Ex vivotissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

Abstract

Pulmonary fibrosis develops as a consequence of failed regeneration after injury. Analyzing mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques. In this work, we coupledex vivocytokine and drug perturbations of human precision-cut lung slices (hPCLS) with scRNAseq and induced a multi-lineage circuit of fibrogenic cell states in hPCLS, which we show to be highly similar to thein vivocell circuit in a multi-cohort lung cell atlas from pulmonary fibrosis patients. Using micro-CT staged patient tissues, we characterized the appearance and interaction of myofibroblasts, an ectopic endothelial cell state and basaloid epithelial cells in the thickened alveolar septum of early-stage lung fibrosis. Induction of these states in theex vivohPCLS model provides evidence that the basaloid cell state was derived from alveolar type-2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in the hPCLS model and anti-fibrotic drug treatments showed highly cell type specific effects. Our work provides an experimental framework for perturbational single cell genomics directly in human lung tissue that enables analysis of tissue homeostasis, regeneration and pathology. We further demonstrate that hPCLS offers novel avenues for scalable, high-resolution drug testing to accelerate anti-fibrotic drug development and translation.

Gene-level alignment of single cell trajectories

“…5d, Supplementary Table 3). While the majority of EMT genes show mismatches only at later pseudotime points, consistent with dysregulated EMT being implicated in ABC development in IPF 27,33,34,36,37 , it is interesting to note that some EMT genes also show differences at early/mid differentiation stages (e.g. NNMT, CXCL1, CXCL8).…”

“…Using the Adams et al (2020) dataset 27 , we investigated the differentiation of alveolar type 2 (AT2) cells into alveolar type 1 (AT1) cells in the healthy lung versus AT2 differentiation into aberrant basaloid cells (ABC) in the IPF lung (Fig. 5a), as suggested by several studies 33,34 . ABCs have only recently been described in single-cell studies from IPF patients 33,[35][36][37] , and their origin and role in IPF pathogenesis is still not understood.…”

Integrated multiomic analysis identifiesTRIP13as a mediator of alveolar epithelial type II cell dysfunction in idiopathic pulmonary fibrosis

“…To further explore the potential antifibrogenic effect of DCZ0415, we treated control human lung tissue slices with a fibrotic cocktail (FC) consisting of 5 ng/ml TGF-β, 10 ng/ml platelet-derived growth factor-AB (PDGF-AB), 10 ng/ml tumor necrosis factor-alpha (TNF-α) and 5 μM lysophosphatidic acid (LPA) in the presence or absence 5 µM DCZ0415. FC promotes EMT and has also been suggested to promote the transdifferentiation of AT2 cells to airway-like epithelial cells (55) . DCZ0415 maintained the AT2 cell phenotype as shown by increased HTII-280 expression in the presence of FC (Figure 4F).…”

Lung injury shifts pulmonary capillary endothelial cells towards regeneration-associated Lrg1+ subpopulations with delayed dynamics in aged mice

“…It is therefore tempting to hypothesize that in the context of the human pathology, SV EC are deficient in this differentiation process and accumulate in the injured tissue. Interestingly, a recent preprint, using an ex vivo human precision-cut lung slice (hPCLS) model of lung fibrogenesis, also described a transcriptomic proximity between alveolar PCEC and a systemic venous EC state [69]. In this case, the authors presented a differentiation trajectory in the opposite direction, proposing that PCEC in injured alveoli give rise to a VWA1+/PLVAP+ ectopic EC state with transcriptional similarities to the systemic vasculature.…”