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Paramétrages

Paramétrages

1. US20170175082 - Method for generating pancreatic bud cells and therapeutic agent for pancreatic disease containing pancreatic bud cells

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

Claims

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.