iPSC-derived human sensory neurons reveal a subset of TRPV1 antagonists as anti-pruritic compounds

Differentiation of hiPSC toward sensory neurons

We used the hiPSC line (BJ-iPS) that was reprogrammed from BJ fibroblasts (ATCC) using modified mRNAs²³. These were directed towards a sensory neuron fate according to our protocol described previously²⁴. Briefly, hiPSCs were plated onto Matrigel-coated plates at a density of 50,000 cells/cm² on day 1 and induced into an anterior neuroectodermal lineage with 0.5 µM LDN-193,189 (Stemcell Technologies) and 10 µM SB431542 (Miltenyi Biotec) for synergistic dual SMAD inhibition. At day 3, 3 µM Wnt agonist CHIR99021 (Miltenyi Biotec) coupled with 5 µM FGF inhibitor, SU5402 (Stemcell Technologies) were added to pattern neural progenitors towards a neural crest fate. 5 µM DAPT (Stemcell Technologies) was additionally used to block Notch signalling and encourage differentiation of progenitors into neurons. From day 6 to 11, LDN-193,189 and SB431542 were withdrawn. Depending on the downstream experiments, differentiating SNs were passaged between day 6 and 8 using Accutase (Stemcell Technologies) and seeded onto Matrigel-coated plates of different formats and at various densities (24-well MEA plate: 125,000 cells/cm²; 8-chamber, 12-, 24-well plate: 80,000 cells/cm²). Finally to promote maturation of sensory neurons, neurotrophic factors including 20 ng/mL BDNF (Miltenyi Biotec), 20 ng/mL GDNF (Miltenyi Biotec), 20 ng/mL NGF-β (Miltenyi Biotec) and 10 ng/mL ascorbic acid (Sigma Aldrich) were added from day 11 onwards. 0.5 µM AraC treatment was performed between day 11 and 13 to eliminate progenitor cells in the culture. Sensory neuron basal media was prepared using 250 mL of DMEM/F12 (Bio-Rev), 250 mL of Neurobasal media (Miltenyi Biotec), supplemented with MACS Neurobrew-21 w/o Vitamin A, 0.5% GlutaMAX and 1% N2 supplement.

Differentiation of hiPSC toward motor neurons

Human induced pluripotent stem cells (hiPSCs; BJ-iPSC cell line) were directed towards a motor neuron fate according to our protocol described previously²⁴.

Single-cell dissociation

For cell detachment, 46-day old hiPSC-SNs were treated with Accutase for 8 min at 37 °C and transferred into 15 mL conical tube before topping up with dPBS to 10 mL and centrifuging at 1000 rpm for 3 min. The supernatant was discarded and hiPSC-SNs within the pellet were dissociated using papain (Worthington Biochemical) according to manufacturer’s instructions. Briefly, papain digestion was carried out at 37 °C with constant agitation for 30 min and with manual pipetting done every 10 min. Cells were spun down at 1000 rpm for 3 min and the supernatant was discarded. The reaction was stopped by re-suspension and incubation in diluted DNase/albumin-inhibitor solution at 37 °C for 5 min before centrifugation at 1000 rpm for 3 min and discarding the supernatant. Finally, dissociated cells were resuspended into 500 µL Sensory neuron maturation medium to a concentration of ~ 320 cells/µL and filtered through a 40 μm cell strainer to remove any remaining undissociated cell clumps.

Single cell RNA-seq library preparation and sequencing

The single-cell suspensions were assessed by acridine orange/propidium iodide staining to contain a cell viability of 97.2%. Using the GEXSCOPE kit, the cells were loaded into microwells of a microfluidic SCOPE-chip and lysed to release mRNA hybridizing to unique barcode sequences on the bead in the same well. The hybridized mRNA was reverse transcribed into cDNA and amplified. The resulting amplified cDNA was fragmented, ligated to adaptors and processed to construct a sequencing library compatible with the Illumina sequencing platform. The raw single cell transcriptomics data is publicly available at Gene Expression Omnibus (GEO) with an accession number GSE256435.

