable marker gene operably linked to a cardiomyocyte-specific regulatory sequence.

3. The method of claim 1, wherein said selectable marker gene confers resistance to puromycin.

4. The method of claim 1, wherein the first population of pluripotent cells comprises a reporter gene operably linked to a cardiomyocyte-specific regulatory sequence.

5. The method of claim 4, wherein the first population of pluripotent cells further comprises a selectable marker gene operably linked to a cardiomyocyte-specific regulatory sequence, and wherein said cardiomyocyte-specific regulator sequence operably linked to the reporter gene is the same as said cardiomyocytes-specific regulatory sequence operably linked to the selective marker gene.

6. The method of claim 5, wherein said reporter gene is selected from different color versions of enhanced green fluorescent protein (EGFP).

7. The method of claim 5, wherein said marker gene and said reporter gene are contained in the same recombinant nucleic acid molecule.

8. The method of claim 7, wherein said marker gene and said reporter gene are contained in the same cistron.

9. The method of claim 2, wherein said cardiomyocyte-specific regulatory sequence is an atrial-specific regulatory sequence, ventricular specific regulatory sequence, or both an atrial and ventricular-specific regulatory sequence.

10. The method of claim 8, wherein said regulatory sequence is a promoter sequence and wherein said promoter sequence is an α

MHC or MLC2V promoter sequence.

11. The method of claim 1, further comprising analyzing the physiological or developmental status or both of the cardiomyocytes and fibroblasts.

12. The method of claim 11, wherein the status is analyzed by monitoring the differentiation of electrical activity of the cardiomyocytes and fibroblasts on an array.

13. The method of claim 12, wherein said status is analyzed by recording the extracellular field potentials with a microelectrode array (MEA).

14. The method of claim 1, wherein said first and/or second population of pluripotent cells is genetically engineered to overexpress or inhibit the expression of a target gene.

15. The method of claim 1, wherein the cardiac tissue is produced and contained in a container.

16. The method of claim 15, comprising taking three or more measurements, optionally at different positions within the container.

17. The method of claim 15, wherein said container is a well in a microtiter plate.

18. The method of claim 17, wherein said microtiter plate is a 24-, 96-, 384- or 1586-well plate.

19. The method of claim 1, further comprising culturing the cardiomyocytes and fibroblasts in the presence of endothelial cells.

20. The method of claim 19, further comprising analyzing the physiological and/or developmental status of the cardiomyocytes, fibroblasts, and endothelial cells.

21. The method of claim 20, wherein the status is analyzed by monitoring the differentiation of electrical activity of the cardiomyocytes, fibroblasts, and endothelial cells on an array.