Generation of human pluripotent stem cell derived functional beta cells showing a glucose-dependent mitochondrial respiration and two-phase insulin secretion response
AssigneeJanssen Biotech, Inc.
InventorsSebastian Rieck, Alireza Rezania
Life of this patent
Estimating the term…
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.
Claims97
A method of differentiating pancreatic endoderm cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 comprising culturing pancreatic endocrine cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.
An in vitro population of functional beta cells expressing single hormonal insulin, PDX1, NKX6.1 and MAFA obtained by in vitro differentiation of pancreatic endocrine cells.
An in vitro population of pancreatic beta cells that express single hormonal insulin, PDX1, NKX6.1 and MAFA obtained by culturing pancreatic endocrine cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.
A method of differentiating pluripotent stem cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A:
- a. Differentiating pluripotent stem cells into immature beta-cells;
- and
- b. Differentiating the immature beta-cells into functional beta-cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 by culturing immature beta-cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.
The method of 54, 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.
The method of 54, wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.
The method of 61, 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.
The method of 61, wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.
A method of differentiating pluripotent stem cells into functional beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A:
- a. Differentiating pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm obtained by culturing pluripotent stem cells in a medium supplemented with activin A and WNT3A;
- b. Differentiating the cells expressing markers characteristic of the definitive endoderm into immature beta-cells;
- and
- c. Differentiating the immature beta-cells into functional beta-cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 by culturing immature beta-cells in a medium supplemented with one or more of UNC0638, UNC0642, UCN0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568.
Dependencies are inferred from the claim text.