Probes for INS

The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes (JXP) of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study we compared two mouse models of constitutive Kv1.6 knock-out achieved by different methods: traditional gene trap via homologous recombination, and CRISPR-mediated excision. Both Kv1.6 knock-out mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6lacZ ) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of 'meganeurites' and impaired transmission to dorsal horn wide dynamic range (WDR) neurons. The anatomical defects were absent in CRISPR-mediated Kv1.6 knock-out mice (Kcna6 -/-) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathological defects in transgenic mice, which has confounding implications for the interpretation of gene knock-outs using lacZ Nonetheless, in Kcna6 -/- mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury.SIGNIFICANCE STATEMENTIn recent decades the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels, one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioural data outcomes. To summarise we highlight the importance of: Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.

Purpose: As skeletal maturity is approached, long bone elongation draws to a close and the cartilaginous growth plate (GP) ossifies and fuses as bone bridges form. This is likely a pivotal moment for the appendicular skeleton, but our mechanistic appreciation of how this process is orchestrated is limited. We have been studying how chondrocytes integrate biological cues, such as growth factor signalling, and mechanical forces, and have investigated the mechanosensitivity of epiphyseal fusion and roles for putative mechanotransduction machinery, including the primary cilium, in these contexts. Here we asked whether primary cilia have a mechanotransduction role in the juvenile GP and adolescent epiphyseal fusion. Methods: We used an inducible aggrecan (ACAN) Cre mouse model, enabling temporal deletion of the core ciliary protein IFT88 in cartilage to investigate GP narrowing dynamics and closure from 4 - 10 weeks of age. Both control (Ift88fl/fl) and cKO (Ift88fl/fl;ACANCreERT2) were injected with tamoxifen (I.P.). Cre activity was validated using a ROSA26TdTomato reporter line. Animals were exposed to (i) sciatic and femoral double neurectomy (DN) to off-load the right hind limb (immobilised DN) whilst the left bears full weight (contralateral DN) at 8 weeks of age, or (ii) voluntary wheel exercise between 8 and 10 weeks of age. Joints were scanned by μCT before histomorphometric analyses of tibial GP using Safranin-O/fast green, TUNEL, Collagen type X (ColX) immunohistochemistry, Von Kossa and TRAP. Cryosections of mouse GPs were analysed by confocal microscopy to investigate primary cilia prevalence and RNA scope was used to identify molecular mechanisms in situ. Medians +/- 95% confidence intervals quoted throughout below, Two-way ANOVA statistical comparisons. Results: We have, for the first time, investigated the role of cilia beyond 4 weeks of age. μCT analysis showed GP length in wild-type mice reduces from ∼260 μm to 130 μm between 4 and 10 weeks of age. Deletion of IFT88 in juvenile mice at 4 or 6 weeks of age resulted in longer GPs in cKO mice at every timepoint compared with control mice (Fig. 1A, 1st and 2nd panel and 1B). Thus, two weeks after tamoxifen, cKO GP lengths were not statistically significantly different to controls at time of treatment, indicating inhibition of GP closure. Deleting IFT88 at 8 weeks of age also resulted in longer GPs (p< 0.0001, n=12 controls, n=23 cKO). Interestingly, some cKO mice exhibited extremely elongated GPs at the edges of the tibia, which appeared as large holes by μCT (Fig. 1A), whilst the centre of the GP appeared less affected. Histology confirmed longer GPs were predominantly characterised by increases in hypertrophic chondrocyte populations. The large, often bi-lateral “holes”, observed by μCT were largely filled with disorganised hypertrophic chondrocytes, as indicated by IHC labelling for ColX. Interestingly limb immobilisation, (DN), at 8 weeks of age, rescued the GP phenotype observed in IFT88 cKO mice (Fig. 1A, 2nd and 3rd panels, and 1C), whilst the contralateral, unoperated (increased load-bearing) limb exhibited bi-lateral failure of ossification, similar to that observed in IFT88 cKO mice. Compared with naïve controls, wheel-exercised mice also displayed elongated GP (p< 0.0001, n=12 controls, n=10 wheel