NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC-Derived Nociceptor Maturation.

Nociceptors are primary afferent neurons serving the reception of acute pain but also the transit into maladaptive pain disorders. Since native human nociceptors are hardly available for mechanistic functional research, and rodent models do not necessarily mirror human pathologies in all aspects, human induced pluripotent stem cell-derived nociceptors (iDN) offer superior advantages as a human model system. Unbiased mRNA::microRNA co-sequencing, immunofluorescence staining, and qPCR validations, reveal expression trajectories as well as miRNA target spaces throughout the transition of pluripotent cells into iDNs. mRNA and miRNA candidates emerge as regulatory hubs for neurite outgrowth, synapse development, and ion channel expression. The exploratory data analysis tool NOCICEPTRA is provided as a containerized platform to retrieve experimentally determined expression trajectories, and to query custom gene sets for pathway and disease enrichments. Querying NOCICEPTRA for marker genes of cortical neurogenesis reveals distinct similarities and differences for cortical and peripheral neurons. The platform provides a public domain neuroresource to exploit the entire data sets and explore miRNA and mRNA as hubs regulating human nociceptor differentiation and function.

Population Coding of Capsaicin Concentration by Sensory Neurons Revealed Using Ca2+ Imaging of Dorsal Root Ganglia Explants from Adult pirt-GCaMP3 Mouse.

BACKGROUND/AIMS: Nociceptors detect noxious capsaicin (CAPS) via the transient receptor potential vanilloid 1 (TRPV1) ion channel, but coding mechanisms for relaying CAPS concentration [CAPS] remain obscure. Prolonged (up to 1h.) exposure to CAPS is used clinically to desensitise sensory fibres for treatment of neuropathic pain, but its signalling has typically been studied in cultures of dissociated sensory neurons employing low cell numbers and very short exposure times. Thus, it was pertinent to examine responses to longer CAPS exposures in large populations of adult neurons. METHODS: Confocal fluorescence microscopy was used to monitor the simultaneous excitation by CAPS of neuronal populations in intact L3/4 dorsal root ganglia (DRG) explants from adult pirt-GCaMP3 mice that express a cytoplasmic, genetically-encoded Ca2+ sensor in almost all primary sensory neurons. Peak analysis was performed using GraphPad Prism 9 to deconstruct the heterogenous and complex fluorescence signals observed into informative, readily-comparable measurements: number of signals, their lag time, maximum intensity relative to baseline (Max.) and duration. RESULTS: Exposure for 5 min. to CAPS activated plasmalemmal TRPV1 and led to increased fluorescence due to Ca2+ entry into DRG neurons (DRGNs), as it was prevented by capsazepine or removal of extracellular Ca2+. Increasing [CAPS] (0.3, 1 and 10 µM, respectively) evoked signals from more neurons (123, 275 and 390 from 5 DRG) with shorter average lag (6.4 ± 0.4, 3.3 ± 0.2 and 1.9 ± 0.1 min.) and longer duration (1.4 ± 0.2, 2.9 ± 0.2 and 4.8 ± 0.3 min.). Whilst raising [CAPS] produced a modest augmentation of Max. for individual neurons, those with large increases were selectively expedited; this contributed to a faster onset and higher peak of cumulative fluorescence for an enlarged responding neuronal population. CAPS caused many cells to fluctuate between high and low levels of fluorescence, with consecutive pulses increasing Max. and duration especially when exposure was extended from 5 to 20 min. Such signal facilitation counteracted tachyphylaxis, observed upon repeated exposure to 1 µM CAPS, preserving the cumulative fluorescence over time (signal density) in the population. CONCLUSION: Individual neurons within DRG differed extensively in the dynamics of response to CAPS, but systematic changes elicited by elevating [CAPS] increased signal density in a graded manner, unveiling a possible mechanism for population coding of responses to noxious chemicals. Signal density is sustained during prolonged and repeated exposure to CAPS, despite profound tachyphylaxis in some neurons, by signal facilitation in others. This may explain the burning sensation that persists for several hours when CAPS is used clinically.

Differentiation of the 50B11 dorsal ganglion cells into NGF and GDNF responsive nociceptor subtypes.

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.

Zebrafish oxytocin neurons drive nocifensive behavior via brainstem premotor targets.

