In Vitro Production of Foregut Stem Cells

Patent metadata

Field	Value
Publication number	US-2016237401-A1
Application number	US-201415028085-A
Country	US
Kind code	A1
Filing date	Oct 6, 2014
Priority date	Oct 9, 2013
Publication date	Aug 18, 2016
Grant date	—

Abstract

This invention relates to the differentiation of pluripotent cells (PSCs) into foregut stem cells (FSCs) using a definitive endoderm induction medium comprising a TGFfi ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor to differentiate the pluripotent cells into definitive endoderm cells and a foregut induction medium comprising a TGFβ ligand to differentiate the definitive endoderm cells into foregut stem cells (FSCs). Methods of differentiation, populations of foregut stem cells, culture media and kits are provided.

First claim

1 . A method for producing a population of foregut stem cells (FSCs) comprising:
i) providing a population of definitive endoderm cells (DECs), ii) culturing the DECs in a foregut induction medium comprising a TGFβ ligand to produce a population of foregut stem cells (FSCs).

2 . A method according to claim 1 wherein the population of DECs is produced by a method comprising culturing a population of pluripotent cells (PSCs) in a definitive endoderm (DE) induction medium comprising a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor and allowing the PSCs to differentiate into DECs.

3 . A method according to claim 2 wherein the pluripotent cells are human pluripotent cells.

4 . A method according to claim 2 or claim 3 wherein the pluripotent cells are IPSCs.

5 . A method according to claim 4 wherein the iPSCs are derived from antecedent cells obtained from an individual.

6 . A method according to any one of claims 2 to 5 wherein the pluripotent cells express one or more of the following pluripotency associated markers: Oct4, Sox2, Alkaline Phosphatase, POU5f1, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc.

7 . A method according to any one of claims 2 to 6 wherein the DE induction medium comprises a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor.

8 . A method according to claim 7 wherein the DE induction medium consists of a chemically defined nutrient medium supplemented with a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor.

9 . A method according to claim 7 or claim 8 wherein the TGFβ ligand is activin and/or the PI3K inhibitor is LY294002.

10 . A method according to any one of the preceding claims wherein the population of DECs is a homogeneous population or a heterogeneous population.

11 . A method according to any one of the preceding claims wherein the DECs express one or more of the following endoderm associated markers: Sox17, foxA2, GSC, Mixl1, Lhx1, CXCR4, GATA4, eomesodermin (EOMES), Mixl1, HNF-3 beta, Cerberus, OTX4, goosecoid, C-kit, CD99, and Hex.

12 . A method according to any one of the preceding claims wherein the foregut induction medium consists of a chemically defined nutrient medium supplemented with a TGFβ ligand.

13 . A method according to any one of the preceding claims wherein the TGFβ ligand is activin.

14 . A method according to any one of the preceding claims wherein the FSCs express one or more of the following markers: SOX2, HHEX, HOXA3, HNF4α, SOX17, CXCR4, EpCAM, HNF1β, GATA4, Cer, HNF6, and HNF1bet.

15 . A method according to any one of the preceding claims comprising culturing the population of FSCs in an FSC maintenance medium comprising an effective amount of TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), HGF, EGF, and heparin.

16 . A method according to claim 15 wherein the FSC maintenance medium consists of a chemically defined nutrient medium supplemented with a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), HGF, EGF, and heparin.

17 . A method according to claim 15 or claim 16 wherein the TGF ligand is activin.

18 . A method according to any one of the preceding claims wherein the FSCs are multipotent and self-renewing in vitro.

19 . A method according to any one of the preceding claims wherein the FSCs are differentiated into endoderm cells.

20 . A method according to claim 19 comprising differentiating the FSCs into hepatic endoderm cells or hepatic progenitor cells.

21 . A method according to claim 19 comprising differentiating the FSCs into pancreatic progenitor cells or pancreatic endoderm cells.

22 . A method according to claim 19 comprising differentiating the FSCs into pulmonary progenitor cells or pulmonary endoderm cells.

23 . A method according to claim 22 comprising;
i) culturing the population of FSCs in a first pulmonary induction medium comprising RA and FGF;
ii) culturing the cells from step i) in a second pulmonary induction medium comprising FGF and HGF;
thereby producing a population of pulmonary progenitor cells.

24 . A method of producing pulmonary progenitor cells comprising;
i) providing a population of FSCs ii) culturing the population of FSCs in a first pulmonary induction medium comprising RA and FGF; iii) culturing the cells from step i) in a second pulmonary induction medium comprising FGF and HGF; thereby producing a population of pulmonary progenitor cells.

25 . A method according to claim 23 or 24 wherein the first pulmonary induction medium consists of a chemically defined nutrient medium supplemented with retinoic acid and fibroblast growth factor (FGF).

26 . A method according to any one of claims 23 to 25 wherein the second pulmonary induction medium consists of a chemically defined nutrient medium supplemented with hepatocyte growth factor (HGF) and fibroblast growth factor (FGF).

27 . A population of isolated FSCs produced by a method according to any one of claims 1 to 18 .

28 . A population according to claim 27 wherein said isolated FSCs have a disease associated genotype.

29 . A method for maintaining a population of foregut stem cells (FSCs), the method comprising:
i) providing a population of foregut stem cells (FSCs); ii) culturing the population of FSCs in an FSC maintenance medium comprising a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), HGF, EGF, and Heparin.

30 . A method according to claim 29 wherein the FSCs are produced by a method according to any one of claims 1 to 18 .

31 . Use of a population of isolated FSCs according to claim 27 or 28 for the in vitro production of endodermal cells.

32 . A population according to claim 27 for use in a method of treatment of the human or animal body.

33 . A population according to claim 27 for use in the treatment of a pulmonary, hepatic or pancreatic condition.

34 . A method of screening a compound comprising;
contacting a population of isolated FSCs according to claim 27 or claim 28 with a test compound, and; determining the effect of the test compound on said foregut stem cells and/or the effect of said foregut stem cells on the test compound.

35 . A kit for production of FSCs comprising;
a foregut induction medium consisting of a chemically defined nutrient medium supplemented with a TGFβ ligand.

36 . A kit according to claim 35 further comprising a DE induction medium consisting of a chemically defined nutrient medium supplemented with a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor.

37 . A kit according to claim 35 or claim 36 further comprising an FSC maintenance medium consisting of a chemically defined nutrient medium supplemented with a TGFβ ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), HGF, EGF, and heparin.

38 . Use of a foregut induction medium for the production of a population of FSCs,
said foregut induction medium consisting of a chemically defined nutrient medium supplement

Assignees

• Cambridge Entpr Ltd

Inventors

• Vallier Ludovic
• Hannan Nicholas

Classifications

• A61P11/00

  Drugs for disorders of the respiratory system · CPC title

• A61P1/16

  for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics · CPC title

• A61P1/18

  for pancreatic disorders, e.g. pancreatic enzymes · CPC title

• C12N2501/12

  Hepatocyte growth factor [HGF] · CPC title

• C12N2506/02

  from embryonic cells · CPC title