Culture method for organoids

• US 10,597,633 B2
• Filed: 05/15/2015
• Issued: 03/24/2020
• Est. Priority Date: 05/16/2014
• Status: Active Grant

• Current Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Original Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Inventors: Huch Ortega, Meritxell, Clevers, Johannes Carolus, Fernandez Boj, Sylvia
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US15/310,905
• Publication Number:
  US 20170191030A1
• Time in Patent Office: 1,775 Days
• Field of Search: None
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• 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 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/30 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C12N2501/85
• CPC Class Codes: C12N 2501/345 Gastrin; Cholecystokinins [CCK]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 : Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to improved culture methods for expanding epithelial stem cells and obtaining organoids, to culture media involved in said methods, and to uses of said organoids.

Claims

1. A method for culturing epithelial stem cells, wherein said method comprises:

culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of a first expansion medium, the first expansion medium comprising a basal medium for animal or human cells to which is added;

one or more receptor tyrosine kinase ligands, a Wnt agonist that is a Lgr5 agonist, a TGF-beta inhibitor and a cAMP pathway activator,

wherein the TGF-beta inhibitor is able to reduce the activity of the TGF-beta signaling pathway that acts via Smad2 and/or Smad3.

2. The method of claim 1, wherein the Lgr5 agonist is Rspondin.

3. The method of claim 1, wherein the method further comprises culturing in a second expansion medium, wherein the second expansion medium comprises a basal medium for animal or human cells to which is added:

one or more receptor tyrosine kinase ligands, a Wnt agonist that is a Lgr5 agonist, a TGF-beta inhibitor and a cAMP pathway activator,

wherein the TGF-beta inhibitor is able to reduce the activity of the TGF-beta signaling pathway that acts via Smad2 and/or Smad3;

and wherein the second expansion medium further comprises a BMP pathway activator.

4. The method of claim 1, wherein the first expansion medium further comprises one or more components selected from a further Wnt agonist, a BMP inhibitor, nicotinamide, gastrin, B27, N2, and N-Acetylcysteine.

5. The method of claim 1, wherein in the first expansion medium:

the one or more receptor tyrosine kinase ligands are selected from FGF, HGF and EGF;

the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7;

the cAMP pathway activator is an adenylyl cyclase activator, a cAMP analog, or NKH477.

6. The method of claim 1, wherein the method further comprises a culturing step in a differentiation medium comprising a basal medium for animal or human cells to which is added one or more receptor tyrosine kinase ligands and a Notch inhibitor.

7. The method of claim 6, wherein the differentiation medium further comprises one or more of:

a TGF-beta inhibitor, gastrin, dexamethasone and a BMP pathway activator.

8. The method of claim 7, wherein in the differentiation medium:

the one or more receptor tyrosine kinase ligands are selected from FGF, HGF, EGF, and combinations thereof;

the Notch inhibitor is a gamma-secretase inhibitor;

the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7; and

/or

the BMP pathway activator is selected from BMP7, BMP4, BMP2, and combinations thereof.

10. The method of claim 1, wherein the method further comprises isolating one or more adult stem cells or obtaining and isolating an organoid.

11. The method of claim 1, wherein the epithelial stem cells are from the liver, pancreas, intestine, stomach, prostate, lung, breast, ovary, salivary gland, hair follicle, skin, oesophagus or thyroid.

12. The method of claim 1, wherein the epithelial stem cells are from the liver and wherein said method comprises:

culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of a first expansion medium comprising a basal medium for animal or human cells to which is added;

EGF, FGF10, HGF, Rspondin, Nicotinamide, a TGF-beta inhibitor, forskolin, gastrin, N-Acetylcysteine, and N2 and/or B27, and which is supplemented with Noggin, a further Wnt agonist, and a ROCK inhibitor;

culturing the one or more epithelial stem cells in a second expansion medium comprising a basal medium for animal or human cells to which is added;

EGF, FGF10, HGF, Rspondin, Nicotinamide, a TGF-beta inhibitor, forskolin, gastrin, N-Acetylcysteine, and N2 and/or B27.

13. The method of claim 1, wherein the epithelial stem cells are from the pancreas and wherein said method comprises:

culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of a first expansion medium comprising a basal medium for animal or human cells to which is added;

EGF, FGF10, Rspondin, Noggin, a further Wnt agonist, Nicotinamide, a TGF-beta inhibitor, forskolin, PGE2, a p38 inhibitor, gastrin, N-Acetylcysteine and N2 and/or B27.

14. The method of claim 2, wherein the Rspondin is selected from R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

15. The method of claim 3, wherein in the second expansion medium:

the one or more receptor tyrosine kinase ligands are selected from FGF, HGF, EGF, and combinations thereof;

the Lgr5 agonist is Rspondin;

the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7;

the cAMP pathway activator is an adenylyl cyclase activator, a cAMP analog, or NKH477; and

the BMP pathway activator is selected from BMP7, BMP4, BMP2, and combinations thereof.

