# US 8,785,192 B2 · Endothelial cell production by programming

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- Number: US 8,785,192 B2
- Document ID: US8785192B2
- Status: Granted
- Kind: B2
- Application number: 13/178,239
- Filed: 2011-07-07
- Published: 2012-01-12
- Granted: 2014-07-22
- Earliest claimed: 2010-07-07
- Assignees: Cellular Dynamics International, Inc.
- Applicants: Junying Yu; Maksym A. Vodyanyk
- Inventors: Junying Yu; Maksym A. Vodyanyk
- Web page: https://patentgrep.com/patent/US8785192B2

## CPC

- C12N 15/85: for animal cells
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/069: {Vascular Endothelial cells}
- C12N 5/0692: {Stem cells; Progenitor cells; Precursor cells}
- G01N 33/5064: {Endothelial cells}
- C12N 2501/40: Regulators of development
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- G01N 2500/10: involving cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention generally regards methods for providing endothelial cells and precursors of endothelial cells from a variety of cell sources, such as pluripotent stem cells. Also provided are therapeutic compositions including the provided endothelial cells, and methods of using them for the treatment of subjects.

## Claims

### Claim 1

A method of providing endothelial cells by forward programming of pluripotent stem cells, comprising contacting the pluripotent stem cells with one or more endothelial programming factor genes, or products of such genes, in an amount sufficient to cause forward programming of the pluripotent stem cells into endothelial cells, said one or more endothelial programming factors or gene products of such genes comprises an ETV2 and/or ERG gene or gene product or an isoform thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with an expression cassette encoding one or more endothelial reprogramming factor genes, wherein said expression cassette is introduced into said pluripotent stem cells and expressed by said pluripotent stem cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with said products of such genes, wherein said products of such genes are polypeptide products of said one or more endothelial programming factor genes.

### Claim 5 (depends on 1)

The method of claim 1, wherein said contacting said pluripotent stem cells comprises contacting said pluripotent stem cells with said products of such genes, wherein said products of such genes are RNA transcript products of said one or more endothelial programming factor genes.

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more endothelial programming factor genes or products of such genes comprises an ERG gene or gene product or isoform thereof.

### Claim 7 (depends on 6)

The method of claim 6, wherein the ERG gene or gene product or isoform thereof is ERG isoform 3.

### Claim 8 (depends on 1)

The method of claim 1, wherein the one or more endothelial programming factor genes or products of such genes comprises an ETV2 gene or gene product or isoform thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the endothelial cells comprise one or more characteristics of endothelial cells comprising:
(i) expression of one or more endothelial cell markers;
(ii) the ability to take up acetylated low density lipoprotein (ac-LDL), form tube-like structures in a three dimensional matrix, upregulate expression of one or more cell adhesion molecules in response to one or more pro-inflammatory stimuli, or a combination of two or more of these abilities; or
(iii) endothelial cell morphological features.

### Claim 10 (depends on 9)

The method of claim 9, wherein the one or more endothelial cell markers are selected from the group consisting of: CD144, CD31, CD34, ESAM, CD102, CD143, CD93, CD184, CD105, CD146, von Willebrand factor, ZO-1, CD304, and DLL4.

### Claim 11 (depends on 9)

The method of claim 9, wherein the one or more pro-inflammatory stimuli are selected from the group consisting of TNF and IL-1, and the one or more cell adhesion molecules are selected from the group consisting of CD54, CD62E, and CD106.

### Claim 12 (depends on 1)

The method of claim 1, wherein the provided endothelial cells are mature endothelial cells.

### Claim 13 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells or progeny cells thereof are cultured in a medium comprising one or more growth factors.

### Claim 14 (depends on 13)

The method of claim 13, wherein the one or more growth factors include basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both.

### Claim 15 (depends on 1)

The method of claim 1, further defined as a method of generating hematopoietic cells, said method comprising culturing the pluripotent stem cells contacted with the one or more endothelial programming factor genes, or products of such genes, in a hematopoietic culture medium, wherein said one or more endothelial programming factors genes, or products of such genes comprises an EVT2 gene or gene product or isoform thereof, thereby producing endothelial cells comprising hematopoietic cells that are CD31+, CD43+, CD235a/CD41a-, and CD45+.

### Claim 16 (depends on 1)

The method of claim 1, further defined as a method of generating mesenchymal cells, said method comprising culturing the pluripotent stem cells contacted with the one or more endothelial programming factor genes, or products of such genes, in a mesenchymal medium comprising FGF and a TGFβ inhibitor, thereby producing endothelial cells comprising mesenchymal cells that that are CD31−, CD73+, and CD105+.