DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO PANCREATIC ENDOCRINE CELLS USING HB9 REGULATORS

• US 20190211309A1
• Filed: 11/26/2018
• Published: 07/11/2019
• Est. Priority Date: 12/31/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Granted Patent: US 10,947,511 B2
• Time in Patent Office: Days
• 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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

Abstract

The present invention provides methods to promote differentiation of pluripotent stem cells to pancreatic endoderm cells expressing PDX1, NKX6.1, and HB9. In particular, the methods encompass culturing Stage 4 to Stage 6 cells with a thyroid hormone (e.g. T3), an ALK5 inhibitor, or both.

Claims

1. An in vitro cell culture for differentiating cells derived from human pluripotent stem cells comprising:

a. a culture vessel;

b. a volume of differentiation medium; and

c. a population of differentiated cells derived from human pluripotent stem cells wherein at least ten percent of said differentiated cells co-express PDX1, NKX6.1 and HB9, wherein said differentiation medium comprises a growth medium supplemented with a thyroid hormone selected from triiodothyronine, thyroxine, analogues of triiodothyronine, analogues of thyroxine and mixtures thereof, or both a thyroid hormone and ALK5 inhibitor.

2. The cell culture of claim 1, wherein said ALK5 inhibitor is SD208, ALK5 inhibitor II, or ALX-270-445.

3. The cell culture of claim 2, wherein said growth medium is MCDB131.

4. The cell culture of claim 1, wherein said differentiated cells comprise cells expressing markers characteristic of pancreatic endocrine cells.

5. The cell culture of claim 2, wherein said growth medium is further supplemented with one or more of:

a. a smoothened receptor inhibitor selected from MRT10 or cyclopamine;

b. a SHH signaling pathway antagonist selected from SANT-1 or HPI-1;

c. a BMP Receptor Inhibitor selected from LDN-193189, Noggin or Chordin;

d. a PKC activator selected from TPB, PDBu, PMA, and ILV;

e. a fibroblast growth factor selected from FGF7 or FGF10;

f. retinoic acid;

g. ascorbic acid;

h. heparin; and

i. zinc sulfate.

6. The cell culture of claim 5, wherein said growth medium is further supplemented with SANT-1, retinoic acid and ascorbic acid.

11. The cell culture of claim 4, wherein said differentiated cells comprise cells expressing markers characteristic of β

cells.

12. The cell culture of claim 11, wherein said differentiated cells produce insulin.

7. An in vitro cell culture comprising a population of differentiated human pluripotent stem cells expressing markers characteristic of pancreatic endocrine cells wherein at least ten percent of said cells express HB9, PDX1 and NKX6.1.

8. The cell culture of claim 7, wherein at least thirty percent of the cells that express both NKX6.1 and PDX1 also express HB9.

9. The cell culture of claim 7, wherein at least fifty percent of the cells that express both NKX6.1 and PDX1 also express HB9.

10. The cell culture of claim 7, wherein at least eighty percent of the cells that express both NKX6.1 and PDX1 also express HB9.