Figure 4. Single-cell RNA analysis identifies altered epithelial gene expression and epithelial cell types in IPF.

(A) Single cells from human IPF (n = 6) and donor (n = 3) distal lung (CD326⁺) were prepared using the Fluidigm C1 system. RNA was prepared and analyzed from a total of 325 single cells from IPF and 215 cells from donor lungs. Shown are lung epithelial cell markers: EPCAM and CDH1; alveolar type 1 cell markers: AGER and HOPX; alveolar type 2 cell markers: SFTPC, SLC34A2, and ABCA3; proximal lung epithelial cell markers: SOX2, PAX9, TP63, KRT5, KRT14, MUC5B, and SCGB1A1. Expression values were measured in TPM and square root (sqrt) normalized. Cells are shown in solid colors if the expressions of the markers were greater than 1 (TPM). (B) MUC5B, PAX9, and SOX2 were selectively expressed in subsets of IPF cells (MUC5B: n = 24, PAX9: n = 65; SOX2: n = 24) but not present in C1 control cells. Representative genes clustering with MUC5B, PAX9, and SOX2 in IPF cells are shown in the heatmaps. Equal numbers of control cells were randomly selected. IPF cells expressed a diversity of conducting airway epithelial markers not present in control cells, the latter expressing RNAs characteristic of AT2 cells. (C) Only 9 of 325 IPF cells clustered with control cells, the heatmap indicating “AT2”-like expression patterns; however, these 9 normal IPF cells also coexpressed some of the of IPF-associated disease markers. Expression data (TPM) were log₁₀ transformed.