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To develop in vitro respiratory models, it is crucial to identify the factors involved in epithelial cell differentiation. In this study, we comprehensively analyzed the effects of air–liquid interface (ALI) culture on epithelial cell differentiation using single-cell RNA sequencing (scRNA-seq). ALI culture induced a pronounced shift in cell composition, marked by a fivefold increase in ciliated cells and a reduction of more than half in basal cells. Transcriptional signatures associated with epithelial cell differentiation, analyzed using iPathwayGuide software, revealed the downregulation of VEGFA and upregulation of CDKN1A as key signals for epithelial differentiation. Our findings highlight the efficacy of the ALI culture for replicating the human lung airway epithelium and provide valuable insights into the crucial factors that influence human ciliated cell differentiation. [ABSTRACT FROM AUTHOR]

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air–liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems. [ABSTRACT FROM AUTHOR]

The bitter taste receptors (T2Rs) expressed in human sinonasal mucosae are known to elicit innate immune responses involving the release of nitric oxide (NO). We investigated the expression and distribution of two T2Rs, T2R14 and T2R38, in patients with chronic rhinosinusitis (CRS) and correlated the results with fractional exhaled NO (FeNO) levels and genotype of the T2R38 gene (TAS2R38). Using the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) phenotypic criteria, we identified CRS patients as either eosinophilic (ECRS, n = 36) or non-eosinophilic (non-ECRS, n = 56) patients and compared these groups with 51 non-CRS subjects. Mucosal specimens from the ethmoid sinus, nasal polyps, and inferior turbinate were collected from all subjects, together with blood samples, for RT-PCR analysis, immunostaining, and single nucleotide polymorphism (SNP) typing. We observed significant downregulation of T2R38 mRNA levels in the ethmoid mucosa of non-ECRS patients and in the nasal polyps of ECRS patients. No significant differences in T2R14 or T2R38 mRNA levels were found among the inferior turbinate mucosae of the three groups. Positive T2R38 immunoreactivity was localized mainly in epithelial ciliated cells, whereas secretary goblet cells generally showed lack of staining. The patients in the non-ECRS group showed significantly lower oral and nasal FeNO levels compared with the control group. There was a trend towards higher CRS prevalence in the PAV/AVI and AVI/AVI genotype groups as compared to the PAV/PAV group. Our findings reveal complex but important roles of T2R38 function in ciliated cells associated with specific CRS phenotypes, suggesting the T2R38 pathway as a potential therapeutic target for promotion of endogenous defense mechanisms. [ABSTRACT FROM AUTHOR]

Ciliated cell markers expressed in epithelial ovarian cancers (EOC) are associated with improved survival. We examined the distribution of cells expressing ciliated cell markers in various EOC histologies and stages. Immunohistochemistry and/or multiplex immunofluorescence were used to determine the expression of FOXJ1 and/or CAPS (ciliated cell markers) in tissue microarrays including 4 normal fallopian tubes, 6 normal endometria, 16 cystadenomas, 25 borderline tumors, 21 low-grade carcinomas, and 118 high-grade carcinomas (HGSOC) (46 serous, 29 endometrioid, 30 clear cell, 13 mucinous). CAPS+ cells were observed in normal fallopian tubes and endometria and in ~85% of serous benign and borderline tumors and low-grade carcinomas but only in <40% of HGSOC. mRNA data from an independent cohort showed higher FOXJ1 and CAPS expression in serous borderline tumors and low-grade carcinomas compared to HGSOC. In HGSOC, ciliated cell-positive markers were observed in 52% primary tumors compared to 26% of patient-matched synchronous metastases, and 24% metachronous metastases (p = 0.009). mRNA data from an independent HGSOC cohort showed lower levels of CAPS in metastases than in primary tumors (p = 0.03). Overall, the study revealed that ciliated cells were less common in mucinous EOC, the percentage of ciliated cell marker-positive cases decreased with increasing grade, and the percentage of ciliated cells decreased in HGSOC metastases compared to patient-matched primary tumors. [ABSTRACT FROM AUTHOR]

Smoking is a widely recognized risk factor in the emergence of cancers and other lung diseases. Studies of non-cancer lung diseases typically investigate the role that smoking has in chronic changes in lungs that might predispose patients to the diseases, whereas most cancer studies focus on the mutagenic properties of smoking. Large-scale cancer analysis efforts have collected expression data from both tumor and control lung tissues, and studies have used control samples to estimate the impact of smoking on gene expression. However, such analyses may be confounded by tumor-related micro-environments as well as patient-specific exposure to smoking. Thus, in this paper, we explore the utilization of mutational signatures to study environment-induced changes of gene expression in control lung tissues from lung adenocarcinoma samples. We show that a joint computational analysis of mutational signatures derived from sequenced tumor samples, and the gene expression obtained from control samples, can shed light on the combined impact that smoking and tumor-related micro-environments have on gene expression and cell-type composition in non-neoplastic (control) lung tissue. The results obtained through such analysis are both supported by experimental studies, including studies utilizing single-cell technology, and also suggest additional novel insights. We argue that the study provides a proof of principle of the utility of mutational signatures to be used as sensors of environmental exposures not only in the context of the mutational landscape of cancer, but also as a reference for changes in non-cancer lung tissues. It also provides an example of how a database collected with the purpose of understanding cancer can provide valuable information for studies not directly related to the disease. [ABSTRACT FROM AUTHOR]

Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wild-type mice, then studied EpCAM+ lung epithelial cells using single-cell RNA-seq analysis. We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge altered the relative abundance of epithelial cell types and resulted in cell type-specific transcriptomic changes. Bulk and cell type-specific analysis also showed that loss of Tet1 led to the altered expression of genes linked to augmented HDM-induced lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and tissue repair genes. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, which may promote allergen-induced lung inflammation. [ABSTRACT FROM AUTHOR]

Cell-based therapies hold great promise in re-establishing organ function for many diseases, including untreatable lung diseases such as idiopathic pulmonary fibrosis (IPF). However, many hurdles still remain, in part due to our lack of knowledge about the disease-driving mechanisms that may affect the cellular niche and thereby possibly hinder the function of any transplanted cells by imposing the disease phenotype onto the newly generated progeny. Recent findings have demonstrated increased ciliation of lung cells from IPF patients, but how this affects ciliated cell function and the airway milieu is not well-known. Here, we performed single-cell RNA sequencing on primary ciliated (FOXJ1+) cells isolated from IPF patients and from healthy control donors. The sequencing identified multiple biological processes, such as cilium morphogenesis and cell signaling, that were significantly changed between IPF and healthy ciliated cells. Ferritin light chain (FTL) was downregulated in IPF, which suggests that iron metabolism may be affected in the IPF ciliated cells. The RNA expression was confirmed at the protein level with histological localization in lung tissue, prompting future functional assays to reveal the potential role of FTL. Taken together, our data demonstrate the importance of careful analyses in pure cell populations to better understand the IPF disease mechanism. [ABSTRACT FROM AUTHOR]

Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air–liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation. [ABSTRACT FROM AUTHOR]

High-grade serous epithelial ovarian cancer (HGSOC) is the fifth leading cause of cancer death in women and the first among gynecological malignancies. Despite an initial response to standard chemotherapy, most HGSOC patients relapse. To improve treatment options, we must continue investigating tumor biology. Tumor characteristics (e.g., risk factors and epidemiology) are valuable clues to accomplish this task. The two most frequent risk factors for HGSOC are the lifetime number of ovulations, which is associated with increased oxidative stress in the pelvic area caused by ovulation fluid, and a positive family history due to genetic factors. In the attempt to identify novel genetic factors (i.e., genes) associated with HGSOC, we observed that several genes in linkage with HGSOC are expressed in the ciliated cells of the fallopian tube. This finding made us hypothesize that ciliated cells, despite not being the cell of origin for HGSOC, may take part in HGSOC tumor initiation. Specifically, malfunction of the ciliary beat impairs the laminar fluid flow above the fallopian tube epithelia, thus likely reducing the clearance of oxidative stress caused by follicular fluid. Herein, we review the up-to-date findings dealing with HGSOC predisposition with the hypothesis that fallopian ciliated cells take part in HGSOC onset. Finally, we review the up-to-date literature concerning genes that are located in genomic loci associated with epithelial ovarian cancer (EOC) predisposition that are expressed by the fallopian ciliated cells. [ABSTRACT FROM AUTHOR]

Objective: To investigate morphological changes in the endometrial epithelial cells of patients with infertility problems. Materials and methods: Endometrial biopsies were obtained from 10 women who have undergone several unsuccessful in vitro fertilisation (IVF) procedures. Endometrial biopsies were performed between luteinizing hormone surge days LH+6 to +10 of the natural menstrual cycle. Each sample was divided into three parts, which were processed for histological, transmission (TEM), and scanning electron microscopy (SEM) investigations. Results: Histological investigations demonstrated significant alterations in the apical part of epithelial cells of one patient; in four patients, the gland maturity was low, not matching the cycle day, and thus a phase lag had developed. By TEM examination, we ascertained changes in secretory and ciliated cells in three patients (decreased amount or missing microvilli, irregular cilia in ciliated cells). SEM examination found pinopodes in five patients: three samples contained fully developed pinopodes-larger and completely smooth, with only some wrinkles; one sample contained regressing small pinopodes, with wrinkled surfaces; and one sample had both developed and regressing pinopodes. Conclusions: To conclude, our study shows that the endometrium of patients with poor IVF outcome has either significant changes in the morphology or the endometrial maturation is inhibited and a phase lag often develops. Our study shows that endometrial pinopodes are found throughout the mid-luteal phase up to day LH+10.