Method of producing pancreatic hormone-producing cells

• US 9,157,069 B2
• Filed: 08/08/2011
• Issued: 10/13/2015
• Est. Priority Date: 08/09/2010
• Status: Active Grant

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Takeda Pharmaceutical Company Limited
• Inventors: Hosoya, Masaki, Shoji, Masanobu
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/814,878
• Publication Number:
  US 20130210060A1
• Time in Patent Office: 1,527 Days
• US Class Current: 1/1
• 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 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: G01N 33/507 Pancreatic cells

Abstract

Disclosed is a production method of pancreatic hormone-producing cells in a form that mimicks the pancreatogenesis, the method comprising subjecting stem cells to the following steps:

• (1) cultivating stem cells in a medium containing a Rho kinase inhibitor,
• (2) cultivating the cells obtained in (1) in a medium containing a GSK3 inhibitor,
• (3) cultivating the cells obtained in (2) in a medium containing GSK3 inhibitor and an activator of activin receptor-like kinase-4,7,
• (4) forming a cell mass from the cells obtained in (3), and cultivating the cell mass in a suspension state in a medium,
• (5) cultivating the cells obtained in (4) in a medium containing a retinoic acid receptor agonist, an inhibitor of AMP-activated protein kinase and/or activin receptor-like kinase-2,3,6, an inhibitor of activin receptor-like kinase-4,5,7 and a cell growth factor, and
• (6) cultivating the cells obtained in (5).

Claims

1. A method of producing human pancreatic hormone-producing cells, comprising subjecting human pluripotent stem cells to the following steps (1)-(6):

(1) a step of cultivating human pluripotent stem cells in a medium containing (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanamide dihydrochloride,

(2) a step of cultivating the cells obtained in the aforementioned step (1) in a medium containing a 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile or (2′

Z,3′

E)-6-bromoindirubin-3′

-oxime,

(3) a step of cultivating the cells obtained in the aforementioned step (2) in a medium containing 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile and activin

(4) a step of forming a cell mass from the cells obtained in the aforementioned (3), and cultivating the cell mass in a suspension state in a medium

(5) a step of cultivating the cells obtained in the aforementioned step (4) in a medium containing dorsomorphin, retinoic acid, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide or hydrate thereof and basic fibroblast growth factor,

(6) a step of cultivating the cells obtained in the aforementioned step (5) to produce human pancreatic hormone-producing cells.

2. The production method according to claim 1, wherein the steps (1)-(6) do not substantially use a feeder cell.

3. The production method according to claim 1, wherein the medium in steps (1)-(6) does not substantially contain a serum.

4. The production method according to claim 1, wherein the pancreatic hormone-producing cells are any selected from the group consisting of insulin-producing cells, glucagon-producing cells, somatostatin-producing cells, pancreatic polypeptide (PP)-producing cells and ghrelin-producing cells.