Differentiation of human pluripotent stem cells

• US 9,752,126 B2
• Filed: 12/09/2015
• Issued: 09/05/2017
• 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, Fryer, Benjamin
• Primary Examiner(s): Hibbert, Catherine S
• Application Number:
  US14/963,436
• Publication Number:
  US 20160160182A1
• Time in Patent Office: 636 Days
• Field of Search: None
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/405 Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• 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 a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of MAFA.

Claims

1. A method for increasing the expression of insulin and MAF bZIP transcription factor A (“

MAFA”

) in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of;

a. sequentially differentiating human pluripotent stem cells to obtain cells expressing markers characteristic of the pancreatic endocrine lineage; and

b. culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of insulin and MAFA as compared to cells expressing markers characteristic of the pancreatic endocrine lineage that are not cultured in medium comprising the added cyclin-dependent kinase inhibitor;

wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

2. The method of claim 1, wherein the cyclin-dependent kinase inhibitor is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 μ

M to about 10 μ

M for about one to seven days.

3. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

4. The method of claim 1, wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

5. The method of claim 1, wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

6. A method for increasing the expression of insulin and MAF bZIP transcription factor A (“

MAFA”

) in a population of cells comprising pancreatic endocrine cells comprising culturing the population of cells in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in the expression of insulin and MAFA in the population as compared to a population of cells comprising pancreatic endocrine cells not cultured in medium comprising the added cyclin-dependent kinase inhibitor, wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

7. The method of claim 6, wherein the method comprises adding the cyclin-dependent kinase inhibitor at a concentration from about 0.1 μ

M to about 10 μ

M for about one to seven days.

8. The method of claim 6, wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

9. The method of claim 6, wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

10. The method of claim 6, wherein the pancreatic endocrine cells are obtained by differentiating human pluripotent stem cells.

11. The method of claim 10, wherein the human pluripotent stem cells are human embryonic cells.