Alveolar regeneration through a Krt8+ transitional stem cell state that persists in human lung fibrosis

Abstract

The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

Single-Cell Transcriptomics Identifies Dysregulated Metabolic Programs of Aging Alveolar Progenitor Cells in Lung Fibrosis

“…Pseudotime analysis suggested a trasition from AEC2-1 to AEC2-2 and further to AEC2-3. A recent study showed a cluster of Krt8 positive AEC2s in bleomycin injured mouse lung are transitional stem cells that precedes the regeneration of AT1 cells (Strunz et al, 2020). The AEC2 in subset AEC2-3 we identified here might include the Krt8+ cells reported.…”

“…Heterogeneity of lung epithelial cells has been recognized with recently developed single cell RNA-seq technique (Reyfman et al, 2019;Strunz et al, 2020). Reyfman et al reported subcluters of alveolar epithelial cells from human lung and these clusters distributed differently between fibrotic and healthy donor lungs (Reyfman et al, 2019).…”

“…Both claudins (Kage et al, 2014) and keratins are tight junction proteins and the markers of epithelial differentiation. Recent reports suggest Krt8+ or Krt19+ AEC2s are progenitor cells in a transitional state from AEC2 to AEC1 in the lung (Kobayashi et al, 2020;Strunz et al, 2020). Much detailed lineage tracing studies and the progenitor characterization especially in IPF are needed to confirm the role of this subpopulation in lung repair.…”

CellRank for directed single-cell fate mapping

“…Formalin-fixed paraffin-embedded lung sections (3.5 μm thick) from bleomycin-treated mice at day 10 (n=2) and day 22 (n=2) after bleomycin instillation, and from phosphate-buffered saline (PBS)-treated controls (n=2) were stained as previously described 15 . In brief, after deparaffinization, rehydration and heat-mediated antigen retrieval with citrate buffer (10 mM, pH = 6.0), sections were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-Bpifb1 (kindly provided by C. Bingle 62 , 1:500), mouse anti-Trp63 (abcam, ab735, clone A4A, 1:50) and chicken anti-Krt5 (BioLegend, Poly9059, 1:1,000).…”

CellRank for directed single-cell fate mapping

“…Formalin-fixed paraffin-embedded lung sections (3.5 µm thick) from bleomycin-treated mice at day 10 (n=2) and day 22 (n=2) after bleomycin instillation, and from phosphate-buffered saline (PBS)treated controls (n=2) were stained as previously described 15 . In brief, after deparaffinization, rehydration and heat-mediated antigen retrieval with citrate buffer (10 mM, pH = 6.0), sections were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-Bpifb1 (kindly provided by C. Bingle 62 , 1:500), mouse anti-Trp63 (abcam, ab735, clone A4A, 1:50) and chicken anti-Krt5 (BioLegend, Poly9059, 1:1,000).…”