Gene expression profile at single cell level of Basal Cells (BCs) from nasal scrapings of patients with and without cystic fibrosis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE200342 | GEO | 2022/06/30
REPOSITORIES: GEO
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Project description:We describe a human nasal epithelial (HNE) organoid model derived directly from patient samples that is well-differentiated and recapitulates the airway epithelium, including the expression of cilia, mucins, tight junctions, the cystic fibrosis transmembrane conductance regulator (CFTR), and ionocytes. This model requires few cells compared to airway epithelial monolayer cultures, with multiple outcome measurements depending on the application. A novel feature of the model is the predictive capacity of lumen formation, a marker of baseline CFTR function that correlates with short-circuit current activation of CFTR in monolayers and discriminates the cystic fibrosis (CF) phenotype from non-CF. Our HNE organoid model is amenable to automated measurements of forskolin-induced swelling (FIS), which distinguishes levels of CFTR activity. While the apical side is not easily accessible, RNA- and DNA-based therapies intended for systemic administration could be evaluated in vitro, or it could be used as an ex vivo biomarker of successful repair of a mutant gene. In conclusion, this highly differentiated airway epithelial model could serve as a surrogate biomarker to assess correction of the mutant gene in CF or other diseases, recapitulating the phenotypic and genotypic diversity of the population.
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Project description:The gold standard for diagnosing cystic fibrosis (CF) is a sweat chloride value above 60 mEq/L. However, this historical and important tool has limitations; other techniques should be studied, including the nasal potential difference (NPD) test. CFTR gene sequencing can identify CFTR mutations, but this method is time-consuming and too expensive to be used in all CF centers. The present study compared CF patients with two classes I-III CFTR mutations (10 patients) (G1), CF patients with classes IV-VI CFTR mutations (five patients) (G2), and 21 healthy subjects (G3). The CF patients and healthy subjects also underwent the NPD test. A statistical analysis was performed using the Mann-Whitney, Kruskal-Wallis, χ(2), and Fisher's exact tests, α = 0.05. No differences were observed between the CF patients and healthy controls for the PDMax, Δamiloride, and Δchloride + free + amiloride markers from the NPD test. For the finger value, a difference between G2 and G3 was described. The Wilschanski index values were different between G1 and G3. In conclusion, our data showed that NPD is useful for CF diagnosis when classes I-III CFTR mutations are screened. However, if classes IV-VI are considered, the NPD test showed an overlap in values with healthy subjects.
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Project description:The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.
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Project description:This multi-center study will compare multi-target DNA and quantitative FIT stool-based testing to colonoscopy in individuals with Cystic Fibrosis (CF) undergoing colon cancer screening with colonoscopy. The primary endpoint is detection of any adenomas, including advanced adenomas and colorectal cancer (CRC).
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Project description:The human airway epithelium is regenerated by basal cells. Thus, basal cell therapy has the potential to cure cystic fibrosis (CF) lung disease. We previously reported that the human basal cells repopulated the mouse airway epithelium after transplantation, and we estimated that 60 million cells would be needed to treat a human patient. To further develop cell therapy, we compared the proliferation potential of non-CF and CF tissue-derived bronchial basal cells. Three methods were used: regenerative cell frequency, burst size, and cell division frequency. Second, we used a serial passage strategy to determine if CF basal cells could be amplified to the estimated therapeutic dose. These studies evaluated that tissue-derived bronchial basal cells and the basal cells that were recovered by brushing bronchial airways or the nasal respiratory epithelium. Finally, we used the limiting dilution method to isolate non-CF and CF basal cell clones. The proliferation assays and the air-liquid-interface differentiation method were used to determine if cell amplification altered the proliferation and/or differentiation potential of clonal isolates. We demonstrate that: (a) non-CF and CF basal cell proliferation is similar, (b) CF basal cells can be amplified to a therapeutic cell dose, and (c) amplified non-CF and CF basal cell clones differentiate normally. Despite these encouraging findings, we also find that the cell amplification process depletes the regenerative basal cell pool. Analysis of basal cell clones indicates that serial passage selects for long-lived basal cells and raise the possibility that prospective isolation of these stem-like cells will improve the efficacy of cell replacement therapy. Stem Cells Translational Medicine 2019;8:225&235.
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Project description:Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl- channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl- efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/F508del) were treated with buserelin and we show here that the treatment alleviates Cl- channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.
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Project description:AimInhaled hypertonic saline increases mucociliary clearance, improves pulmonary function, and decreases exacerbations in cystic fibrosis (CF) but contributes to the already significant treatment burden of CF. Overnight delivery of inhaled medications via a specially designed nasal cannula-aerosol device (Trans-nasal Pulmonary Aerosol Delivery [tPAD]) is an alternative approach. Here, we test whether overnight inhalation of hypertonic saline via tPAD improves mucociliary clearance and assess the tolerability of the device.MethodIn this study, 12 CF subjects inhaled 7% hypertonic saline (HS) for 8 h overnight using the tPAD system. Safety and tolerability were assessed and measurements of mucociliary and absorptive clearance (MCC/ABS) were performed after the treatment. Comparisons were made versus sham treatment where the same subjects wore the nasal cannula overnight but did not receive aerosol.ResultsBoth the HS and sham treatments were well-tolerated. Only one subject did not complete the overnight HS treatment. There were no significant differences in MCC associated with HS inhalation at any time point (90 min, 3 h, 6 h) in any lung zone. Changes in FEV1 on both study days were similar. There were no differences in quality of sleep between HS and sham nights as assessed with the modified Leeds Sleep Evaluation Questionnaire (mLSEQ). Sino-Nasal Outcome Test (SNOT-14) questionnaires demonstrated significant increases (worsening) in 2/14 symptom categories with HS.ConclusionsThe most likely cause for the failure to accelerate MCC was under-dosing of HS relative to the active transport of salt from the airways.
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Project description:The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air-liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.
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Project description:Cystic fibrosis is caused by genetic defects that impair the CFTR channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described CFTR function assays in 3D airway organoids were not fully optimal, because of inefficient organoid differentiation and limited scalability. In this report, we therefore describe an alternative method of culturing nasal-brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1<i>β</i> and interleukin-1<i>β</i>, which enabled consistent detection of CFTR modulator responses in nasal-airway organoid cultures from subjects with cystic fibrosis.
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