Method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells
Provided is a method for generating pancreatic bud cells, having the step of culturing PDX1 + /NKX6.1 − 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 nonmuscle 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, wherein the steps (1)-(3) are under an adherent culture condition; and
(4) dissociating the cells obtained in step (3) into single cells and culturing the dissociated cells under a suspension culture condition in a medium containing KGF, EGF and a BMP inhibitor.
2. The method according to claim 1 , wherein the medium containing an activin in step (1) further contains a GSK3 inhibitor.
3. The method according to claim 2 , wherein the GSK3 inhibitor is CHIR99021.
4. The method according to claim 1 , wherein the BMP inhibitor used in step (3) is Noggin.
5. The method according to claim 1 , wherein the retinoic acid derivative is TTNPB.
6. The method according to claim 1 , wherein the hedgehog pathway inhibitor is KAAD-cyclopamine.
7. The method according to claim 1 , wherein the medium in step (4) further contains a ROCK inhibitor or a nonmuscle myosin II inhibitor.
8. The method according to claim 7 , wherein the ROCK inhibitor selected from the group consisting of Y-27632, Fasudil, SR3677, GSK269962, and H-1152; or the nonmuscle myosin II inhibitor is blebbistatin.
9. The method according to claim 8 , wherein the ROCK inhibitor is Y-27632 and the concentration of Y-27632 in the medium is 20-100 μM.
10. The method according to claim 1 , wherein the medium in step (4) further comprises a TGFβ inhibitor.
11. The method according to claim 1 , wherein the TGFβ inhibitor is ALK5 inhibitor II.
12. The method according to claim 1 , wherein the pancreatic bud cells are PDX1+and NKX6.1+.
13. The method according to claim 1 , wherein the pluripotent stem cells are human cells.