exercised) (Fig. 1A, 4th panel, and 1C) at 10 weeks of age. These expanded GP were, again, most pronounced at the edges of the tibia, whilst the centre of the GP appeared less affected and again was largely filled with disorganised, differentiated, ColX positive hypertrophic chondrocytes. In both wheel exercised and IFT88 cKO mice, regions of failed ossification, but not middle regions, were associated with loss of osteoclast activity. Confocal imaging and analysis revealed a statistically significant (p< 0.001) decrease in cilia positive cells in wheel exercised mice (32.9%, n=5) compared with control (40.7%, n=4) and IFT88cKO mice (p< 0.0001, (23.4%, n=4) compared with controls (40.7% n=4) at 10 weeks of age. Ongoing experiments are investigating 3D spatial analysis of fusion mechanisms, and the status of ciliary Hh signalling (Gli1, by RNAscope) within GP from control, DN, exercised, and cKO mice to dissect the apparently negative, regulatory role IFT88 is plays in the mechanical regulation of epiphyseal fusion. Conclusions: We conclude that IFT88 unequivocally plays a role in GP closure, its removal resulting in failed ossification of the GP, without disruption to chondrocytic lineage differentiation. This phenomenon, observed in cKO animals, is mechanosensitive with limb immobilisation rescuing the phenotype, suggesting, paradoxically, that IFT88 is dampening a mechanically-induced signal in the GP. Wheel exercise also resulted in impaired ossification thus these data collectively unveil both the acute response of the adolescent mouse GP to exercise and, through Ift88 deletion (cKO), a novel mechanoregulatory mechanism orchestrated by ciliary IFT. The effects of altered mechanics and mechanotransduction are most pronounced in the hypertrophic zone where cells are apparently trapped short of transdifferentiation. Osteoclast recruitment and/or activity is impaired, and bone formation inhibited. These Results may have implications for our understanding of hypertrophic chondrocyte biology in articular cartilage in OA. Moreover, it has been proposed that changes to mechanical inputs during adolescence and associated cam morphology contribute to hip OA development. In adolescent patient cohorts, high levels of exercise lead to cartilaginous hypertrophy, epiphyseal extension, cam development, and reduced rates of GP closure. Femoral and tibial epiphyseal extension has also been observed in adolescent athletes that sustain repetitive trauma through high intensity exercise. This research is crucial to a holistic understanding of skeletal mechano-biological health, and the effects of exercise, on the maturing appendicular skeleton

Introduction: Four randomized controlled trials studying fecal microbiota transplantation (FMT) in active ulcerative colitis (UC) patients showed variable success rates. The efficacy of FMT appears to be influenced by various factors including donor- and procedure-specific characteristics. Aim: We hypothesized that the outcome of FMT in patients with active UC could be improved by donor preselection on microbiota level, by using a strict anaerobic approach, and by repeated FMT administration. Methods: The RESTORE-UC trial (NCT03110289) was a national, multi-centric double-blind, sham-controlled randomized trial. Active UC patients (Total Mayo score 4-10 with endoscopic sub-score > or = 2) were randomly allocated (1:1) to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT by permutated blocks (2- 4) and stratified for weight, concomitant steroid use, and therapy refractoriness. S-FMTs were selected after a rigorous screening excluding samples with Bacteroides 2 enterotype, high abundances of Fusobacterium, Escherichia coli and Veillonella and the lowest microbial loads (Q1). A futility analysis after 66% (n=72) of inclusions was planned per protocol including a modified intention-to-treat (mITT) analysis using non-responder imputation (NRI) for patients receiving at least one FMT. The primary endpoint was steroid-free clinical remission (Total Mayo ≤ 2, with no subscore >1) at week 8. Secondary outcomes included steroid-free PRO-2 remission (Combined Mayo subscores of ≤1 for rectal bleeding plus stool frequency) and response (≥3 points or/and ≥50% reduction from baseline in combined Mayo subscores for rectal bleeding plus stool frequency) and steroid-free endoscopic remission (Mayo endoscopic subscore ≤1) and response (Mayo endoscopy subscore ≤1 and ≥1 point reduction from baseline). Results: