Human pluripotent stem cell derived endocardial endothelium

Patent metadata

Field	Value
Publication number	US-10612002-B2
Application number	US-201715437786-A
Country	US
Kind code	B2
Filing date	Feb 21, 2017
Priority date	Feb 19, 2016
Publication date	Apr 7, 2020
Grant date	Apr 7, 2020

Abstract

Provided herein are methods that enable the polarization of hPSC mesoderm such that closely related yet distinct cardiovascular populations can be generated efficiently without the need of post-facto enrichment.

First claim

The invention claimed is:

1. A method for generating human endocardial endothelial cells, comprising:
(i) culturing adherent human pluripotent stem cells in defined medium comprising B27 and at least 100 ng/mL Activin A in the absence of insulin for 12-20 hours,
(ii) culturing the cells of step (i) in defined medium in the absence of insulin, in the presence of Bone Morphogenetic Protein 4 (BMP4) in a concentration that is less than 30 ng/mL, and CHIR-99021 for 20-28 hours,
(iii) culturing the cells of step (ii) in defined medium in the absence of Wnt inhibitor, comprising vascular endothelial growth factor (VEGF), BMP-4 and bFGF for 3 days or until NFATc1 expression is detectable,
thereby generating human endocardial endothelial cells.

2. The method of claim 1 , wherein the pluripotent stem cells comprise human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs).

3. The method of claim 1 , wherein the BMP4 is present in defined medium at a concentration less than 10 ng/mL.

4. The method of claim 1 , which generates a culture comprising at least 60% endocardial endothelial cells, without the need for a cell sorting or enrichment step.

5. The method of claim 4 , wherein the endocardial endothelial cells are NFATc1+.

6. The method of claim 1 , wherein less than 1% of the resulting cells are CD43 + and CD235a + .

7. The method of claim 1 , wherein the concentration of BMP4 is selected from the group consisting of: 1-30 ng/mL, 5-30 ng/mL, 5-20 ng/mL, 10-30 ng/mL, or 10-20 ng/mL.

8. The method of claim 1 , wherein the concentration of Activin A is selected from the group consisting of: at least 105 ng/mL, at least 110 ng/mL, at least 120 ng/mL or at least 150 ng/mL.

Assignees

• Univ Washington

Inventors

• Murry Charles E
• Pabon Lil
• Palpant Nathan

Classifications

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2501/16

  Activin; Inhibin; Mullerian inhibiting substance · CPC title

• C12N2501/155

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

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title

• C12N2501/165

  Vascular endothelial growth factor [VEGF] · CPC title