Method of forming pancreatic beta cells from mesenchymal cells

• US 20050208029A1
• Filed: 04/16/2003
• Published: 09/22/2005
• Est. Priority Date: 04/17/2002
• Status: Abandoned Application

• Current Assignee: Otsuka Pharmaceutical Company Limited (Otsuka Holdings Company Limited)
• Original Assignee: Otsuka Pharmaceutical Company Limited (Otsuka Holdings Company Limited)
• Inventors: Umezawa, Akihiro, Izawa, Yoshikane
• Application Number:
  US10/511,629
• Publication Number:
  US 20050208029A1
• Time in Patent Office: Days
• US Class Current: 424/93.210
• 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: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• 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 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/305 Growth hormone [GH], aka. somatotropin
• CPC Class Codes: C12N 2501/315 Prolactin
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/90 Polysaccharides
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/1353 from bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: G01N 33/507 : Pancreatic cells
• CPC Class Codes: G01N 33/5073 : Stem cells

Abstract

It is intended to provide a method of forming pancreatic β cells from mesenchymal cells characterized by comprising using mammal-origin mesenchymal cells as starting cells, culturing these cells in the presence of, for example, a pancreatic β cell-forming agent, and selecting and separating the thus obtained pancreatic βcells with the use of a gene expressed specifically in such cells as a selection marker; a remedy for glucose intolerance which comprises pancreatic β cells obtained by the above method as the active ingredient; a pancreatic β cell-forming agent such as a cytokine to be used in the above method; a method of screening a candidate compound promoting the formation of pancreatic β cells from mesenchymal cells; and a pancreatic β cell formation promoter obtained by this screening method.

Claims

1. A method of forming pancreatic β

cells from mesenchymal cells which comprises subjecting mammal-derived mesenchymal cells to at least one step selected from the following steps (a) to (e);

a) the step of proliferating mesenchymal cells obtained from a mammalian sample and capable of differentiating into pancreatic β

cells;

b) the step of selecting and isolating those mesenchymal cells which are capable of differentiating into pancreatic β

cells from among mesenchymal cells obtained from a mammalian sample or from among the mesenchymal cells obtained in step a) using an antibody or antibodies capable of binding to a cell membrane antigen;

c) the step of cultivating the mesenchymal cells capable of differentiating into pancreatic β

cells as obtained in step a) or b) or cells including such mesenchymal cells in an adhesion molecule/extracellular matrix-coated reaction vessel which enables the cells to contact with the adhesion molecules/extracellular matrix;

d) the step of cultivating the mesenchymal cells capable of differentiating into pancreatic β

cells as obtained in step a), b) or c) or cells including such mesenchymal cells in contact with a pancreatic β

cell-forming agent; and

e) the step of selecting and separating the pancreatic β

cells obtained in the step c) or d) using a gene specifically expressed in pancreatic β

cells as a selective marker.

2. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 1, wherein the mesenchymal cells are obtained from bone marrow, muscle, pancreas, liver, small intestine, large intestine, kidney, subcutaneous tissue, endometrium, blood, cord blood or placenta.

3. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 1, wherein the selection of mesenchymal cells in step b) is carried out using a CD140-positive antibody.

4. A method of forming pancreatic β

cells from mesenchymal cells as defined in arm) of claim 1, wherein, in step e), the gene specifically expressed in pancreatic β

cells is the insulin gene.

5. A method of forming pancreatic β

cells from mesenchymal cells as defined in an) of claim 1, wherein, in step d), the pancreatic β

cell-forming agent comprises at least one member selected from the group consisting of cytokines, physiologically active substances, transcription factors and adhesion molecules/extracellular matrices.

6. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 5, wherein the cytokine selected comprises at least one member selected from the group consisting of hepatocyte growth factor (HGF), fibroblast growth factor (bFGF)/FGF-2, insulin, transferrin, heparin-binding EGF, gastrin, TGF-β

, insulin-like growth factor (IGF-1), parathyroid hormone-related proteins (PTHrP), growth hormone, prolactin, placental lactogen, glucagon-like peptide-1, exendin-4 and KGF (keratinocyte growth factor).

7. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 5, wherein the physiologically active substance selected comprises at least one member selected from the group consisting of nicotinamide, betacellulin, activin A, progesterone, putrescine and selenium.

8. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 5, wherein the transcription factor selected comprises at least one member selected from the group consisting of PTF1a/PTF-P48, Is1-1, Pdx-1/IPF-1, Beta2/neuroD, ngn3, PAX-6, PAX-4, H1xb-9, Nkx2.2, Nkx6.1, HNF1α

, HNF1β and

HNF4α

.

9. A method of forming pancreatic β

cells from mesenchymal cells as defined in claim 5, wherein the adhesion molecule/extracellular matrix selected comprises at least one member selected from the group consisting of gelatin, laminin, collagen, agarose, fibronectin and ornithine.

