ABSTRACT: Transcription profiling of human airway epithelial cells cultured at an air-liquid interface (ALI) and sampled at multiple time points to examine mucociliary differentiation
Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. In this study, we wanted to investigate mRNA expression patterns during airway epithelium differentiation. By using microarrays, we studied the changes in expression of mRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.
Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs of 20-23 nucleotides that down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation. They are phylogenetically well conserved, which probably implies an important role of miRNAs in biological processes. In this way, we wanted to shed some light on miRNA specific roles and the relationship with their mRNA targets during airway epithelium differentiation. By using microarrays, we studied the changes in expression of microRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA (including small RNAs) extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.
Project description:Human airway epithelial cells cultured in vitro at air-liquid interface (ALI) form a pseudostratified epithelium that forms tight junctions and cilia, and produces mucin, and are widely used as a model of differentiation, injury, and repair. To assess how closely the transcriptome of ALI epithelium matches that of in vivo airway epithelial cells, we used microarrays to compare the transcriptome of human large airway epithelial cells cultured at ALI with the transcriptome of large airway epithelium obtained via bronchoscopy and brushing. Gene expression profiling showed global gene expression correlated well between ALI cells and brushed cells, but there were some differences. Gene expression patterns mirrored differences in proportions of cell types (ALI have higher percentages of basal cells, brushed cells have higher percentages of ciliated cells), with ALI cells expressing higher levels of basal cell-related genes and brushed cells expressing higher levels of cilia-related genes. Pathway analysis showed ALI cells had increased expression of cell cycle and proliferation genes, while brushed cells had increased expression of cytoskeletal organization and humoral immune response genes. Overall, ALI cells are a good representation of the in vivo airway epithelial transcriptome, but for some biologic questions, the differences in the in vitro vs in vivo environments need to be considered. Affymetrix arrays were used to assess the gene expression of large airway cells cultured in vitro at air-liquid interface (12 samples) and large airway epithelial cells obtained by fiberoptic bronchoscopy of 20 healthy nonsmokers. *** Air-liquid interface Samples not provided in this Series. ***
Project description:Mouse tracheal epithelial cells were cultured at air-liquid interface (ALI), RNA was harvested at days 0, 2, and 7 post-ALI, and hybridized to two-channel MEEBO arrays. The experiment was designed to allow investigators to identify genes differentially expressed during airway epithelial cell differentiation and development, including ciliogenesis.
Project description:We report the application of RNA sequencing technology for high-throughput profiling of gene expression responses to human rhinovirus infection at 24 hours in air-liquid interface human airway epithelial cell cultures derived from 6 asthmatic and 6 non-asthmatic donors. RNA-seq analysis identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1), and novel ones that were identified for the first time in this study (e.g. CCRL1, CDHR3). We concluded that air liquid interface cultured human airway epithelial cells challenged with live HRV are a useful in vitro model for the study of rhinovirus induced asthma exacerbation, given that our findings are consistent with clinical data sets. Furthermore, our data suggest that abnormal airway epithelial structure and inflammatory signaling are important contributors to viral induced asthma exacerbation. Differentiated air-liquid interface cultured human airway epithelial cell mRNA profiles from 6 asthmatic and 6 non-asthmatic donors after 24 hour treatment with either HRV or vehicle control were generated by deep sequencing, using Illumina HiSeq 2000.
Project description:Platform: iPSC-derived airway plated on 2D-air liquid interface through basal cell intermediate. Purpose of experiment: To examine the role of mutant CFTR on IPSC-derived airway epithelium (ie. immune dysregulation; dysregulated calcium channel signaling etc). Description of samples:Typical phenotype CF F508del (1565) and syngeneic CFTR-corrected (1564) iPSCs- derived airway epithelium at D90 (D15 CD47hi/CD26lo; replated in 3DMG cultured in 210DCIY x 2 weeks, single-cell passaged and cultured in PNExPlus+dual smad media in 3D, s/p 2 NGFR sorts, cultured at air-liquid interface at 2D)
Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. In this study, we wanted to investigate mRNA expression patterns during airway epithelium differentiation. By using microarrays, we studied the changes in expression of mRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers.
Project description:Microarray analysis was performed to identify transcriptional changes that occur during mucociliary differentiation of human primary bronchial epithelial cells cultured at an air-liquid interface (ALI). Experiment Overall Design: Cells from three different donors were cultured and collected at 11 different time points from day 0 to day 28 of ALI culture.
Project description:Rat airway epithelial cells were cultured at the air-liquid interface and their total RNA was extracted for transcriptomic sequence analysis.
Project description:The goal of this study is to determine the transcriptional response of primary normal human bronchial epithelial (NHBE) cells following interaction with A. fumigatus conidia at 2, 6, and 12 hours in order to examine the infection of airway epithelial cells at multiple time points of co-incubation. After total RNA isolation, the Illumina® HiSeq 2000 next generation sequencing system was used to characterize the transcriptional response, in untreated and A. fumigatus exposed cells.