Endocrine precursor cells, pancreatic hormone-expressing cells and methods of production

• US 8,129,182 B2
• Filed: 03/02/2007
• Issued: 03/06/2012
• Est. Priority Date: 03/02/2006
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin A., Bang, Anne, Baetge, Emmanuel E.
• Primary Examiner(s): Bertoglio, Valarie
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US11/681,687
• Publication Number:
  US 20070259421A1
• Time in Patent Office: 1,831 Days
• Field of Search: 435/325, 435/347
• US Class Current: 435/325
• 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 2500/38 Vitamins
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/33 Insulin
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/58 Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
• CPC Class Codes: C12N 2501/734 Proteases (EC 3.4.)
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 : from embryonic cells
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

Disclosed herein are cell cultures and enriched cell populations of endocrine precursor cells, immature pancreatic hormone-expressing cells and mature pancreatic hormone-expressing cells. Also disclosed herein are methods of producing such cell cultures and cell populations.

Claims

1. An in vitro cell culture comprising human cells differentiated from human pluripotent stem cells, wherein at least 5% of said human cells are endocrine precursor cells that express neurogenin 3 (NEUROG3), paired box 4 (PAX4) and NKX2 transcription factor related locus 2 (NKX2.2), said endocrine precursor cells being multipotent cells that can differentiate into pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of insulin, somatostatin, ghrelin, pancreatic polypeptide and glucagon.

2. The in vitro cell culture of claim 1, wherein at least 10% of said human cells are endocrine precursor cells.

3. The in vitro cell culture of claim 1, wherein at least 50% of said human cells are endocrine precursor cells.

4. The in vitro cell culture of claim 1, wherein said endocrine precursor cells do not substantially express a pancreatic hormone selected from the group consisting of ghrelin, insulin, somatostatin and glucagon.

5. The in vitro cell culture of claim 1, wherein said endocrine precursor cells are non-recombinant cells.

6. The in vitro cell culture of claim 1 further comprising a retinoid.

22. The in vitro cell culture of claim 1 wherein said endocrine precursor cells are multipotent cells that can differentiate into pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of somatostatin, ghrelin, pancreatic polypeptide and glucagon.

23. The in vitro cell culture of claim 1 wherein said endocrine precursor cells can differentiate into pancreatic islet hormone-expressing cells that express insulin.

24. The in vitro cell culture of claim 1 wherein said endocrine precursor cells are multipotent cells that can differentiate into at least two types of pancreatic islet hormone-expressing cells selected from the group consisting of pancreatic hormone-expressing cells expressing somatostatin, pancreatic hormone-expressing cells expressing ghrelin, pancreatic hormone-expressing cells expressing pancreatic polypeptide and pancreatic hormone-expressing cells expressing glucagon.

25. The in vitro cell culture of claim 1 wherein said endocrine precursor cells are multipotent cells that can differentiate into any pancreatic islet hormone-expressing cells selected from the group consisting of pancreatic hormone-expressing cells expressing somatostatin, pancreatic hormone-expressing cells expressing ghrelin, pancreatic hormone-expressing cells expressing pancreatic polypeptide and pancreatic hormone-expressing cells expressing glucagon.

7. An in vitro cell culture comprising human cells differentiated from human pluripotent stem cells, wherein at least 5% of said human cells are endocrine precursor cells that express neurogenin 3 (NEUROG3), said endocrine precursor cells being multipotent cells that can differentiate into pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of insulin, somatostatin, ghrelin, pancreatic polypeptide and glucagon.

8. The in vitro cell culture of claim 7, wherein at least about 10% of said human cells are endocrine precursor cells.

9. The in vitro cell culture of claim 8, wherein said endocrine precursor cells express NCAM.

10. The in vitro cell culture of claim 9, wherein said endocrine precursor cells comprise a reagent bound to NCAM.

11. The in vitro cell culture of claim 10, wherein said reagent comprises a molecule selected from the group consisting of an anti-NCAM antibody, an anti-NCAM antibody fragment and an NCAM ligand.

12. The in vitro cell culture of claim 11, wherein said reagent is an anti-NCAM antibody.

13. The in vitro cell culture of claim 12, wherein said anti-NCAM antibody is labeled.

14. The in vitro cell culture of claim 7, wherein at least about 25% of said human cells are endocrine precursor cells.

15. The in vitro cell culture of claim 7, wherein at least about 50% of said human cells are endocrine precursor cells.

16. The in vitro cell culture of claim 7, wherein at least about 10% of said human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

17. The in vitro cell culture of claim 7, wherein at least about 25% of said human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

18. The in vitro cell culture of claim 7, wherein at least about 50% of said human cells are human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

19. The in vitro cell culture of claim 7, wherein said endocrine precursor cells express a marker selected from the group consisting of paired box 4 (PAX4) and NKX2 transcription factor related locus 2 (NKX2.2).

20. The in vitro cell culture of claim 7, wherein said endocrine precursor cells are non-recombinant cells.

21. The in vitro cell culture of claim 7 comprising a medium which comprises a factor selected from retinoic acid (RA) and exendin 4 (Ex4).

26. The in vitro cell culture of claim 7 wherein said endocrine precursor cells are multipotent cells that can differentiate into pancreatic islet hormone-expressing cells that express at least one pancreatic hormone selected from the group consisting of somatostatin, ghrelin, pancreatic polypeptide and glucagon.

27. The in vitro cell culture of claim 7 wherein said endocrine precursor cells can differentiate into pancreatic islet hormone-expressing cells that express insulin.

28. The in vitro cell culture of claim 7 wherein said endocrine precursor cells are multipotent cells that can differentiate into at least two types of pancreatic islet hormone-expressing cells selected from the group consisting of pancreatic hormone-expressing cells expressing somatostatin, pancreatic hormone-expressing cells expressing ghrelin, pancreatic hormone-expressing cells expressing pancreatic polypeptide and pancreatic hormone-expressing cells expressing glucagon.

29. The in vitro cell culture of claim 7 wherein said endocrine precursor cells are multipotent cells that can differentiate into any pancreatic islet hormone-expressing cells selected from the group consisting of pancreatic hormone-expressing cells expressing somatostatin, pancreatic hormone-expressing cells expressing ghrelin, pancreatic hormone-expressing cells expressing pancreatic polypeptide and pancreatic hormone-expressing cells expressing glucagon.