Culture Medium for Expanding Breast Epithelial Stem Cells

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 - 30 . (canceled) 
     
     
         31 . A breast organoid comprising a population of breast epithelial stem cells obtained from primary breast cancer or metastatic breast cancer. 
     
     
         32 . A breast organoid comprising a population of breast epithelial stem cells in a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands, and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a. 
     
     
         33 . The breast organoid of  claim 32 , wherein the culture medium is suitable for expanding a population of breast epithelial stem cells for at least 4 passages. 
     
     
         34 . The breast organoid of  claim 32 , wherein the breast organoid has been cultured or can be cultured for more than 4 passages. 
     
     
         35 . The breast organoid of  claim 32 , wherein the breast organoid has been cultured for at least 2 months, and wherein the breast epithelial stem cells have been passaged approximately every 1 to 4 weeks. 
     
     
         36 . The breast organoid of  claim 32 , wherein the breast organoid has a cyst-like structure. 
     
     
         37 . The breast organoid of  claim 36 , wherein the cyst-like structure has a central lumen. 
     
     
         38 . The breast organoid of  claim 37 , wherein the central lumen of the cyst-like structure: (a) does not comprise any cells; or (b) comprises breast epithelial stem cells, optionally wherein the central lumen is filled with breast epithelial stem cells. 
     
     
         39 . The breast organoid of  claim 37 , wherein the breast epithelial cells surround the central lumen, optionally wherein the breast epithelial cells surrounding the central lumen are polarized. 
     
     
         40 . The breast organoid of  claim 32 , comprising:
 (a) a continuous epithelium and intercellular adhesion, and/or   (b) luminal cells but no basal cells.   
     
     
         41 . The breast organoid of  claim 32 , wherein the breast epithelial stem cells are multipotent, oligopotent, or unipotent organ-specific adult stem cells. 
     
     
         42 . The breast organoid of  claim 32 , wherein the breast organoid consists essentially of epithelial cells. 
     
     
         43 . The breast organoid of  claim 32 , wherein non-epithelial stem cells are absent from the breast organoids. 
     
     
         44 . The breast organoid of  claim 32 , wherein the breast organoid:
 (a) has a three dimensional structure, and/or   (b) comprise breast epithelial stem cells which are able to actively divide, optionally wherein the breast epithelial stem cells are able to differentiate to all major differentiated cell lineages present in the corresponding in vivo tissue, and/or   (c) have a section which is formed of multiple layers, and/or   (d) has a monolayer of pseudo-stratified cells folds so that it encloses lumina between the folds.   
     
     
         45 . The breast organoid of  claim 32 , wherein the breast organoid is a breast cancer organoid. 
     
     
         46 . The breast organoid of  claim 32 , wherein the breast organoid is obtained from normal tissue. 
     
     
         47 . The breast organoid of  claim 32 , wherein the ErbB3/4 ligand is a neuregulin polypeptide. 
     
     
         48 . The breast organoid of  claim 47 , wherein the neuregulin polypeptide is encoded by NRG. 
     
     
         49 . The breast organoid according to  claim 47 , wherein the neuregulin polypeptide comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
         50 . The breast organoid of  claim 32 , wherein the one or more FGFR2b ligands is FGF7 and/or FGF10. 
     
     
         51 . The breast organoid of  claim 32 , wherein the Wnt agonist is an Lgr5 agonist. 
     
     
         52 . The breast organoid of  claim 51 , wherein the Lgr5 agonist is a Rspondin or a Rspondin mimic. 
     
     
         53 . The breast organoid of  claim 32 , wherein the culture medium further comprises a BMP inhibitor. 
     
     
         54 . The breast organoid of  claim 32 , wherein the culture medium further comprises one or more components selected from: B27, N-acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF-beta inhibitor, and a p38 inhibitor. 
     
     
         55 . The breast organoid of  claim 32 , wherein the culture medium further comprises B27, N-acetylcysteine, Nicotinamide, a ROCK inhibitor, a TGF-beta inhibitor, and a p38 inhibitor. 
     
     
         56 . The breast organoid of  claim 32 , 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.   
     
     
         57 . The breast organoid of  claim 32 , wherein the culture medium further comprises one or more components selected from: one or more further receptor tyrosine kinase ligands and a p53 stabilizing agent. 
     
     
         58 . The breast organoid of  claim 57 , wherein:
 (a) the one or more further receptor tyrosine kinase ligands are EGF, PDGF and/or amphiregulin, and/or   (b) the p53 stabilizing agent is a member of the Nutlin family.   
     
     
         59 . The breast organoid of  claim 58 , wherein:
 (a) the PDGF is PDGF-CC, and/or   (b) the member of the Nutlin family is Nutlin-3.   
     
     
         60 . The breast organoid of  claim 32  obtained by a method comprising:
 providing a population of breast epithelial stem cells; 
 providing a culture medium comprising an ErbB3/4 ligand, one or more FGFR2b ligands and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a; and wherein the culture medium is suitable for expanding a population of breast epithelial stem cells for at least 6 passages; 
 contacting the breast epithelial stem cells with the culture medium; and 
 culturing the breast epithelial stem cells under appropriate conditions to form a breast organoid. 
 
     
     
         61 . The breast organoid of  claim 60 , wherein the ErbB3/4 ligand is a neuregulin polypeptide. 
     
     
         62 . The breast organoid of  claim 60 , wherein the culture medium comprises an ErbB3/4 ligand, one or more FGFR2b ligands, and a Wnt agonist, wherein the Wnt agonist is an Lgr5 agonist or Wnt3a, and wherein the ErbB3/4 ligand is a neuregulin polypeptide encoded by NRG. 
     
     
         63 . A composition comprising a breast organoid of  claim 32 . 
     
     
         64 . The composition of  claim 63 , wherein the composition further comprises an extracellular matrix. 
     
     
         65 . A method for a drug discovery screen; toxicity assay; research of tissue embryology, cell lineages, or differentiation pathways; gene expression study including recombinant gene expression; research of mechanisms involved in tissue injury or repair; research of inflammatory and infectious diseases; studies of pathogenetic mechanisms; or study of mechanisms of cell transformation or aetiology of cancer, comprising utilizing the breast organoid of  claim 32 . 
     
     
         66 . A method of treating a disease in a subject, comprising administering an effective amount of the breast organoid of  claim 32 , or cells derived therefrom to a subject in need thereof.