Methods and compositions for generating epicardium cells

Patent metadata

Field	Value
Publication number	US-10711246-B2
Application number	US-201414915992-A
Country	US
Kind code	B2
Filing date	Sep 12, 2014
Priority date	Sep 13, 2013
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

Provided are methods and products for obtaining cardiovascular lineage cells from hPSCs. The method for obtaining a cardiomyocyte lineage or an epicardial lineage cell population from human pluripotent stem cells (hPSCs) comprises one or more of the following steps: (a) contacting BMP component primed hPSCs with a cardiovascular mesoderm programming cocktail suitable for inducing the hPSCs to differentiate to a cardiovascular mesoderm cell population under conditions suitable for the programming cocktail to penetrate the hPSCs and culturing the contacted hPSCs for a period of time to generate a KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population; (b) contacting the cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail suitable to specify a NKX2-5+ or WT1+ cardiovascular progenitor cell population under conditions suitable for the specification cocktail to penetrate the cardiovascular mesoderm cell population and culturing the contacted cardiovascular mesoderm cell population for a period of time to generate a NKX2-5+ or WT1+ cardiovascular progenitor cell population; and (d) contacting the cardiovascular progenitor cell population with a maturation cocktail under conditions suitable for the maturation cocktail to penetrate the cardiovascular progenitor cell population and culturing the contacted cardiovascular progenitor population for a period of time to produce a cardiovascular population optionally cardiomyocyte lineage cells expressing cardiac troponin T (cTnT) and/or SIRPA and/or epicardial lineage cells expressing WT1.

First claim

The invention claimed is:

1. A method of specifying a WT1+ cardiovascular progenitor cell population comprising the step of contacting an hPSC-derived KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail comprising human BMP4 at a concentration of between 0.63 ng/ml and 10 ng/ml.

2. The method of claim 1 , wherein the cardiovascular progenitor specification cocktail further comprises CHIR99021.

3. The method of claim 2 , wherein CHIR99021 is present at a concentration of between 1 μM and 4 μM.

4. The method of claim 3 , wherein the cardiovascular progenitor specification cocktail further comprises SB431542.

5. The method of claim 4 , wherein the cardiovascular progenitor specification cocktail further comprises human VEGF.

6. The method of claim 5 , wherein the human VEGF is present at a concentration of 5 ng/ml and the SB431542 is present at a concentration of 5.4 μM.

7. The method of claim 3 , wherein the cardiovascular mesoderm cell population is contacted with the cardiovascular progenitor specification cocktail for at least 12 hours to about 48 hours.

8. The method of claim 1 , wherein the hPSC-derived KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population is dissociated prior to the step of contacting with the cardiovascular progenitor specification cocktail.

9. The method of claim 1 , wherein the cardiovascular mesoderm cell population is contacted with the cardiovascular progenitor specification cocktail for at least 12 hours to about 48 hours.

10. The method of claim 9 , wherein the BMP4 concentration in the cardiovascular progenitor specification cocktail is at least 1.25 ng/ml.

11. The method of claim 10 , wherein the BMP4 concentration in the cardiovascular progenitor specification cocktail is 10 ng/ml.

12. A method of producing an epicardial lineage cell population comprising the step of contacting a WT1+ cardiovascular progenitor cell population with a maturation cocktail comprising suitable cell culture components in a suitable cell culture format for a period of from 2 to 20 days.

13. The method of claim 12 , wherein the maturation cocktail further comprises VEGF.

14. The method of claim 13 , wherein the contacting takes place for a period of from 2 to 9 days.

15. The method of claim 12 , wherein the step of contacting takes place in a 96-well plate and, after 2-9 days, the cell population is passaged and cultured in a larger cell culture format than the 96-well-plate.

16. The method of claim 15 , wherein the cell population is cultured in a larger cell culture format until they exhibit an epithelial morphology.

17. The method of claim 15 , wherein the cell population is cultured in a larger cell culture format for 4 days.

18. The method of claim 15 , wherein the larger cell culture format is a 6-well plate.

19. The method of claim 14 comprising contacting the WT1+ cardiovascular progenitor cell population with the maturation cocktail for 9 days, further comprising passaging the cells, then culturing the cells in a larger format in a suitable medium for one day, followed by one of the following regimens comprising the step or steps of:
(a) causing the cells to progress toward a vascular smooth muscle-like fate by treating them with human TGFβ-1 for a period of time;
(b) causing the cells to progress toward a vascular smooth muscle-like fate by treating them with human TGFβ-1 for a period of time, followed by treating them with human bFGF for a period of time;
(c) causing the cells to progress toward a fibroblast-like fate by treating them with human bFGF.

20. The method of claim 19 , wherein:
step (a) is performed and the cells are treated with human TGFβ-1 at a concentration of 5 ng/ml for 4 days, or
step (b) is performed and the cells are treated with human TGFβ-1 at a concentration of 5 ng/ml for 4 days followed by treatment of the cells with human bFGF at a concentration of 10 ng/ml for a period of 4 days, or
step (c) is performed and the cells are treated with human bFGF at a concentration of 10 ng/ml for 8 days.

21. A method of preparing an epicardial lineage cell population and causing the population to further differentiate comprising the steps of:
(a) specifying a WT1+ cardiovascular progenitor cell population by a method comprising contacting an hPSC-derived KDR+ and PDGFRalpha+ cardiovascular mesoderm cell population with a cardiovascular progenitor specification cocktail comprising human BMP4 at a concentration of between 0.63 ng/ml and 10 ng/ml;
(b) contacting the WT1+ cardiovascular progenitor cell population with a maturation cocktail comprising suitable cell culture components in a suitable cell culture format for a period of from 2-20 days;
(c) passaging the cell population, then culturing the cell population in a larger cell culture format in a suitable medium for one day, optionally followed by one of the following regimens comprising:
(i) causing the cell population to progress toward a vascular smooth muscle-like fate by treating it with human TGFβ-1 for a period of time;
(ii) causing the cell population to progress toward a vascular smooth muscle-like fate by treating it with human TGFβ-1 for a period of time, followed by treating it with human bFGF for a period of time;
(iii) causing the cell population to progress toward a fibroblast-like fate by treating it with human bFGF.

22. The method of claim 21 , wherein
(i) is performed and the cells are treated with human TGFβ-1 at a concentration of 5 ng/ml for 4 days, or
(ii) is performed and the cells are treated with human TGFβ-1 at a concentration of 5 ng/ml for 4 days followed by treatment of the cells with human bFGF at a concentration of 10 ng/ml for a period of 4 days, or
(iii) is performed and the cells are treated with human bFGF at a concentration of 10 ng/ml for 8 days.

Assignees

• Univ Health Network

Inventors

• Keller Gordon
• Witty Alec Drake
• Kattman Steven James

Classifications

• G01N33/5044

  involving specific cell types · CPC title

• G01N33/5061

  Muscle cells · CPC title

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N2501/155

  Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor · CPC title