Methods for producing insulin-secreting beta cells from human pluripotent stem cells

A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:

• (a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;
• i) fibroblast growth factor (FGF),
• ii) Activin A, and
• iii) bone morphogenetic protein (BMP);
• (b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;
• i) insulin, transferrin, and selenium (ITS), and
• ii) fibroblast growth factor (FGF);
• (c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium under conditions that induce formation of posterior foregut cells, wherein the medium comprises an effective amount of;
• i) insulin, transferrin, and selenium (ITS), and
• ii) Noggin-Nicotinamide-Retinoic acid (NNR);
• and
• (d) culturing the cells from step (c) for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;
• i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and
• ii) Noggin,
• wherein the cells are cultured on an extracellular matrix coated onto a membrane, and
• wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin+ cells.