Figure 4.

Using IPSC-derived nociceptors to develop therapeutics for pain disorders. (A) Illustration of the protocol and timeline of events to generate mature IPSC-derived nociceptors (adapted from Chambers et al.¹⁰² and Meentz et al.¹⁰⁴; immunomicrograph adapted from Clark et al.¹²¹). [B(i)] The use of CRISPR/cas9 gene editing to generate IPSC-derived nociceptors edited to constituently express HA-epitope tagged Na_v1.7. (ii) Current-clamp analysis of the current require to elicit and action potential of IPSC-derived nociceptors derived from healthy participants and congenital insensitivity to pain (CIP, due to Na_v1.7 loss-of-function) participants. CIP participant IPSC-derived nociceptors were hypoexcitable. The CIP participant hypoexcitability was corrected using CRISPR/Cas9 IPSC editing. CRISPR/Cas9 was used to generate Na_v1.7-KO IPSCs that were differentiated into nociceptors that mimicked CIP hypoexcitability (adapted from McDermott et al.¹⁰⁶). (C) Current-clamp analysis of the rheobase (minimum current required to elicit and action potential) of IPSC-derived nociceptors from wild-type participants, and participants carrying the F139WfsX24 TRESK loss-of-function mutation. IPSC-derived nociceptors from F139WfsX24 are hyperexcitable compared to wild-type nociceptors. This nociceptor hyperexcitability was rescued when using CRISPR/cas9 to correct the TRESK mutation (adapted from Pettingill et al.¹⁰³). (D) IPSC-derived nociceptors transduced with the chemogenetic silencing tool GluCl. Scale bar = 50 µm. Patch clamp analysis revealed that following application of the GluCl agonist ivermectin (IVM), GluCl+ nociceptors were rendered either fully silent or partially silent to depolarizing current injections (adapted from Weir et al.¹²²). (E) Example of stem cell derived nociceptors that have been used to model a chemical injury model through the addition of H₂O₂. H₂O₂ treatment leads increased caspase expression and neurite retraction/disassembly (adapted from Jones et al.¹¹⁹) [F(i)] IPSC-derived nociceptors derived from healthy control (HC1) or CRIPSR/cas9 engineered Na_v1.7 KO lines. The Na_v1.7 blocker BIIB074 promoted action potential failure in both HC1 and Na_v1.7 KO lines. (ii) Current clamp recordings of HC1 and Na_v1.7 KO nociceptors in the presence of vehicle or PF-05089771. Only HC1 but not Na_v1.7 KO nociceptors demonstrated changes in excitability in response to the PF-05089771 compound (adapted from McDermott et al.¹⁰⁶). (G) Multi-electrode arrays (MEAs) used to assess spontaneous activity of IPSC-derived nociceptors from an IEM participant. Activity at electrodes was reduced after addition of the TRESK activator cloxyquin. Spontaneous activity recovery was lost following co-addition of TPA (TRESK inhibitor) and cloxyquin (adapted from Pettingill et al.¹⁰³).