IPSCs were initially maintained on irradiated MEFs (MTI-GlobalStem) in media as previously described. For experimentation, iPSCs were exposed to Accutase (Stem Cell Technology) to aid in detachment from irradiated MEFs and plated onto gelatin-coated plates. The media was changed to ESGRO 2i media (Millipore) and the cells were passaged every other day for 1 week to deplete irradiated MEFs.

Alkaline phosphatase staining was performed after the cells were fixed with 10% neutral buffered formalin solution (Wako). Basal iPSC experiments were performed after iPSCs were grown for 16 hours in DMEM high-glucose, high-pyruvate media supplemented with 0.1% BSA (Wako) and 1 unit/mL of LIF (Millipore). For iPSC signaling experiments, cells were split and seeded at a density of 2 × 105 onto gelatin-coated 6-well plates. Cells were grown in DMEM/F-12 media with 1 unit/mL of LIF (Millipore) for 16 hours. Then 100 nM of insulin (Sigma–Aldrich), 100 nM of IGF1 (Sigma–Aldrich), or 100 units/mL of LIF (Millipore) were added to each well for 15 min.

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