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

• US 10,472,610 B2
• Filed: 05/20/2015
• Issued: 11/12/2019
• Est. Priority Date: 05/21/2014
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Osafune, Kenji, Toyoda, Taro
• Primary Examiner(s): Kim, Taeyoon
• Application Number:
  US15/311,759
• Publication Number:
  US 20170175082A1
• Time in Patent Office: 1,637 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: Y02A 20/402 River restoration

Abstract

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.

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 μ

M.

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.