Culture Medium

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.

1 .- 45 . (canceled) 
     
     
         46 . An organoid comprising a population of epithelial stem cells in a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands, and a BMP inhibitor. 
     
     
         47 . The organoid according to  claim 46 , wherein:
 (a) the organoid has a three-dimensional structure, and/or   (b) the organoid comprises epithelial stem cells which are able to actively divide, optionally wherein the epithelial stem cells are able to differentiate to all major differentiated cell lineages present in the corresponding in vivo tissue, and/or   (c) the organoid has a section which is formed of multiple layers,   (d) the organoid has a monolayer of pseudo-stratified cells folds so that it encloses lumina between the folds; and/or   (e) the organoid consists essentially of epithelial cells.   
     
     
         48 . The organoid according to  claim 46 , wherein the organoid has a cyst-like structure, optionally wherein the cyst-like structure has a central lumen, further optionally wherein the central lumen of the cyst-like structure:
 (a) does not comprise any cells, or   (b) comprises epithelial stem cells, optionally wherein the central lumen is filled with epithelial stem cells.   
     
     
         49 . The organoid of  claim 46 , wherein:
 (a) the organoid has an anastomosing cyst-like structure containing multiple lumina, optionally wherein at one position in the organoid, the boundary of at least one lumen branches out from the boundary of at least one other distinct lumen and the boundaries reconnect with each other at another position in the organoid; or   (b) the culture medium is suitable for expanding a population of epithelial stem cells for at least 4 passages; or   (c) the organoid has been cultured for at least 2 months and/or wherein the epithelial stem cells have been passaged approximately every 1 to 4 weeks.   
     
     
         50 . The organoid according to  claim 46 , wherein the organoid is a lung organoid. 
     
     
         51 . The organoid of  claim 50 , wherein:
 (a) the lung organoid is obtained from normal lung tissue and has a cystic structure, optionally wherein the lung organoid comprises a monolayer of basal and luminal cells surrounding a central lumen; or   (b) the lung organoid comprises one or more of the following cell types: Clara cells, basal cells, ciliated cells and goblet cells; or   (c) the lung organoid is a single layered pseudostratified epithelial culture of a heterogeneous cell population that comprises Clara cells, basal cells, ciliated cells, and goblet cells, wherein the organoid represents the proximal lung epithelium.   
     
     
         52 . The organoid of  claim 50 , wherein the lung organoid is obtained from cancerous lung tissue and has a cystic structure, optionally wherein:
 (a) the lung organoid comprises: (i) a central lumen, multiple small lumina, or no lumen; and/or (ii) a monolayer of epithelial cells or a multi-layered epithelium; or   (b) the lung organoid comprises solid balls of tumour cells; or   (c) the lung organoid displays cribiform, comedo, or solid morphologies.   
     
     
         53 . The organoid according to  claim 50 , wherein the lung organoid comprises ciliated cells, optionally wherein:
 (a) the cilia of the ciliated cells project into the lumen of the lung organoid; or   (b) the ciliated cells are capable of synchronous movement or the ciliated cells are moving in a synchronous fashion.   
     
     
         54 . The organoid according to  claim 50 , wherein the lung organoid displays upregulated expression, compared to whole lung samples, of one or more of the following sets of markers: (i) one or more respiratory lineage markers, (ii) one or more epithelial cell markers, (iii) one or more basal cell markers, (iv) one or more ciliated cell markers, (v) one or more Clara cell markers, and (vi) one or more goblet cell markers. 
     
     
         55 . The organoid according to  claim 54 , wherein:
 (a) the one or more respiratory lineage markers is NKX2 1; and/or   (b) the one or more epithelial cell markers is CDH1 and/or CLDN1; and/or   (c) the one or more basal cell markers is KRT5; and/or   (d) the one or more ciliated cell markers is DNAH5 and/or NPHP1; and/or   (e) the one or more Clara cell markers is SCGB1A1; and/or   (f) the one or more goblet cell markers is AGR2.   
     
     
         56 . The organoid according to  claim 50 , wherein the lung organoid displays downregulated expression, compared to whole lung samples, of one or more of the following sets of markers: (i) one or more lung mesenchyme markers, (ii) one or more neuroendocrine cell markers, (iii) one or more distal epithelial cell markers and (iv) one or more type II alveolar cell markers. 
     
     
         57 . The organoid according to  claim 56 , wherein:
 (a) the one or more lung mesenchyme markers is HOXA5; and/or   (b) the one or more neuroendocrine cell markers is UCHL1; and/or   (c) the one or more distal epithelial cell markers is ID2; and/or   (d) one or more type II alveolar cell markers is ABCA3 and/or SFTPA1.   
     
     
         58 . The organoid according to  claim 46 , wherein the ErbB3/4 ligand is a neuregulin polypeptide, optionally wherein the neuregulin polypeptide is encoded by NRG1, and further optionally wherein the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
         59 . The organoid according to  claim 46 , wherein:
 (a) the one or more FGFR2b ligands is FGF7 and/or FGF10;   (b) the Wnt agonist is an Lgr5 agonist; and/or   (c) the Lgr5 agonist is a Rspondin or a Rspondin mimic.   
     
     
         60 . The organoid according to  claim 46 , wherein the culture medium further comprises:
 (a) a BMP inhibitor;   (b) one or more components selected from: B27, N-acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF-beta inhibitor and a p38 inhibitor; and/or   (c) B27, N-acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF-beta inhibitor, and a p38 inhibitor.   
     
     
         61 . The organoid according to claim  76 , wherein:
 (a) the BMP inhibitor is Noggin, and/or   (b) the ROCK inhibitor is Y-27632, and/or   (c) the TGF-beta inhibitor is A83-01, and/or   (d) the p38 inhibitor is SB 202190.   
     
     
         62 . The organoid according to  claim 46 , wherein the culture medium further comprises one or more components selected from:
 (a) one or more further receptor tyrosine kinase ligands, optionally wherein the one or more further receptor tyrosine kinase ligands are EGF, PDGF, and/or amphiregulin;   (b) a p53 stabilizing agent, optionally wherein the p53 stabilizing agent is a member of the Nutlin family; and   (c) an extracellular matrix.   
     
     
         63 . The organoid according to  claim 62 , wherein:
 (a) the PDGF is PDGF-CC, and/or   (b) the member of the Nutlin family is Nutlin-3.   
     
     
         64 . An organoid according to  claim 46 , obtainable or obtained by a method comprising:
 providing a population of epithelial stem cells;   providing a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands and a BMP inhibitor; and wherein the culture medium is suitable for expanding a population of epithelial stem cells for at least 6 passages;   contacting the epithelial stem cells with the culture medium; and   culturing the epithelial stem cells under appropriate conditions to form an organoid.   
     
     
         65 . The organoid according to  claim 64 , wherein the ErbB3/4 ligand is a neuregulin polypeptide, optionally wherein the ErbB3/4 ligand is a neuregulin polypeptide encoded by NRG1.