Generation and differentiation of adult stem cell lines

• US 20060093586A1
• Filed: 10/20/2005
• Published: 05/04/2006
• Est. Priority Date: 10/20/2004
• Status: Active Grant

• Current Assignee: Vitro Diagnostics, Inc.
• Original Assignee: Vitro Diagnostics, Inc.
• Inventors: Musick, James
• Granted Patent: US 7,527,971 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention teaches a new method of stem cell generation involving the establishment of a mesenchymal cell culture, induction of epithelial marker expression and isolation and cloning of the cells expressing epithelial markers and expansion of these cell lines. Further processes are described for a two-step method to induce the differentiation of terminally differentiated cell types such as insulin-producing beta cells of the human pancreas gland. The invention indicates that mesenchymal to epithelial transition may be an important component in the development, renewal and regeneration of animal organ systems and provides a reliable method for in-vitro generation of adult stem cells and terminally differentiated cell types for various uses in medical research and cell therapy of disease.

Claims

1. A method for generation of adult stem cells with capacity to differentiate into endocrine cells of the pancreas gland comprising the following steps:

a) Obtaining a culture of mesenchymal cells;

b) Inducing the expression of epithelial cell markers in said culture of mesenchymal cells;

c) Isolating and cloning cells expressing epithelial cell markers; and

d) Maintaining and expanding said isolated and cloned cells to generate stem cell lines.

2. The method of claim 1, wherein said mesenchymal cells are derived from the human fetal pancreas gland.

3. The method of claim 1, wherein said mesenchymal cells are the VIT1 cell line.

4. The method of claim 1, wherein expression of epithelial markers is induced by exposure to glucagon-like peptide 1 and derivatives thereof.

5. The method of claim 1, wherein expression of epithelial markers is induced by exposure to exendin-4 and derivatives thereof.

6. The method of claim 1, wherein said stem cell lines are differentiated into fully differentiated beta islet cells.

7. The method of claim 6, wherein said differentiated beta islet cells are human beta cells.

8. A cell line generated according to the method of claim 1.

12. A method to provide cell therapy to a human being, comprising transplanting stem cell lines generated by the method according to claim 1.

13. A method to provide cell therapy, comprising transplanting the stem cell line generated by the method according to claim 1 to an animal species selected from the group consisting of:

canine, feline, equine, bovine, ovine, rat and mouse.

14. A method to treat diabetes in humans, comprising transplanting the cells produced by the method of claim 7 into a human who has diabetes.

15. The method according to claim 14, further comprising encapsulation of the transplanted cells to prevent immune rejection.

16. The method according to claim 14, further comprising the use of immuno-privileged cells that are not rejected by the immune system of the recipient

17. A method to treat diabetes, comprising transplanting fully differentiated beta islet cells produced by the method of claim 6 into an animal species selected from the group consisting of:

canine, feline, equine, bovine, ovine, rat and mouse.

18. The method according to claim 17, further comprising encapsulation of the transplanted cells to prevent immune rejection.

19. Stem cell lines generated according to the method of claim 1 that are self-renewing through activation of endogenous cell proliferation processes and inhibition of apoptosis.

9. The VIT1, LT2, VIT2, VIT3, VIT4, VIT5, VIT2-A2, VIT2-A11, VIT2-B4, VIT2-C12, VIT2-G1, VIT2-G6, VIT2-H9, VIT3-B4, VIT3-C8, VIT3-F1, VIT3-F4, VIT3-F7, VIT3-G12, VIT3-H11, VIT4-C4, VIT4-D10, VIT4-F9, VIT4-G9, VIT4-H9, VIT5-D12, VIT5-H2, VIT5-H9, VIT5-H12 cell lines and derivatives thereof.

10. The VIT4-G9 cell line and derivatives thereof.

11. The VIT3-G12 cell line and derivatives thereof.