# US 10,119,121 B2 · Ex vivo proliferation of epithelial cells

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

- Number: US 10,119,121 B2
- Document ID: US10119121B2
- Status: Granted
- Kind: B2
- Application number: 15/296,920
- Filed: 2016-10-18
- Published: 2017-02-02
- Granted: 2018-11-06
- Earliest claimed: 2015-04-03
- Assignees: Propagenix Inc.
- Applicants: Propagenix Inc.
- Inventors: Chengkang Zhang
- Web page: https://patentgrep.com/patent/US10119121B2

## CPC

- C12N 5/0688: {Cells from the lungs or the respiratory tract}
- C12N 5/0629: {Keratinocytes; Whole skin}
- C12N 5/0683: {Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells (spermatogonia C12N5/061)}
- C12N 2500/14: Calcium; Ca chelators; Calcitonin
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/99: Serum-free medium
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/113: Acidic fibroblast growth factor (aFGF, FGF-1)
- 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 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2533/54: Collagen; Gelatin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The technology relates in part to methods and compositions for ex vivo proliferation and expansion of epithelial cells.

## Claims

### Claim 1

A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.

### Claim 2 (depends on 1)

The method of claim 1, wherein the TGF-beta inhibitor is an ALK5 inhibitor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the ALK5 inhibitor is selected from A83-01, GW788388, RepSox, and SB 431542.

### Claim 4 (depends on 1)

The method of claim 1, wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, and a myosin II inhibitor.

### Claim 5 (depends on 1)

The method of claim 1, wherein the agent that disrupts cytoskeletal structure is a Rho-associated protein kinase inhibitor.

### Claim 6 (depends on 5)

The method of claim 5, wherein the Rho-associated protein kinase inhibitor is selected from Y-27632, SR 3677, thiazovivin, HA1100 hydrochloride, HA1077 and GSK-429286.

### Claim 7 (depends on 1)

The method of claim 1, wherein the agent that disrupts cytoskeletal structure is a PAK inhibitor.

### Claim 8 (depends on 7)

The method of claim 7, wherein the PAK inhibitor is IPA3.

### Claim 9 (depends on 1)

The method of claim 1, wherein the agent that disrupts cytoskeletal structure is a myosin II inhibitor.

### Claim 10 (depends on 9)

The method of claim 9, wherein the myosin II inhibitor is blebbistatin.

### Claim 11 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise a beta-adrenergic receptor agonist.

### Claim 12 (depends on 11)

The method of claim 11, wherein the beta-adrenergic receptor agonist is isoproterenol.

### Claim 13 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise calcium at a concentration below 200 μM.

### Claim 14 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.

### Claim 15 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise one or more mitogenic growth factors.

### Claim 16 (depends on 15)

The method of claim 15, wherein the one or more mitogenic growth factors comprise EGF, FGF, or EGF and FGF.

### Claim 17 (depends on 1)

The method of claim 1, wherein the originating cell population is capable of 25 population doublings or more when cultured under the expansion culture conditions; and the originating cell population is capable of no more than 20 population doublings when cultured under control culture conditions that do not include the transforming growth factor beta (TGF-beta) inhibitor and the agent that disrupts cytoskeletal structure.

### Claim 18 (depends on 17)

The method of claim 17, wherein the originating cell population is capable of 50 population doublings or more when cultured under the expansion culture conditions.

### Claim 19 (depends on 17)

The method of claim 17, wherein the originating cell population is capable of 80 population doublings or more when cultured under the expansion culture conditions.

### Claim 20 (depends on 17)

The method of claim 17, wherein the originating cell population is capable of 100 population doublings or more when cultured under the expansion culture conditions.

### Claim 21 (depends on 1)

The method of claim 1, wherein the originating cell population is derived from differentiated tissue.

### Claim 22 (depends on 1)

The method of claim 1, wherein the originating cell population is derived from embryonic stem (ES) cells.

### Claim 23 (depends on 1)

The method of claim 1, wherein the originating cell population is derived from induced pluripotent stem cells (iPSCs).

### Claim 24 (depends on 1)

The method of claim 1, wherein the originating cell population does not comprise pluripotent cells.

### Claim 25 (depends on 1)

The method of claim 1, wherein the expansion culture conditions do not comprise one or more of extracellular matrix comprising heterogeneous components, a Wnt agonist or a beta-catenin agonist.

### Claim 26 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a Rho-associated protein kinase inhibitor.

### Claim 27 (depends on 26)

The method of claim 26, wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and Y-27632.

### Claim 28 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a p21-activated kinase (PAK) inhibitor.

### Claim 29 (depends on 28)

The method of claim 28, wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and IPA3.

### Claim 30 (depends on 1)

The method of claim 1, wherein the expansion culture conditions comprise inhibitors consisting of an ALK5 inhibitor and a myosin II inhibitor.

### Claim 31 (depends on 30)

The method of claim 30, wherein the expansion culture conditions comprise inhibitors consisting of A83-01 and blebbistatin.

### Claim 32 (depends on 1)

The method of claim 1, wherein the originating cell population comprises differentiated epithelial cells.

### Claim 33 (depends on 1)

The method of claim 1, wherein the originating cell population comprises transit-amplifying epithelial cells.

### Claim 34 (depends on 1)

The method of claim 1, wherein the originating cell population comprises lineage-committed epithelial cells.

### Claim 35 (depends on 1)

The method of claim 1, wherein the originating cell population comprises basal epithelial cells.

### Claim 36

A method for proliferating epithelial cells ex vivo, comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise components consisting essentially of i) a transforming growth factor beta (TGF-beta) signaling inhibitor; ii) a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, or a myosin II inhibitor; iii) a mitogenic growth factor; iv) a beta-adrenergic receptor agonist; v) calcium at a concentration below 100 μM; and vi) a serum-free base medium; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.

### Claim 37

A population of ex vivo expanded epithelial cells produced by a method comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.

### Claim 38 (depends on 37)

The population of ex vivo expanded epithelial cells of claim 37, wherein the TGF-beta inhibitor is an ALK5 inhibitor.

### Claim 39 (depends on 37)

The population of ex vivo expanded epithelial cells of claim 37, wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a PAK inhibitor, and a myosin II inhibitor.

### Claim 40 (depends on 37)

The population of ex vivo expanded epithelial cells of claim 37, wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.

### Claim 41

A method for genetically modifying cells, comprising contacting cells with one or more agents that introduce a genetic modification to the cells, wherein the cells have been expanded by a method comprising:
expanding the number of cells in an originating cell population comprising one or more of differentiated epithelial cells, transit-amplifying epithelial cells, lineage-committed epithelial cells, and basal epithelial cells under expansion culture conditions, thereby generating an expanded epithelial cell population, wherein:
the expansion culture conditions comprise a transforming growth factor beta (TGF-beta) inhibitor and an agent that disrupts cytoskeletal structure; and
the expansion culture conditions are serum-free and feeder-cell free culture conditions.

### Claim 42 (depends on 41)

The method of claim 41, wherein the TGF-beta inhibitor is an ALK5 inhibitor.

### Claim 43 (depends on 41)

The method of claim 41, wherein the agent that disrupts cytoskeletal structure is chosen from a Rho-associated protein kinase inhibitor, a PAK inhibitor, and a myosin II inhibitor.

### Claim 44 (depends on 41)

The method of claim 41, wherein the expansion culture conditions comprise calcium at a concentration below 100 μM.