A reduced sense of smell is a common condition in people with cystic fibrosis (CF) that negatively impacts their quality of life. While often attributed to nasal mucosa inflammation, the underlying causes of the olfactory loss remain unknown. Here, we characterized gene expression in olfactory epithelium cells from CF patients using single-nuclei RNA sequencing and found altered expression of olfactory receptors (ORs) and genes related to progenitor cell proliferation. We confirmed these findings in newborn, inflammation-free samples of a CF animal model, and further identified ultrastructural alterations in the olfactory epithelium and bulbs of these animals. We established that CFTR, the anion channel whose dysfunction causes CF, is dispensable for odor-evoked signaling in sensory neurons, yet CF animals displayed defective odor-guided behaviors consistent with the morphological and molecular alterations. Our study highlights CF's major role in modulating epithelial structure and OR expression, shedding light on the mechanisms contributing to olfactory loss in CF.

Experimental design and participants

The objective of this study was to establish the causes of smell alterations observed in cystic fibrosis patients. In a prospective study design, patients who presented themselves with CF were recruited from CF outpatient clinic consultations in the sense of regular disease-specific follow-up. Healthy subjects were recruited for comparison. For the Sniffin’ Sticks test, a total of 3 healthy participants (aged 23 to 52 years, 1 woman) and 10 CF patients (aged 18-52 years, 5 women) participated in the study from 07/04/2023 to 07/25/2023. For the SNOT-22 test, 17 healthy controls (23-52 years old, 10 women) and the previous 10 CF were used. For snRNA-seq, samples from 7 CF and 9 controls were sequenced. The information on the genetic form of the disease comes from the documentation of earlier DNA tests. For healthy participants, exclusion criteria were neurological diseases, systemic diseases associated with smell disorders like chronic renal failure, subjective smell impairment, chronic rhinosinusitis, allergic rhinitis, alcohol or drug abuse, and pregnancy. The study was conducted according to the declaration of Helsinki and had been approved by the Ethics Committee at the TU Dresden (EK 552122022). All participants gave written informed consent.

Olfactory testing

A detailed medical history was taken including age, gender, CF symptoms, medication, surgery, and questions regarding olfactory function. Further, the questionnaire sino-nasal outcome test (SNOT-22) was filled out to focus on lists of symptoms and social/emotional consequences of rhinosinusitis. Patients underwent olfactory tests, using the Sniffin’ Sticks test battery (Burghart Messtechnik, Holm, Germany) to categorize the olfactory function regarding odor threshold and identification (1, 2). A nasal endoscopy by an ENT specialist was performed to categorize endonasal diseases such as polyps or any form of chronic rhinosinusitis according to the Lildholdt score (3). Nasal brushing was performed on the olfactory cleft on both sides under constant endoscopic control using a sterile cotton swab (CLASSIQSwabs™, Brescia, Italy). The tips of the brushes were stored in CryoStor® cell cryopreservation media (~1-2 ml) for 10 min at 4°C and then transferred to a cooling device (Thermo Fisher Scientific, Dreieich, Germany) and stored at -80°C until further processing. Patient records were assigned to codes and anonymized.

Human single-nuclei RNA sequencing

Sample preparation: Cellular heterogeneity of human cystic fibrosis olfactory epithelium was analyzed by single-nuclei RNA sequencing (snRNA-seq). Frozen individual olfactory epithelium swabs were thawed and pooled for nuclei extraction in four pools containing 3 distinct samples from 3 individuals each (16 total individuals, 9 controls and 7 CF). This pooling ensured a sufficient amount of material for nuclei isolation. Genetic polymorphisms between samples was then used to discriminate individual samples (see below). Nuclei extraction was performed according to (4), with some modifications. In brief, each cryotube was thawed at 37°C, supplemented with 400 µl pre-heated saline medium. Brushes were cleared on ice by pipetting, transferred into 15 ml tubes, and centrifuged for 5 min 290xg at 4°C. After removal of the supernatant, the cell pellet was resuspended into 1 ml of a buffer containing 25 mM citric acid and 0.25M sucrose. The suspension was homogenized by 15 strokes with loose, then tight pestles, interrupted by 2 x 3 min incubation on ice. After a filtration on a 20 µm filter (Miltenyi, Paris, France), nuclei were centrifuged for 5 minutes 500xg, resuspended in 500µl of previous citric buffer, and centrifuged for 5 minutes at 500xg. Nuclei were resuspended in a buffer containing 25mM KCl, 3mM MgCl₂, 5mM Tris-buffer pH8, 0.4U/µl RNAsin® Plus ribonuclease inhibitor, 0.4U/µl SUPERaseIn® RNase inhibitor, and 1mM dithiothreitol (DTT). After a control of morphology on a Floid cell imaging station (PicoQuant, Berlin, Germany), nuclei were counted with a Countess II FL Automated Cell Counter (Thermo Fisher Scientific, Waltham, USA). Nuclei were then diluted at a concentration of 1200 nuclei /µl to target a capture of ~10,000 nuclei, and the suspension was rapidly loaded on a 10x Genomics Next GEM Single Cell 3ʹ v3.1 system (10X Genomics, Pleasanton, USA). Libraries were prepared according to the constructor specifications, with sequencing on a Nextseq 2000 (Illumina, San Diego, USA).

