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: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.
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: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:Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using exon microarrays and RNAsequencing. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. In conclusion, In vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium.
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). We used microarrays to detail the global programme of gene expression that occurs during regeneration and ciliogenesis of the human airway mucociliary epithelium. The four time points of regeneration of the airway epithelium (ALI-D0 which corresponds to the end of proliferation step; ALI-D7 which corresponds to the polarization step; ALI-D14 which corresponds to the onset of ciliogenesis and ALI-D21 corresponding to the terminal differentiation step) for RNA extraction and hybridization on Affymetrix microarrays.
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.
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). The airway mucociliary epithelium is consituted of three main cell types : ciliated cells, secretory cells and basal cells. We used microRNA microrrays to investigate the signature of microRNA during the four step of regeneration of the airway epithelium. Four time points (ALI-D0, ALI-D7, ALI-D14, ALI-D21) of regeneration of the airway epithelium for 3 donors.
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). The airway mucociliary epithelium is consituted of three main cell types : ciliated cells, secretory cells and basal cells. We used microRNA microrrays to investigate the signature of microRNA during the four step of regeneration of the airway epithelium. Four time points (ALI-D0, ALI-D7, ALI-D14, ALI-D21) of regeneration of the airway epithelium for 3 donors.