METHOD FOR PRODUCING INTESTINAL CELLS

• US 20140199700A1
• Filed: 10/31/2011
• Published: 07/17/2014
• Est. Priority Date: 11/02/2010
• Status: Active Grant

• Current Assignee: Tokyo Institute of Technology (National University Corporation Tokyo Institute of Technology)
• Original Assignee: Lsip, LLC
• Inventors: Kume, Shoen, Shiraki, Nobuaki, Kume, Kazuhiko, Ogaki, Soichiro
• Granted Patent: US 9,376,665 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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, 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.

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

3. The method according to claim 1, wherein, in step (A), the pluripotent stem cells are cultured in the presence of feeder cells and in the presence of activin and/or bFGF to thereby induce of differentiation of the pluripotent stem cells into the definitive endoderm cells.

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

5. The method according to claim 3, wherein the feeder cells are M15 cells, MEF cells, or ST2 cells.

6. The method according to claim 1, wherein the definitive endoderm cells are cultured in the presence of M15 cells or MEF cells in step (B).

7. The method according to claim 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

8. The method according to claim 1, wherein the pluripotent stem cells are human embryonic stem cells or mouse embryonic stem cells.

9. Intestinal cells which are obtained by inducing differentiation of pluripotent stem cells and which are obtained by the method according to claim 1.

10. A method of screening for substances which promote or inhibit induction of differentiation of pluripotent stem cells into intestinal cells, the method comprising:

culturing pluripotent stem cells in the presence of a test substance in inducing differentiation of the pluripotent stem cells into intestinal cells by the method according to claim 1; and

comparing a level of differentiation of the pluripotent stem cells into intestinal cells in a case where the pluripotent stem cells are cultured in the presence of the test substance with a level of differentiation of pluripotent stem cells into intestinal cells in a case where the pluripotent stem cells are cultured in the absence of the test substance.

11. The screening method according to claim 10, wherein the test substance is a growth factor or a low-molecular-weight compound.

12. The screening method according to claim 10, wherein an amount of maker transcript or a protein thereof expressed in intestinal cells, or both of them are used as indicators to thereby determine the levels of differentiation into intestinal cells.