Between March 2017-2021, 72 patients signed the ICF and 66 were randomly allocated to S-FMT (n=30) or A-FMT (N=36) and received at least one FMT. Both study arms were matched for baseline characteristics, yet a trend (p= 0,07) towards higher concomitant biological use in the S-FMT arm was observed. A remarkably high proportion of patients were previously exposed to biologicals (58.3% and 60.0% for the A-FMT and S-FMT group respectively). In the S-FMT and the A-FMT respectively 4 and 5 patients terminated the trial early due to worsening of colitis (4 in both arms) or FMT enema intolerance (1 A-FMT). They were included in the mITT analysis using NRI, showing after 66% of intended inclusions, the primary endpoint was reached in 3/30 (mITT with NRI 10.0%) S-FMT and 5/31 (13.9%) patients randomized to A-FMT (p=0.72). As the predefined minimum difference of 5% between both treatment arms was not attained, the study was stopped due to futility. Steroid-free PRO-2 remission was achieved in 7/30 (23,3%) patients on S-FMT and 10/36 (27,8%) on A-FMT (p= 0,78). Steroid-free PRO-2 response was attained by respectively 9/30 (30,0%) patients in the S-FMT arm and 12/36 (33,3%) patients in the A-FMT arm (p= 0,80). Steroid-free endoscopic response and remission were noted in 5/30 (16,7%) assigned to the S-FMT arm compared with 7/36 (19,4%) allocated to the A-FMT arm (p= 1.0). Of note, no patients on concomitant biologicals reached the primary endpoint, and there were 2 serious adverse events in the A-FMT arm: dysuria requiring hospitalization and worsening of UC requiring colectomy. Conclusions: In this double-blind sham-controlled trial comparing repeated administrations of anaerobic-prepared S-FMT with A-FMT in patients with active UC, no significant difference in steroid-free remission rates at week 8 were observed. The FMT procedure was generally well tolerated, and no new safety signals were observed.

Introduction: Four randomized controlled trials studying fecal microbiota transplantation (FMT) in active ulcerative colitis (UC) patients showed variable success rates. The efficacy of FMT appears to be influenced by various factors including donor- and procedure-specific characteristics. Aim: We hypothesized that the outcome of FMT in patients with active UC could be improved by donor preselection on microbiota level, by using a strict anaerobic approach, and by repeated FMT administration. Methods: The RESTORE-UC trial (NCT03110289) was a national, multi-centric double-blind, sham-controlled randomized trial. Active UC patients (Total Mayo score 4-10 with endoscopic sub-score > or = 2) were randomly allocated (1:1) to receive 4 anaerobic-prepared superdonor (S) FMT or autologous (A) FMT by permutated blocks (2- 4) and stratified for weight, concomitant steroid use, and therapy refractoriness. S-FMTs were selected after a rigorous screening excluding samples with Bacteroides 2 enterotype, high abundances of Fusobacterium, Escherichia coli and Veillonella and the lowest microbial loads (Q1). A futility analysis after 66% (n=72) of inclusions was planned per protocol including a modified intention-to-treat (mITT) analysis using non-responder imputation (NRI) for patients receiving at least one FMT. The primary endpoint was steroid-free clinical remission (Total Mayo ≤ 2, with no subscore >1) at week 8. Secondary outcomes included steroid-free PRO-2 remission (Combined Mayo subscores of ≤1 for rectal bleeding plus stool frequency) and response (≥3 points or/and ≥50% reduction from baseline in combined Mayo subscores for rectal bleeding plus stool frequency) and steroid-free endoscopic remission (Mayo endoscopic subscore ≤1) and response (Mayo endoscopy subscore ≤1 and ≥1 point reduction from baseline). Results: Between March 2017-2021, 72 patients signed the ICF and 66 were randomly allocated to S-FMT (n=30) or A-FMT (N=36) and received at least one FMT. Both study arms were matched for baseline characteristics, yet a trend (p= 0,07) towards higher concomitant biological use in the S-FMT arm was observed. A remarkably high proportion of patients were previously exposed to biologicals (58.3% and 60.0% for the A-FMT and S-FMT group respectively). In the S-FMT and the A-FMT respectively 4 and 5 patients terminated the trial early due to worsening of colitis (4 in both arms) or FMT enema intolerance (1 A-FMT). They were included in the mITT analysis using NRI, showing after 66% of intended inclusions, the primary endpoint was reached in 3/30 (mITT with NRI 10.0%) S-FMT and 5/31 (13.9%) patients randomized to A-FMT (p=0.72). As the predefined minimum difference of 5% between both treatment arms was not attained, the study was stopped due to futility. Steroid-free PRO-2 remission was achieved in 7/30 (23,3%) patients on S-FMT and 10/36 (27,8%) on A-FMT (p= 0,78). Steroid-free PRO-2 response was attained by respectively 9/30 (30,0%) patients in the S-FMT arm and 12/36 (33,3%) patients in the A-FMT arm (p= 0,80). Steroid-free endoscopic response and remission were noted in 5/30 (16,7%) assigned to the S-FMT arm compared with 7/36 (19,4%) allocated to the A-FMT arm (p= 1.0). Of note, no patients on concomitant biologicals reached the primary endpoint, and there were 2 serious adverse events in the A-FMT arm: dysuria requiring hospitalization and worsening of UC requiring colectomy. Conclusions: In this double-blind sham-controlled trial comparing repeated administrations of anaerobic-prepared S-FMT with A-FMT in patients with active UC, no significant difference in steroid-free remission rates at week 8 were observed. The FMT procedure was generally well tolerated, and no new safety signals were observed.

Background: Mucus buildup in multiple mucin-producing organs, including the lungs and the intestine, is the hallmark of CF. In the gastrointestinal (GI) tract, aberrant mucus properties may play a critical role in bowel obstruction, inflammation, and bacterial overgrowth, as well as reduced nutrient absorption. Interestingly, all animal models of CF (e.g., mice, rats, ferrets, pigs) experience gastrointestinal complications, with poor growth and intestinal obstruction, as a result of excessive mucus accumulation. However, the precise biochemical change(s) within the CF gut mucus is still undetermined. Disrupted transepithelial Cl− and HCO3 − secretions due to CFTR malfunction affect the viscoelastic properties of airway mucus and may affect gut mucus similarly. Using WT and F508delCftr (CF) mice, we explored how interactions between Cftr and Muc2, the dominant GI mucin, resulted in the intestinal obstructive phenotype observed in CF mice. Methods: CF mice were weaned from laxative for 48 h prior to experiments. Intestinal sections (duodenum, jejunum, ileum, and colon) from WT and F508del-cftr CF mice were analyzed using H&E and AB-PAS staining to examine tissue structure, cell morphology, and mucus accumulation. RNAScope and qRT-PCR were used to determine Muc2 and Cftr mRNA expression along the proximal-to-distal and crypt-villus axes of the murine gut. IHC was used to determine the mucin compositions of gut sections, and fluorescent in-situ hybridization (FISH) was used to observe bacterial penetration of the mucus layer in the mouse intestine. Comparing WT and CF mice, we measured % solids of the luminal content and relative changes in gut mucus concentration via Western blotting on intestinal lavages. Results: Gross pathology revealed that intestinal blockage frequently emerged from the distal ileum. In parallel, we demonstrated that Muc2 expression increased from proximal to distal, while Cftr expression remained uniform throughout the GI tract of WT animals. However, Cftr expression changed gradually along the crypt-villus axis, with a homogenous signal distribution in the duodenum that progressively concentrated to the crypts in the ileum. FISH confirmed that a layer of mucus devoid of bacteria protected the WT murine epithelium from pathogen invasion. Histological and IHC examinations of obstructed ileal regions revealed that distal regions of intestinal plugs were mainly composed of mucus and inflammatory cells, while more proximal regions of the blockages were contaminated by feces and other luminal debris. Luminal content % solids was significantly increased in the CF (32.7%) compared to the WT ileum (22.3%), with concentrations approaching that of the WT colon (37.3%). Western blot analysis of intestinal lavages revealed that highmolecular-weight Muc2 was more concentrated (fold increase ∼2) in the small intestine of CF compared to WT mice. Conclusion: In CF mice, elevated mucus concentrations are caused by the combination of high Muc2 expression and lack of fluid secretion due to defective Cftr protein in the distal ileum, which creates optimal conditions for bowel obstruction in this particular region. Unlike the colon, the ileum is not adapted to high friction and is prone to villi sloughing, further exacerbating the gut phenotype in CF animals.

