Biology subjects

Brody, S. L.

Publications and source records attributed to Brody, S. L..

3 recordsLinked to original sources

bioRxiv2020

Single nucleus RNASeq profiling of mouse lung: reduced dissociation bias and improved detection of rare cell types compared with single cell RNASeq

RATIONALESingle cell RNA-sequencing (scRNASeq) has led to multiple recent advances in our understanding of lung biology and pathophysiology, but utility is limited by the need for fresh samples, loss of cell types due to death or inadequate dissociation, and the induction of transcriptional stress responses during tissue digestion. Single nucleus RNASeq (snRNASeq) has addressed these deficiencies in some other tissues, but no protocol exists for lung. We sought to develop such a protocol and compare its results with scRNA-seq. METHODSSingle nucleus suspensions were prepared rapidly (45 min) from two mouse lungs in lysis buffer on ice while a single cell suspension from an additional mouse lung was generated using a combination of enzymatic and mechanical dissociation (1.5 h). Cells and nuclei were processed using the 10x Genomics platform, and following sequencing of cDNA libraries single cell data was analyzed by Seurat. RESULTS16,656 single nucleus and 11,934 single cell transcriptomes were generated. Despite reduced mRNA levels in nuclei vs. cells, gene detection rates were equivalent in snRNASeq and scRNASeq ([~]1,750 genes and 3,000 UMI per cell) when mapping intronic and exonic reads. snRNASeq identified a much greater proportion of epithelial cells than scRNASeq (46% vs 2% of total), including basal and neuroendocrine cells, while reducing immune cells from 54% to 15%. snRNASeq transcripts are enriched for transcription factors and signaling proteins, with reduced detection of housekeeping genes, mitochondrial genes, and artifactual stress response genes. Both techniques improved mesenchymal cell detection over previous studies, and analysis of fibroblast diversity showed two transcriptionally distinct populations of Col13a1+ cells, termed Bmper+ and Brinp1+ fibroblasts. To define homeostatic signaling relationships among cell types, receptor-ligand mapping of was performed for alveolar compartment cells using snRNASeq data, revealing complex interplay among epithelial, mesenchymal, and capillary endothelial cells. CONCLUSIONSingle nucleus RNASeq can be readily applied to snap frozen, archival murine lung samples, improves dissociation bias, eliminates artifactual gene expression and provides similar gene detection compared to scRNASeq.

genomics↗

bioRxiv2020

Chemokine receptor 2-targeted molecular imaging in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, inflammatory lung disease that is monitored clinically by measures of lung function, without effective molecular markers of disease activity or therapeutic efficacy. Lung immune cells active in the pro-fibrotic process include inflammatory monocyte and interstitial macrophages that express the C-C motif chemokine receptor 2 (CCR2). CCR2+ monocyte lung influx is essential for disease phenotypes in models of fibrosis and identified in lungs from subjects with IPF. Here, we show that our peptide-based radiotracer 64Cu-DOTA-ECL1i identifies CCR2+ inflammatory monocytes and interstitial macrophages in multiple preclinical mouse models of lung fibrosis, using positron emission tomography (PET) imaging. Mice with bleomycin-induced fibrosis treated with blocking antibodies to interleukin-1{beta}, a mediator of fibrosis associated with CCR2+ cell inflammation, or with pirfenidone, an approved anti-fibrotic agent, demonstrated decreased CCR2-dependent interstitial macrophage accumulation and reduced 64Cu-DOTA-ECL1i PET uptake, compared to controls. Lung tissues from patients with fibrotic lung disease demonstrated abundant CCR2+ cells surrounding regions of fibrosis, and an ex vivo tissue-binding assay showed correlation between radiotracer localization and CCR2+ cells. In a phase 0/1 clinical study of 64Cu-DOTA-ECL1i PET, healthy volunteers showed little lung uptake, while subjects with pulmonary fibrosis exhibited increased uptake, notably in zones of subpleural fibrosis, reflecting the distribution of CCR2+ cells in the profibrotic niche. These findings support a pathologic role of inflammatory lung monocytes/macrophages in fibrotic lung disease and the translational use of 64Cu-DOTA-ECL1i PET to track CCR2-specific inflammation for image-guided therapy. One Sentence SummaryPET imaging of CCR2+ cells in lung fibrosis identifies a therapeutic response in mouse models and displays a perifibrotic signal in subjects with IPF.

cell biology↗

bioRxiv2020

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

The derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells ("iBCs"), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker NGFR. However, in response to primary basal cell medium, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.

developmental biology↗