In Vitro Production of Foregut Stem Cells

• US 20160237401A1
• Filed: 10/06/2014
• Published: 08/18/2016
• Est. Priority Date: 10/09/2013
• Status: Active Grant

• Current Assignee: Cambridge Enterprises Limited
• Original Assignee: Cambridge Enterprises Limited
• Inventors: Vallier, Ludovic, Hannan, Nicholas
• Granted Patent: US 10,000,740 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors
• CPC Class Codes: C12N 5/0689 : Stem cells; Progenitors
• CPC Class Codes: G01N 33/5023 : on expression patterns

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.

Claims

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.

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.

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.

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.

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.

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 supplemented with a TGFβ

ligand.

39. Use of first and a second pulmonary induction media for the production of a population of pulmonary progenitor cells, the first pulmonary induction medium consisting of a chemically defined nutrient medium supplemented with retinoic acid and fibroblast growth factor (FGF) and the second pulmonary induction medium consists of a chemically defined nutrient medium supplemented with hepatocyte growth factor (HGF) and fibroblast growth factor (FGF).