Insulin producing cells derived from pluripotent stem cells

• US 9,404,087 B2
• Filed: 12/15/2011
• Issued: 08/02/2016
• Est. Priority Date: 12/15/2010
• Status: Active Grant

• Current Assignee: Kadimastem Ltd
• Original Assignee: Kadimastem Ltd
• Inventors: Molakandov, Kfir, Revel, Michel, Chebath, Judith, Slutsky, Guy, Levy, Alon, Izrael, Michal, Hasson, Arik, Kaufman, Rosalia
• Primary Examiner(s): Leavitt, Maria
• Assistant Examiner(s): Aron, Kimberly A
• Application Number:
  US13/989,805
• Publication Number:
  US 20130273013A1
• Time in Patent Office: 1,692 Days
• US Class Current: 1/1
• 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 2500/38 Vitamins
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

A method of generating islet cells from pluripotent stem cells is disclosed. The method comprises: (a) culturing the pluripotent stem cells in a differentiation medium so as to differentiate the pluripotent stem cells into endoderm cells; and (b) culturing the endoderm cells in a medium comprising at least one growth factor, a cAMP inducer and retinoic acid (RA), said at least one growth factor being selected from the group consisting of FGF10, bFGF and FGF7 so as to generate further differentiated cells; and (c) culturing the further differentiated cells in a medium comprising a maturation factor selected from the group consisting of nicotinamide, GLP-1 and exendin 4, thereby generating islet cells from pluripotent stem cells. Further methods of generating islet cells are also disclosed, isolated cell populations comprising same and uses thereof.

Claims

1. A method of generating human islet cells or human islet progenitor cells from human pluripotent stem cells and selecting for insulin producing cells, the method comprising:

(a) culturing the human pluripotent stem cells for a period ranging from 5 to 8 days in a differentiation medium comprising glucose so as to differentiate the human pluripotent stem cells into endoderm cells expressing Sox17 and FoxA2;

(b) after obtaining said endoderm cells, culturing said endoderm cells for a period ranging from 2 to 10 days in a medium comprising forskolin, retinoic acid, glucose and at least one growth factor, the least one growth factor selected from the group consisting of FGF10,bFGF and FGF7 so as to generate PDX-1 positive cells;

(c) culturing said PDX-1 positive cells in a medium comprising glucose and at least one maturation factor selected from the group consisting of nicotinamide, GLP-1 and exendin 4, thereby generating islet cells or islet progenitor cells from pluripotent stem cells;

wherein the generating of human islet cells or human islet progenitor cells is effected without the generating of embryoid bodies;

(d) dispersing said islet cells or islet progenitor cells to generate dispersed human islet cells or dispersed human islet progenitor cells;

(e) contacting said islet cells or islet progenitor cells with an agent that binds to EpCAM;

(f) selecting cells which bind to said agent;

thereby generating human islet cells or human islet progenitor cells that produce insulin; and

(g) reaggregating said dispersed human islet cells or dispersed human islet progenitor cells that produce insulin.

2. The method of claim 1, wherein said differentiation medium comprises activin A.

3. The method of claim 2, wherein said differentiation medium comprises serum or serum replacement substitute.

4. The method of claim 2, wherein said differentiation medium is devoid of serum.

5. The method of claim 4, wherein said medium of step (b) further comprises noggin.

6. The method of claim 2, wherein said differentiation medium further comprises Wnt3.

7. The method of claim 1, wherein said culturing the pluripotent stem cells is effected by culturing collagenase-detached clusters of pluripotent stem cells on a gelatin coated surface.

8. The method of claim 1, wherein the human pluripotent stem cells comprise human induced pluripotent cells (iPP) cells.

9. The method of claim 1, wherein said re-aggregating is effected in a presence of an agent that chelates calcium selected from the group consisting of EDTA, EGTA, BAPTA, citrate, and phosphate.

10. The method of claim 1, further comprising seeding said dispersed islet cells on a scaffold.

11. The method of claim 1, wherein said re-aggregating is effected in a medium comprising glucose which is lower than that used in steps (a), (b) or (c).

12. The method of claim 1, wherein a glucose concentration of each of said media is between 5 mM-100 mM.

13. The method of claim 1, wherein the islet cells are glucose responsive, and synthesize insulin, glucagon or somatostatin.

14. The method of claim 1, wherein said endoderm cells do not express Oct4.

15. The method of claim 1, wherein step (a) or (b) is effected for about 5 days.

16. A population of islet cells or islet progenitor cells generated according to the method of claim 1.

17. A pharmaceutical composition comprising the population of cells of claim 16 as an active ingredient and a pharmaceutically acceptable carrier.

18. A method of treating diabetes in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the population of cells of claim 16 into the subject, thereby treating the diabetes.

19. The method of claim 1, wherein the culturing of said PDX-1 positive cells of step further comprises a maturation factor, wherein said maturation factor is nicotinamide, exendin 4, or a combination thereof.