Chemically defined albumin-free conditions for cardiomyocyte differentiation of human pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9765299-B2
Application number	US-201514850451-A
Country	US
Kind code	B2
Filing date	Sep 10, 2015
Priority date	Sep 10, 2014
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

Methods for generating high-yield, high-purity cardiomyocyte progenitors or cardiomyocytes from pluripotent cells are described. Wnt/β-catenin signaling is first activated in pluripotent cells, by, for example, inhibiting Gsk-3 to obtain a first population of cells. Wnt/β-catenin signaling is then inhibited in the first cell population to induce cardiogenesis. One or more of these steps is performed under defined, albumin-free culture conditions.

First claim

The invention claimed is:

1. A method for generating a population of cardiomyocyte progenitors from human pluripotent stem cells, comprising:
(i) activating Wnt/β-catenin signaling in human pluripotent stem cells by culturing the human pluripotent stem cells in the presence of a Gsk3 inhibitor to obtain a first cell population; and
(ii) subsequently inhibiting Wnt/β-catenin signaling in the first cell population while culturing the first cell population to obtain a second cell population comprising human cardiomyocyte progenitors,
wherein the first cell population in step (ii) is cultured under albumin-free conditions.

2. The method of claim 1 , wherein the Gsk3 inhibitor is a small molecule selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.

3. The method of claim 2 , wherein the small molecule Gsk3 inhibitor is selected from the group consisting of CHIR 99021, CHIR 98014, and BIO-acetoxime.

4. The method of claim 2 , wherein in step (i), the small molecule Gsk3 inhibitor is present in a concentration of from 0.2 to 9 μM.

5. The method of claim 1 , wherein the step of inhibiting the Wnt/β-catenin signaling in the first cell population comprises contacting the first cell population with a small molecule that inhibits Wnt/β-catenin signaling.

6. The method of claim 5 , wherein the small molecule that inhibits Wnt/β-catenin signaling stabilizes axin and stimulates β-catenin degradation.

7. The method of claim 6 , wherein the small molecule that stimulates β-catenin degradation and stabilizes axin is XAV939 or IWR-1.

8. The method of claim 5 , wherein the small molecule that inhibits Wnt/β-catenin signaling is a porcupine inhibitor that prevents palmitylation of Wnt proteins by porcupine.

9. The method of claim 8 , wherein the porcupine inhibitor is IWP2, IWP4, or a combination thereof.

10. The method of claim 9 , wherein in step (ii), the porcupine inhibitor is present in a concentration of from 1 to 4 μM.

11. The method of claim 1 , wherein the step of inhibiting the Wnt/β-catenin signaling in the first cell population comprises contacting the first cell population with at least one antibody that blocks activation of a Wnt ligand receptor.

12. The method of claim 11 , wherein the at least one antibody binds to one or more Wnt ligand family members.

13. The method of claim 1 , wherein the step of inhibiting the Wnt/β-catenin signaling in the first cell population comprises reducing β-catenin expression in the first cell population.

14. The method of claim 13 , wherein reducing β-catenin expression comprises expressing shRNA for β-catenin in the first cell population.

15. The method of claim 1 , wherein the pluripotent stem cells in step (i), the first cell population in step (ii), or both are cultured under conditions that are free of one or more of the group consisting of L-ascorbic acid 2-phosphate, transferrin, sodium selenite, progesterone, and putrescine.

16. The method of claim 1 , wherein step (ii) of inhibiting Wnt/β-catenin signaling in the first cell population is performed immediately after step (i) of activating Wnt/β-catenin signaling in the human pluripotent stem cells is completed.

17. The method of claim 1 , further comprising culturing the second cell population after ending the inhibition of Wnt/β-catenin signaling initiated during step (ii) to obtain a cell population comprising cardiomyocytes.

18. The method of claim 17 , wherein no cell separation or selection step is used to obtain the cell population comprising cardiomyocytes.

19. The method of claim 18 , wherein at least 85% of the cells in the cell population comprising cardiomyocytes are cardiac troponin T (cTnT)-positive.

20. A method for culturing pluripotent stem cells to obtain a population of cardiomyocytes, the method comprising the steps of:
sequentially inhibiting Gsk3 in the pluripotent cells and then inhibiting Wnt signaling in the Gsk3 inhibited cells under albumin-free conditions; and
culturing the sequentially inhibited cells in an albumin-free culture medium to form a differentiated cell population comprising cardiomyocytes.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Palecek Sean Paul
• Lian Xiaojun
• Bao Xiaoping

Classifications

• C12N2506/02

  from embryonic cells · CPC title

• C12N2500/46

  Amines, e.g. putrescine · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2501/392

  Sexual steroids · CPC title