# US 11,299,712 B2 · Method for inducing differentiation of alveolar epithelial cells

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- Number: US 11,299,712 B2
- Document ID: US11299712B2
- Status: Granted
- Kind: B2
- Application number: 15/555,183
- Filed: 2016-03-02
- Published: 2018-02-22
- Granted: 2022-04-12
- Earliest claimed: 2015-03-06
- Assignees: Kyoto University
- Applicants: Kyoto University
- Inventors: Shimpei Gotoh; Yuki Yamamoto; Satoshi Konishi; Michiaki Mishima
- Web page: https://patentgrep.com/patent/US11299712B2

## CPC

- C12N 5/0688: {Cells from the lungs or the respiratory tract}
- C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/15: Transforming growth factor beta (TGF-β)
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance
- C12N 2501/39: Steroid hormones
- C12N 2501/415: Wnt; Frizzeled
- C12N 2501/50: Cell markers; Cell surface determinants
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/73: Hydrolases (EC 3.)
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2513/00: 3D culture
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention provides a method for stably producing type II alveolar epithelial cells from pluripotent stem cells. Specifically, the invention relates to a method for producing type II alveolar epithelial cells from pluripotent stem cells comprising steps of: (1) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor; (2) culturing the cells obtained in Step (1) in a medium containing a BMP inhibitor and a TGFβ inhibitor; (3) culturing the cells obtained in Step (2) in a medium containing BMP4, retinoic acid, and a GSK3β inhibitor; (4) culturing the ventral anterior foregut cells obtained in Step (3) in a medium containing a GSK3β inhibitor, FGF10, KGF, and a NOTCH signal inhibitor; and (5) subjecting the alveolar epithelial progenitor cells obtained in Step (4) to three-dimensional culture in a medium containing a steroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF.

## Claims

### Claim 1

A method for producing a cell population comprising type II alveolar epithelial cells from pluripotent stem cells comprising Steps (1) to (5) below:
(1) culturing pluripotent stem cells in a medium containing activin A and a glycogen synthase kinase 3β (GSK3β) inhibitor;
(2) culturing the cells obtained in Step (1) in a medium containing a morphogenetic protein (BMP) inhibitor and a transforming growth factor beta (TGFβ) inhibitor;
(3) culturing the cells obtained in Step (2) in a medium containing bone morphogenetic protein 4 (BMP4), retinoic acid, and a GSK3β inhibitor;
(4) culturing the ventral anterior foregut cells obtained in Step (3) in a medium containing a GSK3β inhibitor, Fibroblast Growth Factor 10 (FGF10), keratinocyte growth factor (KGF), and a NOTCH signal inhibitor for a duration of time until alveolar epithelial progenitor cells are induced; followed by a step of isolating carboxypeptidase M-positive (CPM-positive) cells as alveolar epithelial progenitor cells; and
(5) subjecting the induced alveolar epithelial progenitor cells obtained in Step (4) to three-dimensional culture in a basal medium supplemented with additives consisting of a steroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF;
following Step (5), a further step of isolating cells positive for one or more type II alveolar epithelial cell markers selected from the group consisting of surfactant protein C (SFTPC), epithelial cell adhesion molecule (EpCAM), and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) as type II alveolar epithelial cells;
wherein cells positive for staining of acidic fractions for live cells are isolated as type II alveolar epithelial cells;
thereby obtaining a cell population wherein the type II alveolar epithelial cells are at least 50% of the cell population, relative to total epithelial cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the GSK3β inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is SB431542, the NOTCH signal inhibitor is N—[N-(3,5-Difluorophenylacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester (DAPT), the steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP, and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX).

### Claim 3 (depends on 1)

The method according to claim 1, wherein the medium of Step (1) further comprises Rho kinase (ROCK) inhibitor and/or a histone deacetylase (HDAC) inhibitor.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the ROCK inhibitor is Y-27632 and/or the HDAC inhibitor is sodium butyrate.

### Claim 5 (depends on 1)

The method according to claim 1, wherein, following Step (4), the alveolar epithelial progenitor cells are cryopreserved and the alveolar epithelial progenitor cells cultured in Step (5) are obtained by thawing the cryopreserved alveolar epithelial progenitor cells.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the medium of Step (5) further comprises a ROCK inhibitor.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the ROCK inhibitor is Y-27632.

### Claim 8 (depends on 1)

The method according to claim 1, wherein, following Step (5), the type II alveolar epithelial cells obtained are cryopreserved.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the alveolar epithelial progenitor cells induced in Step (4) are carboxypeptidase M-positive (CPM-positive).

