Method for differentiating stem cells into insulin-producing cells

• US 20050054102A1
• Filed: 04/19/2002
• Published: 03/10/2005
• Est. Priority Date: 04/19/2001
• Status: Abandoned Application

• Current Assignee: Anna Wobus, Luc St-Onge, Przemyslaw Blyszczuk, Ursula Hoffmann
• Original Assignee: Anna Wobus, Luc St-Onge, Przemyslaw Blyszczuk, Ursula Hoffmann
• Inventors: Hoffmann, Ursula, Blyszczuk, Przemyslaw, St-Onge, Luc, Wobus, Anna
• Application Number:
  US10/475,021
• Publication Number:
  US 20050054102A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/04 from germ cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: G01N 2333/62 Insulins
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/507 Pancreatic cells
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

The present invention relates a novel method for differentiating stem cells into insulin-producing cells by culturing such cells in specially defined media and optimally, activating one or more genes involved in beta-cell differentiation. The present invention further relates to applications in the medical (particularly diabetes) field that directly arise from the method of the invention. Additionally, the present invention relates to applications for identifying and characterising compounds with therapeutic medical effects or toxicological effects that directly arise from the method of the invention.

Claims

1. A method for differentiating stem cells into insulin-producing cells comprising:

culturing stem cells in a suitable medium and activating at least one gene involved in beta-cell differentiation.

2. The method of claim 1 further comprising:

aggregating said cultivated stem cells to form embryoid bodies, cultivating said embryoid bodies in a differentiation medium enhancing β

-cell differentiation, identifying, and optionally selecting insulin-producing cells.

3. The method of claim 1, wherein said stem cells are selected from embryonic stem cells, adult stem cells, somatic stem cells and primordial germ cells.

4. The method of claim 1, wherein said stem cells are of human origin.

5. The method of claim 1, wherein the genes involved in β

-cell differentiation are selected from the group consisting of Pdx1, Pax4, Pax6, ngn3, Nkx6.1, Nkx6.2, Nkx2.2, HB9, BETA2, NeuroD, IsI1, HNF1-alpha, HNF1-beta, HNF3, and combinations thereof.

6. The method of claim 5, wherein the genes are selected from Pdx1, Pax4, Pax6, ngn3, and combinations thereof.

7. The method of claim 5, wherein the genes are of human origin.

8. The method of claim 1, wherein the gene activation comprises a delivery of a pancreatic gene into stem cells.

9. The method of claim 8, wherein said gene delivery comprises a transfection of stem cells with a cDNA of at least one pancreatic gene under the control of a regulatory region allowing the initiation of transcription.

10. The method of claim 8, wherein said gene delivery comprises a DNA transfer using a viral delivery system.

11. The method of claim 1, wherein the gene activation comprises a delivery of a protein product of a pancreatic gene into stem cells.

12. The method of claim 2, wherein said embryoid bodies are formed by a hanging drop method.

13. The method of claim 2, wherein said differentiation medium is based on Iscove'"'"'s modified Dulbecco'"'"'s medium (IMDM) supplemented with fetal calf serum, L-glutamine, non-essential amino acids, and à

-monothioglycerol optionally containing EGF, bFGF, progesterone, growth hormone, follistatin, and/or activin.

14. The method of claim 13, wherein said differentiation medium further contains extracellular matrix proteins, collagens, and/or mixtures of growth factors and extracellular matrix proteins.

15. The method of claim 1, wherein at least 20% insulin-producing cells are obtained after a differentiation time of 15 days.

16. The method of claim 15, wherein at least 40% insulin-producing cells are obtained.

17. The method of claim 1 further comprising a selection of nestin-positive cells.

18. The method of claim 1, wherein the insulin-producing cells are used for pharmaceutical applications.

19. The method of claim 18 for the treatment of pancreatic diseases, metabolic syndrome and metabolic disorders with impaired glucose levels, such as diabetes, hyperglycaemia, and/or impaired glucose tolerance.

20. The method of claim 18, wherein between 3000 and 100000 equivalent differentiated insulin-producing cells are administered per kilogram body weight.

21. A cell composition comprising insulin-producing cells obtainable by the method of claim 1.

22. The composition of claim 21 comprising at least 20% insulin-producing cells after a differentiation time of 15 days.

23. The composition of claim 22 comprising at least 40% insulin-producing cells.

24. The composition of claim 21 comprising a ratio of insulin-producing cells versus glucagon-producing cells of at least 2:

1.

25. The composition of claim 24 comprising a ratio of at least 5:

1.

26. The composition of claim 21 exhibiting an increase in the insulin secretion of at least 2-fold 15 min after stimulation with 27.7 mM glucose.

27. The composition of claim 21, which is a pharmaceutical composition.

28. The composition of claim 27 for the treatment of pancreatic diseases, metabolic syndrome and metabolic disorders with impaired glucose levels, such as diabetes, hyperglycaemia, and/or impaired glucose tolerance.

29. The composition of claim 27, which is administered by transplantation or used in a medical device.

30. A method for identifying and/or characterizing compounds capable of modulating the differentiation of stems cells into insulin-producing cells comprising:

contacting a compound to be tested with stem cells under conditions wherein the stem cells are capable of being differentiated into insulin-producing cells and determining the effect of the compound on the differentiation process.

31. The method of claim 30 comprising transfecting stem cells with a DNA construct containing a reporter gene under regulatory control of a gene involved in β

-cell differentiation, contacting said transfected cells with a compound to be tested and determining the activity of the reporter gene.

32. The method of claim 30 comprising contacting embryoid bodies which are cultivated in a differentiation medium enhancing β

-cell differentiation with a compound to be tested and determining differentiation into insulin-producing cells.