Differentiation of human embryonic stem cells

• US 9,752,125 B2
• Filed: 05/11/2011
• Issued: 09/05/2017
• Est. Priority Date: 05/12/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Xu, Jean
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/105,285
• Publication Number:
  US 20110281355A1
• Time in Patent Office: 2,309 Days
• 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 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• 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 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/385 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells expressing markers characteristic of the pancreatic endoderm lineage, wherein greater than 50% of the cells in the population co-express PDX1 and NKX6.1.

Claims

1. A cell culture comprising:

a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;

human definitive endoderm cells; and

a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,

wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,

wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and

wherein cells in the isolated PDX1 and NKX6.1 co-expressing population express CDX2.

2. The cell culture of claim 1 wherein greater than 70% of the cells in the population co-express PDX1 and NKX6.1.

3. The cell culture of claim 1 wherein greater than 80% of the cells in the population co-express PDX1 and NKX6.1.

4. The cell culture of claim 1 wherein greater than 90% of the cells in the population co-express PDX1 and NKX6.1.

5. The cell culture of claim 1, wherein cells in the cell population express ISL1 and/or NEUROD1.

6. The cell culture of claim 1, wherein the protein kinase C activator is selected from the group consisting of (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB), phorbol-12-myristate-13-acetate (PMA), and phorbol-12,13-dibutyrate (PDBu).

13. The cell culture of claim 1, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

16. The cell culture of claim 1, wherein cells in the cell population express NEUROD1.

17. The cell culture of claim 1, wherein the cells are obtained by culturing a population of cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a protein kinase C activator and lacking FGF10.

19. The cell culture of claim 1, wherein the cells are obtainable by culturing a population of cells expressing markers characteristic of the definitive endoderm lineage in a medium supplemented with a protein kinase C activator and lacking FGF10.

21. The cell culture of claim 1, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

7. A cell culture comprising:

a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;

human definitive endoderm cells; and

a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,

wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,

wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and

wherein the PDX1 and NKX6.1 co-expressing cells in the isolated population differentiate into insulin secreting cells in vivo.

8. The cell culture of claim 7, wherein cells in the cell population express ISL1 and/or NEUROD1.

9. The cell culture of claim 7, wherein greater than 70% of the cells in the population co-express PDX1 and NKX6.1.

10. The cell culture of claim 7, wherein greater than 80% of the cells in the population co-express PDX1 and NKX6.1.

11. The cell culture of claim 7, wherein greater than 90% of the cells in the population co-express PDX1 and NKX6.1.

12. The cell culture of claim 7, wherein the protein kinase C activator is selected from the group consisting of (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB), phorbol-12-myristate-13-acetate (PMA), and phorbol-12,13-dibutyrate (PDBu).

14. The cell culture of claim 7, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

18. The cell culture of claim 7, wherein cells in the cell population express NEUROD1.

22. The cell culture of claim 7, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

15. A cell culture comprising:

a medium supplemented with ALK5 inhibitor II and a protein kinase C activator;

human definitive endoderm cells; and

a population comprising pancreatic endoderm cells,

wherein the presence of ALK5 inhibitor II and the protein kinase C activator in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 90% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1, and

wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells.

23. The cell culture of claim 15, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.

20. A cell culture comprising:

a medium supplemented with (2S, 5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam (TPB);

human definitive endoderm cells; and

a population of human cells expressing markers characteristic of the pancreatic endoderm lineage,

wherein the presence of TPB in the culture increases the percentage of pancreatic endoderm cells that co-express PDX1 and NKX6.1, and results in a culture wherein greater than 60% of the cells in the population are pancreatic endoderm cells co-expressing PDX1 and NKX6.1,

wherein the human definitive endoderm cells are derived from established lines of human pluripotent cells, and

wherein cells in the isolated population express CDX2.

24. The cell culture of claim 20, wherein the culturing of the first population of cells expressing markers characteristic of the definitive endoderm does not require purification.