Project description:Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a severe syndrome affecting more than 200,000 patients annually in the U.S. New studies are needed to understand the biological and clinical mechanisms that impair alveolar epithelial function. Also, innovative therapies are needed for the resolution of pulmonary edema in ARDS. We and other investigators have reported that bone marrow derived mesenchymal stem cells (MSCs) are effective in preclinical models of ALI due to their ability to secrete several paracrine factors that can regulate lung endothelial and epithelial permeability, including growth factors, anti-inflammatory cytokines, and antimicrobial peptides. So in this study we will test the therapeutic value of human MSCs in an in vitro model of acute lung injury induced by pro-inflammatory cytokines. We will identify differentially expressed genes in primary cultures of human alveolar epithelial type II cells and human bone marrow derived mesenchymal stem cells using Affymetrix gene expression arrays. Human mesenchymal stem cells (MSCs) and human alveolar epithelial type II cells were co-cultured in a transwell system. The cells were stimulated with cytomix (a combination of different pro-inflammatory cytokines) under different conditions. Cells were harvested for Affymetrix gene expression arrays. Total 25 samples are analyzed, 3~5 replicates are included.
Project description:Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a severe syndrome affecting more than 200,000 patients annually in the U.S. New studies are needed to understand the biological and clinical mechanisms that impair alveolar epithelial function. Also, innovative therapies are needed for the resolution of pulmonary edema in ARDS. We and other investigators have reported that bone marrow derived mesenchymal stem cells (MSCs) are effective in preclinical models of ALI due to their ability to secrete several paracrine factors that can regulate lung endothelial and epithelial permeability, including growth factors, anti-inflammatory cytokines, and antimicrobial peptides. So in this study we will test the therapeutic value of human MSCs in an in vitro model of acute lung injury induced by pro-inflammatory cytokines. We will identify differentially expressed genes in primary cultures of human alveolar epithelial type II cells and human bone marrow derived mesenchymal stem cells using Affymetrix gene expression arrays.
Project description:We investigated whether in vitro expansion of human alveolar epithelial type II cells is possible. We found that human endogenous human alveolar epithelial type II cells can be cultured and passaged. The culture system enabled retroviral gene transduction into human alveolar epithelial type II cells. We performed RNA sequencing of human alveolar epithelial type II cells transduced with mutant surfactant protein C or control vector.
Project description:We used RNA sequencing to comprehensively map the expression of coding and non-coding RNAs in primary human alveolar epithelial type II cells (AECIIs), alveolar macrophages (AMs), human lung tissue, and the epithelial cell line A549 during infection with IAV strain H3N2 Panama
Project description:We established a new protocol for negative immunomagnetic isolation of murine primary Type II alveolar epithelial cells (AEC II) yielding untouched primary murine AEC II. AEC II were collected from mice 24h after Aspergillus fumigatus or mock infection (9 replicates per experimental group) and analyzed by label-free quantitative proteomics.
Project description:In 3D cultures of hPSC-derived lung progenitors withdrawal of the GSK3 inhibitor, CHIR99021, induces multilineage maturation, including of proximal cells, type I alveolar epithelial cells and morphologically mature type II cells.
Project description:To test whether vitamin D has a functionally important effect upon primary alveolar epithelial type II cells we used gene expression microarray to identify genes that are regulated by 25-dihydroxyvitamin D in adult alveolar type II cells.
Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.
Project description:Pandemic influenza H1N1 (pdmH1N1) virus causes mild disease in humans but occasionally leads to severe complications and even death, especially in those who are pregnant or have underlying disease. Cytokine responses induced by pdmH1N1 viruses in vitro are comparable to other seasonal influenza viruses, suggesting the cytokine dysregulation as seen in H5N1 infection is not a feature of the pdmH1N1 virus. However, a comprehensive gene expression profile of pdmH1N1 in relevant primary human cells in vitro has not been reported. Type I alveolar epithelial cells are a key target cell in pdmH1N1 pneumonia. We carried out a comprehensive gene expression profiling using the Affymetrix microarray platform to compare the transcriptomes of primary human alveolar type I-like alveolar epithelial cells infected with pdmH1N1 or seasonal H1N1 virus. Primary type II alveolar epithelial cells were isolated from human non-malignant lung tissue of three patients who underwent lung resection, and cells were differentiated to type I-like before use. Type I-like alveolar epithelial cells were mock infected, or infected with pdmH1N1 or seasonal H1N1 viruses at a multiplicity of infection (MOI) of two. Total RNA was extracted from cells after 8h post-infection, and gene expression profiling was performed using an Affymetrix Human Gene 1.0 ST microarray platform.
Project description:Comparison of rat freshly-isolated alveolar epithelial type I cells, freshly-isolated type II cells, and type II cells cultured for 7 days Keywords = rat, alveolar epithelial type I cells, cultured type II cells Keywords: parallel sample