snRNA-seq processing and analysis: Human raw data were analyzed using Cell Ranger Single-cell software v6.0.0, with inclusion of the intron sequences. The raw gene-barcode matrices were aligned to the hg38 genome. Demultiplexing was performed using Demuxafy within a Singularity container to accurately assign reads to individual samples (5). After demultiplexing, each sample data was processed using Seurat package v.4.0 (6) in R v.4.0.2. Cells with less than 200 genes or more than 5% mitochondrial content, and genes expressed in fewer than 3 cells were excluded from the analysis. Each individual Seurat object was normalized and highly variable features were identified using the variance-stabilizing transformation (vst) method, selecting the top 2000 variable features. Integration features were selected from the variable features identified in each object. Each Seurat object was then scaled and subjected to Principal Component Analysis (PCA) using the selected integration features. Integration anchors were identified using Reciprocal PCA (rPCA), and the data was integrated based on these anchors. The integrated data was further processed by setting the default assay to “integrated”, scaling the data, running PCA, and performing Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction on the first 30 principal components (PCs). A neighbor graph was then constructed, and clustering was performed at a resolution of 1.0. Differentially expressed genes were identified for each cluster using the FindMarkers function. Pseudobulk analysis was conducted using the edgeR−LRT from Libra R package (7) to aggregate single-cell data into bulk-like samples for differential expression analysis between CF and control. Codes used for all analyses can be found here: https://github.com/ymbouamboua/Human_Pig_Olfactory_CF. Numbers for all cell types are: B cells (n = 106); Bowman's gland (n = 84); Club (n = 2600); Deuterosomal cells (n = 405); GBCs (n = 315); Goblet cells (n = 1785); Ionocytes/microvillar cells (n = 430); Macrophages (n = 166); Monocytes (n = 345); Neutrophils (n = 32); NK cells (n = 308); olfactory HBCs (n = 4104); OSNs (n = 31); pDCs (n = 192); Respiratory HBCs (n = 183); Respiratory multiciliated cells (n = 10298); Sustentacular cells (n = 383); T cells (n = 1147); Tuft cells (n = 18).

Pig single-nuclei RNA sequencing

Sample preparation: Pig main olfactory epithelia were snap frozen in liquid nitrogen and included in OCT. 300 µm sections were made using a cryostat, and kept at -80°C. In a glass dounce tissue grinder previously cooled on ice, 7 tissue slices and 1ml of citric acid-based buffer (0.25 M sucrose, 25 mM citric acid) were added. Tissue samples were homogenized with 3-5 strokes of loose pestle and incubated on ice for 5 min. After 5 more strokes using a loose pestle, samples were incubated on ice for 5 min, homogenized again with 3 strokes using a loose pestle and 5 strokes using a tight pestle (4). The suspension was filtered through a 40 µm cell strainer, and 1ml of citric acid-based buffer was used to wash the containers. After a centrifugation for 5 min at 500xg at 4°C, the supernatant was carefully removed and the sample was resuspended in 1ml of Wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1% BSA, 0.1%Tween 20, 1 mM DTT, 0.6 U/μl RNaseIn®, 0.2 U/μl SuperaseIn®). Sample was filtered through a 5 µm cell strainer and centrifuged for 5min at 500 g at 4°C. Nuclei were resuspended in 50-100 µl of diluted nuclei buffer (Nuclei Buffer® 1X; Multiome kit, 10X Genomics), DTT 1 mM, RNaseIn® 0.6U/µl, SuperaseIn® 0.2 U/μL). The nuclei morphology was verified using a Floid cell imaging station. Nuclei were counted on a Countess II FL Automated Cell Counter, diluted to the desired concentration (for a target capture of 10,000 nuclei). An RNA quality test was performed for each preparation before the 10x sequencing library was made. To do so, an aliquot of purified nuclei was placed in QIAzol Lysis Reagent® and RNA was isolated using miRNeasy Micro kit®. The RNA integrity number (RIN) was checked using the Agilent Bioanalyzer System. snNucSeq was performed according to the protocol provided by 10X Genomics.

