# US 10,370,644 B2 · Method for making human pluripotent suspension cultures and cells derived therefrom

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- Number: US 10,370,644 B2
- Document ID: US10370644B2
- Status: Granted
- Kind: B2
- Application number: 14/307,100
- Filed: 2014-06-17
- Published: 2014-10-02
- Granted: 2019-08-06
- Earliest claimed: 2012-12-31
- Assignees: Janssen Biotech, Inc.
- Applicants: Janssen Biotech, Inc.
- Inventors: Benjamin Fryer; Daina Laniauskas; Marcia Blackmoore; Haiyun Wang; Kostadinka Lilova; Shelley Nelson; Elizabeth Rosocha
- Web page: https://patentgrep.com/patent/US10370644B2

## CPC

- C12N 5/0676: {Pancreatic cells}
- C12N 2500/25: Insulin-transferrin; Insulin-transferrin-selenium
- C12N 2500/34: Sugars
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/19: Growth and differentiation factors [GDF]
- C12N 2501/385: of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
- C12N 2501/415: Wnt; Frizzeled
- 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

The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation. Specifically, the invention discloses methods of differentiating pluripotent cells into beta cell, cardiac cell and neuronal cell lineages using suspension clustering. The methods involve preparing the aggregated cell clusters followed by differentiation of these clusters.

## Claims

### Claim 1

A method of differentiating pluripotent cells comprising:
a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture;
b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells;
c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml;
d. transferring the suspension of cell aggregates to a dynamic suspension culture;
e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate pluripotent stem cell clusters; and
f. differentiating the pluripotent stem cell clusters in the dynamic suspension culture system, wherein the step of differentiating comprises differentiating posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in a medium supplemented with a Cyp26 inhibitor.

### Claim 2 (depends on 1)

The method claim 1, wherein the method increases the percentage of cells in GO/G1 phase of the cell cycle.

### Claim 3 (depends on 1)

The method of claim 1, wherein the expanding comprises an environment that includes from about 0.1% to about 2% of bovine serum albumin.

### Claim 4 (depends on 1)

The method of claim 1, wherein the Rho kinase inhibitor in (i) step b., (ii) step. c, or (iii) step b. and step c. is Y-27632.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 6)

The method of claim 6, wherein the human pluripotent stem cells are human embryonic stem cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are treated with a chelating agent.

### Claim 9 (depends on 8)

The method of claim 8, wherein the chelating agent is EDTA.

### Claim 10 (depends on 1)

The method of claim 1, wherein the pluripotent stem cells are treated with an enzyme.

### Claim 11 (depends on 10)

The method of claim 10, wherein the enzyme is (A) a collagenolytic enzyme, (B) a proteolytic enzyme or (C) both a collagenolytic and proteolytic enzyme.

### Claim 12 (depends on 1)

The method of claim 1, wherein after step e and prior to step f, microcarriers are added to the dynamic suspension culture system.

### Claim 13 (depends on 1)

The method of claim 1, wherein the medium supplemented with the Cyp26 inhibitor is further supplemented with an ALK5 inhibitor.

### Claim 14 (depends on 13)

The method of claim 13, where the medium supplemented with the Cyp26 inhibitor and the ALK5 inhibitor is further supplemented with a PKC activator.

### Claim 15 (depends on 1)

The method of claim 1, wherein the medium supplemented with a Cyp26 inhibitor further comprises about 8 mM glucose.

### Claim 16 (depends on 1)

The method of claim 1, wherein the medium supplemented with a Cyp26 inhibitor further comprises about 25 mM glucose.

### Claim 17 (depends on 1)

The method of claim 1, wherein the medium supplemented with the Cyp26 inhibitor is further supplemented with SANT, TPPB and either LDN or SCIO.

### Claim 18 (depends on 1)

The method of claim 1, wherein prior to the differentiating of step (f), the method comprises differentiating the pluripotent stem cell clusters to definitive endoderm (Stage 1) cells in a culture medium supplemented with a cyclic aniline-pyridinotriazine and GDF8.

### Claim 19 (depends on 18)

The method of claim 18, wherein the cyclic aniline-pyridinotriazine is cyclic aniline-pyridinotriazine is 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6-˜.1˜8,12.˜[heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-non-aen-16-one.

### Claim 20 (depends on 19)

The method of claim 19, wherein the culture medium supplemented with 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6-˜.1˜8,12.˜[heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-non-aen-16-one and GDF8 is further supplemented with 8 mM glucose.

### Claim 21 (depends on 18)

The method of claim 18, wherein the Stage 1 cells are subsequently cultured in a medium supplemented with FGF7 to differentiate the Stage 1 cells into Stage 2 cells.

### Claim 22 (depends on 21)

The method of claim 21, wherein the method further comprises culturing the Stage 2 cells in a medium supplemented with FGF7, retinoic acid, SANT-1, TPPB and optionally LDN to differentiate the Stage 2 cells into Stage 3 cells.

### Claim 23 (depends on 22)

The method of claim 22, wherein the medium supplemented with FGF7, retinoic acid, SANT-1, TPPB and optionally LDN to differentiate the Stage 2 cells into Stage 3 cells is further supplemented with activin A.

### Claim 24 (depends on 22)

The method of claim 22, wherein the method of differentiating the posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in the medium supplemented with a Cyp26 inhibitor further comprises an ALK5 inhibitor and a PKC activator.

### Claim 25 (depends on 22)

The method of claim 22, wherein the method of differentiating the posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in the medium supplemented with a Cyp26 inhibitor further comprises SANT, TPPB and either LDN or SCIO.

### Claim 26 (depends on 22)

The method of claim 22, wherein the medium used for differentiation into Stage 1 cells, Stage 2 cells, Stage 3 cells, and Stage 4 cells is supplemented with about 8 mM glucose.

### Claim 27 (depends on 22)

The method of claim 22, wherein the medium used for differentiation into Stage 3 cells and Stage 4 cells is supplemented with about 25 mM glucose.

### Claim 28 (depends on 1)

The method of claim 1, wherein the cells are expanded to aggregated cell clusters in an environment that includes from about 0.1% to about 2% of bovine serum albumin.