Animals have evolved specialized neural circuits to defend themselves from pain- and injury-causing stimuli. Using a combination of optical, behavioral and genetic approaches in the larval zebrafish, we describe a novel role for hypothalamic oxytocin (OXT) neurons in the processing of noxious stimuli. In vivo imaging revealed that a large and distributed fraction of zebrafish OXT neurons respond strongly to noxious inputs, including the activation of damage-sensing TRPA1 receptors. OXT population activity reflects the sensorimotor transformation of the noxious stimulus, with some neurons encoding sensory information and others correlating more strongly with large-angle swims. Notably, OXT neuron activation is sufficient to generate this defensive behavior via the recruitment of brainstem premotor targets, whereas ablation of OXT neurons or loss of the peptide attenuates behavioral responses to TRPA1 activation. These data highlight a crucial role for OXT neurons in the generation of appropriate defensive responses to noxious input.

Millimeter Wave Radiation Activates Leech Nociceptors via TRPV1-Like Receptor Sensitization.

There is evidence that millimeter waves (MMWs) can have an impact on cellular function, including neurons. Earlier in vitro studies have shown that exposure levels well below the recommended safe limit of 1 mW/cm2 cause changes in the action potential (AP) firing rate, resting potential, and AP pulse shape of sensory neurons in leech preparations as well as alter neuronal properties in rat cortical brain slices; these effects differ from changes induced by direct heating. In this article, we compare the responses of thermosensitive primary nociceptors of the medicinal leech under thermal heating and MMW irradiation (80-170 mW/cm2 at 60 GHz). The results show that MMW exposure causes an almost twofold decrease in the threshold for activation of the AP compared with thermal heating (3.9 ± 0.4 vs. 8.3 ± 0.4 mV, respectively). Our analysis suggests that MMWs-mediated threshold alterations are not caused by the enhancement of voltage-gated sodium and potassium conductance. We propose that the reduction in AP threshold can be attributed to the sensitization of the transient receptor potential vanilloid 1-like receptor in the leech nociceptor. In silico modeling supported our experimental findings. Our results provide evidence that MMW exposure stimulates specific receptor responses that differ from direct thermal heating, fostering the need for additional studies.

Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA.

In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.

A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites.

Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.

Pain relief in a neuropathy patient by lacosamide: Proof of principle of clinical translation from patient-specific iPS cell-derived nociceptors.

BACKGROUND: Small fiber neuropathy (SFN) is a severe and disabling chronic pain syndrome with no causal and limited symptomatic treatment options. Mechanistically based individual treatment is not available. We report an in-vitro predicted individualized treatment success in one therapy-refractory Caucasian patient suffering from SFN for over ten years. METHODS: Intrinsic excitability of human induced pluripotent stem cell (iPSC) derived nociceptors from this patient and respective controls were recorded on multi-electrode (MEA) arrays, in the presence and absence of lacosamide. The patient's pain ratings were assessed by a visual analogue scale (10: worst pain, 0: no pain) and treatment effect was objectified by microneurography recordings of the patient's single nerve C-fibers. FINDINGS: We identified patient-specific changes in iPSC-derived nociceptor excitability in MEA recordings, which were reverted by the FDA-approved compound lacosamide in vitro. Using this drug for individualized treatment of this patient, the patient's pain ratings decreased from 7.5 to 1.5. Consistent with the pain relief reported by the patient, microneurography recordings of the patient's single nerve fibers mirrored a reduced spontaneous nociceptor (C-fiber) activity in the patient during lacosamide treatment. Microneurography recordings yielded an objective measurement of altered peripheral nociceptor activity following treatment. INTERPRETATION: Thus, we are here presenting one example of successful patient specific precision medicine using iPSC technology and individualized therapeutic treatment based on patient-derived sensory neurons.

Development and validation of an in vitro model system to study peripheral sensory neuron development and injury.

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

Somatotopic organization of central arbors from nociceptive afferents develops independently of their intact peripheral target innervation.

Functionally important regions of sensory maps are overrepresented in the sensory pathways and cortex, but the underlying developmental mechanisms are not clear. In the spinal cord dorsal horn (DH), we recently showed that paw innervating Mrgprd+ nonpeptidergic nociceptors display distinctive central arbor morphologies that well correlate with increased synapse transmission efficiency and heightened sensitivity of distal limb skin. Given that peripheral and central arbor formation of Mrgprd+ neurons co-occurs around the time of birth, we tested whether peripheral cues from different skin areas and/or postnatal reorganization mechanisms could instruct this somatotopic difference among central arbors. We found that, while terminal outgrowth/refinement occurs during early postnatal development in both the skin and the DH, postnatal refinement of central terminals precedes that of peripheral terminals. Furthermore, we used single-cell ablation of Ret to genetically disrupt epidermal innervation of Mrgprd+ neurons and revealed that the somatotopic difference among their central arbors was unaffected by this manipulation. Finally, we saw that region-specific Mrgprd+ central terminal arbors are present from the earliest postnatal stages, before skin terminals are evident. In summary, we find that region-specific organization of Mrgprd+ neuron central arbors is present shortly after initial central terminal formation, which likely develops independently of peripheral target innervation. Our data suggest that either cell-intrinsic and/or DH prepatterning mechanisms are likely to establish this somatotopic difference.

