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Media compostions containing low concentrations of glucose useful for human embryonic stem cells, differentiation method of human embryonic stem cells into insulin-producing cells or cell clusters using thereof, and insulin-producing cells or cell clusters differentiated thereby
Media compostions containing low concentrations of glucose useful for human embryonic stem cells, differentiation method of human embryonic stem cells into insulin-producing cells or cell clusters using thereof, and insulin-producing cells or cell clusters differentiated thereby
A medium composition containing low concentration of glucose for culturing human embryonic stem cells is provided to reduce insulin tolerance of cells shown by long-term exposure to high concentration of glucose environment during cultivation of cells and produce insulin-producing cells having enhanced insulin production yield. A DMEM/F12(Dulbecco's modified eagle medium/Ham's F12) medium composition for inducing differentiation of human embryonic stem cells into insulin-producing cells optionally contains low concentration such as 5-10 mM of glucose, and further contains 10mM of nicotinamide. A method for producing insulin-producing cells or cell clusters differentiated thereby comprises the steps of: (1) culturing undifferentiated human embryonic stem cells in a DMEM/F12 medium containing 5-10mM of glucose, 15% of serum replacement and 2 ng/mL of FGF-2; (2) floating culturing the cultured cells in a DMEM/F12 medium containing 5-10mM of glucose and 15% of serum replacement to form embryoid body; (3) inducing differentiation from embryoid body to endodermal stem cells by using a DMEM/F12 medium containing 5-10mM of glucose, 15% of serum replacement and 50-100 ng/mL of activin A; (4) selecting the differentiated cells to the endodermal stem cells by using a DMEM/F12 medium containing 5-10mM of glucose, 0.5-2.0 mg/mL of insulin, 0.25-1.0 mg/mL of transferring and 0.25-1.0 mug/mL of sodium selenite; (5) inducing differentiation into precursor cells of insulin-producing cells and their proliferation by using a DMEM/F12 medium containing 5-10mM of glucose and 5-20ng/mL of betacellulin; (6) inducing differentiation from the precursor cells to insulin-producing cells by using a DMEM/F12 medium containing 5-10mM of glucose and 10mM of nicotinamide; and (7) inducing insulin-producing cell clusters by using a DMEM/F12 medium containing 5-10mM of glucose and 10mM of nicotinamide.
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