Modelling neuropathic pain in vitro: a multimodal approach using induced-pluripotent stem cell-derived sensory neurons and mechanically compressed keratinocytes

Neuropathic pain is a chronic pain disorder arising from a dysfunction of the somatosensory nervous system, often resulting in altered activity of voltage-gated sodium channels (Nav). However, the molecular mechanism is complex and not fully understood. Therefore, this thesis investigated the development of advanced in vitro models for neuropathic pain, with a focus on sensory neurons and the keratinocytes surrounding them in the skin. Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells of healthy individuals and neuropathic pain patients offer a unique opportunity to obtain human sensory neurons harboring the donor’s genotype. IPSC-derived sensory neurons have been successfully used to model phenotypes of small fiber neuropathy (SFN) patients with mutations in Nav1.7. The tetrodotoxin-resistant (TTXr) channels Nav1.8 and Nav1.9 critically modulate nociceptor excitability, and variants have been linked to neuropathic pain. However, the functional presence of Nav1.8 and Nav1.9 in iPSC-derived sensory neurons is unclear. Here, iPSCs from two SFN patients with a putative pathogenic Nav1.9-Y66S variant were employed to evaluate iPSC-sensory neurons as neuropathic pain models, focusing on TTX-resistant currents, neuronal excitability, and action potential characteristics, as well as modelling of the Nav1.9-Y66S phenotype. The three iPSC-sensory neuron differentiation protocols employed – small-molecule- or transcription factor overexpression-driven – yielded comparable TTXr currents. However, no persistent Nav1.9-like current or clearly-identifiable Nav1.8 current could be detected, indicating that the TTXr currents largely consist of Nav1.5 current. This is consistent with a hyperpolarizing shift of activation and inactivation of iPSC-sensory neurons compared to mouse dorsal root ganglia (DRG) and indicates a developmental stage of the iPSC-sensory neurons distinct from mature DRG neurons. In contrast, the neuronal excitability and action potential characteristics were comparable to native human DRG neurons. Disease modelling of Nav1.9-Y66S revealed increased firing frequency and narrowed action potentials in patient-derived sensory neurons in the small molecule-driven differentiation, raising the possibility of marginal Nav1.9 activity below the detection limit or additional modulatory factors contributing to the phenotype. Recent studies have attributed an essential role to keratinocytes in touch and pain sensation. Patients with pachyonychia congenita (PC) carry mutations in keratins, resulting in palmoplantar keratosis, which increases the mechanical load locally, and neuropathic pain in the affected regions. Shaving of the callus does not alleviate the pain, indicating a chronic pain phenotype. Therefore, this thesis aimed to investigate whether mechanically compressed keratinocytes could sensitize sensory nerves. For this, a keratinocyte monolayer cyclic compression system was developed. Keratinocytes from PC patients intrinsically showed a pro-inflammatory phenotype trend on the mRNA level, which was exacerbated by cyclic mechanical compression for 1 h at 150 mHz at 0.9 kPa compressive force. However, conditioned media from compressed keratinocytes failed to alter the excitability of mouse DRG neurons. In conclusion, iPSC-sensory neurons, despite displaying limited Nav1.8 or Nav1.9 currents, demonstrate potential for disease modelling, but the model requires further advancements to allow for precise modelling of native human nociceptors. Mechanically stressed keratinocytes may contribute to pain in PC through sensory nerve sensitization via inflammation, but further experiments are required to understand the extent and molecular mechanisms behind it. Both iPSC- sensory neurons and mechanically compressed keratinocytes present valuable models to answer burning questions in pain research, but will benefit from further optimization and ultimately integration into larger, more complex models featuring multiple cell types.