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Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis
Yan Xu, … , Barry R. Stripp, Jeffrey A. Whitsett
Yan Xu, … , Barry R. Stripp, Jeffrey A. Whitsett
Published December 8, 2016
Citation Information: JCI Insight. 2016;1(20):e90558. https://doi.org/10.1172/jci.insight.90558.
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Research Article Inflammation

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease.

Authors

Yan Xu, Takako Mizuno, Anusha Sridharan, Yina Du, Minzhe Guo, Jie Tang, Kathryn A. Wikenheiser-Brokamp, Anne-Karina T. Perl, Vincent A. Funari, Jason J. Gokey, Barry R. Stripp, Jeffrey A. Whitsett

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Figure 3

Single-cell RNA sequencing analysis from human IPF and normal lung epithelial cells.

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Single-cell RNA sequencing analysis from human IPF and normal lung epith...
CD326+ epithelial cells were isolated from peripheral lung tissues by FACS as described in Methods, followed by single-cell isolation using Fluidigm C1 system and RNA sequencing. (A) Hierarchical clustering and principal component analysis (PCA) of 540 single cells from control (n = 3) and IPF patients (n = 6) reveals 4 major cell types (C1–C4), termed as normal AT2 (C1, green), indeterminate (C2, yellow), basal (C3, red), and club/goblet (C4, blue) cells. Single cells are colored by cluster on a 3D space. (B) Heatmap represents the expression of distinct RNAs that identify each of the 4 cell types. (C) Hierarchical clustering of all IPF and control cells using differentially expressed genes involved in epithelial proliferation (GO:0050673), “response to cytokines” (GO:0034097), and “response to growth factors” (GO:0034097) is shown. Minimum expression values were set to 0.01 TPM. Genes (n = 9,154) with specificity >0.7 and with TPM >1 in at least 10 cells in at least 6 samples were selected for hierarchical clustering using Z score–transformed expression.
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