Human peptidergic nociceptive sensory neurons generated from human epidermal neural crest stem cells (hEPI-NCSC).

Here we provide new technology for generating human peptidergic nociceptive sensory neurons in a straightforward and efficient way. The cellular source, human epidermal neural crest stem cells (hEPI-NCSC), consists of multipotent somatic stem cells that reside in the bulge of hair follicles. hEPI-NCSC and primary sensory neurons have a common origin, the embryonic neural crest. For directed differentiation, hEPI-NCSC were exposed to pertinent growth factors and small molecules in order to modulate master signalling networks involved in differentiation of neural crest cells into postmitotic peptidergic sensory neurons during embryonic development. The neuronal populations were homogenous in regard to antibody marker expression. Cells were immunoreactive for essential master regulatory genes, including NGN1/2, SOX10, and BRN3a among others, and for the pain-mediating genes substance P (SP), calcitonin gene related protein (CGRP) and the TRPV1 channel. Approximately 30% of total cells responded to capsaicin, indicating that they expressed an active TRPV1 channel. In summary, hEPI-NCSC are a biologically relevant and easily available source of somatic stem cells for generating human peptidergic nociceptive neurons without the need for genetic manipulation and cell purification. As no analgesics exist that specifically target TRPV1, a ready supply of high-quality human peptidergic nociceptive sensory neurons could open the way for new approaches, in a biologically relevant cellular context, to drug discovery and patient-specific disease modelling that is aimed at pain control, and as such is highly desirable.

Differential aging-related changes in neurophysiology and gene expression in IB4-positive and IB4-negative nociceptive neurons.

Despite pain prevalence altering with age, the effects of aging on the properties of nociceptors are not well understood. Nociceptors, whose somas are located in dorsal root ganglia, are frequently divided into two groups based on their ability to bind isolectin B4 (IB4). Here, using cultured neurons from 1-, 3-, 5-, 8-, 12-, and 18-month-old mice, we investigate age-dependent changes in IB4-positive and IB4-negative neurons. Current-clamp experiments at physiological temperature revealed nonlinear changes in firing frequency of IB4-positive, but not IB4-negative neurons, with a peak at 8 months. This was likely due to the presence of proexcitatory conductances activated at depolarized membrane potentials and significantly higher input resistances found in IB4-positive neurons from 8-month-old mice. Repetitive firing in nociceptors is driven primarily by the TTX-resistant sodium current, and indeed, IB4-positive neurons from 8-month-old mice were found to receive larger contributions from the TTX-resistant window current around the resting membrane potential. To further address the mechanisms behind these differences, we performed RNA-seq experiments on IB4-positive and IB4-negative neurons from 1-, 8-, and 18-month-old mice. We found a larger number of genes significantly affected by age within the IB4-positive than IB4-negative neurons from 8-month-old mice, including known determinants of nociceptor excitability. The above pronounced age-dependent changes at the cellular and molecular levels in IB4-positive neurons point to potential mechanisms behind the reported increase in pain sensitivity in middle-aged rodents and humans, and highlight the possibility of targeting a particular group of neurons in the development of age-tailored pain treatments.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity.

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor-encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an "ur-" bHLH gene.

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.

Sparse genetic tracing reveals regionally specific functional organization of mammalian nociceptors.

The human distal limbs have a high spatial acuity for noxious stimuli but a low density of pain-sensing neurites. To elucidate mechanisms underlying regional differences in processing nociception, we sparsely traced non-peptidergic nociceptors across the body using a newly generated MrgprdCreERT2 mouse line. We found that mouse plantar paw skin is also innervated by a low density of Mrgprd+ nociceptors, while individual arbors in different locations are comparable in size. Surprisingly, the central arbors of plantar paw and trunk innervating nociceptors have distinct morphologies in the spinal cord. This regional difference is well correlated with a heightened signal transmission for plantar paw circuits, as revealed by both spinal cord slice recordings and behavior assays. Taken together, our results elucidate a novel somatotopic functional organization of the mammalian pain system and suggest that regional central arbor structure could facilitate the "enlarged representation" of plantar paw regions in the CNS.

