Method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells

Abstract

Provided is a method for generating pancreatic bud cells, having the step of culturing PDX1 + /NKX 6.1 + cells in a medium containing KGF, EGF and a BMP inhibitor. The culturing step may be performed in suspension cultures or in adherent cultures. When the cells are cultured in adherent cultures, the cells may be cultured in a medium further containing a ROCK inhibitor or a nonmuscie myosin II inhibitor.

1 . A method for generating pancreatic bud cells from pluripotent stem cells, comprising the following steps:
 (1) culturing the pluripotent stem cells in a medium containing an activin;   (2) culturing the cells obtained in step (1) in a medium containing KGF;   (3) culturing the cells obtained in step (2) in a medium containing KGF, a BMP inhibitor, a retinoic acid derivative and a hedgehog pathway inhibitor; and   (4) dissociating the cells obtained in step (3) into single cells and culturing the dissociated cells in a medium containing KGF, a BMP inhibitor, and an agent selected from the group consisting of ROCK inhibitor and nonmuscle myosin II inhibitor, wherein the steps (1)-(4) are under an adherent culture condition.   
     
     
         2 . The method according to  claim 1 , wherein the ROCK inhibitor is selected from the group consisting of Y-27632, Fasudil, SR3677, GSK269962 and H-1152; or the nonmuscie myosin II inhibitor is Blebbistatin. 
     
     
         3 . The method according to  claim 2 , wherein the ROCK inhibitor is Y-27632. 
     
     
         4 . The method according to  claim 3 , wherein the concentration of Y-27832 in the medium is 10-200 μM. 
     
     
         5 . The method according to  claim 2 , wherein the nonmuscie myosin II inhibitor is Blebbistatin. 
     
     
         6 . The method according to  claim 5 , wherein the concentration of Blebbistatin in the medium is 5-20 μM. 
     
     
         7 . The method according to  claim 1 , wherein the medium containing an activin in step (1) further contains a GSK3 inhibitor. 
     
     
         8 . The method according to  claim 7 , wherein the GSK3 inhibitor is CHIR93021. 
     
     
         9 . The method according to  claim 1 , wherein the BMP inhibitor is Noggin. 
     
     
         10 . The method according to  claim 1 , wherein the retinoic acid derivative is TTNPB. 
     
     
         11 . The method according to claim wherein the hedgehog pathway inhibitor is KAAD-cyclopamine. 
     
     
         12 . The method according to  claim 1 , wherein the medium in step (4) further comprises a TGFβ inhibitor. 
     
     
         13 . The method according to  claim 11 , wherein the TGFβ inhibitor is ALK5 inhibitor II. 
     
     
         14 . The method according to  claim 1 , wherein the pancreatic bud cells are PDX1 + /NKX6.1 + . 
     
     
         15 . The method according to  claim 1 , wherein the pluripotent stem cells are human cells. 
     
     
         16 . A method for generating pancreatic bud cells from pluripotent stem cells, comprising the following steps:
 (1) culturing the pluripotent stem cells in a medium containing an activin and a GSK3 inhibitor;   (2) culturing the cells obtained in step (I) in a medium containing KGF;   (3) culturing the cells obtained in step (2) in a medium containing NSF, a BMP inhibitor, a retinoic acid derivative and a hedgehog pathway inhibitor; and   (4) dissociating the cells obtained in step (3) into single cells and culturing the dissociated cells in a medium containing KGF, a BMP inhibitor, a TGFβ inhibitor and an agent selected from the group consisting of ROCK inhibitor and nonmuscie myosin II inhibitor,   wherein the steps (1)-(4) are under an adherent culture condition.