10. A pancreatic β

cell-forming agent for use in the method as defined in claim 1 which comprises, as an active ingredient, at least one member selected from the group consisting of cytokines, physiologically active substances, transcription factors and adhesion molecules/extracellular matrices.

11. A pancreatic β

cell-forming agent as defined in claim 10, wherein the cytokine selected comprises at least one member selected from the group consisting of hepatocyte growth factor (HGF), fibroblast growth factor (bFGF)/FGF-2, insulin, transferrin, heparin-binding EGF, gastrin, TGF-β

, insulin-like growth factor (IGF-1), parathyroid hormone-related proteins (PTHrP), growth hormone, prolactin, placental lactogen, glucagon-like peptide-1, exendin-4 and KGF.

12. A pancreatic β

cell-forming agent as defined in claim 10, wherein the physiologically active substance selected comprises at least one member selected from the group consisting of nicotinamide, betacellulin, activin A, progesterone, putrescine and selenium.

13. A pancreatic β

cell-forming agent as defined in claim 10, wherein the transcription factor selected comprises at least one member selected from the group consisting of PTF1a/PTF-P48, Is1-1, Pdx-1/IPF-1, Beta2/neuroD, ngn3, PAX-6, PAX-4, H1xb-9, Nkx2.2, Nkx6.1, HNF1α

, HNF1β and

HNF4α

.

14. A pancreatic β

cell-forming agent as defined in claim 10, wherein the adhesion molecule/extracellular matrix selected comprises at least one member selected from the group consisting of gelatin, laminin, collagen, agarose, fibronectin and ornithine.

15. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells which comprises causing the formation of pancreatic β

cells from mesenchymal cells according to the method as defined in claim 1 in the presence of each candidate substance and selecting a candidate substance showing a pancreatic β

cell formation-promoting effect in comparison with pancreatic β

cells formed in the absence of the candidate substance.

16. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 15, wherein the mesenchymal cells are obtained from bone marrow, muscle, pancreas, liver, small intestine, large intestine, kidney, subcutaneous tissue, endometrium, blood, cord blood or placenta.

17. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 15, wherein the selection of mesenchymal cells in step b) is carried out using a CD140-positive antibody.

18. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 15, wherein, in step e), the gene specifically expressed in pancreatic β

cells is the insulin gene.

19. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 15, wherein, in step d), the pancreatic β

cell-forming agent comprises at least one member selected from the group consisting of cytokines, physiologically active substances, transcription factors and adhesion molecules/extracellular matrices.

20. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 19, wherein the cytokine selected comprises at least one member selected from the group consisting of hepatocyte growth factor (HGF), fibroblast growth factor (bFGF)/FGF-2, insulin, transferrin, heparin-binding EGF, gastrin, TGF-β

, insulin-like growth factor (IGF-1), parathyroid hormone-related proteins (PTHrP), growth hormone, prolactin, placental lactogen, glucagon-like peptide-1, exendin-4 and KGF.

21. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 19, wherein the physiologically active substance selected comprises at least one member selected from the group consisting of nicotinamide, betacellulin, activin A, progesterone, putrescine and selenium.

22. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 19, wherein the transcription factor selected comprises at least one member selected from the group consisting of PTF1α

/PTF-P48, Is1-1, Pdx-1/IPF-1, Beta2/neuroD, ngn3, PAX-6, PAX-4, H1xb-9, Nkx-2.2, Nkx6.1, HNF1α

, HNF1β and

HNF4α

.

23. A method of screening candidate compounds promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 19, wherein the adhesion molecule/extracellular matrix selected comprises at least one member selected from the group consisting of gelatin, laminin, collagen, agarose, fibronectin and ornithine.

24. A method of screening candidate substances promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claim 15, wherein the candidate substance is a cultivation-derived composition.

25. A substance capable of promoting the formation of pancreatic β

cells from mesenchymal cells as obtained by the method of screening candidate compounds promoting the formation of pancreatic β

cells from mesenchymal cells as defined in claims 15.

26. A method for treating an impaired glucose tolerance-due disease of a patient which comprises administering an effective amount of the pancreatic β

cells obtained by the method as defined in claim 1 to the patient.

27. A therapeutic agent for an impaired glucose tolerance-due disease which comprises, as an active ingredient, the pancreatic β

cells obtainable by the method as defined in claim 1.

28. A method of causing differentiation into insulin-secreting cells which comprises scattering cells capable of differentiating into insulin-secreting cells on the layer of mesenchymal cells as obtained by monolayer culture on a culture dish and carrying out the cocultivation thereof.

29. A method of causing differentiation into insulin-secreting cells by cocultivation as defined in claim 28, wherein the cells capable of differentiating into insulin-secreting cell comprise at least one member selected from the group consisting of embryonic stem cells, pancreatic stem cells, small intestinal epithelial stem cells, liver-derived stem cells and amniotic cells.