Compositions and methods for differentiating stem cells into cell populations comprising beta-like cells
Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce populations of cells comprising beta-like cells (e.g., pancreatic lineage, glucose-responsive, and/or insulin-producing). In particular, culture conditions are provided that result in the generation of beta-like cells from a starting culture of human pluripotent stem cells.
1. A method of culturing human pluripotent stem cells to produce a cell population comprising pancreatic β-like cells comprising:
(a) culturing the stem cells on an extracellular matrix for 2-4 days in a chemically defined medium, basic fibroblast growth factor, Activin A, and BMP4;
(b) culturing the cells on an extracellular matrix from step (a) for 2-4 days in the presence of chemically defined insulin, transferrin and selenium (ITS) medium, FGF7, and nicotinamide;
(c) culturing the cells on an extracellular matrix from step (b) for 3-5 days in the presence of a chemically defined ITS medium; retinoic acid; Noggin; and nicotinamide; and
(d) culturing the cells on an extracellular matrix from step (c) for 6-10 days in the presence of a B27 serum-free medium, IGF I, IGF II, FGF7, insulin, nicotinamide, exendin-4, an ALK5i II, and forskolin, thereby producing insulin+glucagon−cells that exhibit in vitro glucose stimulated insulin secretion.
2. The method of claim 1 , further comprising:
(e) maintaining the cells from step (d) for 1-14 days by culturing the cells in the presence of B27 serum-free medium, IGF I, FGF7, insulin, nicotinamide, exendin-4, and forskolin.
3. The method of claim 1 , wherein said step (c) is performed in two steps.
4. The method of claim 3 , wherein said first step of step (c) comprises culturing in the presence of chemically defined ITS medium; retinoic acid; and Noggin for 2 days and then culturing in in the presence of chemically defined ITS medium, EGF, and FGF7 for two days.
5. The method of claim 1 , wherein said step (d) further comprises EGF.
6. The method of claim 1 , wherein said step (e) further comprises Warfarin.
7. A method of culturing human pluripotent stem cells to produce a cell population comprising pancreatic β-like cells comprising:
(a) culturing the stem cells on an extracellular matrix for 2-4 days in a chemically defined medium, basic fibroblast growth factor, Activin A, BMP4, LY294002;
(b) culturing the cells from step (a) on an extracellular matrix for 2-4 days in the presence of chemically defined ITS medium, FGF7, nicotinamide;
(c) culturing the cells from step (b) on an extracellular matrix for 2 days in the presence of a chemically defined ITS medium, retinoic acid, Noggin, nicotinamide;
(d) culturing the cells of step (c) for 2 days in the presence of chemically defined insulin, transferrin and selenium (ITS) medium, EGF, and FGF7;
(e) culturing the cells from step (d) on an extracellular matrix for 6-10 days in the presence of a B27 serum-free medium, IGF I, IGF II, FGF7, insulin, Noggin, nicotinamide, exendin-4, ALK5i II, forskolin, thereby producing insulin+glucagon−cells that exhibit in vitro glucose stimulated insulin secretion; and
(f) maintaining the cells of step (e) for 1-50 days by culturing the cells in suspension culture in the presence of a serum-free medium, ALK5, Forskolin, ZnS04, T3, B27, heparin, and antioxidant, Warfarin, and a P13 kinase inhibitor.