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

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.

1 - 23 . (canceled) 
     
     
         24 . A composition comprising a three-dimensional organoid obtained by in vitro expansion of one or more adult epithelial stem cells, wherein the organoid has a sealed central lumen lined by epithelial cells, wherein the organoid comprises adult epithelial 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. 
     
     
         25 . The composition of  claim 24 , further comprising an exogenous extracellular matrix. 
     
     
         26 . The composition of  claim 25 , wherein the extracellular matrix is obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. 
     
     
         27 . The composition of  claim 25 , wherein the extracellular matrix comprises collagen and/or laminin. 
     
     
         28 . The composition of  claim 24 , further comprising a cell culture medium. 
     
     
         29 . The composition of  claim 28 , wherein the cell culture medium comprises a bone morphogenetic protein (BMP) inhibitor and a mitogenic growth factor. 
     
     
         30 . The composition of  claim 29 , wherein the cell culture medium further comprises a Wnt agonist. 
     
     
         31 . The composition of  claim 29 , wherein the BMP inhibitor is noggin. 
     
     
         32 . The composition of  claim 29 , wherein the mitogenic growth factor is one or more of epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF). 
     
     
         33 . The composition of  claim 30 , wherein the Wnt agonist is R-spondin 1-4 and/or a Wnt ligand, such as Wnt-3a. 
     
     
         34 . The composition of  claim 28 , wherein the cell culture medium comprises EGF, R-spondin 1-4, FGF10, HGF and nicotinamide. 
     
     
         35 . The composition of  claim 24 , wherein the adult epithelial stem cells are mammalian cells, preferably human cells. 
     
     
         36 . The composition of  claim 24 , wherein the adult epithelial stem cells are cancer stem cells. 
     
     
         37 . The composition of  claim 24 , wherein the adult epithelial stem cells are obtained from pancreatic, small intestinal, large intestinal, corneal, olfactory, respiratory, gastric, liver, esophageal, or skin tissue. 
     
     
         38 . The composition of  claim 24 , wherein the adult epithelial stem cells express Lgr5. 
     
     
         39 . The composition of  claim 24 , wherein the cells lining the central lumen are polarised. 
     
     
         40 . The composition of  claim 24 , wherein the cells lining the central lumen are randomly oriented towards either the periphery or the central lumen. 
     
     
         41 . The composition of  claim 24 , wherein epithelial cells within the organoid comprise nuclear beta-catenin. 
     
     
         42 . The composition of  claim 24 , wherein the composition is frozen at below −5° C. 
     
     
         43 . The composition of  claim 24 , wherein the stem cells in the organoid display the same karyotype as the stem cells from which they are obtained. 
     
     
         44 . A method for treating a patient by cellular therapy, wherein the method comprises administering the composition of  claim 24  to said patient by injection or implantation. 
     
     
         45 . The method of  claim 43 , wherein the patient has tissues damaged by disease, injury, trauma, an autoimmune reaction or by a viral or bacterial infection, or wherein the patient has diabetes. 
     
     
         46 . A method for testing drugs, wherein the method comprises contacting a composition of  claim 24  with a candidate drug and detecting changes in the composition that are indicative of drug efficacy or toxicity.