Generation of human nociceptor-enriched sensory neurons for the study of pain-related dysfunctions

Abstract

In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically-useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly-enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9,000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Bridging the Translational Gap in Chemotherapy-Induced Peripheral Neuropathy with iPSC-Based Modeling

“…Oxaliplatin affects specific ion channels on peripheral neurons leading to cold hypersensitivity, a unique feature of oxaliplatin-induced peripheral neuropathy [ 46 ]. Platinum-based agents have a delayed onset of symptoms that often worsen after treatment cessation, a phenomenon called coasting [ 47 ].…”

EFSA Pilot Project on New Approach Methodologies (NAMs) for Tebufenpyrad Risk Assessment. Part 2. Hazard characterisation and identification of the Reference Point

“…The differentiation was essentially performed as described before (Holzer 2022a;Holzer 2022b;Hoelting 2016). Details are given Annex A (supplementary methods and Annex A5 (ToxTemp UKN5 (Blum 2022))).…”

Paclitaxel- and vincristine-induced neurotoxicity and drug transport in sensory neurons

“…Paclitaxel and vincristine, two antitubulin agents with opposite effects on microtubules, have both been proposed to exhibit neurotoxicity by targeting the distal nerve terminals on sensory neurons (15,16). Oxaliplatin on the other hand affects specific ion channels in cold-sensing sensory neurons, leading to neuronal hyperexcitability (17), while bortezomib primarily targets the myelin sheaths causing structural and functional alterations in sensory neurons (14). This explains why patients present with differences in symptom onset and clinical presentation when treated with distinct chemotherapies.…”