Exploring the Role of Insulin and IGF-I Receptor Signaling in the Pluripotency, Differentiation, and Maintenance of Stem Cells

Pluripotent stem cells (PSCs) express insulin receptors (IR) and insulin-like growth factor-1 receptors (IGF-1R). In human embryonic stem cells (hESCs), IGF-1R is necessary for cell survival, self-renewal and differentiation into endoderm. Data on the role of IR in hESCs is limited; it’s been linked to self-renewal and pluripotency of murine neural stem cells. What’s still unclear is the significance of IR and IGF-1R in modulating stem cell function, pluripotency and differentiation. We created murine induced PSCs (iPSCs) with double knockout (DKO) of IGF-1R and IR. Analysis of 4 individual iPSC clones from 4 control and DKO mice showed similar cell proliferation and morphology. QPCR of pluripotency markers showed significant decreases in Lin28a (p<0.005) and Tbx3 (p<0.0005) expression in DKOs; Lin28a protein levels were also decreased. IPSCs from both groups developed into embryoid bodies in vitro and formed teratomas in vivo, indicating equal abilities to differentiate. With directed differentiation towards a neural lineage, DKOs showed upregulation of neural markers Brn2, Pax6, Sox1, Ngn2 and NeuN. We next examined downstream signaling of IR and IGF-1R. Although both groups showed mTor, Akt and Erk phosphorylation in the basal state, mTor phosphorylation in DKOs was significantly lower (28% of controls; p<0.05). Total mTor and Akt protein levels in DKOs were also lower (31% and 59% of controls, respectively; p<0.05). Prior to stimulation, basal phosphorylation of Stat3 was evident only in DKOs. It was increased 3-fold (p<0.05) upon treatment with IGF-1 or insulin (100 nM) compared to controls. Stimulation with LIF (100 unit/mL) led to Stat3 phosphorylation in both groups; this was 1.5-fold greater in DKOs compared to controls (p<0.005). These data suggest activation of a receptor independent of IR and IGF-1R and/or alterations in downstream proteins in the canonical IR/IGF-1R pathway that allow DKO iPSCs to maintain pluripotency and undergo differentiation.