Method for inducing cardiac differentiation of pluripotent stem cell

Patent metadata

Field	Value
Publication number	US-9587220-B2
Application number	US-201314374453-A
Country	US
Kind code	B2
Filing date	Jan 25, 2013
Priority date	Jan 27, 2012
Publication date	Mar 7, 2017
Grant date	Mar 7, 2017

Abstract

The present invention relates to a method for inducing cardiac differentiation of a pluripotent stem cell, which comprises the steps of
(1) culturing a pluripotent stem cell in a medium containing one or more WNT signaling activators, and (2) culturing a cell produced in the step (1) in a medium containing one or more WNT signaling inhibitor.

First claim

What is claimed is:

1. A method for inducing cardiac differentiation of a pluripotent stem cell, which comprises the steps of
(1) culturing the pluripotent stem cell in a medium containing one or more WNT signaling activators, and
(2) replacing the medium from step (1) with a medium containing one or more WNT signaling inhibitors wherein a plurality of cardiac differentiated cells are produced,
wherein said one or more WNT signaling inhibitors comprise the compound of Formula (I):

wherein
R 1 to R 5 are each independently a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 , wherein R 12 and R 13 are each independently a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; wherein two adjacent groups among R 1 to R 5 may join together to form —O—CH 2 —O—or —O—(CH 2 ) 2 —O—,
R 6 to R 9 are each independently a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; a linear or branched alkoxy group having 1 to 5 carbon atoms which is substituted with a group —C(O)A, wherein A is a saturated or unsaturated 5- or 6-membered ring which is unsubstituted or substituted with a linear or branched alkyl group having 1 to 5 carbon atoms and the ring may contain 1 or 2 atoms independently selected from a nitrogen atom, an oxygen atom and a sulfur atom; a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 , wherein R 12 and R 13 are each independently a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; wherein two adjacent groups among R 6 to R 9 may join together to form —O—CH 2 —O—or —O—(CH 2 ) 2 —O—,
R 10 to R 11 are each independently a hydrogen atom; or a linear or branched alkyl group having 1 to 5 carbon atoms,
X is a sulfur atom, and
n is an integer of 0 to 6,

or a salt thereof.

2. The method of claim 1 , wherein said compound of Formula (I) is selected from the group consisting of

or a salt thereof.

3. The method of claim 2 , wherein the one or more WNT signaling inhibitors comprise

or a salt thereof.

4. The method of claim 1 , wherein the one or more WNT signaling inhibitors further comprise a WNT signaling inhibitor selected from XAV939 and IWP-2.

5. The method of claim 1 , wherein the one or more WNT signaling activators comprise a GSK3β inhibitor.

6. The method of claim 5 , wherein the GSK3β inhibitor is selected from BIO and CHIR99021.

7. The method of claim 1 , wherein the medium in the step (1) and the medium in the step (2) do not comprise serum.

8. The method of claim 1 , wherein the medium in the step (1) and the medium in the step (2) do not comprise a cytokine.

9. The method of claim 1 , wherein the medium in the step (1) and the medium in the step (2) comprise albumin.

10. The method of claim 1 , wherein the medium in the step (1) and the medium in the step (2) do not comprise a protein other than albumin.

11. The method of claim 1 , wherein the two culturing steps are conducted in the absence of a xenogeneic component.

12. The method of claim 1 , wherein the pluripotent stem cell is a monkey or human pluripotent stem cell.

13. The method of claim 1 , wherein the differentiated cells include a cardiomyocyte.

14. A kit for promoting cardiac differentiation comprising one or more WNT signaling activators and/or one or more WNT signaling inhibitors, wherein the kit is used for the method of claim 1 ,
wherein said one or more WNT signaling inhibitors comprise the compound of Formula (I):

wherein
R 1 to R 5 are each independently a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 , wherein R 12 , and R 13 are each independently a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; wherein two adjacent groups among R 1 to R 5 may join together to form —O—CH 2 —O—or —O—(CH 2 ) 2 —O—,
R 6 to R 9 are each independently a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; a linear or branched alkoxy group having 1 to 5 carbon atoms which is substituted with a group —C(O)A, wherein A is a saturated or unsaturated 5- or 6-membered ring which is unsubstituted or substituted with a linear or branched alkyl group having 1 to 5 carbon atoms and the ring may contain 1 or 2 atoms independently selected from a nitrogen atom, an oxygen atom and a sulfur atom; a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 , wherein R 12 , and R 13 are each independently a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; wherein two adjacent groups among R 6 to R 9 may join together to form —O—CH 2 —O—or —O—(CH 2 ) 2 —O—,
R 10 to R 11 are each independently a hydrogen atom; or a linear or branched alkyl group having 1 to 5 carbon atoms,
X is a sulfur atom, and
n is an integer of 0 to 6,

or a salt thereof.

15. The method of claim 1 , wherein
R 1 , R 4 , R 5 , R 6 and R 9 each are a hydrogen atom,
R 2 and R 3 are a methoxy group, an ethoxy group or a propoxy group,
R 10 and R 11 are a hydrogen atom, and
n is an integer of 0 to 4.

16. The method of claim 15 , wherein
R 7 is a halogen atom, and
R 8 is a hydrogen atom.

17. The method of claim 16 , wherein n is 2 or 3.

18. The method of claim 17 , wherein R 2 and R 3 are a methoxy group.

Assignees

• Univ Kyoto

Inventors

• Nakatsuji Norio
• Minami Itsunari
• Uesugi Motonari
• Aiba Kazuhiro

Classifications

• C07D495/04

  Ortho-condensed systems · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C07D277/82

  Nitrogen atoms · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title