Single-cell RNA-seq data analysis

Raw sequencing reads were processed by Celescope pipeline with default parameters to generate filtered single-cell gene expression matrix files (https://github.com/singleron-RD/CeleScope). R package Seurat (v4.1.1) was used to further process the gene expression matrix files²⁵. Quality control was first performed, and cells containing between 100 and 7500 unique feature counts with less than 20% mitochondrial transcripts were kept. Genes expressed in less than 5 cells were also removed. After quality control, 13,410 cells remained, and their expression data was normalized and scaled. FindVariableFeatures was used to identify the highly variable genes which was used to conduct principal component analysis. The ElbowPlot function was used to suggest the dimensionality of the dataset, and a value of 20 was used. FindNeighbors and FindClusters were used to cluster the cells using the shared nearest neighbor graphs. A range of resolutions were tested, and the value of 0.3 was used, which resulted in 8 clusters. Uniform Manifold Approximation and Projection dimensionality reduction was performed using RunUMAP for visualization. To infer the identities of the clusters, FindAllMarkers was used to identify the most upregulated genes in each cluster. DotPlot was used to aid in the visualization in the most upregulated genes from each cluster. In addition, commonly accepted gene markers (such as PRPH, ISL1, STMN2) was used to further support the identities of the neuronal clusters. Clusters 0, 3 and 7 were determined to be neuronal, and contained a total of 5031 cells.

RNA extraction and qPCR

Cells were harvested in Trizol reagent for RNA extraction using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. cDNA conversion was performed using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystem, USA). Quantitative PCR (qPCR) was performed on QuantStudio 5 Real-Time PCR system using Fast SYBR Master Mix (Applied Biosystems). Gene expression was normalized to ACTINB and GAPDH. Primers used are listed in Supplementary Table S1.

Immunostaining and image acquisition

Human iPSC-derived sensory neurons were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 20 min and incubated in blocking buffer consisting of 5% FBS and 1% BSA for 2 h at room temperature. Blocking solution were replaced with primary antibody diluted in blocking solution and incubated at 4 °C overnight. The primary antibodies used in this study were rabbit anti-TrkA (1:400; CST, 2510 S), mouse anti-Brn-3a (1:100; Merck Millipore, MAB1585), rabbit anti-Peripherin (1:500; Merck Millipore, AB1530), rabbit anti-Islet 1 (1:500; Abcam, ab109517), rabbit anti-H1 Histamine Receptor (1:500; Alomone, AHR-001), rabbit anti-TRPV1 (1:500; Alomone, ACC-030) and followed by Alexa Fluor-conjugated secondary antibodies (1:1250; Invitrogen). DAPI (0.5 ug/mL) was used for nuclear staining. To capture H1R and TRPV1 co-expression, sequential staining was carried out with thorough washes in between to avoid cross-reactivity of secondary antibodies to the primary antibodies. Fluorescence images were captured with either ZOE Fluorescent Cell Imager (Biorad) or FV1000 confocal microscope (Olympus) using 20x objective.

Preparation of small molecule agonists and antagonists

For agonist preparation, histamine (Sigma, H7125) and capsaicin (Sigma, M2028) were reconstituted in ddH2O and DMSO to stock concentrations of 100 mM first and then further diluted in media to working concentrations of 25 µM and 0.5 µM, respectively, unless otherwise stated. For antagonist preparation, mepyramine/pyrilamine maleate salt (Sigma, P5514), QX 314 bromide (Hello bio, HB1029), AMG9810 (Sigma, A2731), AMG517 (Cayman, Cay26191), ABT102 (Axon Medchem, Axon 1504), SB366791 (Sigma, S0441), SB705498 (Alomone, S-160) and PAC14028 (MedChemExpress, HY-12777) were reconstituted in DMSO to stock concentrations of 50 mM and then diluted in media to their stated concentrations.

