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In the multi-step differentiation protocol used to generate pancreatic endocrine cells from human pluripotent stem cells, the induction of NGN3+ endocrine precursors from the PDX1+/NKX6.1+ pancreatic endoderm is crucial for efficient endocrine cell production. Here, we demonstrate that transient, not prolonged FOXO1 inhibition results in enhanced NGN3+ endocrine precursors and hormone-producing cell production. FOXO1 inhibition does not directly induce NGN3 expression but stimulates PDX1+/NKX6.1+ cell proliferation. NOTCH activity, whos
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