snRNA-seq processing and analysis: Pig raw data were analyzed using Cell Ranger Single-cell software v6.0.0. The raw gene-barcode matrices were aligned to the Sscrofa11-1‎ genome. Cell Ranger output was analyzed with the Seurat package v.4.0(6) using R v.4.0.2. Each individual data was filtered to keep cells with 200-7,000 genes, less than 99% of dropouts, and less than 5% of mitochondrial sequences. Doublet cells were removed with DoubletFinder (8). After normalization and variance stabilization with SCTransform, and dimensionality reduction by Principal Component Analysis, data was visualized using UMAP embedding based on the first 20 principal components. Global integration was performed after normalization and identification of the 4000 most variable features of each dataset. Features that varied across datasets were selected, and experiments were integrated with the matching mutual nearest neighbors method as described (9). Cell clusters were annotated according to canonical gene markers: Bowman’s gland (SOX9, SLC6A11, TMEM163),  Endothelial (MMRN1, CLVS1, CCL21), Fibroblast/Stromal (DCN, LUM, PTPRD), GBCs (EZH2, CXCR4, HES6), Immature neurons (GNG8, GAP43, TPD52), Immune (CD163, ARHGAP15, MRC1), Mature neurons (GNG13, STXBP5L, PEX5L), Olfactory ensheathing glia (SORCS1, ZNF536), Olfactory HBCs (CCDC129, CAPN13, MGAM2), Olfactory microvillar (STAP1, CLNK, SLC35F3), Pericytes (NOSTRIN, LRFN5, CXCL2), Respiratory ciliated (CFAP126, FOXJ1), Respiratory epithelial (NDAL, GPS, EIF1), Respiratory HBCs (KRT5, MET, TP63), Respiratory secretory (TTC6, EIF2AK2), Sustentacular (MOCOS, GLDN, ERMN), Vascular smooth muscle (TAGLN, TPM2, MYL9). Bowman's gland (n = 460); Endothelial cells (n = 167); Fibroblasts/stromal cells (n = 1475); GBCs (n = 313); Immature neurons (n = 1241); Immune cells (n = 144); Mature neurons (n = 1835); Olfactory ensheathing glia (n = 360); Olfactory HBCs (n = 108); Olfactory microvillar/ionocyte cells (n = 128); Pericytes (n = 129); Respiratory multiciliated cells (n = 314); Respiratory epithelial cells (n = 73); Respiratory HBCs (n = 222); Respiratory secretory cells (n = 546); Sustentacular cells (n = 1115); Vascular smooth muscle cells (n = 293).