Calcium imaging

Human iPSC-derived sensory neurons that are at least 28 days old were pre-loaded with Calcium 6 Assay Dye (Molecular Devices, R8190) and incubated for 2 h at 37 °C in a 5% CO₂ incubator. All calcium imaging was performed directly in the dye without washing. Fluorescent images were obtained on a Nikon Ti-E Motorized Inverted microscope (SBIC-Nikon Imaging Centre at Biopolis) with a 20x objective and at 488 nm excitation. Exposure timing was set as 30 ms and interval as 500 ms. Baseline activities were measured for 20 s before addition of agonists and recorded for another 3 min. For antagonist testing, neurons were pre-incubated with the inhibitors for 10 min prior to recording. Quantitation was carried out on NIS-Elements software using the General Analysis module. Briefly, the original video file was used to generate an intermediate file containing three channels, i.e. baseline (B), maximum intensity projected (P) and maximum minus baseline (P-B) intensity. Next, the General Analysis function to increase the image contrast, threshold and count was applied to (P) and (P-B) channels in the intermediate file. This generated binary layers which were then converted to regions of interest (ROIs) corresponding to total cell count in (B) channel and agonist-responsive only cells in (P-B) channel. Finally, the ROIs were applied back to the original video file to prune off ROIs that did not accurately map to sensory neurons excitabilities. For experiment involving non-differentiated cells, BJ-iPSC cells were pre-loaded with Calcium 6 Assay Dye and incubated for 2 h at 37 °C in a 5% CO₂ incubator. Fluorescent images were captured using both the EVOS M5000 Imaging System and the Varioskan LUX Multimode Microplate Reader, employing an excitation wavelength of 488 nm. Exposure durations were configured at 30 ms for the EVOS M5000 and 100 ms for the Varioskan LUX, with a common interval of 1 s.

Multielectrode array (MEA) electrophysiology

All MEA extracellular potential recordings were carried out on the 24-well glass bottom Multiwell-MEA system (Multi Channel Systems), which contains 12 electrodes per well. MEA plates were pre-coated with Matrigel to promote neural cell attachment, and differentiating neurons between day 6 and 8 were seeded at 125,000 cells/cm² and allowed to continue to mature until at least 28 days old, when recordings were taken. For standard assay without noxious heat (recorded at a consistent temperature of 37 °C), baseline condition was recorded for 5 min prior to recording for another 5 min in the presence of agonist. Neurons were rested for 45 min before being incubated with antagonists for 10 min and then recorded for another 5 min in the additional presence of an agonist. This is with the exception for QX 314 bromide experiments where the neurons were incubated with both antagonist and agonist to silence selective subsets of neurons before excitation with a second agonist. The MEA plates were always stabilized on the reader for 3–5 min prior to recording. For assay involving single time heating, extracellular potentials were recorded for 20 min as the neurons were heated from 37 to 42 °C and gradually cooled back to 37 °C. For repeated heating assays, 45 min rest intervals were added in between heating and burst spike count was only tabulated between the 8 and 9th minute where temperature is stabilized at 42 °C. All raw data recorded was analyzed on the Multiwell-Analyzer software. Burst spike count was quantified as the total number of spikes that occurred during burst events. Bursts were identified as at least four densely packed spikes with a maximum inter-spike interval of 50 ms, minimum inter-burst interval of 100 ms and a minimum burst duration of 50 ms. Spikes are set as signals with rising and falling edges beyond the threshold of 5 standard deviation.

Statistical analyses

All data shown represent at least three biological replicates except for experiments described in Fig. 3C, F,I. Shapiro–Wilk tests were performed on all datasets to determine for normality. When the normality assumption is not met, non-parametric tests were performed instead. For comparisons between three or more conditions, statistical significance was determined by Friedman test with Dunn’s multiple comparisons test using Graphpad Prism, unless otherwise specified. For comparisons between two conditions, statistical significance was determined by unpaired parametric Student T-test or by non-parametric Mann-Whitney test.

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• Source: https://www.nature.com/articles/s41598-024-82549-7