Improved culture method for organoids

Patent metadata

Field	Value
Publication number	US-2017191030-A1
Application number	US-201515310905-A
Country	US
Kind code	A1
Filing date	May 15, 2015
Priority date	May 16, 2014
Publication date	Jul 6, 2017
Grant date	—

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.

First claim

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 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, one or more Wnt agonist wherein the Wnt agonist is an Lgr5 agonist, a TGF-beta inhibitor and a cAMP pathway activator.

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

3 . The method of claim 1 , wherein the expansion medium further comprises a BMP pathway activator.

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

5 . The method of claim 1 , wherein in the expansion medium:
the one or more receptor tyrosine kinase ligands are selected from the group consisting of: FGF, HGF and EGF, wherein the FGF is preferably an FGF able to bind to FGFR2 or FGFR4 and is preferably FGF10; the TGF-beta inhibitor is a small molecule inhibitor of ALK4, ALK5 or ALK7, optionally selected from the group consisting of: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208 and SJN 2511; the cAMP pathway activator is an adenylyl cyclase activator, for example, forskolin, a forskolin analog or cholera toxin, or a cAMP analog, for example 8-bromo-cAMP, or NKH477; when Rspondin is present, the Rspondin is selected from R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4; when a further Wnt agonist is present, the further Wnt agonist is selected from one or more of Wnt-3a, Wnt-5, Wnt-6a, Norrin, and a GSK-inhibitor; and/or when a BMP pathway activator is present, it is selected from one or more of BMP7, BMP4 and BMP2.

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 6 , wherein in the differentiation medium:
the one or more receptor tyrosine kinase ligands are selected from one or more of the group: FGF, HGF and EGF, wherein the FGF is preferably an FGF able to bind to FGFR2 or FGFR4 and is preferably FGF19; the Notch inhibitor is a gamma-secretase inhibitor, optionally DAPT or dibenzazepine (DBZ) or benzodiazepine (BZ) or LY-411575; when a TGF-beta inhibitor is present, it is a small molecule inhibitor of ALK4, ALK5 or ALK7 optionally selected from the group consisting of: A83-01, SB-431542, SB-505124, SB-525334, LY 364947, SD-208, SJN 2511; and/or when a BMP pathway activator is present, it is selected from one or more of BMP7, BMP4 and BMP2.

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, FGF19, DAPT, dexamethasone, HGF and A8301.

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 . (canceled)

13 . 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 an expansion medium, the 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, and optionally BMP7; and optionally, 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, and optionally BMP7.

14 . 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 an expansion medium, the 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, and optionally BMP7; and optionally 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, and optionally a BMP7.

15 . An expansion medium, comprising a basal medium for animal or human cells and:
one or more receptor tyrosine kinase ligands, one or more Wnt agonist wherein the Wnt agonist is an Lgr5 agonist, a TGF-beta inhibitor, and a cAMP pathway activator.

16 .- 21 . (canceled)

22 . A differentiation medium comprising a basal medium for animal or human cells and EGF, gastrin, FGF19, DAPT, dexamethasone, HGF and A8301 and optionally a BMP pathway activator.

23 . An organoid obtainable or obtained by a method of claim 1 .

24 . An organoid of claim 23 which has been cultured for at least 6, 8, 10, 12, 14, 16, 18 or 20 weeks.

25 . (canceled)

26 . An organoid in a culture medium according to claim 15 .

27 . (canceled)

28 . A liver organoid according to claim 23 , wherein the liver organoid is derived from a human and:
expresses at least one, preferably all, of the following stem cell signature genes: LGR4, TACSTD1/Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7 and ELF3; and/or expresses at least one, preferably all, of the following reprogramming genes: KLF4, MYC, POU5F1 and SOX2; and/or expresses at least one, preferably all, of the following hepatocyte/cholangiocyte specific genes: HNF1A, HNF1B, HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19 and MET; and/or does not express at least one, preferably all, of the following hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R and CD34, AFP, GCG and PTF1A, for example, it does not express NEUROG2, IGF1R and CD34; and/or expresses at least one, preferably all, of the following hepatocyte specific genes: TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2 and SCARB1.

29 . A liver organoid according to claim 23 , wherein the liver organoid is derived from a mouse and:
expresses at least one, preferably all, of the following stem

Assignees

• Koninklijke Nederlandse Akademie Van Wetenschappen

Inventors

• Huch Ortega Meritxell
• Clevers Johannes Carolus
• Fernandez Boj Sylvia

Classifications

• C12N5/0676

  Pancreatic cells · CPC title

• C12N2501/345

  Gastrin; Cholecystokinins [CCK] · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N2501/19

  Growth and differentiation factors [GDF] · CPC title