Methods for inducing differentiation of pluripotent cells

• US 20050042748A1
• Filed: 02/27/2004
• Published: 02/24/2005
• Est. Priority Date: 08/19/2003
• Status: Active Grant

• Current Assignee: ECI Incorporated
• Original Assignee: Effector Cell Institute
• Inventors: Ochiya, Takahiro, Teratani, Takumi
• Granted Patent: US 7,332,336 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 object of the present invention is to provide methods for identifying factors involved in inducing the differentiation of pluripotent cells, and methods for using those factors.

Using cDNA microarray technology, variations in gene expression between placebo-treated and CCl₄-treated mice livers were analyzed 24 hours after treatment, and several growth factors were selected. The effects of matrix and growth factors on hepatocyte differentiation rates of ES cells were investigated. As a result, 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 pluripotent cells comprising the following steps (a) and (b):

(a) culturing the pluripotent cells in a medium comprising any one of the following growth factors (i) to (iii);

(i) acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor;

(ii) acidic fibroblast growth factor, and growth factor(s) selected from activin A, epidermal growth factor, and β

-nerve growth factor; and

(iii) fibroblast growth factor 4, and growth factor(s) selected from activin A and hepatocyte growth factor; and

,

(b) culturing the cell cultured in step (a) in a medium comprising oncostatin M.

2. The method according to claim 1, wherein a gelatin-coated culture dish is used in step (a), and a collagen type I-coated culture dish or laminin-coated culture dish is used in step (b).

3. The method according to claim 1, wherein a collagen type I-coated culture dish is used.

5. The method according to claim 3, wherein gelatin-coated culture dishes are used in steps (a) and (b).

8. A method according to claim 1, wherein the pluripotent cells are derived from a mammal.

9. The method according to claim 8, wherein the mammal is a human, monkey, mouse, rat or pig.

10. A method according to claim 1, wherein the pluripotent cells are embryonic stem cells, adult stem cells, mesenchymal stem cells, or umbilical cord blood cells.

11. A method for producing hepatocytes, wherein the method comprises steps (a) and (b) according to claim 1, or steps (a) to (c) according to claim 6.

12. The method according to claim 11, wherein the hepatocytes are mature hepatocytes.

13. The method according to claim 11, wherein the pluripotent cells are derived from a mammal.

14. The method according to claim 13, wherein the mammal is a human, monkey, mouse, rat or pig.

15. A method according to claim 11, wherein the pluripotent cells are embryonic stem cells, adult stem cells, mesenchymal stem cells, or umbilical cord blood cells.

16. A hepatocyte produced by a method according to claim 11.

17. A therapeutic agent for a liver disease comprising the hepatocyte according to claim 16.

18. The therapeutic agent according to claim 17, wherein the liver disease is cirrhosis, fulminant hepatitis, biliary atresia, liver cancer, or hepatitis.

4. A method for inducing differentiation of pluripotent cells comprising the following steps (a) and (b):

(a) culturing the pluripotent cells in a medium comprising at least one growth factor selected from retinoic acid, leukemia inhibitory factor, and hepatocyte growth factor; and

,

(b) culturing the cell cultured in step (a) in a medium comprising any one of the following growth factors (i) to (iii);

(i) acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor;

(ii) acidic fibroblast growth factor, and growth factor(s) selected from activin A, epidermal growth factor and β

-nerve growth factor; and

(iii) fibroblast growth factor 4, and growth factor(s) selected from activin A and hepatocyte growth factor.

6. A method for inducing differentiation of pluripotent cells comprising the following steps (a) to (c):

(a) culturing the pluripotent cells in a medium comprising at least one of the growth factors selected from retinoic acid, leukemia inhibitory factor and hepatocyte growth factor;

(b) culturing the cell cultured in step (a) in a medium comprising any one of the following growth factors (i) to (iii);

(i) acidic fibroblast growth factor, fibroblast growth factor 4 and hepatocyte growth factor;

(ii) acidic fibroblast growth factor, and growth factor(s) selected from activin A, epidermal growth factor and α

-nerve growth factor; and

(iii) fibroblast growth factor 4, and growth factor(s) selected from activin A and hepatocyte growth factor; and

,

(c) culturing the cells cultured in step (b) in a medium comprising oncostatin M.

7. The method according to claim 6, wherein gelatin-coated culture dishes are used in steps (a) and (b), and a collagen type I-coated culture dish or laminin-coated culture dish is used in step (c).

19. A kit comprising any one of the following (a) to (c):

(a) acidic fibroblast growth factor, fibroblast growth factor 4, and hepatocyte growth factor;

(b) acidic fibroblast growth factor, and growth factor(s) selected from activin A, epidermal growth factor, and β

-nerve growth factor; and

(c) fibroblast growth factor 4, and growth factor(s) selected from activin A and hepatocyte growth factor.

20. The kit according to claim 19 further comprising oncostatin M.

21. The kit according to claim 20 further comprising at least one growth factor selected from the group consisting of retinoic acid, leukemia inhibitory factor, and hepatocyte growth factor.