Composition for promoting cardiac differentiation of pluripotent stem cell comprising EGFR inhibitor

Patent metadata

Field	Value
Publication number	US-10196609-B2
Application number	US-201414772991-A
Country	US
Kind code	B2
Filing date	Feb 5, 2014
Priority date	Mar 8, 2013
Publication date	Feb 5, 2019
Grant date	Feb 5, 2019

Abstract

The present invention provides a composition for promoting cardiac differentiation of a pluripotent stem cell containing an EGFR inhibitor. The present invention also provides a kit for promoting cardiac differentiation containing an EGFR inhibitor and a method for inducing cardiac differentiation of a pluripotent stem cell comprising culturing the pluripotent stem cell in a medium containing an EGFR inhibitor.

First claim

The invention claimed is:

1. A method for inducing cardiac differentiation of a pluripotent stem cell in vitro, comprising the steps in a sequential order of:
(1) first culturing the pluripotent stem cell in a medium containing a Wnt signaling activator, and then
(2) culturing the cell in a medium containing an EGFR inhibitor after the step (1), wherein the cell is cultured in a medium containing an EGFR inhibitor and a Wnt signaling inhibitor, and optionally before or after, or both before and after the culture in the medium containing an EGFR inhibitor and a Wnt signaling inhibitor, the cell is cultured in a medium containing an EGFR inhibitor but not a Wnt signaling inhibitor and wherein the cell is cultured in floating culture,
wherein the pluripotent stem cell is a monkey or human ES or iPS cell,
the step (1) is conducted at Day 0 to Day 2 or Day 0 to Day 3 of culture for cardiac differentiation, and
the step (2) is conducted for 3 to 10 days in the period from Day 2, Day 3, or Day 4 to Day 14 of the culture for cardiac differentiation when the step (1) is conducted at Day 0 to Day 2, or conducted for 3 to 10 days in the period from Day 3 or Day 4 to Day 14 of the culture for cardiac differentiation when the step (1) is conducted at Day 0 to Day 3.

2. The method according to claim 1 , wherein the medium contains no protein other than albumin.

3. The method according to claim 1 , which is a method for preparing a cardiomyocyte.

4. The method according to claim 1 , wherein the EGFR inhibitor is selected from the group consisting of AG1478, gefitinib, afatinib, ARRY334543, AST1306, AZD8931, BIBU1361, BIBX1382, BPDQ, BPIQ-I, BPIQ-II, canertinib, CL-387,785, CUDC101, dacomitinib, vandetanib, EGFR Inhibitor III (CAS 733009-42-2), EGFR/ErbB-2 Inhibitor (CAS 179248-61-4), erlotinib, GW583340, GW2974, HDS029, lapatinib, WHI-P154, OSI-420, PD153035, PD168393, PD174265, pelitinib, Compound 56, XL657, PP3, AG-490, AG555, tyrphostin B42, tyrphostin B44, AG556, AG494, AG825, RG-13022, DAPH, EGFR Inhibitor (CAS 879127-07-8), erbstatin analog (CAS 3177-57-1), JNJ28871063, tyrphostin 47, lavendustin A, lavendustin C, lavendustin C methylate, LFM-A12, TAK165, TAK285, tyrphostin 51, tyrphostin AG183, tyrphostin AG528, tyrphostin AG99, tyrphostin RG14620, WZ3146, WZ4002, WZ8040, Butein, and tyrphostin AG112.

5. The method according to claim 1 , wherein the EGFR inhibitor is selected from the group consisting of AG1478, gefitinib, and PP3.

6. The method according to claim 1 , wherein the Wnt signaling inhibitor is a compound represented by 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 13 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 —CR 14 , wherein R 14 is a hydrogen atom, a halogen atom, a hydroxyl group, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a linear or branched alkyl group having 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; an oxygen atom; a sulfur atom; a selenium atom; or a group —NR 15 , wherein R 15 is a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or a linear or branched acyl group having 1 to 5 carbon atoms, and
n is an integer of 0 to 6,

or a salt thereof.

7. The method according to claim 6 , wherein in Formula (I)
R 1 , R 4 , R 5 , R 6 , R 8 , R 9 , R 10 , and R 11 are a hydrogen atom,
R 2 and R 3 is a methoxy group, an ethoxy group or a propoxy group,
R 7 is a halogen atom,
X is a sulfur atom, and
n is an integer of 0 to 4.

8. The method according to claim 7 , wherein in Formula (I)
R 2 is a methoxy group, and
R 3 is a methoxy group, an ethoxy group or a propoxy group.

9. The method according to claim 1 , wherein the Wnt signaling inhibitor is a compound selected from the group consisting of

or a salt thereof.

10. The method according to claim 1 , wherein the Wnt signaling inhibitor is selected from the group consisting of IWP2, XAV939, and IWR1.

11. The method according to claim 1 , wherein the Wnt signaling activator is selected from the group consisting of BIO and CHIR99021.

12. The method according to claim 1 , wherein two or more Wnt signaling inhibitors and two or more Wnt signaling activators are used in combination.

13. The method according to claim 12 , wherein
the EGFR inhibitor is selected from the group consisting of AG1478, gefitinib, and PP3;
the Wnt signaling inhibitors are a compound represented by Formula (I):

wherein
R 1 , R 4 , R 5 , R 6 R 8 , R 9 , R 10 , and R 11 are a hydrogen atom,
R 2 and R 3 is a methoxy group, an ethoxy group or a propoxy group,
R 7 is a halogen atom,
X is a sulfur atom, and
n is an integer of 0 to 4,

and a Wnt signaling inhibitor selected from the group consisting of IWP2, XAV939, and IWR1; and
the Wnt signaling activators are BIO and CHIR99021.

14. The method according to claim 13 , wherein
the EGFR inhibitor is AG1478 or gefitinib; and
the Wnt signaling inhibitors are the compound represented by Formula (I) and XAV939.

Assignees

• Univ Kyoto

Inventors

• Nakatsuji Norio
• Minami Itsunari

Classifications

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2500/95

  Protein-free medium and culture conditions · CPC title