Differentiation of human embryonic stem cells

• US 9,388,387 B2
• Filed: 03/18/2015
• Issued: 07/12/2016
• Est. Priority Date: 10/31/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/661,647
• Publication Number:
  US 20150191700A1
• Time in Patent Office: 482 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• 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 2501/155 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides a method to increase the expression of markers associated with the pancreatic endocrine lineage.

Claims

1. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising the steps of:

a) Culturing the pluripotent stem cells;

b) Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;

c) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;

d) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and

e) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist to increase the number of pancreatic endocrine cells in the population,

wherein the TGF-β

receptor agonist is GDF-8, GDF-11 or GDF-15.

2. The method of claim 1, wherein the step of treating with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist causes an increase in expression of markers associated with the pancreatic endocrine lineage.

3. The method of claim 1, wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.

4. The method of claim 1, wherein the method comprises an increase in expression of PDX-1, NKX6.1 and PAX4.

5. The method of claim 1, wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cells.

11. The method of claim 1, wherein step e) comprises treating the pancreatic endocrine cells with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist for about three to about five days.

13. The method of claim 1, wherein the step of treating increases expression of PDX-1, NKX6.1 and PAX4.

6. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising:

a) Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;

b) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;

c) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and

d) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist to increase the number of pancreatic endocrine cells in the population,

wherein the TGF-β

receptor agonist is GDF-8, GDF-11 or GDF-15.

7. The method of claim 6, wherein the step of treating with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist causes an increase in expression of markers associated with the pancreatic endocrine lineage.

8. The method of claim 6, wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.

9. The method of claim 6, wherein the method comprises an increase in expression of PDX-1, NKX6.1 and PAX4.

10. The method of claim 6, wherein the pancreatic endocrine cell is a pancreatic hormone expressing or pancreatic hormone secreting cells.

12. The method of claim 6, wherein step d) comprises treating the pancreatic endocrine cells with the medium comprising a cyclic aniline-pyridinotriazine and a TGF-β

receptor agonist for about three to about five days.

14. The method of claim 6, wherein the step of treating increases expression of PDX-1, NKX6.1 and PAX4.

15. A method for differentiating pluripotent stem cells into pancreatic endocrine cells comprising:

a) Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage;

b) Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage;

c) Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into a population comprising pancreatic endocrine cells; and

d) Treating the population comprising pancreatic endocrine cells with a medium comprising a cyclic aniline-pyridinotriazine and GDF-8, GDF-11 or GDF-15 to increase expression of PDX-1, NKX6.1 and PAX4 in the population.

16. The method of claim 15, wherein the step of treating also increases NKX2.2, insulin and glucagon expression in the population.

17. The method of claim 15, wherein the method further comprises enriching the pancreatic endocrine cells by obtaining a suspension of the cells and separating the suspension of cells through a strainer, wherein the enriched cells are retained by the strainer.