Use of small molecules to enhance MAFA expression in pancreatic endocrine cells

• US 10,870,832 B2
• Filed: 06/13/2018
• Issued: 12/22/2020
• Est. Priority Date: 05/16/2014
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): Ton, Thaian N.
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US16/007,600
• Publication Number:
  US 20180291348A1
• Time in Patent Office: 923 Days
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/375 Thyroid stimulating hormone [TSH]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/72 Transferases (EC 2.) acetylation of histones C12N2501/065
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 2506/22 from pancreatic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods, cell cultures and differentiation media to promote differentiation of pluripotent stem cells to pancreatic endocrine cells of a mature phenotype. The resulting pancreatic endocrine cells express single hormonal insulin, PDX1, NKX6.1, and MAFA. In one or more differentiation stages, culturing may be carried out in a culture vessel at the air-liquid interface.

Claims

1. A method for producing pancreatic endocrine cells expressing MAFA comprising:

(a) obtaining a population of cells comprising pancreatic endoderm and endocrine precursor cells;

(b) differentiating the population of cells comprising pancreatic endoderm and endocrine precursor cells into a pancreatic endocrine cell population in a medium comprising (i) a SHH signaling pathway antagonist, (ii) a BMP inhibitor, (iii) T3, a T3 analogue, T4, a T4 analogue or a mixture thereof, and (iv) an ALK5 inhibitor; and

(c) culturing the pancreatic endocrine cell population in a further medium comprising an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT 1L, or a combination thereof.

2. The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the aurora kinase inhibitor.

3. The method of claim 2, wherein the aurora kinase inhibitor is aurora kinase inhibitor II, SNS 314 mesylate, GSK1070916 or TAK-901.

4. The method of claim 3, wherein the aurora kinase inhibitor is aurora kinase inhibitor II.

5. The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the RSK inhibitor.

6. The method of claim 5, wherein the RSK inhibitor is RSK inhibitor II.

7. The method of claim 1, wherein the method comprises culturing the pancreatic endocrine cell population in the further medium comprising the inhibitor of protein methyltransferase DOT1L.

8. The method of claim 7, wherein the inhibitor of protein methyltransferase DOT1L is EPZ-5676.

9. The method of claim 1, wherein the a) the population of pancreatic endoderm and endocrine precursor cells and/or b) the pancreatic endocrine cell population, are cultured in suspension cultures.

10. A method for producing a pancreatic endocrine cell population expressing MAFA, comprising:

(a) obtaining a pancreatic endoderm and endocrine precursor cell population; and

(b) culturing the pancreatic endoderm and endocrine precursor cell population in a medium, wherein the medium comprises (i) a SMO inhibitor or a SHH signaling pathway antagonist, (ii) a BMP inhibitor, (iii) T3, an analogue of T3, T4, an analogue of T4 or a mixture thereof, (iv) an ALK5 inhibitor, and (v) an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, or a combination thereof,

thereby producing the pancreatic endocrine cell population, wherein cells in the pancreatic cell endocrine population express MAFA.

11. The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising with the aurora kinase inhibitor.

12. The method of claim 11, wherein the aurora kinase inhibitor is aurora kinase inhibitor II, SNS 314 mesylate, GSK1070916 or TAK-901.

13. The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising the RSK inhibitor.

14. The method of claim 13, wherein the RSK inhibitor is RSK inhibitor II.

15. The method of claim 10, wherein step (b) comprises culturing the pancreatic endoderm and endocrine precursor cell population in the medium comprising the inhibitor of protein methyltransferase DOT1L.

16. The method of claim 15, wherein the inhibitor of protein methyltransferase DOTIL is EPZ-5676.