Method for differentiating rat hepatic stem cells to insulin-producing cells

• US 7,029,915 B2
• Filed: 02/24/2003
• Issued: 04/18/2006
• Est. Priority Date: 02/22/2002
• Status: Expired due to Fees

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Yang, Lijun
• Primary Examiner(s): Saucier, Sandra E.
• Assistant Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US10/373,124
• Publication Number:
  US 20030223974A1
• Time in Patent Office: 1,149 Days
• Field of Search: 424/93.7, 435/325, 435/370, 435/377, 435/404
• US Class Current: 435/377
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

Highly purified hepatic stem cells are trans-differentiated into pancreatic endocrine hormone-producing cells by culturing them in vitro in a medium containing high levels of glucose. These trans-differentiated cells express insulin, glucagon, and pancreatic polypeptide, but not hepatocyte protein Hep-par. When stimulated with glucose, these cells synthesize and secrete insulin, a response enhanced by nicotinamide. Transplantation of these trans-differentiated cells into a hyperglycemic animal normalizes blood sugar levels in the animal.

Claims

1. A method for making cells having a pancreatic endocrine cell phenotype, the method comprising the steps of:

(a) isolating hepatic stem cells from the livers of 2-acetylamino-fluorene-treated adult rats; and

(b) culturing said hepatic stem cells in vitro in a medium comprising greater than 5.5 mM glucose until said hepatic stem cells differentiate into cells having a pancreatic endocrine cell phenotype.

2. The method of claim 1, wherein the medium comprises at least 10 mM glucose.

3. The method of claim 1, wherein the medium comprises RPMI 1640 supplemented with fetal calf serum.

4. The method of claim 1, wherein the cells having a pancreatic endocrine cell phenotype produce insulin.

5. The method of claim 1, wherein the cells having a pancreatic endocrine cell-like phenotype produce somatostatin.

6. The method of claim 1, wherein the cells having a pancreatic endocrine cell-like phenotype produce glucagon.

7. The method of claim 1, wherein the cells having a pancreatic endocrine cell-like phenotype produce pancreatic polypeptide.

8. The method of claim 1, wherein the cells having a pancreatic endocrine cell phenotype contain higher levels of insulin mRNA encoding polypeptide than do the hepatic stem cells from which they were differentiated.

9. Cells having a pancreatic endocrine cell phenotype made according to the method of claim 1.

10. The cells of claim 9, wherein the cells produce insulin.

11. The cells of claim 9, wherein the cells produce somatostatin.

12. The cells of claim 9, wherein the cells produce glucagon.

13. The cells of claim 9, wherein the cells produce pancreatic polypeptide.

14. The cells of claim 9, wherein the cells contain higher levels of mRNA encoding insulin polypeptide than do the hepatic stem cells from which they were differentiated.