Bulk RNA-seq processing and analysis

Olfactory epithelia from CFTR-null and control piglets were dissected and immediately frozen on liquid N₂. RNA was extracted using the RNeasy mini kit (Qiagen) with on-column DNAse digestion. mRNA was prepared for sequencing using the TruSeq RNA sample preparation kit (Illumina). All samples were multiplexed together and sequenced on four lanes on the Illumina HiSeq 2500 platform, to generate 100 bp paired-end reads. Sequencing reads were mapped using STAR 2.3 to the 11.1 Sus scrofa reference genome, annotation version 11.1 in the Ensembl pig genome database. The number of fragments aligned to each gene was counted using the HTSeq package, with the script htseq-count (mode intersection-nonempty). Any read that maps to multiple locations in the genome (also called multireads) was not counted towards the expression estimates as it cannot be assigned to any gene unambiguously. To compare the expression values across samples, raw count data was normalized to account for the depth of sequencing. Size factors were calculated using DESeq2’s function estimateSizeFactorsForMatrix, and raw counts were divided by the corresponding size factor for each sample. To test for differential expression, DESeq2 was used with standard parameters. Genes were considered to be differentially expressed if they had an adjusted P value of ≤0.05 (equivalent to a false discovery rate of 5 %). A total of 11 DE expressed genes located on the Y chromosome were excluded from the analysis to prevent sex-specific bias. To find terms that are enriched in our list of DE genes the over-/under-representation algorithm from GeneTrail (http://genetrail.bioinf.uni-sb.de/) was used. The background provided were all those genes tested for differential expression. To assess whether the DE genes form putative regulatory networks, STRING (http://string-db.org/) was used with default settings, for the 212 DE genes only. All normalized data and detailed results of the DE and enrichment analyses can be found in the Data S1. Heatmaps of genes with significantly changed expression were generated using OriginPro 2021b program. Functional classification was analyzed by Gene Ontology Consortium (Panther Classification System) and UniProt database (https://www.uniprot.org) using the biological process terms. Classification into OSN- or non-OSN-specific genes in Fig. 4B-C was performed by direct comparison with the dataset published by Saraiva et al.(10) obtained by RNA-seq of mouse OMP+ mature olfactory sensory neurons (mOSNs). Cell type assignments for specific marker genes was based on previously published mouse and human expression studies (10, 11).

Human olfactory neuroepithelium cell culture.

Human olfactory cell samples were obtained independently from snRNAseq samples using a slightly different technique. Cells from the olfactory mucosa were collected by nasal brushings from 10 healthy control, non-smoker, SARS-CoV-2 negative subjects between 18 and 45 years old (both males and females) as previously described (12). Every subject gave written informed consent for the study and the procedures involved. The study was approved by the local institutional ethics committee CEIm Ethical Committee Parc de Salut MAR, IMIM-Hospital del Mar Research Institute, Barcelona: study no. 2018/7942/I. Samples from the middle and upper turbinates were maintained in 250 µl of cold Dulbecco’s Modified Eagle Medium/Ham F-12 (DMEM/F12) enriched with 10% fetal bovine serum (FBS), 2% glutamine and 1% streptomycin–penicillin (GibcoBRL), as previously described(12). At 80% confluence, cells were expanded using 0.25% trypsin (GibcoBRL) and replated in 75 cm² flasks. Cells were then expanded until a maximum of 5 passages. Cells from 2 healthy individuals were treated with 0, 10, 20 or 30 µM of the CFTR_inh172 selective CFTR blocker (Tocris) on the culture media and incubated for 24 hours. Cells were washed with phosphate buffered saline (PBS), fixed with paraformaldehyde (PFA) 4% for 15 min and in situ hybridization and/or immunolabeling was performed as described below.

Quantitative RT-PCR

RNA from cultured human olfactory cells from 10 controls was purified with the PureLink^® RNA Mini Kit (Invitrogen) and stored at − 20 °C. Extracted total RNA was converted to cDNA by reverse transcription of 20 ng of RNA using the Nucleospin® RNA XS kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol. The cDNA was applied in TakyonTM No ROX SYBR 2X MasterMix blue dTTP (Eurogentec) and the primers used are listed in Data S1. All RT-qPCR reactions were performed in 96-microwell plates using The LightCycler® 480 Real-Time PCR System (Roche Applied Science). Three technical replicates were averaged and the quantitative RT-PCR data were analyzed using the 2ΔΔCt method. GAPDH and RLP-19 genes were used as a control for normalization. The results are expressed as relative fold change.

CFTR-deficient pigs

Male and female CFTR^+/− heterozygous transgenic pigs (13) were provided by the LMU Munich, Germany, transferred to France. Pigs (Large White breed) were housed together and had access to a standard grain-based diet and water ad libitum. Heterozygous pigs were mated to generate CFTR^+/+, CFTR^+/− and CFTR^−/− piglets. Newborn piglets (0.8-1.5 kg) were genotyped within 6 hours after birth and sacrificed for olfactory tissue collection. E75 pig fetuses were collected from amniotic sacs by c-section in pregnant dams. Pig littermates homozygous for CFTR (CFTR^+/+) served as controls. Littermates were randomly assigned to experimental groups keeping the same sex ratio between groups. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at INRAE. All experimental procedures were evaluated by the Ethics Committee of the Val de Loire (CEEA VdL, committee N°19) and approved by the Ministry of Higher Education and Research (APAFIS#1166-2015071615392426 Notification and APAFIS#10125-20170602162555 Notification).

