# US 10,767,162 B2 · Methods for differentiation

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,767,162 B2
- Document ID: US10767162B2
- Status: Granted
- Kind: B2
- Application number: 16/222,184
- Filed: 2018-12-17
- Published: 2019-04-11
- Granted: 2020-09-08
- Earliest claimed: 2014-06-10
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Victor Chun Li; Marc W. Kirschner
- Web page: https://patentgrep.com/patent/US10767162B2

## CPC

- C12N 5/0619: {Neurons}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 2501/405: Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
- C12N 2501/60: Transcription factors
- C12N 2501/606: c-Myc
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2506/02: from embryonic cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Described herein are methods relating to the differentiation of stem cells to more differentiated phenotypes, e.g. to terminally differentiated cell types and/or precursors thereof. In some embodiments, the methods relate to contacting the stem cells with differentiation factors and halting the cell cycle, thereby increasing the rate of differentiation.

## Claims

### Claim 1

A method of differentiating a pluripotent stem cell, the method comprising:
i) contacting the pluripotent stem cell with one or more nucleic acids encoding one or more terminal transcription factors; and
ii) inhibiting the cell cycle of the pluripotent stem cell, wherein the cell cycle is inhibited by one or more of the following:
reducing serum levels; reducing serum levels below 5%; contacting the cell with a PI3K inhibitor; contacting the cell with a Myc inhibitor; contacting the cell with a MAPK inhibitor; contacting the cell with a MEK1/2 inhibitor; contacting the cell with a CDK inhibitor; contacting the cell with an Id inhibitor; contacting the cell with a Ink family agonist; contacting the cell with a Cip/Kip family agonist; and culturing the cell in a media lacking a factor selected from the group consisting of: Bmp; Fgf; Activin; or TGFβ;
wherein steps i) and ii) occur either:
a) concurrently; or
b) within 15 days of each other in either order; and
whereby a differentiated cell is generated.

### Claim 2 (depends on 1)

The method of claim 1, wherein steps i) and ii) occur within 14 days of each other.

### Claim 3 (depends on 1)

The method of claim 1, wherein steps i) and ii) occur within 7 days of each other.

### Claim 4 (depends on 1)

The method of claim 1, wherein steps i) and ii) occur within 2 days of each other.

### Claim 5 (depends on 1)

The method of claim 1, wherein steps i) and ii) occur within 24 hours of each other.

### Claim 6 (depends on 1)

The method of claim 1, wherein steps i) and ii) occur simultaneously.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cell cycle is inhibited by reducing or removing growth factors.

### Claim 8 (depends on 1)

The method of claim 1, wherein the PI3K inhibitor is LY294002.

### Claim 9 (depends on 1)

The method of claim 1, wherein the E2F transcription factor inhibitor is HLM006474.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Myc inhibitor is JQ1 or 10058-F4.

### Claim 11 (depends on 1)

The method of claim 1, wherein the MAPK inhibitor is PD98059.

### Claim 12 (depends on 1)

The method of claim 1, wherein the CDK inhibitor is a CDK4 inhibitor, a CDK2 inhibitor, p16, p15, p18, p19, p21, p27, or p57.

### Claim 13 (depends on 1)

The method of claim 1, wherein the pluripotent stem cell is an embryonic stem cell.