METHODS FOR GENERATING STEM CELL-DERIVED BETA CELLS AND USES THEREOF

• US 20160175363A1
• Filed: 12/18/2015
• Published: 06/23/2016
• Est. Priority Date: 12/18/2014
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Melton, Douglas A., Millman, Jeffrey R.
• Application Number:
  US14/975,383
• Publication Number:
  US 20160175363A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 9/0024 Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue compositions for intravenous administration, normal injectable solutions or dispersions for, e.g. subcutaneous administration A61K9/0019; brain implants A61K9/0085; (coated) prostheses, catheters or stents A61L
• CPC Class Codes: A61K 9/50 Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals A61K9/2081 takes precedence; particles with a single coating comprising drug A61K9/167
• CPC Class Codes: A61K 9/7007 Drug-containing films, membranes or sheets A61K9/0041, A61K9/0043, A61K9/006, A61K9/0063 take precedence
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/07 from endocrine cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

Disclosed herein are methods for generating SC-β cells, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

Claims

1. A method for generating stem cell-derived β

(SC-β

) cells, the method comprising contacting a cell population comprising endocrine progenitor cells or precursors thereof under conditions suitable for directing differentiation of said cells into SC-β

cells with an effective amount of a first agent that specifically inhibits the level and/or activity of at least one activin receptor-like kinase (ALK), thereby generating SC-β

cells.

2. The method of claim 1, wherein the endocrine progenitor cells comprise PDX1+/NKX6.1+/NEUROD1+/insulin+/glucagon−

/somatostatin−

cells.

3. The method of claim 1, wherein the precursors are selected from the group consisting of pluripotent stem cells, SOX17+ definitive endoderm cells, PDX1+ primitive gut tube cells, PDX1+/NKX6.1+ pancreatic progenitor cells, PDX1+/NKX6.1+/NEUROD1+ endocrine progenitor cells.

4. The method of claim 1, wherein the endocrine progenitor cells are directed to differentiate into SC-β

cells by contacting the endocrine progenitor cells under conditions that promote cell clustering with i) a transforming growth factor β

(TGF-β

) signaling pathway inhibitor and ii) a thyroid hormone signaling pathway activator to induce the in vitro maturation of at least some of the endocrine progenitor cells into SC-β

cells.

5. The method of claim 1, wherein the effective amount of the first agent comprises a concentration range of between 0.1 μ

M and 110 μ

M.

6. The method of claim 1, further comprising contacting the cell population with an effective amount of at least a second agent that specifically inhibits the level and/or activity of at least one ALK.

7. The method of claim 6, wherein the effective amount of the second agent comprises a concentration range of between 0.1 μ

M and 110 μ

M.

8. The method of claim 1, wherein the at least one ALK is selected from the group consisting of ALK1, ALK2, ALK3, ALK4, ALK5, ALK6 and ALK7.

9. The method of claim 6, wherein the first agent and/or the second agent is selected from the group consisting of SB431542, DMH-1, and Alk5 inhibitor II.

10. The method of claim 1, wherein the at least one ALK is selected from the group consisting of ALK1, ALK2, ALK3, ALK4, ALK6 and ALK7.

11. The method of claim 1, wherein the at least one ALK is selected from the group consisting of SB431542 and DMH-1.

12. The method claim 6, wherein the first agent and/or the second agent is not ALK5 inhibitor II.

13. The method of claim 6, wherein the first agent and/or the second agent exhibits an IC₅₀ for the at least one ALK that is less than or equal to 500 nm or wherein the cells are contacted with a concentration of the first agent and/or the second agent that is equal to or greater than its IC50 value for at least one ALK.

14. The method claim 6, wherein the first agent and/or the second agent is more selective for the at least one ALK than for at least one mitogen activated protein kinase (MAPK) or other kinases.

15. The method of claim 1, wherein between at least 5% and 65% of the endocrine cells in the population differentiate into SC-β

cells.

16. An isolated non-native SC-β

cell or population thereof generated according to the method of claim 1 that exhibits a glucose stimulated insulin secretion (GSIS) response both in vitro and in vivo.

17. An isolated non-native SC-β

cell or population thereof according to claim 16 that exhibits a stimulation index that is at least between 1.5-fold and 10-fold greater than the stimulation index of a control SC-β

cell.

18. An isolated non-native SC-β

cell or population thereof according to claim 16 that produces between approximately 300 uIU and 4000 uIU per 30 minute incubation at a high glucose concentration.

19. An isolated non-native SC-β

cell or population thereof according to claim 16 that two weeks after transplantation into a subject in vivo releases between 3 uIU/mL and 81 uIU/mL of insulin within 30 minutes of administering 2 g/kg glucose to the subject.

20. A microcapsule comprising the isolated non-native SC-β

cell or population thereof according to claim 16 encapsulated therein.

21. A microencapsulation device comprising the isolated non-native SC-β

cell or population thereof according to claim 16 encapsulated therein.

22. A method for the treatment of a subject in need thereof, the method comprising administering to a subject in need thereof an isolated population of non-native SC-β

cells produced according to the method of claim 1.

23. An artificial islet or pancreas comprising of non-native SC-β

cells produced according to the method of claim 1.