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

• US 8,603,811 B2
• Filed: 10/17/2011
• Issued: 12/10/2013
• 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): Paras, Jr., Peter
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US13/275,274
• Publication Number:
  US 20120034692A1
• Time in Patent Office: 785 Days
• Field of Search: 435/325, 435/347, 435/366, 435/377
• 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 together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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. A first and second cell population comprising:

a) a first in vitro cell population comprising human pluripotent cells; and

b) a second in vitro cell population comprising progeny of a portion of the first cell population, wherein the progeny comprise at least 5% endocrine precursor cells that express a marker selected from the group consisting of neurogenin 3 (NEUROG3), paired box 4 (PAX4) and NKX2 transcription factor related locus 2 (NKX2.2).

2. The first and second cell population 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 insulin, somatostatin, ghrelin, pancreatic polypeptide and glucagon.

3. The first and second cell population of claim 1, wherein said endocrine precursor cells express NEUROG3, PAX4 and NKX2.2.

4. The first and second cell population of claim 1, wherein the progeny comprise at least 50% endocrine precursor cells.

5. The first and second cell population 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.

6. The first and second cell population of claim 1, wherein said endocrine precursor cells are non-recombinant cells.

7. The first and second cell population of claim 1, wherein said endocrine precursor cells express NCAM.

8. The first and second cell population of claim 1, wherein said endocrine precursor cells comprise a reagent bound to NCAM.

9. The in vitro cell culture of claim 8, 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.

10. The first and second cell population of claim 9, wherein said reagent is an anti-NCAM antibody.

11. The first and second cell population of claim 10, wherein said anti-NCAM antibody is labeled.

12. The first and second cell population of claim 1, wherein said first in vitro cell population comprises at least 10% human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

13. The first and second cell population of claim 1, wherein said first in vitro cell population comprises at least 50% human pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic endoderm cells.

14. The first and second cell population of claim 1, wherein the first in vitro cell population further medium-comprises a factor selected from retinoic acid (RA) and exendin 4 (Ex4).

15. A first and second cell population comprising

a) a first in vitro cell population comprising human pluripotent cells;

and

b) a second in vitro cell population comprising progeny of a portion of the first cell population, wherein the progeny are obtained by;

(1) providing said first in vitro cell population with a TGFβ

superfamily growth factor;

(2) withdrawing the TGFβ

superfamily growth factor from said cell population;

(3) providing said cell population with a retinoid; and

(4) incubating said cell population in a culture medium for a sufficient time to permit formation of human endocrine precursor cells expressing a marker selected from the group consisting of neurogenin 3 (NEUROG3), paired box 4 (PAX4) and NKX2 transcription factor related locus 2 (NKX2.2).

16. The first and second cell population of claim 15, wherein step (2) further comprises providing said cell population with FGF.

17. The first and second cell population of claim 15, wherein said retinoid comprises retinoic acid.

18. The first and second cell population of claim 15, wherein step (4) further comprises providing said cell population with a gamma secretase inhibitor.