Culture medium for epithelial stem cells and organoids comprising the stem cells

• US 9,752,124 B2
• Filed: 07/29/2011
• Issued: 09/05/2017
• Est. Priority Date: 02/03/2009
• Status: Active Grant

• Current Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Original Assignee: Koninklijke Nederlandse Akademie Van Wetenschappen
• Inventors: Sato, Toshiro, Clevers, Johannes C., Huch Ortega, Meritxell
• Primary Examiner(s): SCHUBERG, LAURA J
• Application Number:
  US13/194,866
• Publication Number:
  US 20120196312A1
• Time in Patent Office: 2,230 Days
• Field of Search: None
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/119 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 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• 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 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/345 Gastrin; Cholecystokinins [CCK]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

The invention relates to a method for culturing epithelial stem cells, isolated tissue fragments comprising the epithelial stem cells, or adenoma cells, and culturing the cells or fragments in the presence of a Bone Morphogenetic Protein (BMP) inhibitor, a mitogenic growth factor, and a Wnt agonist when culturing epithelial stem cells and isolated tissue fragments. The invention further relates to a cell culture medium comprising a BMP inhibitor, a mitogenic growth factor, and a Wnt agonist, to the use of the culture medium, and to crypt-villus organoids, gastric organoids, pancreatic organoids, liver organoids, colon organoids, Barrett'"'"'s Esophagus organoids, adenocarcinoma organoids and colon carcinoma organoids that are formed in the culture medium.

Claims

1. A composition comprising a three-dimensional organoid obtained by in vitro expansion of one or more adult adenoma stem cells, wherein the organoid has a sealed central lumen lined by epithelial cells, wherein the organoid comprises adult adenoma stem cells which are capable of expansion for at least three months, and wherein non-epithelial cells are absent from said organoid and said composition.

2. The composition of claim 1, wherein the three-dimensional organoid comprises cells expressing Lgr5.

3. The composition of claim 1, wherein every epithelial cell within the three-dimensional organoid comprises nuclear beta-catenin.

4. The composition of claim 1, wherein the cells lining the central lumen are polarised.

5. The composition of claim 4, wherein the lumen contains apoptotic cell bodies.

6. The composition of claim 1, comprising differentiated cell types.

7. The composition of claim 1, wherein the organoid is derived from human epithelial stem cells.

8. The composition of claim 1, wherein the composition further comprises an exogenous extracellular matrix.

9. The composition of claim 8, wherein the composition further comprises a cell culture medium comprising a Bone Morphogenetic Protein (BMP) inhibitor;

between 5 and 500 ngram/ml of a mitogenic growth factor; and

a Wnt agonist.

10. The composition of claim 9, wherein the BMP inhibitor is selected from the group consisting of Noggin, DAN, Cerberus and Gremlin.

11. The composition of claim 9, wherein the Wnt agonist is selected from the group consisting of one or more of Wnt, R-spondin 1 through R-spondin 4, Norrin, and a GSK-inhibitor.

12. The composition of claim 9, wherein the BMP inhibitor is Noggin, the mitogenic growth factor is Epidermal Growth Factor, and the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4.

13. The composition of claim 1, wherein the cells lining the lumen are randomly oriented towards either the periphery or the central lumen.