Stem cells of the islets of langerhans and their use in treating diabetes mellitus

• US 20070071730A1
• Filed: 04/24/2006
• Published: 03/29/2007
• Est. Priority Date: 12/06/1999
• Status: Active Grant

• Current Assignee: General Hospital Corporation, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Abraham, Elizabeth, Vallejo, Mario, Habener, Joel, Zulewski, Henryk, Thomas, Melissa
• Granted Patent: US 7,544,510 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• 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: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin has been identified as a molecular marker for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

Claims

1. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet; and

(c) selecting a nestin-positive cell from the culture.

6. The method of claim 1 comprising the additional step of:

expanding the nestin-positive clone by treatment with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

16. An isolated nestin-positive stem cell isolated by the method of claim 1, 2 or 3.

2. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet, wherein said cultured cells that form a monolayer comprise nestin-positive cells.

3. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet under conditions wherein said cultured cells comprise nestin-positive cells which have migrated from said islet; and

(c) selecting said nestin-positive cells from the culture.

4. The method of claim 3, wherein said migrated cells from step (b) form a monolayer.

5. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet in a first vessel coated with concanavalin A to separate concanavalin A adherent and non-adherent cells;

(c) transferring said non-adherent cells from step (b) to a second vessel not coated with concanavalin A;

(d) culturing cells from step (c) in said second vessel to produce a cell culture; and

(e) selecting said nestin-positive cells from step (d) to produce an isolated stem cell.

7. A method of inducing the differentiation of an isolated nestin-positive pancreatic stem cell into a pancreatic progenitor cell, comprising the step of:

treating a nestin-positive pancreatic stem cell with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β

, and combinations thereof, whereby the stem cell subsequently differentiates into a pancreatic progenitor cell.

8. The method of claim 7, wherein the pancreatic progenitor cell subsequently forms pseudo-islet like aggregates.

9. An isolated, nestin-positive human pancreatic or liver stem cell that is not a neural stem cell.

10. The isolated stem cell of claim 9 that differentiates to form insulin-producing beta cells.

11. The isolated stem cell of claim 9 that differentiates to form glucagon- producing alpha cells.

12. The isolated stem cell of claim 9 that differentiates to form pseudo-islet like aggregates.

13. The isolated stem cell of claim 9 that differentiates to form hepatocytes.

14. The isolated stem cell of claim 9 that does not express class I MHC antigens.

15. A pharmaceutical composition comprising the isolated stem cell of claim 9 admixed with a physiologically compatible carrier.