Methods for inducing differentiation of pluripotent cells

• US 7,332,336 B2
• Filed: 02/27/2004
• Issued: 02/19/2008
• Est. Priority Date: 08/19/2003
• Status: Expired due to Fees

• Current Assignee: ECI Incorporated
• Original Assignee: Effector Cell Institute
• Inventors: Ochiya, Takahiro, Teratani, Takumi
• Primary Examiner(s): Saucier; Sandra
• Assistant Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US10/789,159
• Publication Number:
  US 20050042748A1
• Time in Patent Office: 1,453 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present invention provides methods for inducing the differentiation of pluripotent cells. The present inventors succeeded in differentiating hepatocytes from ES cells without EB formation, using simple adherent monoculturing in media comprising several growth factors in culture dishes with two different matrices.

Claims

1. A method for inducing differentiation of mesenchymal stem cells to hepatocytes comprising

(a) culturing the mesenchymal stem cells in a medium comprising acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor;

and then

(b) culturing the cells cultured in step (a) in a medium comprising oncostatin M, thereby differentiating the mesenchymal stem cells into hepatocytes.

2. The method according to claim 1, wherein the cells are cultured on a gelatin-coated culture dish in step (a), and the cells are cultured on a collagen type I-coated culture dish or laminin-coated culture dish in step (b).

3. The method according to claim 1, wherein the cells are cultured on a gelatin-coated culture dish.

4. A method according to claim 1, wherein the mesenchymal stem cells are derived from a mamnnal.

5. The method according to claim 3, wherein the mammal is a human, monkey, mouse, rat or pig.

8. The method of claim 1 or 6 wherein the hepatocytes are mature hepatocytes.

6. A method for inducing differentiation of embryonic stem cells to hepatocytes comprising

(a) culturing the embryonic stem cells in a medium comprising retinoic acid, leukemia inhibitory factor, and hepatocyte growth factor;

and then

(b) culturing the cells cultured in step (a) in a medium comprising acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor; and

then

(c) culturing the cells cultured in step (b) in a medium comprising oncostatin M, thereby differentiating the embryonic stem cells into hepatocytes.

7. The method according to claim 6, wherein the cells are cultured on a gelatin-coated culture dish in steps (a) and (b), and the cells are cultured on a collagen I-coated culture dish or laminin-coated culture dish in step (c).