Methods for production of functional beta cells

• US 10,633,635 B2
• Filed: 06/14/2017
• Issued: 04/28/2020
• Est. Priority Date: 06/21/2016
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rieck, Sebastian, Rezania, Alireza
• Primary Examiner(s): Ton, Thaian N.
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US15/622,931
• Publication Number:
  US 20170362572A1
• Time in Patent Office: 1,049 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/345 Gastrin; Cholecystokinins [CCK]
• CPC Class Codes: C12N 2501/375 Thyroid stimulating hormone [TSH]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/068 : Stem cells; Progenitors
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The invention provides for methods of differentiating pancreatic endocrine cells into pancreatic beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A. These pancreatic beta cells may be obtained by step-wise differentiation of pluripotent stem cells. The pancreatic beta cells exhibit glucose-dependent mitochondrial respiration and glucose-stimulated insulin secretion similar to islet cells.

Claims

1. A method of maturing an immature pancreatic beta cell into a mature pancreatic beta cell expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 comprising:

culturing the immature pancreatic beta cell in a medium supplemented with one or more of UNC0638, UNC0642, UNC0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568, thereby maturing the immature pancreatic beta cell into the mature beta cell.

2. The method of claim 1, wherein the medium is further supplemented with i) heparin, ii) N-Acetyl Cysteine, and iii) one or more of T3, T4 and an analogue thereof.

3. The method of claim 1, wherein the medium lacks an ALK5 inhibitor.

4. The method of claim 1, wherein the medium is further supplemented with an ALK5 inhibitor.

5. The method of claim 1, wherein the medium is supplemented with

i) ZM447439;

ii) heparin;

iii) N-Acetyl Cysteine; and

iv) one or more of T3, T4 and an analogue thereof;

and wherein the medium lacks an ALK5 inhibitor.

6. The method of claim 5, wherein the medium is supplemented with T3.

7. The method of claim 6, wherein the medium is supplemented with AZT.

8. The method of claim 7, wherein the medium is supplemented with DEZA.

9. The method of claim 1, wherein the immature beta cell is obtained by step-wise differentiation of definitive endoderm cells, primitive gut tube cells, foregut endoderm cells, pancreatic endoderm cells, or pancreatic endocrine precursor cells.

10. The method of claim 1, wherein the method comprises culturing the immature pancreatic beta cell at the air-liquid interface.

11. The method of claim 1, wherein the method comprises culturing the immature pancreatic beta cell in suspension clusters.

41. The method of claim 6, wherein the medium is supplemented with 1 nM to 100 nM T3.

12. A method of differentiating pluripotent stem cells into mature beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A:

a) differentiating pluripotent stem cells into definitive endoderm cells;

b) differentiating the definitive endoderm cells of step a) into primitive gut tube cells;

c) differentiating the primitive gut tube cells of step b) into foregut endoderm cells;

d) differentiating the foregut endoderm cells of step c) into pancreatic endoderm cells;

e) differentiating the pancreatic endoderm cells of step d) into pancreatic endocrine precursor cells;

f) differentiating the pancreatic endocrine precursor cells of step e) into immature beta cells; and

g) culturing the immature beta-cells of step f) in a medium supplemented with one or more of UNC0638, UNC0642, UNC0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568 to mature the immature beta cells into the mature beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1.

13. The method of claim 12, wherein the mature beta cells exhibit glucose-dependent mitochondrial respiration.

14. The method of claim 12, wherein the mature beta cells exhibit glucose-stimulated insulin secretion.

15. The method of claim 12, wherein the mature beta cells secrete insulin in multiple phases.

16. The method of claim 12, wherein the medium is further supplemented with:

i) heparin;

ii) N-Acetyl Cysteine; and

iii) one or more of T3, T4 or an analogue thereof.

17. The method of claim 12, wherein the medium lacks an ALK5 inhibitor.

18. The method of claim 12, wherein the medium is further supplemented with T3.

19. The method of claim 12, wherein the medium is supplemented with

i) ZM447439;

ii) heparin;

iii) N-Acetyl Cysteine; and

iv) one or more of T3, T4 or and an analogue thereof, and

wherein the medium lacks an ALK5 inhibitor.

20. The method of claim 19, wherein the medium is supplemented with T3.

21. The method of claim 20, wherein the medium is further supplemented with AZT.

22. The method of claim 21, wherein the medium is further supplemented with DEZA.

23. The method of claim 12, wherein the method comprises culturing the immature beta cells at the air-liquid interface.

24. The method of claim 12, wherein the method comprises culturing the immature beta cells in suspension clusters.

25. The method of claim 12, wherein step a) comprises differentiating the pluripotent stem cells into the definitive endoderm cells by culturing the pluripotent stem cells in a medium supplemented with MCX compound and GDF-8.

26. The method of claim 12, wherein step b) comprises differentiating the definitive endoderm cells into the primitive gut tube cells by culturing the definitive endoderm cells in a medium supplemented with FGF7 and ascorbic acid.

27. The method of claim 12, wherein step c) comprises differentiating the primitive gut tube cells into the foregut endoderm cells by culturing the primitive gut tube cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid.

28. The method of claim 12, wherein the step d) comprises differentiating the foregut endoderm cells into the pancreatic endoderm cells by culturing the foregut endoderm cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid.

29. The method of claim 12, wherein step e) comprises differentiating the pancreatic endoderm cells into pancreatic endocrine precursor cells by culturing the pancreatic endoderm cells in a medium supplemented with SANT-1, a PKC activator, a BMP inhibitor, and ascorbic acid.

30. The method of claim 29, wherein the medium is further supplemented one or more of T3, T4 or an analogue thereof.

31. The method of claim 12, wherein step f) comprises culturing the pancreatic endocrine precursor cells into immature beta-cells by culturing the pancreatic endocrine precursor cells in a medium supplemented with a i) a BMP inhibitor, ii) ascorbic acid, and iii) one or more of T3, T4 and an analogue thereof.

32. The method of claim 31, wherein the medium is further supplemented with an ALK 5 inhibitor or a gamma secretase inhibitor.

33. The method of claim 12, wherein step g) comprises culturing immature beta-cells in a medium lacking an ALK5 inhibitor and supplemented with ZM447439, AZT, N-acetyl cysteine, DEZA, Formulation I and one or more of T3, T4 and an analogue thereof.

34. The method of claim 33, wherein the mature beta cells exhibit glucose-dependent mitochondrial respiration.

35. The method of claim 33, wherein the mature beta cells exhibit glucose-stimulated insulin secretion.

36. The method of claim 34, wherein the glucose-dependent mitochondrial respiration has an oxygen consumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate.

37. The method of claim 36, wherein the oxygen consumption rate response occurred at least 15 minutes following glucose stimulation.

38. The method of claim 35, wherein the glucose-stimulated insulin secretion comprises a bi-phasic insulin secretion in response to glucose stimulation.

39. The method of claim 38, wherein a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase has at least a two-fold increase to at least a four-fold increase over the baseline secretion.

40. The method of claim 38, wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.

42. The method of claim 30, wherein the medium is supplemented 1 nM to 1 μ

M T3.

43. The method of claim 12, wherein the pluripotent stem cells are human H1 or H9 cells.