Characterisation of calcitonin receptor expression and signalling in iPSC-derived sensory neurons

Introduction Human induced pluripotent stem cell-derived sensory neurons (iPSC-SNs) provide an accessible and physiologically relevant model for studying neuropeptide systems implicated in migraine and chronic pain. The calcitonin peptide family, including calcitonin gene-related peptide (CGRP), amylin, and their receptors, plays key roles in trigeminal and dorsal root ganglia (DRG) sensory neuron function. However, whether iPSC-SNs replicate the calcitonin family expression and signalling reported in trigeminal ganglia (TG) and DRG sensory neurons remains unknown. Therefore, we characterised the expression and function of the CGRP and amylin receptor system in human iPSC-SNs. Methods Human iPSCs were differentiated into sensory neurons, then assessed for gene and protein expression using RT-qPCR and immunofluorescence with validated antibodies. Receptor function was evaluated using ligand-evoked cAMP assays and compared with COS-7 cells expressing defined receptor constructs. Results iPSC-SNs upregulated peripheral and sensory neuron markers ( TUBB3, PRPH, SCN10A, SCN11A, POU4F1 , TRPV1 , NTRK1 , and ISL1 ) and formed extensive β-tubulin III-positive neurite networks. Compared with undifferentiated iPSCs, iPSC-SNs displayed increased gene expression of calcitonin receptor (CTR), calcitonin receptor-like receptor, and RAMP1, reduced RAMP2, and relatively low RAMP3. Immunofluorescence detected CGRP, amylin, and CTR protein, with amylin predominantly localised to neuronal Soma. Functionally, calcitonin produced robust cAMP responses, whereas CGRP, amylin, and adrenomedullin yielded weaker signalling. This potency hierarchy matched that of CTR-expressing COS-7 cells, indicating that iPSC-SNs chiefly signal via the CTR rather than CGRP or AMY 1 receptor complexes. Conclusion Human iPSC-SNs recapitulate sensory neuron-like co-expression of CTR, CGRP, and amylin and exhibit dominant CTR-mediated signalling. These findings support their utility as a model translational platform for dissecting neuropeptide-driven pain mechanisms and developing receptor-targeted therapeutics.