Pancreatic progenitor cells, methods and uses related thereto

• US 6,326,201 B1
• Filed: 02/10/2000
• Issued: 12/04/2001
• Est. Priority Date: 02/10/1999
• Status: Expired due to Term

• Current Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Curis, Inc.
• Inventors: Pang, Kevin, Kagan, David, Fung, Brenda
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): Davis, Katharine F
• Application Number:
  US09/499,362
• Time in Patent Office: 663 Days
• Field of Search: 435/325, 435/366, 435/375, 435/378, 435/379, 435/381, 435/377, 435/371
• 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: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a substantially pure population of viable pancreatic progenitor cells, and methods for isolating such cells. The present invention further concerns certain therapeutic uses for such progenitor cells, and their progeny.

Claims

1. A method of isolating progenitor cells comprising:

i) obtaining pancreatic ductal tissue substantially free of tissues other than the ductal epithelium and surrounding mesenchyme,

ii) obtaining dissociated pancreatic ductal cells from the pancreatic ductal tissue;

iii) culturing said pancreatic ductal cells in a suitable nutrient medium; and

iv) isolating a population of progenitor cells from said culture.

2. A method of claim 1, wherein obtaining pancreatic ductal cells comprises obtaining pancreatic intralobular ductal epithelial cells.

3. A method of claim 1, wherein said pancreatic ductal cells are obtained by explant or by enzymatic digestion.

4. A method of claim 1, wherein culturing said pancreatic ductal cells comprises growing said cells to confluence.

5. A method of claim 1, wherein said progenitor cells are isolated by mechanical separation.

6. A method for preparing a composition comprising progenitor cells, comprising

i) isolating pancreatic progenitor cells by the method of claim 1, and

ii) combining the pancreatic progenitor cells with a pharmaceutically acceptable excipient.

7. The method of claim 1, wherein the pancreatic ducts are substantially free of islet tissue.

8. A method of claim 4, wherein isolating a population of progenitor cells comprises isolating non-adherent cells, and wherein the method further comprises treating the isolated cells with an agent.

9. The method of claim 6, which progenitor cells are mammalian.

10. The method of claim 6, which progenitor cells differentiate to pancreatic lineages.

11. The method of claim 6, which progenitor cells can be maintained in culture for at least about 7 days.

12. The method of claim 6, which progenitor cells are inducible to differentiate into pancreatic islet cells.

13. The method of claim 6, further comprising combining the cells and/or the excipient with an agent that induces differentiation of the progenitor cells.

14. A method of claim 8, wherein said agent induces differentiation and is selected from forskolin, dibutyryl cAMP, Na-butyrate, dexametbasone, and cholera toxin.

15. A method of claim 8, wherein said agent is a growth factor.

16. The method of claim 9, wherein the progenitor cells are transgenic mammalian cells.

17. The method of claim 9, wherein the progenitor cells are primate cells.

18. The method of claim 9, wherein the progenitor cells are miniature swine cells.

19. The method of claim 10, which progenitor cells differentiate to β

islet cells, α

cells, δ

islet cells, φ

islet cells, or exocrine cells.

20. The method of claim 12, which islet cells are pancreatic β

islet cells.

21. The method of claim 12, which islet cells are pancreatic α

islet cells.

22. The method of claim 12, which islet cells are pancreatic δ

islet cells.

23. The method of claim 12, which islet cells are pancreatic φ

islet cells.

24. The method of claim 13, wherein said agent is selected from forskolin, dibutyryl cAMP, Na-butyrate, dexamethasone, and cholera toxin.

25. A method of claim 15, wherein said growth factor is selected from insulinlike growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGP), hedgehog, and vascular endothelial growth factor (VEGF).

26. A method of claim 15, wherein said growth factor is selected from the transforming growth factor-β

(TGFβ

) superfamily, bone morphogenic protein-2 (BMP2) and bone morphogenic protein-7 (BMP7).

27. The method of claim 17, wherein the progenitor cells are human cells.

28. A method for stimulating the ex vivo proliferation of mammalian pancreatic β

-islet cells, comprising the steps of;

(a) preparing a primary culture of mammalian pancreatic cells; and

(b) contacting said primary culture cells with a cAMP agonist in an amount sufficient to induce the primary culture to differentiate to β

-islet cells.

29. The method of claim 28, wherein the mammalian pancreatic cells are human pancreatic cells.

30. The method of claim 28, wherein inducing the primary culture to differentiate comprises inducing an increase in average cellular insulin production.

31. The method of claim 28, further comprising growing said primary culture cells in a monolayer on an extracellular matrix in the presence of a growth factor.

32. The method of claim 28, further comprising contacting said primary culture cells or β

-islet cells with an agent that upregulates the insulin gene.

33. A method for preparing a composition comprising β

-islet cells, comprising

i) isolating β

-islet cells by the method of claim 28, and

ii) combining the β

-islet cells with a pharmaceutically acceptable excipient.

34. The method of claim 32, wherein the agent is a poly (ADP-ribose) synthetase inhibitor.

35. The method of claim 34, wherein the poly(ADP-ribose)synthetase inhibitor is nicotinamide or a benzamide.

36. A method for stimulating the ex vivo proliferation of human adult pancreatic beta islet-cells, comprising the steps of:

(a) preparing a monolayer culture of primary human adult pancreatic cells; and

(b) culturing said cells with a cAMP agonist in an amount sufficient to induce the primary culture to produce insulin-producing cells.

37. A method for preparing a composition comprising insulin-producing cells, comprising

i) producing insulin-producing cells by the method of claim 36, and

ii) combining the insulin-producing cells with a pharmaceutically acceptable excipient.

38. A method of producing human adult pancreatic islet cells in clinically useful quantities, comprising:

(a) seeding a bioreactor with a human pancreatic cell culture;

(b) perfusing said bioreactor with a complete growth medium supplemented with an amount of a cAMP agonist sufficient to induce cells in the bioreactor to proliferate and differentiate into insulin-secreting cells; and

(c) harvesting insulin-secreting cells from said bioreactor.

39. A method for preparing a composition comprising insulin-secreting cells, comprising

i) producing insulin-secreting cells by the method of claim 38, and

ii) combining the insulin-secreting cells with a pharmaceutically acceptable excipient.