# US 2015/0147299 A1 · Generation of functional and durable endothelial cells from human amniotic fluid-derived cells

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- Number: US 2015/0147299 A1
- Document ID: US20150147299A1
- Status: Published application
- Kind: A1
- Application number: 14/404,739
- Filed: 2013-05-30
- Published: 2015-05-28
- Earliest claimed: 2012-05-30
- Assignees: Cornell University
- Applicants: Cornell University
- Inventors: Shahin Rafii; Sina Y. Rabbany; Michael Ginsberg
- Web page: https://patentgrep.com/patent/US20150147299A1

## CPC

- C12N 5/069: {Vascular Endothelial cells}
- A61K 35/44: Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/60: Transcription factors
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/02: from embryonic cells
- C12N 2506/025: from extra-embryonic cells, e.g. trophoblast, placenta
- C12N 2510/00: Genetically modified cells
- C12N 2510/02: Cells for production
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

This disclosure is directed to methods for reproducibly generating substantial amounts of endothelial cells from amniotic cells. The endothelial cells generated in accordance with the present methodology, as well as therapeutic methods utilizing these cells, are also disclosed.

## Claims

### Claim 1

A method of generating endothelial cells, comprising
obtaining amniotic cells; and
culturing the amniotic cells under conditions where transcription factors ETV2, FLI1 and ERG are expressed in the amniotic cells in the presence of a TGFβ signaling inhibitor, thereby obtaining endothelial cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the expression of ETV2 in the amniotic cells is transient, and the expression of FLI and ERG is constitutive.

### Claim 3 (depends on 2)

The method of claim 2, wherein ETV2 is transiently expressed for 13-15 days.

### Claim 4 (depends on 1)

The method of claim 1, wherein the amniotic cells are transduced with vectors comprising nucleic acids encoding transcription factors ETV2, FLI1 and ERG to achieve expression of the transcription factors.

### Claim 5 (depends on 1)

The method of claim 1, wherein mRNAs encoding the transcription factors are delivered into amniotic cells to achieve expression of the transcription factors.

### Claim 6 (depends on 1)

The method of claim 1, wherein the transcription factors are delivered to amniotic cells in polypeptide forms.

### Claim 7 (depends on 1)

The method of claim 1, wherein the TGFβ signaling inhibitor is present in the cell culture for 20-24 days.

### Claim 8 (depends on 7)

The method of claim 7, wherein the TGFβ signaling inhibitor is an inhibitor specific for the type I TGFβ receptors.

### Claim 9 (depends on 8)

The method of claim 8, wherein said inhibitor is a polypeptide comprising a soluble form of a type I TGFβ receptor, an antibody directed to a type I TGFβ receptor or ligand, or a small molecule compound.

### Claim 10 (depends on 9)

The method of claim 9, wherein said inhibitor is a small molecule compound selected from SB-431542, A 83-01, D 4476, LY 364947, SB 525334, SD 208, and SJN 2511.

### Claim 11 (depends on 10)

The method of claim 10, wherein said inhibitor is SB-431542.

### Claim 12 (depends on 1)

The method of claim 1, wherein amniotic cells are cultured for at least 21 days with the expression of ETV2 in the amniotic cells for the first 13-15 days, the presence of the TGFβ signaling inhibitor for the first 20-21 days, and constitutive expression of FLI1 and ERG.

### Claim 13 (depends on 12)

The method of claim 12, wherein the cells are cultured for a total duration of at least 28 days.

### Claim 14 (depends on 12)

The method of claim 12, wherein the cells are cultured for a total duration of at least 42 days.

### Claim 15 (depends on 1, 2, 12)

The method of any one of claim 1, 2 or 12, wherein ERG is ERG1.

### Claim 16 (depends on 1)

The method of claim 1, wherein cells characterized by expression of surface markers, VE-cadherin, CD31 and VEGFR2, are isolated.

### Claim 17

A substantially pure population of amniotic cell-derived ECs, wherein said ECs are characterized by expression of surface markers, VE-cadherin, CD31 and VEGFR2.

### Claim 18

The substantially pure population of amniotic cell-derived ECs, wherein said ECs comprise an exogenously introduced nucleic acid encoding FLI1.

### Claim 19 (depends on 17)

A composition comprising the substantially pure population of ECs of claim 17 and at least one pharmaceutically acceptable carrier or diluents.

### Claim 20 (depends on 19)

A method for repairing injured tissue in a human subject, comprising administering to the subject the composition of claim 19 to promote vascularization in said tissue.

### Claim 21 (depends on 19)

A method for treating a tumor in a human subject, comprising administering to the subject the composition of claim 19, wherein said ECs are engineered to deliver an anti-tumor agent, and upon administration, said ECs form vessels into said tumor.