Abstract
Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. Here, we identify five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons that recapitulate the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibit TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we show that the technique can reveal novel aspects of human disease phenotypes in vitro.
Highlights
Nociceptor neuron development occurs through dorsalization within the neural tube[18], followed by neural crest induction and migration[19] and nociceptor specification within the still-multipotent neural crest lineage[20]
These factors were chosen using a combination of the prior literature, transcription factor expression profiles in FACS-sorted adult mouse nociceptors (NaV1.8-positive) as compared to proprioceptors (Chiu et al, submitted), an expression profile similarity to NaV1.8 in the BioGPS database[27], and postnatal dorsal root ganglion (DRG) expression in the Allen Brain Atlas[28]
After transducing the fibroblasts with a combination of all 12 individual retroviruses containing the selected transcription factors, we detected a small number of tdTomato-positive cells with a neuronal morphology after two weeks (Supplementary Fig. 3a)
Summary
Nociceptor neuron development occurs through dorsalization within the neural tube[18], followed by neural crest induction and migration[19] and nociceptor specification within the still-multipotent neural crest lineage[20]. The generation of nociceptor progenitors expressing the TrkA neurotrophin receptor (Ntrk1) and postnatal nociceptors expressing TrpV1 requires the basic helix-loop-helix transcription factor Ngn[1] (Neurogenin1), which is normally present from approximately days E9–E13 in the embryonic mouse[21]. Developing nociceptors express multiple Trk-family receptors, maturing nociceptors express only TrkA. For non-peptidergic nociceptors, most of which bind isolectin B4, the glial cell derived neurotrophic factor (GDNF) receptor Ret replaces TrkA in a process dependent on Runx[1], and loss of Runx[1] markedly reduces TrpV1 expression[23]. From an initial set of 12 factors, we find that expressing five factors is sufficient to generate functional mouse nociceptor neurons. We derive human nociceptor neurons from patients with familial dysautonomia (FD) and show that these neurons reveal potentially disease-relevant phenotypes in vitro
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