# US 9,040,694 B1 · Compounds and methods useful for directing stem cell differentiation

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- Number: US 9,040,694 B1
- Document ID: US9040694B1
- Status: Granted
- Kind: B1
- Application number: 14/337,412
- Filed: 2014-07-22
- Granted: 2015-05-26
- Earliest claimed: 2009-08-06
- Assignees: Vanderbilt University
- Applicants: Vanderbilt University
- Inventors: Charles C. Hong; Corey R. Hopkins; Antonis K. Hatzopoulos; Craig W. Lindsley; Jijun Hao
- Web page: https://patentgrep.com/patent/US9040694B1

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- A61K 31/5377: not condensed and containing further heterocyclic rings, e.g. timolol
- C07D 413/14: containing three or more hetero rings
- C07D 487/04: Ortho-condensed systems
- G01N 33/5026: {on cell morphology}
- C12N 2501/40: Regulators of development
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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 presently-disclosed subject matter relates to compounds of the formula: and methods for use thereof. The presently-disclosed subject matter relates methods of selectively differentiating a stem cell, and methods of screening for compounds useful for enhancing terminal differentiation of committed cardiac progenitor cells.

## Claims

### Claim 1

A compound of the formula:
wherein R1 is selected from the group consisting of

### Claim 2 (depends on 1)

The compound according to claim 1 of the formula:

### Claim 3 (depends on 1)

The compound according to claim 1 of the formula:

### Claim 4

A method of selectively differentiating a stem cell, comprising:
providing the stem cell; and
contacting the stem cell with an effective amount of a compound of the formula,
wherein R1 is selected from the group consisting of
and
wherein the stem cell becomes differentiated into a cardiomyocyte.

### Claim 5 (depends on 4)

The method of claim 4, wherein the compound is

### Claim 6 (depends on 4)

The method of claim 4, wherein the compound is

### Claim 7 (depends on 4)

The method of claim 4, wherein the compound is an inhibitor of Bone Morphogenetic Protein (BMP) signaling.

### Claim 8 (depends on 4)

The method of claim 4, wherein contacting the cells with the compound causes the cells to exhibit at least one indicator of cardiomyogenesis.

### Claim 9 (depends on 8)

The method of claim 8, wherein the at least one indicator of cardiomyogenesis is selected from: fluorescence, beating cells, and expression of at least one cardiac-specific gene.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least one cardiac-specific gene is selected from: Nkx2.5, Troponin T, Myh6, Myl2, Myl7, and Mybpc3.

### Claim 11 (depends on 9)

The method of claim 9, wherein the at least one indicator of cardiomyogenesis is beating cells and, the beating cells are first observed from about day 6 to about day 12.

### Claim 12 (depends on 4)

The method of claim 4, further comprising:
providing a second compound that enhances terminal differentiation of committed cardiac progenitor cells; and
contacting the stem cell with the second compound.

### Claim 13 (depends on 4)

The method of claim 4, wherein the stem cell is an embryonic stem cell.

### Claim 14 (depends on 4)

The method of claim 4, wherein the stem cell is a human embryonic stem cell.

### Claim 15

A method of screening for a compound useful for enhancing terminal differentiation of committed cardiac progenitor cells, comprising:
providing a first pluripotent stem cell and a second pluripotent stem cell;
contacting the first and second pluripotent stem cells with an effective amount of a first compound of the formula,
wherein R1 is selected from the group consisting of
contacting the second cells with a test compound;
identifying at least one indicator of cardiomyogenesis exhibited by the first cells and the second cells;
comparing the appearance of the at least one indicator of cardiomyogenesis exhibited by the first cells to the appearance of the at least one indicator of cardiomyogenesis exhibited by the second cells; and
identifying the test compound as a compound useful for enhancing terminal differentiation of committed cardiac progenitor cells if the appearance of the at least one indicator of cardiomyogenesis in the second cells is accelerated and/or augmented as compared to the appearance of the at least one indicator of cardiomyogenesis in the first cells.

### Claim 16 (depends on 15)

The method of claim 15, wherein the first compound is an inhibitor of Bone Morphogenetic Protein (BMP) signaling.