In Vitro Pancreatic Differentiation of Pluripotent Mammalian Cells

• US 20150225698A1
• Filed: 09/16/2013
• Published: 08/13/2015
• Est. Priority Date: 09/20/2012
• Status: Active Grant

• Current Assignee: Cambridge Enterprises Limited
• Original Assignee: Cambridge Enterprises Limited
• Inventors: Vallier, Ludovic, Cho, Hsin-hua
• Granted Patent: US 9,790,470 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• 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 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 33/5038 involving detection of metabolites per se
• CPC Class Codes: G01N 33/507 Pancreatic cells
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

This invention relates to the in vitro differentiation of pluripotent cells into pancreatic progenitors by i) culturing pluripotent cells in a definitive endoderm (DE) medium comprising a TGFp ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), a PI3K inhibitor and optionally a GSK3 β inhibitor to produce a population of definitive endoderm cells, ii) culturing the definitive endoderm cells in a first pancreatic medium comprising an activin antagonist; FGF; retinoic acid; and a BMP inhibitor to produce a population of dorsal foregut cells; iii) culturing the dorsal foregut cells in a second pancreatic medium comprising FGF, retinoic acid, a BMP inhibitor, and a hedgehog signalling inhibitor, and; iv) culturing the endoderm cells in a third pancreatic medium comprising FGF. The progenitor cells thus produced may be further differentiated into pancreatic endocrine cells. These methods may be useful, for example, in producing pancreatic cells for therapy or disease modelling.

Claims

1. A method for producing a population of pancreatic progenitor cells which comprises:

i) providing a population of pluripotent cells;

ii) culturing the population in a definitive endoderm (DE) induction medium to produce a population of definitive endoderm cells, wherein the definitive endoderm (DE) induction medium comprises a TGFβ

ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP), a PI3K inhibitor and optionally a GSK3β

inhibitor;

iii) culturing the population of definitive endoderm cells in a first pancreatic induction medium comprising an activin antagonist;

FGF;

retinoic acid; and

a BMP inhibitor to produce a population of dorsal foregut cells;

iv) culturing the dorsal foregut cells in a second pancreatic induction medium comprising FGF, retinoic acid, a BMP inhibitor, and a hedgehog signalling inhibitor;

v) culturing the endoderm cells in a third pancreatic induction medium comprising FGF;

thereby producing a population of pancreatic progenitor cells.

2. A method according to claim 1 wherein definitive endoderm (DE) induction medium is a chemically defined medium which comprises TGFβ

ligand, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and a PI3K inhibitor.

3. A method according to claim 1 or claim 2 wherein the definitive endoderm (DE) induction medium is a chemically defined medium which consists of a basal medium supplemented with activin, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and LY294002.

4. A method according to claim 3 wherein the cells are cultured in said medium for 2 to 4 days to produce the population of DE cells.

5. A method according to claim 1 or claim 2 wherein step (ii) comprises:

(a) culturing the population of pluripotent cells in said DE induction medium, wherein said DE induction medium further comprises a GSK33 inhibitor,

(b) further culturing the population in the definitive endoderm induction medium lacking GSK3β

inhibitor; and

,

(c) further culturing the population in anterior definitive endoderm (ADE) induction medium which comprises a TGFβ

ligand and fibroblast growth factor to produce the population of definitive endoderm (DE) cells.

6. A method according to claim 5 wherein the definitive endoderm (DE) induction medium is a chemically defined medium which consists of a basal medium supplemented with activin and fibroblast growth factor (FGF).

7. A method according to claim 5 or claim 6 wherein the population is cultured for 24 hours in each of steps a) to c).

8. A method according to any one of the preceding claims wherein the definitive endoderm cells express SOX17, CXCR4 and GSC.

9. A method according to any one of the preceding claims wherein the first pancreatic induction medium is a chemically defined medium which comprises activin/TGFβ

antagonist;

FGF;

retinoic acid; and

a BMP antagonist.

10. A method according to any one of the preceding claims wherein the first pancreatic induction medium is a chemically defined medium which consists of a basal medium supplemented with SB-431542;

FGF;

retinoic acid; and

noggin.

11. A method according to any one of the preceding claims wherein the dorsal foregut cells express RFX6, FOXA2, HNF1b, SOX2, HNF4a, and HLXB9.

12. A method according to any one of the preceding claims wherein the second pancreatic induction medium is a chemically defined medium which comprises FGF, a BMP antagonist, retinoic acid, and a hedgehog signalling inhibitor.

13. A method according to any one of the preceding claims wherein second pancreatic induction medium is a chemically defined medium which consists of a basal medium supplemented with FGF;

retinoic acid;

noggin; and

KAAD-cyclopamine.