Dendritic space-filling requires a neuronal type-specific extracellular permissive signal in Drosophila.

Neurons sometimes completely fill available space in their receptive fields with evenly spaced dendrites to uniformly sample sensory or synaptic information. The mechanisms that enable neurons to sense and innervate all space in their target tissues are poorly understood. Using Drosophila somatosensory neurons as a model, we show that heparan sulfate proteoglycans (HSPGs) Dally and Syndecan on the surface of epidermal cells act as local permissive signals for the dendritic growth and maintenance of space-filling nociceptive C4da neurons, allowing them to innervate the entire skin. Using long-term time-lapse imaging with intact Drosophila larvae, we found that dendrites grow into HSPG-deficient areas but fail to stay there. HSPGs are necessary to stabilize microtubules in newly formed high-order dendrites. In contrast to C4da neurons, non-space-filling sensory neurons that develop in the same microenvironment do not rely on HSPGs for their dendritic growth. Furthermore, HSPGs do not act by transporting extracellular diffusible ligands or require leukocyte antigen-related (Lar), a receptor protein tyrosine phosphatase (RPTP) and the only known Drosophila HSPG receptor, for promoting dendritic growth of space-filling neurons. Interestingly, another RPTP, Ptp69D, promotes dendritic growth of C4da neurons in parallel to HSPGs. Together, our data reveal an HSPG-dependent pathway that specifically allows dendrites of space-filling neurons to innervate all target tissues in Drosophila.

Possible mechanism of bursting suppression in nociceptive neurons.

The use of the mathematical model of rat nociceptive neuron membrane allowed us to predict a new mechanism of suppression of ectopic bursting discharges, which arise in neurons of dorsal root ganglia and are one of the causes of neuropathic pain. The treatment with comenic acid leads to switching off the ectopic bursting discharges due to a decrease in the effective charge transferring via the activation gating structure of the slow sodium channels (Na V1.8a). Comenic acid is a drug substance of a new non-opioid analgesic [1] Thus, this analgesic not only reduces the frequency of rhythmic discharges of nociceptive neuron membrane [2] but also it suppresses its ectopic bursting discharges.

Functional Properties of Sensory Nerve Terminals of the Mouse Cornea.

Purpose: To define the firing properties of sensory nerve terminals innervating the adult mouse cornea in response to external stimuli of differing modality. Methods: Extracellular electrical activity of single corneal sensory nerve terminals was recorded in excised eyes of C57BL/6J mice. Eyes were placed in a recording chamber and were continuously superfused with warm saline solution. Nerve terminal impulse (NTI) activity was recorded by means of a glass pipette (tip ∼ 50 µm), applied on the corneal surface. Nerve terminal impulse discharges were stored in a computer for offline analysis. Results: Three functionally distinct populations of nerve terminals were identified in the mouse cornea. Pure mechanonociceptor terminals (9.5%) responded phasically and only to mechanical stimuli. Polymodal nociceptor terminals (41.1%) were tonically activated by heat and hyperosmolal solutions (850 mOsm·kg-1), mechanical force, and/or TRPV1 and TRPA1 agonists (capsaicin and allyl isothiocyanate [AITC], respectively). Cold-sensitive terminals (49.4%) responded to cooling. Approximately two-thirds of them fired continuously at 34°C and responded vigorously to small temperature reductions, being classified as high-background activity, low-threshold (HB-LT) cold thermoreceptor terminals. The remaining one-third exhibited very low ongoing activity at 34°C and responded weakly to intense cooling, being named low-background activity, high-threshold (LB-HT) cold thermoreceptor terminals. Conclusions: The mouse cornea is innervated by trigeminal ganglion (TG) neurons that respond to the same stimulus modalities as corneal receptors of other mammalian species. Mechano- and polymodal endings underlie detection of mechanical and chemical noxious stimuli while HB-LT and LB-HT cold thermoreceptors appear to be responsible for basal and irritation-evoked tearing and blinking, respectively.

Exploring pain pathophysiology in patients.

Although animal models of pain have brought invaluable information on basic processes underlying pain pathophysiology, translation to humans is a problem. This Review will summarize what information has been gained by the direct study of patients with chronic pain. The techniques discussed range from patient phenotyping using quantitative sensory testing to specialized nociceptor neurophysiology, imaging methods of peripheral nociceptors, analyses of body fluids, genetics and epigenetics, and the generation of sensory neurons from patients via inducible pluripotent stem cells.