16. The method of claim 15, wherein the Rspondin is selected from R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

17. The method of claim 15, wherein the small molecule inhibitor of ALK4, ALK5 or ALK7 is selected from A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

18. The method of claim 15, wherein the adenylyl cyclase activator is forskolin, a forskolin analog, or cholera toxin.

19. The method of claim 15, wherein the cAMP analog is 8-bromo-cAMP.

20. The method of claim 4, wherein the further Wnt agonist is selected from Wnt-3a, Wnt-5, Wnt-6a, Norrin, a GSK-inhibitor, and combinations thereof.

21. The method of claim 5, wherein the FGF is an FGF able to bind to FGFR2 or FGFR4.

22. The method of claim 21, wherein the FGF is FGF10.

23. The method of claim 5, wherein the small molecule inhibitor of ALK4, ALK5 or ALK7 is selected from A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

24. The method of claim 5, wherein the adenylyl cyclase activator is forskolin, a forskolin analog, or cholera toxin.

25. The method of claim 5, wherein the cAMP analog is 8-bromo-cAMP.

27. The method of claim 8, wherein the FGF is an FGF able to bind to FGFR2 or FGFR4.

28. The method of claim 27, wherein the FGF is FGF19.

29. The method of claim 8, wherein the gamma-secretase inhibitor is DAPT, dibenzazepine (DBZ), benzodiazepine (BZ), or LY-411575.

30. The method of claim 8, wherein the small molecule inhibitor of ALK4, ALK5 or ALK7 is selected from A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208, and SJN 2511.

35. The method of claim 12, further comprising adding BMP7 to the second expansion medium.

36. The method of claim 12, further comprising culturing the one or more expanded epithelial stem cells in a differentiation medium comprising a basal medium for animal or human cells to which is added EGF, gastrin, FGF19, DAPT, dexamethasone, HGF, a TGF-beta inhibitor.

37. The method of claim 36, further comprising adding BMP7 to the differentiation medium.

38. The method of claim 13, further comprising culturing the one or more expanded epithelial stem cells in a differentiation medium comprising a basal medium for animal or human cells to which is added EGF, gastrin, FGF19, DAPT, dexamethasone, HGF, a TGF-beta inhibitor.

39. The method of claim 38, further comprising adding BMP7 to the differentiation medium.

9. A method for differentiating epithelial stem cells, wherein said method comprises:

culturing the cells in a differentiation medium comprising a basal medium for animal or human cells to which is added EGF, gastrin, FGF , DAPT, dexamethasone, HGF and a TGF-beta inhibitor, wherein the TGF-beta inhibitor is able to reduce the activity of the TGF-beta signaling pathway that acts via Smad2 and/or Smad3.

26. The method of claim 9, wherein the method further comprises isolating one or more adult stem cells or obtaining and isolating an organoid.

31. The method of claim 9, wherein the FGF is an FGF able to bind to FGFR2 or FGFR4.

32. The method of claim 31, wherein the FGF is FGF19.

33. The method of claim 9, wherein the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7.

34. The method of claim 33, wherein the small molecule inhibitor of ALK4, ALK5 or ALK7 is selected from A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

40. A method for culturing epithelial stem cells, wherein said method comprises:

culturing one or more epithelial stem cells in contact with an extracellular matrix in the presence of an expansion medium, the expansion medium comprising a basal medium for animal or human cells to which is added;

one or more receptor tyrosine kinase ligands, a Wnt agonist that is a Lgr5 agonist, a TGF-beta inhibitor, and a BMP pathway activator,

wherein the TGF-beta inhibitor is able to reduce the activity of the TGF-beta signaling pathway that acts via Smad2 and/or Smad3.

41. The method of claim 40, wherein the expansion medium further comprises a cAMP pathway activator.

42. The method of claim 41, wherein the cAMP pathway activator is an adenylyl cyclase activator, a cAMP analog, or NKH477.

43. The method of claim 42, wherein the adenylyl cyclase activator is forskolin, a forskolin analog, or cholera toxin.

44. The method of claim 42, wherein the cAMP analog is 8-bromo-cAMP.

45. The method of claim 40, wherein:

the one or more receptor tyrosine kinase ligands are selected from FGF, HGF, EGF, and combinations thereof;

the Lgr5 agonist is Rspondin;

the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7; and

the BMP pathway activator is selected from BMP7, BMP4, BMP2, and combinations thereof.

46. The method of claim 45, wherein the Rspondin is selected from R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

47. The method of claim 45, wherein the small molecule inhibitor of ALK4, ALK5 or ALK7 is-selected from A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511.

48. The method of claim 45, wherein the FGF is an FGF able to bind to FGFR2 or FGFR4.

49. The method of claim 48, wherein the FGF is FGF10.

50. The method of claim 40, wherein the expansion medium further comprises one or more components selected from a further Wnt agonist, a BMP inhibitor, nicotinamide, gastrin, B27, N2, and N-Acetylcysteine.

51. The method of claim 50, wherein the further Wnt agonist is selected from Wnt-3a, Wnt-5, Wnt-6a, Norrin, a GSK-inhibitor, and combinations thereof.

52. The method of claim 40, wherein the method further comprises isolating one or more adult stem cells or obtaining and isolating an organoid.