High throughput quantitative whole transcriptome analysis of E18.5 mouse distal lung mesenchyme and endothelium
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ABSTRACT: We used microfluidic single cell RNA-seq on 42 individual mouse lung mesenchymal and 69 endothelial cells at E18.5 to measure the transcriptional state. We were able to classify these cells into distinct populations as well as map their signaling interactions with the distal lung epithelium. In this manner, single cell RNA-seq can be used to unbiasedly determine the signals the dictate construction and maintenance of the alveolus.
Project description:We used microfluidic single cell RNA-seq on 47 individual mouse lung mesenchymal cells at adulthood to measure the transcriptional state. We were able to classify these cells into distinct populations as well as map their signaling interactions with the distal lung epithelium and endothelium. In this manner, single cell RNA-seq can be used to unbiasedly determine the signals the dictate construction and maintenance of the alveolus.
Project description:We used microfluidic single cell RNA-seq on 198 individual mouse lung epithelial cells at 4 different stages throughout development to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We classified 80 cells comprising the distal lung epithelium at E18.5 into distinct populations using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell types. This M-bM-^@M-^\reverse tissue engineeringM-bM-^@M-^] approach confirmed the basic outlines of the conventional model of cell type diversity in the distal lung and led to the discovery of a large number of novel transcriptional regulators and cell type markers that discriminate between the different populations. Moreover, we reconstructed the steps during maturation of bipotential progenitors into both alveolar lineages based on the presence of undifferentiated, differentiated as well as differentiation intermediate cells at the single time point E18.5. Finally, we followed Sftpc-positive cells throughout their lifecycle (E14.5, E16.5, E18.5, adult) and identified 7 gene sets that differentiate between the multipotential, bipotential, mature, as well as intermediate states of the AT2 lineage. 198 single-cell transcriptomes from mouse lung epithelium were analyzed in total, two 200-cell bulk control samples as well as one no-cell control; All single cell and control samples contain 92 external RNA spike-ins; For time point E18.5, three individual experiments were performed using 3 different pregnant mice (3 biological replicates): Replicate 1 (pooled sibling lungs) yielded 20 single cell transcriptomes, replicate 2 (one single embryonic lung) yielded 34 single cell transcriptomes and replicate 3 (pooled siibling lungs) yielded 26 single cell transcriptomes; In addition, a 200-cell bulk control sample was prepared for E18.5 replicate 1 and E18.5 replicate 3 experiments; A no-cell control sample was generated for the E18.5 replicate 1 experiment; For time point E14.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 45 single cell transcriptomes; For time point E16.5, one experiment (one pregnant mouse, pooled sibling lungs) was performed yielding 27 single cell transcriptomes; For the adult time point, one 107 day old mouse was used and transcriptomes of 46 single cells were obtained; All single cell samples were processed on the microfluidic platform, 200-cell-bulk and no-cell control samples were processed in microliter volumes in PCR tubes.
Project description:We used microfluidic single cell RNA-seq on 198 individual mouse lung epithelial cells at 4 different stages throughout development to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We classified 80 cells comprising the distal lung epithelium at E18.5 into distinct populations using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or prior purification of cell types. This “reverse tissue engineering” approach confirmed the basic outlines of the conventional model of cell type diversity in the distal lung and led to the discovery of a large number of novel transcriptional regulators and cell type markers that discriminate between the different populations. Moreover, we reconstructed the steps during maturation of bipotential progenitors into both alveolar lineages based on the presence of undifferentiated, differentiated as well as differentiation intermediate cells at the single time point E18.5. Finally, we followed Sftpc-positive cells throughout their lifecycle (E14.5, E16.5, E18.5, adult) and identified 7 gene sets that differentiate between the multipotential, bipotential, mature, as well as intermediate states of the AT2 lineage.
Project description:Acute exposure to high-dose gamma radiation can result in radiation-induced lung injury (RILI), characterized by acute pneumonitis and subsequent lung fibrosis. A microfluidic organ-on-a-chip lined by human lung alveolar epithelium interfaced with pulmonary endothelium (Lung Alveolus Chip) is used to model acute, early stage RILI in vitro. Both lung epithelium and endothelium exhibit DNA damage, cellular hypertrophy, upregulation of inflammatory cytokines, and loss of barrier function within 6h of radiation exposure, and greater damage is observed in the endothelium. The alveolus chips are exposed to radiation injury at 16 Gy and shows effects that resemble the human lung greater than animal preclinical models. The Alveolus Chip is also used to evaluate the potential ability of two drugs to suppress the effects of acute RILI. These data demonstrate that the Lung Alveolus Chip provides a human relevant alternative approach for studying the molecular basis of acute RILI towards screening radiation countermeasure therapeutics.
Project description:Acute exposure to high-dose gamma radiation often results in radiation-induced lung injury (RILI), characterized by acute pneumonitis and subsequent lung fibrosis. A microfluidic organ-on-a-chip device consisting of human lung alveolar epithelium and pulmonary endothelium (Lung Alveolus Chip) is used to recapitulate acute, early stage RILI in vitro. This RNA-seq data captures that both the lung epithelium and endothelium in this model capture key hallmarks of this disease particularly, DNA damage, cellular hypertrophy, upregulation of inflammatory cytokines, and loss of barrier function within 6h of radiation exposure. The data also suggests that radiation affects the alveolar endothelium more significantly than the epithelium. The alveolus chips are exposed to radiation injury at 16 Gy and shows effects that resemble the human lung greater than animal preclinical models. These data demonstrate that the Lung Alveolus Chip provides a human relevant alternative approach for studying the molecular basis of acute RILI towards screening radiation countermeasure therapeutics.
Project description:ShhCre;Mst1/2flx/flx (Mst1/2 D/D) mice were generated to conditionally delete Mst1 and Mst2 from epithelial progenitors during lung morphogenesis. Lungs from E18.5 control and Mst1/2 D/D mice were mechanically and enzymatically dissociated to generate single cell suspension. Epcam(+) cells were isolated using magnetic microbeads. Microarray analysis of mRNAs isolated from Epcam(+) epithelial cells from E18.5 control and Mst1/2 D/D mice was performed to identify transcriptional changes following deletion of the mammalian Hippo kinases (Mst1 and Mst2) from the embryonic lung. The mammalian Hippo kinases, Mst1 and Mst2, were conditionally deleted in epithelial progenitors of the developing lung using ShhCre. Epcam(+) epithelial cells were isolated from the lungs of E18.5 control and Mst1/2 deleted mice. mRNA isolated from Epcam(+) epithelial cells was analyzed by microarray.
Project description:ShhCre;Mst1/2flx/flx (Mst1/2 D/D) mice were generated to conditionally delete Mst1 and Mst2 from epithelial progenitors during lung morphogenesis. Lungs from E18.5 control and Mst1/2 D/D mice were mechanically and enzymatically dissociated to generate single cell suspension. Epcam(+) cells were isolated using magnetic microbeads. Microarray analysis of mRNAs isolated from Epcam(+) epithelial cells from E18.5 control and Mst1/2 D/D mice was performed to identify transcriptional changes following deletion of the mammalian Hippo kinases (Mst1 and Mst2) from the embryonic lung.