Method for producing intestinal cells

• US 9,376,665 B2
• Filed: 10/31/2011
• Issued: 06/28/2016
• Est. Priority Date: 11/02/2010
• Status: Active Grant

• Current Assignee: Tokyo Institute of Technology (National University Corporation Tokyo Institute of Technology)
• Original Assignee: Kumamoto University, Lsip, LLC
• Inventors: Kume, Shoen, Ogaki, Soichiro, Shiraki, Nobuaki, Kume, Kazuhiko
• Primary Examiner(s): Singh, Anoop
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US13/882,904
• Publication Number:
  US 20140199700A1
• Time in Patent Office: 1,702 Days
• Field of Search: 435/6
• US Class Current: 1/1
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5064 Endothelial cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

An object of the present invention is to provide a method of producing intestinal cells by use of pluripotent stem cells as a starting material. According to the present invention, provided is a method of producing intestinal cells, comprising the steps of: (A) inducing differentiation of pluripotent stem cells into definitive endoderm cells; and (B) culturing the definitive endoderm cells in the presence of (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO) and N-[(3,5-difluorophenyl)acetyl]-L-Ala-2-phenyl-L-Gly-tert-butyl-OH (DAPT) to thereby induce differentiation of the definitive endoderm cells into intestinal cells.

Claims

1. A method of producing intestinal cells expressing Cdx2, comprising the steps of:

(a) obtaining mouse or human embryonic stem (ES) cells,

(b) culturing the ES cells of step (a) with Activin and/or bFGF to produce definitive endoderm cells,

(c) culturing the definitive endoderm cells of step (b) with (2′

Z,3′

E)-6-bromoindirubin-3′

-oxime (BIO) and N-[(3,5-difluorophenyl)acetyl]-L-Ala-2-phenyl-L-Gly-tert-butyl-OH (DAPT) and in the presence of M15 cells or MEF cells, to thereby induce differentiation of the definitive endoderm cells into intestinal cells expressing Cdx2.

2. The method according to claim 1, wherein the definitive endoderm cells are separated from a cell culture obtained in step (b) by flow cytometry using fluorescently-labelled antibodies against E-cadherin (ECD) and CXCR4, and said separated definitive endoderm cells are used in the step (c).

3. The method according to claim 1, wherein the ES cells are cultured in the presence of feeder cells.

4. The method according to claim 3, wherein the feeder cells are cells derived from a mesoderm.