Neonate suckling assay

Newborn piglets were tested for suckling latency right after birth. Delivery was induced after 112 days of gestation by injection of 175 µg of cloprostenol and 20 units (IU) of oxytocin and each piglet was marked right after birth, monitored and latencies to stand up after birth and to suckle were scored (see Movie S1). Testing lasted until the piglet reached the mother’s nipple and started to suckle or for a maximum of 400 min after the birth of each piglet. Each test group contained piglets from one single litter. Each litter was tested only once, to ensure no learning occurred. Time scoring was performed by an experimenter blind to the genotype of the piglets, as biopsies for genotyping were collected after behavior testing.

Calcium imaging

Ca²⁺ imaging was performed in freshly dissociated pig olfactory epithelium cells adapting protocols previously developed for mouse olfactory cells (14, 15). The olfactory neuroepithelium was detached from the cartilage and minced in PBS at 4°C. The tissue was incubated (20 min at 37°C) in PBS supplemented with papain (0.22 U/ml; Worthington) and DNase I (10 U/ml; Fermentas), urea (40 mM; Sigma) gently extruded in DMEM (Invitrogen) supplemented with 10% FBS, and centrifuged at 100 × g (5 min). Dissociated cells were plated on coverslips previously coated with concanavalin-A type IV (0.5 mg/ml, overnight at 4 °C; Sigma). Cells were used immediately for imaging after loading with fura-2/AM (5 µM; Invitrogen) for 60 min. Coverslips containing cells were placed in a laminar-flow chamber (Warner Instruments) and constantly perfused at 22 °C with extracellular solution Hank’s balanced salt solution (HBSS, Invitrogen) supplemented with 10 mM Hepes (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). Cells were alternately illuminated at 340 and 380 nm, and light emitted above 510 nm was recorded using a C10600-10B Hamamatsu camera installed on an Olympus IX71 microscope. Images were acquired at 0.25 Hz and analyzed using ImageJ (NIH), including background subtraction, region of interest (ROI) detection and signal analyses. ROIs were selected manually and always included the whole cell body. Peak signals were calculated from the temporal profiles of image ratio/fluorescent values. Results are based on recordings from 5 piglets for each condition and genotype (n = 10–149 activated cells of a total of 18,781 cells analyzed; 10,479 CFTR^+/+ and 8,302 CFTR^–/– cells). Cells were stimulated successively and in random order using bath application. The following criteria for stimulus-induced Ca²⁺ responses were applied: (1) A response was defined as a stimulus-dependent deviation of fluorescence ratio that exceeded twice the standard deviation of the mean of the baseline fluorescence noise. (2) Cells showing a response to control buffer were excluded from analysis. (3) A response had to occur within 1 min after stimulus application. In time series experiments, ligand application was repeated to confirm the repeatability of a given Ca²⁺ response. Chemostimuli were freshly prepared each day and diluted in extracellular solution to give the following final concentrations: 1-octanol 10 µM (Sigma); 2-heptanone 10 µM (Sigma); octanal 10 µM (Sigma); forskolin (Sigma) 50 µM, 3-isobutyl-1-methylxanthine (IBMX; Sigma) 100 µM, and 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP; Sigma) 500 µM; KCl 90 mM. Volatile odorants were initially prepared in dimethyl sulfoxide (DMSO; Sigma) and further diluted in extracellular solution.