14. A method according to any one of the preceding claims wherein the third pancreatic induction medium is a chemically defined medium which comprises FGF.

15. A method according to any one of the preceding claims wherein third pancreatic induction medium is a chemically defined medium which consists of a basal medium supplemented with FGF.

16. A method according to claim 14 or claim 15 wherein the third pancreatic induction medium further comprises retinoic acid.

17. A method according to any one of the preceding claims wherein the pancreatic progenitor cells express PDX1, SOX9, HNF6, NKX6.1 and PTF1a.

18. A method according to any one of the preceding claims comprising maturing the pancreatic progenitor cells to produce a population of pancreatic endocrine cells.

19. A method according to claim 18 wherein the pancreatic progenitor cells are matured by i) culturing in a first endocrine induction medium and ii) culturing in a second endocrine induction medium to produce the population of pancreatic endocrine cells,

wherein the first endocrine induction medium is a chemically defined medium comprising a Notch signalling inhibitor;

and the second endocrine induction medium is a chemically defined medium devoid of differentiation factors.

20. A method according to claim 19 wherein the first endocrine induction medium is a chemically defined medium consisting of a supplemented basal medium and N—

[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT); and

the second endocrine induction medium is a chemically defined medium consisting of a supplemented basal medium.

21. A method according to claim 18 wherein the pancreatic progenitor cells are matured by i) culturing in a first endocrine induction medium and ii) culturing in a second endocrine induction medium to produce the population of pancreatic endocrine cells,

wherein the first endocrine induction medium is a chemically defined medium comprising a Notch signalling inhibitor and retinoic acid;

and the second endocrine induction medium is a chemically defined medium devoid of differentiation factors other than retinoic acid.

22. A method according to claim 21 wherein the first endocrine induction medium is a chemically defined medium consisting of a supplemented basal medium, N—

[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) and retinoic acid; and

the second endocrine induction medium is a chemically defined medium consisting of a supplemented basal medium and retinoic acid.

23. A method according to any one of claims 16 to 22 wherein the pancreatic endocrine cells express NGN3, INS, SST and GLU.

24. A method according to any one of the preceding claims comprising monitoring and/or detecting the expression of one or more cell markers in the population of differentiating cells.

25. A method according to any one of the preceding claims comprising expanding the population of pancreatic progenitor cells or pancreatic endocrine cells.

26. A method according to any one of the preceding claims comprising culturing or maintaining the population of pancreatic progenitor cells or pancreatic endocrine cells.

27. A method according to any one of the preceding claims comprising storing the population of pancreatic progenitor cells, or pancreatic endocrine cells.

28. A method according to any one of the preceding claims comprising admixing the population of pancreatic progenitor cells, pancreatic endocrine cells or pancreatic endocrine cells with a therapeutically acceptable excipient.

29. A method according to any one of the preceding claims wherein the pluripotent cells are human pluripotent cells.

30. A method according to any one of the preceding claims wherein the pluripotent cells are ESCs or iPSCs.

31. A method according to any one of the preceding claims wherein the pluripotent cells are iPS cells derived from an individual with a genetic background associated with a pancreatic condition.

32. A method according to any one of the preceding claims wherein the pluripotent cells are iPS cells derived from an individual with a genetic disorder associated with a pancreatic condition.

33. A population of isolated pancreatic progenitor cells or pancreatic endocrine cells produced by a method according to any one of claims 1 to 32.

34. A population according to claim 33 wherein said isolated pancreatic progenitor cells or pancreatic endocrine cells have a disease associated genotype.

35. A population according to claim 34 for use in a method of treatment of the human or animal body.

36. A population according to claim 35 for use in the treatment of a pancreatic condition.

37. A method of treating a patient with a pancreatic condition comprising;

administering a population of isolated pancreatic progenitor cells or pancreatic endocrine cells according to claim 33 to an individual in need thereof.

38. Use of a population according to claim 33 in the manufacture of a medicament for use in the treatment of a patient with a pancreatic condition.

39. A population, method or use according to any one of claims 36 to 38 wherein the pancreatic condition is a diabetic condition.

40. A population, method or use according to claim 39 wherein the diabetic condition is type 1 diabetes or type 2 diabetes.

41. A method of screening a compound comprising;

contacting isolated pancreatic progenitor cells or pancreatic endocrine cells according to claim 33 or claim 34 with a test compound, and;

determining the effect of the test compound on said pancreatic progenitor cells or pancreatic endocrine cells and/or the effect of said pancreatic progenitor cells or pancreatic endocrine cells on the test compound.

42. A method according to claim 41 wherein the effect of the test compound on one or more of proliferation, insulin expression, insulin secretion;

glucose responsiveness is determined.