MEL1 and H9 cells were subjected to the pancreatic differentiation protocol of D'Amour et al. [21, 23], with slight modifications. hESCs were inoculated into Matrigel-coated 24-well plates at a density of 2.1 × 105/cm2. The differentiation of cells was directly differentiated by growth factors and small molecules, which mimics in vivo pancreatic organogenesis [23]. The differentiation process comprised four stages: definitive endoderm (stage I), gastrulation (stage II), endocrine precursor (stage III), and pancreatic endocrine cell (stage IV) (see Figure 1(a)). hESCs were differentiated to primitive endoderm using STEMdiff Basal Medium or RPMI as the basic culture medium supplemented with 100 ng/mL activin A and 25 ng/mL Wnt3a.

A four-stage protocol for differentiation of human embryonic stem cells to insulin-producing cells. (a) Flowchart of the cell differentiation process. Differentiation is divided into four stages: definitive endoderm (stage I), gastrulation (stage II), endocrine precursor (stage III), and pancreatic endocrine cell (stage IV). (b) The expression of marker genes at each stage of the differentiation process. ∗∗∗ indicates P < 0.001, ∗∗ indicates P < 0.01, and ∗ indicates P < 0.05. (c) Immunofluorescence detection of PDX-1 (samples collected on D13) and INSULIN (samples collected on D21). Scale bar =100 μm.

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