Methods for in vitro expansion and transdifferentiation of human pancreatic acinar cells into insulin-producing cells

• US 20040127406A1
• Filed: 05/22/2003
• Published: 07/01/2004
• Est. Priority Date: 05/28/2002
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Presnell, Sharon C., Heidaran, Mohammad, Haaland, Perry
• Application Number:
  US10/443,732
• Publication Number:
  US 20040127406A1
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• 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 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• 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 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/315 : Prolactin
• CPC Class Codes: C12N 2501/335 : Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/34 : Calcitonin; Calcitonin-gene related peptide [CGRO]; Amylin
• CPC Class Codes: C12N 2501/345 : Gastrin; Cholecystokinins [CCK]
• CPC Class Codes: C12N 2501/35 : Vasoactive intestinal peptide [VIP]; Pituitary adenylate cyclase activating polypeptide [PACAP]
• CPC Class Codes: C12N 2501/37 : Parathyroid hormone [PTH]
• CPC Class Codes: C12N 2501/39 : Steroid hormones
• CPC Class Codes: C12N 2501/392 : Sexual steroids
• CPC Class Codes: C12N 2501/41 : Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/83 : Tachykinins, e.g. substance P
• CPC Class Codes: C12N 2501/85 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/22 : from pancreatic cells
• CPC Class Codes: C12N 5/0037 : Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0676 : Pancreatic 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

This invention relates, e.g., to a method for expanding mammalian acinar cells, comprising culturing the cells in a cell culture system comprising a cell culture medium and a cell attachment surface, under conditions wherein the acinar cells undergo a 3-4 fold expansion together with transdifferentiation into a modified cell phenotype (IP cells) showing characteristics of acinar cells and liver cells. The invention also relates to a method for transforming these IP cells to insulin-producing cells in vitro, comprising culturing the cells in a novel, defined medium. Also disclosed are suitable culture media for performing these methods, isolated cells having the phenotype of IP cells and/or produced by these methods, and kits for performing the methods.

Claims

1. A method for transforming IP cells that express markers of acinar cells and liver-associated genes into insulin-producing cells in vitro, comprising culturing said IP cells in a cell culture medium comprising an effective amount of at least one differentiation promoting factor selected from the group consisting of C-Natriuretic Peptide (CNP), Calcitonin Gene Related Peptide, Cholera Toxin B Subunit, Dexamethasone, Gastrin-Releasing Peptide, Laminin, Met-Enkephalin, PDGFAA+PDGFBB, Sonic Hedgehog, and Substance P such that the IP cells are transformed into insulin-producing cells.

2. The method of claim 1, wherein the IP cells are derived from a culture of pancreatic acinar cells.

3. The method of claim 2, wherein the cells are human.

4. The method of claim 1, further comprising contacting said cells with a substrate that is coated with one or more extracellular matrix molecules.

5. The method of claim 4, wherein the extracellular matrix molecules are collagen I, collagen VI, collagen IV, vitronectin, and/or fibronectin.

6. The method of claim 4, wherein the substrate is on the surface of a flask, petri dish, plate, well or roller bottle, or is part of a scaffold.

7. The method of claim 1, wherein the medium is serum-free.

8. The method of claim 1, wherein the medium comprises serum.

9. The method of claim 7, wherein the medium comprises BSA, insulin, transferrin, selenium and epidermal growth factor (EGF).

10. The method of claim 3, wherein the cells are seeded on the substrate at a density of 5×

10³ to 20×

10⁵ cells/cm².

11. An isolated insulin-producing cell generated by the method of claim 1.

17. The method of claim 1, wherein the cell culture medium comprises a 1:

1 mixture of DMEM and Hams 12.

19. The method of claim 1, wherein said differentiation promoting factors have the concentrations in the medium as indicated in Table 1.

12. An insulin-producing cell, prepared by differentiating a mammalian acinar cell in vitro, wherein said insulin-producing cell has an expression profile after 16 days ex vivo as shown in Table 6.

13. A serum-free medium comprising at least one differentiation promoting factor selected from the group consisting of C-Natriuretic Peptide (CNP), Calcitonin Gene Related Peptide, Cholera Toxin B Subunit, Dexamethasone, Gastrin-Releasing Peptide, Laminin, Met-Enkephalin, PDGFAA+PDGFBB, Sonic Hedgehog, and Substance P wherein said medium facilitates differentiation of IP cells into insulin-producing cells.

18. The serum-free medium of claim 13 which comprises a 1:

1 mixture of DMEM and Hams 12.

20. The serum-free medium of claim 13, wherein said differentiation promoting factors have the concentrations in the medium as indicated in Table 1.

14. A serum free medium comprising a 1:

1 mixture of DMEM and Hams F12 plus the components listed in Table 2.

15. A kit suitable for differentiating IP cells to insulin-producing cells, comprising

a) a base medium suitable for the cultivation of mammalian epithelial cells;

b) a collagen I coated culture substrate, and, separately packaged,

c) a serum-free medium supplement containing BSA, C-Natriuretic Peptide (CNP), Calcitonin Gene Related Peptide, Cholera Toxin B Subunit, Dexamethasone, Gastrin-Releasing Peptide, Laminin, Met-Enkephalin, PDGFAA+PDGFBB, Sonic Hedgehog, and Substance P or two or more of these components in combination, in suitable amounts to yield final concentrations in the completed medium as indicated in Table 1 herein.

16. The kit of claim 15, wherein the cell culture substrate is contained on the surface of a flask, bottle, petri dish, plate or well suitable for cell culture.