Essential roles of insulin and IGF-1 receptors during embryonic lineage development.

The insulin and insulin-like growth factor-1 (IGF-1) receptors are important for the growth and development of embryonic tissues. To directly define their roles in the maintenance of pluripotency and differentiation of stem cells, we knocked out both the receptors in induced pluripotent stem cells (iPSCs). iPSCs lacking both the insulin and IGF-1 receptors (double knock-out, DKO) exhibited preserved pluripotency potential despite decreased expression of the transcription factors, Lin28a and Tbx3, compared to control iPSCs. While embryoid body and teratoma assays revealed an intact ability of DKO iPSCs to form all three germ layers, the latter were composed of primitive neuroectodermal tumor-like cells in the DKO group. RNAseq analyses of control versus DKO iPSCs revealed differential regulation of pluripotency, developmental, E2F1 and apoptosis pathways. Signaling analyses pointed to downregulation of the AKT/mTOR pathway and an upregulation of the Stat3 pathway in DKO iPSCs in the basal state and following stimulation with insulin/IGF-1. Directed differentiation towards the three lineages was dysregulated in DKO iPSCs with significant downregulation of key markers (Cebpα, Fas, Pparγ, Fsp27) in adipocytes and transcription factors (Ngn3, Isl1, Pax6 and Neurod1) in pancreatic endocrine progenitors. Furthermore, differentiated pancreatic endocrine progenitor cells from DKO iPSCs showed increased apoptosis. We conclude that the insulin and insulin-like growth factor-1 receptors are indispensable for normal lineage development and perturbations in the function and signaling of these receptors leads to upregulation of alternative compensatory pathways to maintain pluripotency.