### Claim 10 (depends on 1)

The method according to claim 1, wherein the culturing of Step (1) is for at least 6 days; the culturing of Step (2) is for at least 2 days; the culturing of Step (3) is for at least 4 days; the culturing of Step (4) is for at least 4 days; and the culturing of Step (5) is for at least 14 days.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the activin A and the GSK3β inhibitor are present in the medium of Step (1) at concentrations, respectively, of 100 ng/ml and 1 μM;
wherein the BMP and TGFβ inhibitor are present in the medium of Step (2) at concentrations, respectively, of 100 ng/ml and 10 μM;
wherein the BMP4, retinoic acid and the GSK3β inhibitor are present in the medium of Step (3) at concentrations, respectively, of 20 ng, 0.05 μM and; 3 μM;
wherein the GSK3β inhibitor, FGF10, KGF and NOTCH signal inhibitor are present in the medium of Step (4) at concentrations, respectively of, 3 μM, 10 ng/ml, 10 ng/ml, and 20 μM,
wherein the steroid drug, cAMP derivative, phosphodiesterase inhibitor, and the KGF are present in the medium of Step (5) at concentrations, respectively, of 50 nM, 100 μM, 100 μM, and 10 ng/ml, without any GSK3 β inhibitor and without any FGF10;
wherein the culturing of Step (1) is for 6 days; the culturing of Step (2) is for 2 to 4 days; the culturing of Step (3) is for 4 to 6 days; the culturing of Step (4) is for 4 to 14 days; and the culturing of Step (5) is for 14 days, and
wherein the GSK3 β inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is SB431542, the NOTCH signal inhibitor is N—[N-(3,5-Difluorophenylacetyl-Lalanyl)]-S-phenylglycine t-Butyl ester (DAPT), the steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP, the retinoic acid is all-trans retinoic acid (ATRA), and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX).

### Claim 12 (depends on 1)

The method according to claim 1, wherein the activin A and the GSK3 β inhibitor are present in the medium of Step (1) at concentrations, respectively, of 100 ng/ml and 1 μM;
wherein the BMP and TGFβ inhibitor are present in the medium of Step (2) at concentrations, respectively, of 100 ng/ml and 10 μM;
wherein the BMP4, retinoic acid and the GSK3β inhibitor are present in the medium of Step (3) at concentrations, respectively, of 20 to 100 ng/ml, 0.05 to 1 μM and 2.5 to 3.5 μM;
wherein the GSK3β inhibitor, FGF10, KGF and NOTCH signal inhibitor are present in the medium of Step (4) at concentrations, respectively of, 1 to 3 μM, 10 to 100 ng/ml, 10 ng/ml and 10 to 50 μM;
wherein the steroid drug, cAMP derivative, phosphodiesterase inhibitor, and the KGF are present in the medium of Step (5) at concentrations, respectively, of 50 nM, 100 μM, 100 μM and 10 to 100 ng/ml;
wherein the culturing of Step (1) is for 6 days; the culturing of Step (2) is for 2 to 4 days; the culturing of Step (3) is for 4 to 6 days; the culturing of Step (4) is for 4 to 14 days; and the culturing of Step (5) is for 14 days, and
wherein the GSK3 β inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is SB431542, the NOTCH signal inhibitor is N—[N-(3,5-Difluorophenylacetyl-Lalanyl)]-S-phenylglycine t-Butyl ester (DAPT), the steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP, and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX).

### Claim 13 (depends on 1)

The method according to claim 1, wherein the activin A and the GSK3β inhibitor are present in the medium of Step (1) at concentrations, respectively, of 100 ng/ml and 1 μM;
wherein the BMP and TGFβ inhibitor are present in the medium of Step (2) at concentrations, respectively, of 100 ng/ml and 10 μM;
wherein the BMP4, retinoic acid and the GSK3β inhibitor are present in the medium of Step (3) at concentrations, respectively, of 20 ng/ml, 1 μM and 2.5 to 3.5 μM;
wherein the GSK3β inhibitor, FGF10, KGF and NOTCH signal inhibitor are present in the medium of Step (4) at concentrations, respectively of, 3 μM, 10 ng/ml, 10 ng/ml and 20 μM;
wherein the steroid drug, cAMP derivative, phosphodiesterase inhibitor, and the KGF are present in the medium of Step (5) at concentrations, respectively, of 50 nM, 100 μM, 100 μM and 10 ng/ml;
wherein the culturing of Step (1) is for 6 days; the culturing of Step (2) is for 2 to 4 days; the culturing of Step (3) is for 4 to 6 days; the culturing of Step (4) is for 4 to 14 days; and the culturing of Step (5) is for 14 days, and
wherein the GSK3 β inhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is SB431542, the NOTCH signal inhibitor is N—[N-(3,5-Difluorophenylacetyl-Lalanyl)]-S-phenylglycine t-Butyl ester (DAPT), the steroid drug is dexamethasone, the cAMP derivative is 8-Br-cAMP, and the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine (IBMX).

### Claim 14 (depends on 1)

The method of claim 1, wherein:
the medium of Step (1) is a basal medium supplemented with additives consisting of the activin A and the glycogen synthase kinase 3β (GSK3β) inhibitor;
the medium of Step (2) is a basal medium supplemented with additives consisting of the BMP inhibitor and the TGFβ inhibitor; and
the medium of Step (3) is a basal medium supplemented with additives consisting of the BMP4, the retinoic acid, and the GSK3β inhibitor.