Method for generating mature β-like cells

• US 10,568,913 B2
• Filed: 03/28/2014
• Issued: 02/25/2020
• Est. Priority Date: 03/28/2013
• Status: Active Grant

• Current Assignee: Healthregen Pty Ltd
• Original Assignee: Healthregen Pty Ltd
• Inventors: Jiang, Fang-Xu
• Primary Examiner(s): Barron, Sean C.
• Application Number:
  US14/780,445
• Publication Number:
  US 20160045554A1
• Time in Patent Office: 2,160 Days
• Field of Search: 424 937, 435 34, 435377, 435 612, 435 71
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2506/22 from pancreatic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention relates to methods for generating mature insulin-producing β-like cells comprising the steps: (a) isolating, purifying and/or enriching β-cell progenitor cells from a population of cells; (b) differentiation of the β-cell progenitor cells into immature insulin-producing β-like cells; and (c) maturation of the immature insulin-producing β-like cells into mature insulin-producing β-like cells, comprising exposing the immature insulin-producing β-like cells to calcitriol or an analogue thereof. More particularly, the present invention relates to methods for generating mature insulin-producing β-like cells for use in treating diabetes.

Claims

1. A method to generate mature insulin-producing β

-like cells for treating a subject with diabetes mellitus, the method consisting essentially of the steps of;

(a) exposing purified, and/or enriched β

cell progenitor cells to a Sirt1 inhibitor at a concentration sufficient to cause differentiation of the β

-cell progenitor cells into immature insulin-producing β

-like cells;

wherein the immature insulin producing β

-like cells have insulin granule numbers equivalent to adult β

cells, and secrete approximately 10-fold more insulin content in response to stimulation with basal glucose and stimulus glucose compared to islet progenitor cells; and

(b) exposing the immature insulin-producing β

-like cells to calcitriol or an analogue thereof in vitro at a concentration sufficient to cause maturation of the immature insulin-producing β

-like cells;

wherein the mature insulin-producing β

-like cells secrete insulin content in response to basal glucose and stimulus glucose equivalent to healthy adult islets and at least 4-fold more compared to immature insulin-producing β

-like cells.

2. The method according to claim 1, wherein said subject has Type 1 or 2 diabetes mellitus.

3. The method according to claim 1, wherein the subject is a human.

4. The method of claim 1, wherein the Sirt1 is inhibited by 10 mM nicotinamide.

5. The method of claim 1, wherein the Sirt1 is inhibited by a nicotinamide analogue, wherein said nicotinamide analogue causes differentiation of β

-cell progenitor cells into immature insulin-producing β

-like cells.

6. The method of claim 1, further comprising a step of analyzing the mature insulin-producing β

-like cells in vitro to determine the levels of expression of insulin in response to exposure of the insulin-producing β

-like cells to glucose.

7. The method of claim 1, wherein the population of β

cell progenitor cells are exposed to the Sirt1 inhibitor between 4 and 8 days.

8. The method of claim 7, wherein the population of β

cell progenitor cells are exposed to the Sirt1 inhibitor between 4 and 6 days.