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 and pancreatic organoids that are formed in the culture medium.

1 . (canceled) 
     
     
         2 . An in vitro method for obtaining a pancreatic epithelial organoid, the method comprising:
 incubating a pancreatic epithelial stem cell or an isolated pancreatic tissue fragment comprising a pancreatic epithelial stem cell with an extracellular matrix; and   culturing the pancreatic epithelial stem cell or isolated pancreatic tissue fragment for a length of time sufficient to be able to observe formation of a pancreatic organoid, in an animal or human cell culture medium comprising:
 a Bone Morphogenetic Protein (BMP) inhibitor, 
 a mitogenic growth factor, and 
 a Wnt agonist, and 
   obtaining from the culture medium a pancreatic epithelial organoid.   
     
     
         3 . The method according to  claim 2 , wherein the BMP inhibitor is Noggin, the mitogenic growth factor is Epidermal Growth Factor (EGF), and the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4. 
     
     
         4 . The method according to  claim 2 , wherein the BMP inhibitor is selected from the group consisting of Noggin, DAN, Cerberus and Gremlin. 
     
     
         5 . The method according to  claim 2 , wherein said 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. 
     
     
         6 . The method according to  claim 2 , wherein the Wnt agonist is one of R-spondin 1 through R-spondin 4 and Wnt-3a. 
     
     
         7 . The method of  claim 2 , wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152. 
     
     
         8 . The method according to  claim 2 , wherein the cell culture medium further comprises a notch agonist. 
     
     
         9 . An in vitro method for obtaining a pancreatic organoid, the method comprising:
 culturing epithelial stem cells or isolated pancreatic tissue fragments comprising pancreatic epithelial stem cells in contact with an extracellular matrix for a length of time sufficient to be able to observe formation of a pancreatic organoid, in a medium comprising Noggin, EGF and Keratinocyte Growth Factor or Fibroblast Growth Factor, any one of R-spondin 1 through R-spondin 4 and/or Wnt-3a, and further comprising one or more supplements selected from, B27, N2, and N-Acetylcystein; and   obtaining from the culture medium a pancreatic organoid.   
     
     
         10 - 18 . (canceled) 
     
     
         19 . The method according to  claim 9 , wherein the cell culture medium further comprises [Leu15]-Gastrin I, Exendin and/or Nicotinamide. 
     
     
         20 . An in vitro method for obtaining a pancreatic epithelial organoid, the method comprising:
 culturing pancreatic epithelial stem cells or isolated pancreatic tissue fragments comprising pancreatic epithelial stem cells in contact with an extracellular matrix for a length of time sufficient to be able to observe formation of a pancreatic organoid, in an animal or human cell culture medium comprising:
 a Bone Morphogenetic Protein (BMP) inhibitor; 
 a Wnt agonist; and 
   Epidermal Growth Factor (EGF); and   obtaining from the culture medium a pancreatic epithelial organoid.   
     
     
         21 . The method according to  claim 2 , wherein the mitogenic growth factor is EGF, FGF and/or KGF. 
     
     
         22 . The method according to  claim 4 , 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. 
     
     
         23 . The method according to  claim 3 , wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a. 
     
     
         24 . The method according to  claim 4 , wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a. 
     
     
         25 . The method according to  claim 5 , wherein the Wnt agonist comprises any one of R-spondin 1 through R-spondin 4 and Wnt-3a. 
     
     
         26 . The method according to  claim 4 , wherein the mitogenic growth factor is EGF, FGF and/or KGF. 
     
     
         27 . The method according to  claim 5 , wherein the mitogenic growth factor is EGF, FGF and/or KGF. 
     
     
         28 . The method according to  claim 3 , wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152. 
     
     
         29 . The method according to  claim 4 , wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152. 
     
     
         30 . The method according to  claim 5 , wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152. 
     
     
         31 . The method according to  claim 6 , wherein the cell culture medium further comprises a Rock (Rho-kinase) inhibitor selected from the group consisting of Y-27632, Fasudil, and H-1152. 
     
     
         32 . An in vitro method for obtaining a pancreatic adenoma organoid, the method comprising:
 incubating a pancreatic epithelial adenoma stem cell or an isolated pancreatic tissue fragment comprising a pancreatic epithelial adenoma stem cell with an extracellular matrix, and   culturing the pancreatic epithelial adenoma stem cell or isolated pancreatic tissue fragment for a length of time sufficient to be able to observe formation of an organoid, in an animal or human cell culture medium comprising:
 a Bone Morphogenetic Protein (BMP) inhibitor, and 
 a mitogenic growth factor, and 
   obtaining from the culture medium a pancreatic adenoma organoid.   
     
     
         33 . The method according to  claim 2 , wherein the pancreatic epithelial stem cell is a pancreatic adenoma stem cell. 
     
     
         34 . The method according to  claim 9 , wherein the pancreatic epithelial stem cells are pancreatic adenoma stem cells. 
     
     
         35 . The method according to  claim 20 , wherein the pancreatic epithelial stem cells are pancreatic adenoma stem cells. 
     
     
         36 . The method according to  claim 2 , wherein the culture medium comprises Noggin as BMP inhibitor, EGF and FGF as mitogenic growth factors, and any one of R-spondin 1 through R-spondin 4 as Wnt agonist, and wherein the culture medium further comprises nicotinamide. 
     
     
         37 . The method according to  claim 36 , wherein the pancreatic epithelial stem cell or isolated pancreatic tissue fragment is subsequently cultured in a second culture medium which is identical to said first medium except there is no Noggin. 
     
     
         38 . The method according to  claim 2 , wherein the culture medium further comprises nicotinamide. 
     
     
         39 . The method according to  claim 9 , wherein the culture medium further comprises nicotinamide. 
     
     
         40 . The method according to  claim 20 , wherein the culture medium further comprises nicotinamide. 
     
     
         41 . The method according to  claim 32 , wherein the culture medium further comprises nicotinamide. 
     
     
         42 . The method according to  claim 2 , wherein the pancreatic epithelial stem cell or isolated pancreatic tissue fragment is cultured for at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, or at least 10 months. 
     
     
         43 . The method according to  claim 21 , wherein the FGF is FGF10. 
     
     
         44 . The method according to  claim 26 , wherein the FGF is FGF10. 
     
     
         45 . The method according to  claim 27 , wherein the FGF is FGF10. 
     
     
         46 . The method according to  claim 1 , wherein the cell culture medium comprises between 5 and 500 ngram/ml of the mitogenic growth factor. 
     
     
         47 . The method according to  claim 20 , wherein the cell culture medium comprises between 5 and 500 ngram/ml of Epidermal Growth Factor (EGF). 
     
     
         48 . The method according to  claim 32 , wherein the cell culture medium comprises between 5 and 500 ngram/ml of the mitogenic growth factor.