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

Patent metadata

Field	Value
Publication number	US-9752124-B2
Application number	US-201113194866-A
Country	US
Kind code	B2
Filing date	Jul 29, 2011
Priority date	Feb 3, 2009
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

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.

First claim

The invention claimed is:

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.

Assignees

• Sato Toshiro
• Clevers Johannes C
• Huch Ortega Meritxell
• Koninklijke Nederlandse Akademie Van Wetenschappen

Inventors

• Sato Toshiro
• Clevers Johannes C
• Huch Ortega Meritxell

Classifications

• C12N2501/15

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

• C12N2501/345

  Gastrin; Cholecystokinins [CCK] · CPC title

• C12N2501/12

  Hepatocyte growth factor [HGF] · CPC title

• C12N2501/155

  Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor · CPC title

• C12N2501/11

  Epidermal growth factor [EGF] · CPC title