The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) of the rodent thalamus process various signals and participate in circadian entrainment In both structures, cells exhibiting infra-slow oscillatory activity as well as non-rhythmically firing neurons can be observed Here, we reveal that only one of these two groups of cells responds to anorexigenic (CCK, GLP-1 and OXM) and orexigenic (Ghrl and OXA) peptides. Neuronal responses vary depending on the time of day (day vs. night) and on the diet (standard vs. high-fat diet) Additionally, we visualized receptors to the tested peptides in the IGL/VLG using in situ hybridisation Our results suggest that two electrophysiologically different subpopulations of IGL/VLG neurons are involved in two separate functions: the one related to body's energy homeostasis and the one associated with the subcortical visual system ABSTRACT: The intergeniculate leaflet and ventral lateral geniculate nucleus (IGL/VLG) are subcortical structures involved in entrainment of the brain's circadian system to photic and non-photic (e.g. metabolic and arousal) cues. Both receive information about environmental light from photoreceptors, exhibit an infra-slow oscillations (ISO) in vivo, and connect to the master circadian clock. Although current evidence demonstrates that the IGL/VLG communicate metabolic information and are crucial for entrainment of circadian rhythms to time-restricted feeding, their sensitivity to food-intake-related peptides has not been investigated yet. We examined the effect of metabolically relevant peptides on the spontaneous activity of IGL/VLG neurons. Using ex vivo and in vivo electrophysiological recordings as well as in situ hybridisation, we tested potential sensitivity of the IGL/VLG to anorexigenic and orexigenic peptides, such as cholecystokinin, glucagon-like peptide 1, oxyntomodulin, peptide YY, orexin A, and ghrelin. We explored neuronal responses to these drugs during day and night, and in standard vs. high-fat diet conditions. We found that IGL/VLG neurons response to all the substances tested, except peptide YY. Moreover, more neurons responded to anorexigenic drugs at night, while a high-fat diet affected the IGL/VLG sensitivity to orexigenic peptides. Interestingly, ISO neurons responded to light and orexin A, but did not respond to the other food-intake-related peptides.. In contrast, non-ISO cells were activated by metabolic peptides, with only some being responsive to light. Our results show for the first time that peptides involved in the body's energy homeostasis stimulate the thalamus and suggest functional separation of the IGL/VLG cells. Abstract figure legend (1) Sprague Dawley rats were fed ad libitum with control (CD) or a high-fat (HFD) chow in a 12:12 h light-dark cycle. (2) The subject of the study was a thalamic intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (VLG) showing spontaneous infra-slow oscillatory (ISO) or nonâ¿¿oscillatory (nonâ¿¿ISO) activity. (3) Neuronal activity of the IGL and VLG was recorded using ex vivo and in vivo electrophysiological techniques. (4) Anorexigenic (in green) and orexigenic (in red) peptides such as cholecystokinin (CCK), glucagon-like peptide 1 (GLPâ¿¿1), oxyntomodulin (OXM), peptide YY (pYY), orexin A (OXA) and ghrelin (Ghrl) were administered during IGL/VLG recordings. (5) We found that non-ISO neurons of the IGL/VLG responded with an increase in firing rate to all the substances tested, except peptide YY. The amplitude (marked with arrows) and frequency of responses (marked with <, >) varied depending on the diet and the phase of the day. (6) In situ hybridization was performed on IGL/VLGâ¿¿containing brain sections to visualise receptors to the tested peptides. This article is protected by

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Xuming Zhu is first author on ‘ FZD2 regulates limb development by mediating β-catenin-dependent and -independent Wnt signaling pathways’, published in DMM. Xuming is an instructor in the lab of Sarah E. Millar at Icahn School of Medicine at Mount Sinai, New York, NY, USA, investigating the molecular mechanisms that underlie the development of appendages, epithelial homeostasis and diseases.