Immunostaining

Human olfactory cells were fixed in PBS containing 4% PFA for 15 min at room temperature, washed 3X in PBS and incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 3% horse serum) for 30 min at room temperature. For newborn pigs, olfactory tissues were removed, postfixed overnight in 0.1 M phosphate buffer (PB) containing 4% PFA and later cryoprotected in 0.1 M PB buffer containing 30% sucrose. Samples were embedded in Tissue-Tek OCT compound, snap-frozen in cold isopentane and processed on a Leica CM 3050S cryostat. Olfactory epithelia were cut in 16-μm thick coronal sections and were directly mounted on SuperFrost Plus slides glasses (Thermo Scientific). Olfactory bulbs were cut in 30-µm serial free-floating sagittal sections in a PBS solution. Sections were treated with 10mM sodium citrate for 5 min at 95-100 ºC for antigen retrieval, washed (3 × 5 min) with PBS, incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 5% normal horse serum) for 2 h at room temperature (RT), incubated overnight at 4 °C in blocking solution supplemented with the primary antibody, washed in PBS solution and incubated in blocking solution supplemented with secondary antibody for 1 h at RT. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Sigma) for 5 min. We used the following primary antibodies: goat anti-SOX2 (RD Systems AF2018, 1:300), rabbit anti-KRT5 (Biolegend 905501, 1:800), goat anti-OMP (Wako 019-22291, 1:2000) and mouse anti-NGFR (Sigma N5408, 1:1000), mouse anti-PCNA (Sigma P8825, 1:1000), rabbit anti-DCX (Abcam AB18723, 1:2000), rabbit anti-Ki67 (Abcam ab9021, 1:100) mouse anti-Gαolf (Santa Cruz sc-55545, 1:500), rabbit anti-ITPR3 (Millipore AB9076, 1:500), rabbit anti-TRPM5 (Alomone Labs ACC-045, 1:400), rat anti-NES (Santa Cruz sc-33677, 1:600). Secondary antibodies used for the corresponding target species were conjugated with Alexa488, Alexa546, and Alexa647 (Thermofisher, 1:500). Epithelium thickness, cell density, areas and olfactory bulb glomeruli quantifications were preformed from images acquired on a Zeiss LSM-780 confocal laser-scanning microscope. Image regions were analyzed in the entire z-axis with 3 µm step intervals, and images were reconstituted using the Maximum Intensity Projection tool of Zen software. Images were analyzed with Fiji/ImageJ (NIH). Demarcations of epithelium limits and cell-specific layers were based on OMP and NGFR immunoreactivities, and DAPI+ cells were counted using the particle analyzer plug-in of Fiji. Counts of the number of cells were evaluated blindly for each animal. Five slices per animal and genotype were used. Cells were counted from five to seven independent animals. For each sample, images were acquired at least from five different anatomical levels. Olfactory bulb glomeruli demarcation was based on OMP immunoreactivity in 5-17 slices per animal from 6 independent animals per genotype. A total of 819 glomeruli (469 CFTR^+/+ and 350 CFTR^−/−) covering all main olfactory bulb topography (dorsal, ventral, lateral and medial) were analyzed.

In situ hybridization (ISH)

Staining for CFTR, NGFR, OR51E2, OR51E1 and NPY mRNAs was performed using multiplex fluorescent ISH. Human olfactory cells and olfactory tissue sections were prepared as described above. RNAscope Fluorescent MultiplexV2 labeling kit (ACDBio 323110) was used to perform the ISH assays according to the manufacturer’s recommendations. Probes used for staining are hs-CFTR (ACDBio 603291), ss-CFTR (ACDBio 541401-C2), ss-NGFR (ACDBio 828841), ss-LOC100625684 (ACDBio 1216061-C2), ss-LOC100737531 (ACDBio 1216070-C3), and ss-NPY (ACDBio 318751). Negative control slides were performed in parallel. After incubation with fluorescent-labeled probes, slides were counterstained with DAPI and mounted with antifade fluorescent mounting medium (Dako). Fluorescent images were captured using sequential laser scanning confocal microscopy (Zeiss LSM-780). CFTR+ cells were counted from four independent human subjects.

Combined ISH and immunohistochemistry

After a standard ISH protocol, cells and tissue sections were directly incubated in blocking solution followed by primary and secondary antibodies for DCX, PCNA, Ki67 and SOX2 as described above.

Quantification and Statistical Analysis

Statistical analyses were performed using the statistical package R version 3.6.0 and the packages ggplot2, drc and ggpubr (R-studio Software, 4.1.1), OriginPro 2021b (OriginLab Corporation, Northampton, MA, USA). Statistical details of experiments can be found in the figure legends, including statistical test used, the exact value of n and dispersion and precision measures. Assumptions of normality and homogeneity of variance were tested before conducting the following statistical approaches. Mann-Whitney U test was used to measure the significance of the differences between two distributions. Multiple groups were compared using a two-way analysis of variance (ANOVA). Kolmogorov–Smirnov test was used to compare cumulative distributions. The probability of error level (alpha) was chosen to be 0.05. P values and the specific statistical test performed for each experiment is included in the appropriate figure legend or main text. Unless otherwise stated, results are presented as individual data points combined with boxplot indicating median lines and 25%-75% ranges.

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