# US 11,898,161 B2 · Stem cell culture medium and method

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- Number: US 11,898,161 B2
- Document ID: US11898161B2
- Status: Granted
- Kind: B2
- Application number: 16/852,361
- Filed: 2020-04-17
- Published: 2020-07-30
- Granted: 2024-02-13
- Earliest claimed: 2006-09-22
- Assignees: Riken
- Applicants: Riken
- Inventors: Yoshiki Sasai; Kiichi Watanabe
- Web page: https://patentgrep.com/patent/US11898161B2

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- C12N 2501/70: Enzymes
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/00: Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides a method of differentiating human pluripotent stem cells to ectodermal cell by treating human pluripotent stem cells, which are dissociated single cells, and inducing differentiation to ectodermal cells under conditions where a ROCK(Rho-kinase) inhibitor is present in a culture medium in contact with the cells after dissociation of the human pluripotent stem cells.

## Claims

### Claim 1

A method of improving the differentiation efficacy of human pluripotent stem cells to ectodermal cells, which comprises culturing human pluripotent stem cells, which are dissociated single cells, in a serum-free culture medium which is suitable for induction of differentiation of pluripotent stem cells into ectodermal cells by a Serum-free Floating culture of Embryoid Body-like aggregates (SFEB) method,
wherein the culture medium comprises a Rho kinase (ROCK) inhibitor for at least 2 days,
wherein, during culturing, induction of differentiation occurs, and
wherein the ectodermal cells are neural cells, or neural precursor cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the ROCK inhibitor is present in a culture medium in contact with the cells during a portion of the period of inducing differentiation to ectodermal cells.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the ectodermal cells are Nestin and Pax6 positive cells.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the induction of differentiation comprises inducing neural cells or neural precursor cells in the presence of a factor selected from the group consisting of Nodal inhibitors, Wnt inhibitors, and BMP inhibitors.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

### Claim 7 (depends on 1)

The method according to claim 1, wherein prior to the culturing of the human pluripotent stem cells, which are dissociated single cells, in a serum-free culture medium,
the pluripotent stem cells are cultured in a culture medium in the presence of the ROCK inhibitor and serum, serum extract, and/or feeders.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the ROCK inhibitor is present in a culture medium for up to six days.

### Claim 9 (depends on 1)

The method according to claim 1, wherein after differentiation to neural cells or neural precursor cells is induced, the ROCK inhibitor is withdrawn from the culture medium.

### Claim 10 (depends on 1)

The method according to claim 1, further comprising the step of dissociating human pluripotent stem cells into single cells prior to the step of culturing the dissociated human pluripotent stem cells in a serum-free culture medium.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

### Claim 12 (depends on 10)

The method according to claim 10, wherein the human pluripotent stem cells are human embryonic stem cells.

### Claim 13 (depends on 10)

The method according to claim 10, wherein prior to the culturing of the human pluripotent stem cells, which are dissociated single cells, in a serum-free culture medium,
the pluripotent stem cells are cultured in a culture medium in the presence of the ROCK inhibitor and serum, serum extract, and/or feeders.

### Claim 14 (depends on 10)

The method according to claim 10, wherein the ROCK inhibitor is present in a culture medium for up to six days.

### Claim 15 (depends on 10)

The method according to claim 10, wherein after differentiation to neural cells or neural precursor cells is induced, the ROCK inhibitor is withdrawn from the culture medium.

### Claim 16 (depends on 10)

The method according to claim 10, wherein the serum-free culture medium comprises a material selected from a group consisting of albumin, transferrin, fatty acids, insulin, collagen precursor, trace elements, 2-mercaptoethanol, and 3′-thiolglycerol.