Mike May, is a freelance writer and editor with more than 30 years of experience. He earned an MS in biological engineering from the University of Connecticut and a PhD in neurobiology and behavior from Cornell University. He worked as an associate editor at American Scientist, and he is the author of more than 1,000 articles for clients that include GEN, Nature, Science, Scientific American and many others. In addition, he served as the editorial director of many publications, including several Nature Outlooks and Scientific American Worldview.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene ( SFTPC ) and the alveolar macrophage marker gene ( MARCO ), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene ( AGER ). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

Neuropathic pain is amongst the most common non-communicable disorders and the poor effectiveness of current treatment is an unmet need. Although pain is a universal experience, there are significant inter-individual phenotypic differences. Developing models that can accurately recapitulate the clinical pain features is crucial to better understand underlying pathophysiological mechanisms and find innovative treatments. Current data from heterologous expression systems that investigate properties of specific molecules involved in pain signaling, and from animal models, show limited success with their translation into the development of novel treatments for pain. This is in part because they do not recapitulate the native environment in which a particular molecule functions, and due to species-specific differences in the properties of several key molecules that are involved in pain signaling. The limited availability of post-mortem tissue, in particular dorsal root ganglia (DRG), has hampered research using human cells in pre-clinical studies. Human induced-pluripotent stem cells (iPSCs) have emerged as an exciting alternative platform to study patient-specific diseases. Sensory neurons that are derived from iPSCs (iPSC-SNs) have provided new avenues towards elucidating peripheral pathophysiological mechanisms, the potential for development of personalized treatments, and as a cell-based system for high-throughput screening for discovering novel analgesics. Nevertheless, reprogramming and differentiation protocols to obtain nociceptors have mostly yielded immature homogenous cell populations that do not recapitulate the heterogeneity of native sensory neurons. To close the gap between native human tissue and iPSCs, alternative strategies have been developed. We will review here recent developments in differentiating iPSC-SNs and their use in pre-clinical translational studies. Direct conversion of stem cells into the cells of interest has provided a more cost- and time-saving method to improve reproducibility and diversity of sensory cell types. Furthermore, multi-cellular strategies that mimic in vivo microenvironments for cell maturation, by improving cell contact and communication (co-cultures), reproducing the organ complexity and architecture (three-dimensional organoid), and providing iPSCs with the full spatiotemporal context and nutrients needed for acquiring a mature phenotype (xenotransplantation), have led to functional sensory neuron-like systems. Finally, this review touches on novel prospective strategies, including fluorescent-tracking to select the differentiated neurons of relevance, and dynamic clamp, an electrophysiological method that allows direct manipulation of ionic conductances that are missing in iPSC-SNs.

The milestone achievement of reprogramming a human somatic cell into a pluripotent stem cell by Yamanaka and Takahashi in 2007 has changed the stem cell research landscape tremendously. Their discovery opened the unprecedented opportunity to work with human-induced pluripotent stem cells and the differentiated progeny thereof, without major ethical restrictions. Additionally, the new method offers the possibility to generate pluripotent stem cells from patients with various genetic diseases which is of great importance (a) to understand the basic mechanisms of a specific disease in a human cellular context and (b) to help find suitable therapies for the persons concerned. In individual cases, this can even help to develop personalized treatment options. Chronic pain is a disease that affects roughly one in five people worldwide, but its onset is rarely based upon genetic alterations. Nevertheless, the work with sensory-like neurons derived from human pluripotent stem cells has become a more widely used tool also in the field of pain research, as during the past years several differentiation procedures have been published that describe the generation of different types of sensory-like neurons and their useful contribution to studying mechanisms of sensitization. Especially also to complement and verify cellular and molecular mechanisms identified in rodent model systems, the model of choice for decades. Although a sole cellular system is not able to mimic a disease as complex as pain, it is a valid tool to understand basic mechanisms of sensitization in specific subsets of human neurons that might be at the onset of the disease. In addition, the creativity of basic researchers and the more and more advanced available technologies will most likely find ways to implement the derived human cells in more complex networks. In this chapter, I want to introduce a selection of published differentiation strategies that result in the generation of human sensory-like neurons. Additionally, I will point out some studies whose results helped to further understand pain-related mechanisms and which were conducted using the aforementioned differentiation procedures.