Method for preparing mesenchymal stem cell-like cells and cardiomyocyte-like cells

Patent metadata

Field	Value
Publication number	US-9598675-B2
Application number	US-201214351798-A
Country	US
Kind code	B2
Filing date	Oct 12, 2012
Priority date	Oct 13, 2011
Publication date	Mar 21, 2017
Grant date	Mar 21, 2017

Abstract

The present invention provides a method for preparing mesenchymal stem cell-like cells and cardiomyocyte-like cells from induced pluripotent stem cells. With the method, embryoid bodies are first formed from induced pluripotent stem cells in a non-adherent substrate. The embryoid bodies are then contacted with a serum-free and insulin-free medium comprising a p38-MAPK inhibitor to form aggregates of contracting embryoid bodies. The contracting embryoid body aggregate(s) are then induced to form mesenchymal stem-cell-like cells and aggregates of cardiomyocyte-like stem cells with a medium comprising ≦5% serum in a cell-culture treated substrate. The cardiomyocyte-like cells and the mesenchymal stem-cell like cells may be separated to give isolated populations of each cell type. The population of cardiomyocyte-like cells and mesenchymal stem-cell like cells and the isolated population of each cell type may be used for replacing cells. In particular, the cells may be used for replacing cells in cardiac repair and/or cardiac therapy.

First claim

The invention claimed is:

1. A method for preparing induced pluripotent stem cell-derived mesenchymal stem cells and induced pluripotent stem cell-derived cardiomyocyte cells comprising the steps of:
(i) providing one or more induced pluripotent stem cells;
(ii) forming one or more embryoid bodies from the induced pluripotent stem cells using a non-adherent substrate;
(iii) contacting the embryoid bodies with serum-free and insulin-free medium comprising a p38-MAPK inhibitor to form aggregates of contracting embryoid body cells;
(iv) transferring at least one contracting embryoid body aggregate to a cell-culture treated substrate and contacting the contracting embryoid body aggregates with a medium comprising ≦5% serum to form a population of cells comprising induced pluripotent stem cell-derived mesenchymal stem cells and aggregates of induced pluripotent stem cell-derived cardiomyocyte cells, wherein the population of cells is substantially adhered to the substrate.

2. The method according to claim 1 , comprising detaching the adhered cells from the substrate and collecting the detached cells as a population of induced pluripotent stem cell-derived mesenchymal stem cells and induced pluripotent stem cell-derived cardiomyocyte cells.

3. The method according to claim 1 , comprising separating the aggregates of induced pluripotent stem cell-derived cardiomyocyte cells from the induced pluripotent stem cell-derived mesenchymal stem cells to give an isolated population of induced pluripotent stem cell-derived cardiomyocyte cells and an isolated population of induced pluripotent stem cell-derived mesenchymal stem cells.

4. The method according to claim 3 , wherein the isolated population of induced pluripotent stem cell-derived mesenchymal stem cells is homogeneous.

5. The method according to claim 3 , comprising culturing the isolated population of induced pluripotent stem cell-derived mesenchymal stem cells.

6. The method according to claim 3 , further comprising maintaining the isolated population of induced pluripotent stem cell-derived mesenchymal stem cells in culture.

7. The method according to claim 3 , wherein the isolated population of induced pluripotent stem cell-derived cardiomyocyte cells comprises ventricular-like, atrial-like and nodal-like cardiomyocytes.

8. The method according to claim 3 , further comprising culturing the isolated population of induced pluripotent stem cell-derived cardiomyocyte cells.

9. The method according to claim 3 , further comprising maintaining the isolated population of induced pluripotent stem cell-derived cardiomyocyte cells in culture.

10. The method according to claim 1 , wherein step (ii) comprises contacting the induced pluripotent stem cells with a suitable culture medium to form one or more embryoid bodies.

11. The method according to claim 1 , wherein step (iii) comprises contacting the embryoid bodies with a serum-free and insulin-free medium comprising a p38-MAPK inhibitor for at least 8 days.

12. The method according to claim 1 , wherein the serum-free and insulin-free medium of step (iii) comprises the p38-MAPK inhibitor 4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine, 1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea, 4-[4-(4-fluorophenyl)-5-pyridin-4-yl-1,3-dihydroimidazol-2-ylidene]cyclohexa-2,5-dien-1-one, 6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide, 5-[2-tert-butyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-3-(2,2-dimethylpropyl)imidazo[4,5-b]pyridin-2-amine; methanesulfonic acid, 5-(2,6-dichlorophenyl)-2-(2,4-difluorophenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one or 3-[3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2-oxopyridin-1-yl]-N,4-dimethylbenzamide.

13. The method according to claim 1 , wherein the medium of step (iv) comprises 0.5 to 5% serum.

14. The method according to claim 1 , wherein the serum of step (iv) comprises fetal bovine serum (FBS).

15. The method according to claim 1 , wherein the induced pluripotent stem cells comprises human induced pluripotent stem cells.

Assignees

• Singapore Health Serv Pte Ltd

Inventors

• Shim Winston Se Ngie
• Wei Heming

Classifications

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• A61K35/34

  Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44) · CPC title

• C12N5/0668

  Mesenchymal stem cells from other natural sources · CPC title

• C12N5/0662

  Stem cells · CPC title

• C12N2500/90

  Serum-free medium, which may still contain naturally-sourced components · CPC title