Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts

Abstract

Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi-dimensional data set was analyzed using visualization software based on the tSNE algorithm. The analysis identified 6 cell clusters; 1 cell cluster was present only after pneumonectomy. This post-pneumonectomy cluster was significantly less transcriptionally active than 3 other clusters and may represent a transitional cell population. A provisional cluster identity for 4 of the 6 cell clusters was obtained by embedding bulk transcriptional data into the tSNE analysis. The transcriptional pattern of the 6 clusters was further analyzed for genes associated with lung repair, matrix production, and angiogenesis. The data demonstrated that multiple cell-types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple cell clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.

Keywords: aerobic glycolysis; cholangiocarcinoma; glucose metabolism; metabolic reprogramming; warburg effect.

Figure 1

Precision-cut lung slices of the cardiac lobe, laser microdissection and microfluidic single-cell isolation. (A–C) The precision-cult lung slices (200 μm thick) examined at 10x and 20x magnification without counterstain. Alveolar ducts in the posterior curvature of the cardiac lobe were harvested by laser microdissection (21). (D) After enzymatic digestion and filtering, the cells were isolated on the C1 chip (Fluidigm). (E) Capture of individual cells without debris was confirmed by light microscopy (red circle).

Figure 2

Violin plot comparison of gene transcription pre- and post-pneumonectomy. The transcription profiles of cells derived from littermate controls were compared to profiles obtained from post-pneumonectomy (PNX) mice in the first week after surgery. The data for 24 genes linked to lung repair, matrix production and angiogenesis are shown. Gene expression is shown as log₁₀. Student's test level of significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

tSNE clustering of the combined single-cell transcriptional data (colored circles) and embedded bulk transcriptional data (black dots) in 2D maps. The analysis was statistically constrained to 6 clusters. The 6 clusters were color-coded for presentation purposes (A). To obtain a provisional cell-type identity for the clusters, previously obtained post-pneumonectomy bulk transcriptional data were embedded into the tSNE analyses. The results of these embedded analyses were projected on the tSNE map (shown as black dots) for 6 cell-types. Note, the bulk transcriptional data is typically not restricted to one cluster, but may be linked to multiple clusters. For this reason, the cell-type identities are considered provisional: Cluster 1: α-smooth muscle actin myofibroblasts (20) (B) and Type II cells (16) (E); Cluster 2: CD11b⁺ monocytes (13) (F) and F4/80⁺ alveolar macrophages (15) (G); Cluster 3: CD31⁺ endothelial cells (4) (C); Cluster 4: CD11b⁻/CD31⁻ epithelial Type I cells (16) (D). There was no provisional identity for Clusters 5 and 6.

Figure 4

Rank-order of highest gene expression within each cluster. The log₂ fold-change in gene expression of the 10 highest expressing genes in each cluster compared to the remaining 5 clusters. The data from all timepoints are shown. The remainder of the 86 genes are presented in Supplementary Data.

Figure 5

The time course distribution of cluster frequency s analyzed by single-cell qPCR. The single-cells in each cluster are shown as a fraction of the total number of cells isolated at each time point. The single-cells isolated from littermate controls (C) were compared to cells isolated from regenerative alveolar ducts on postoperative day 1 (20), day 3 (11), and day 7 (13). Plombage and phrenic nerve controls are not shown. Note the low frequency of Cluster 2 in littermate controls (*).

Figure 6

TSNE map of the combined single-cell transcription of genes classified as lung repair genes. The map is color-coded to reflect relative gene expression. The genes are elastin (Eln), lysyl oxidase (Lox), fibrillin (Fbn1), mitogen-activated protein kinase 3 (Mapk3), fibulin 1 (Fbln1), fibulin 5 (Fbln5), latent-transforming growth factor beta-binding protein 2 (Ltbp2) and 4 (Ltbp4). The linked pattern of gene expression is apparent for Eln, Lox, and Ltbp2. Cluster 1 expression of the lung repair genes is high for all lung repair genes. All timepoints are combined in the analysis. In the first panel, the cluster boundaries are shown for clarity.

Figure 7

The tSNE map of the combined single-cell transcription of genes classified as lung matrix genes. The map is color-coded to reflect relative gene expression. The genes are fibronectin-1 (Fn1), collagen Type XV111 alpha-1 (Col18a1), collagen Type IV alpha-3 (Col4a3), matrix metalloproteinase 2 (Mmp2) and 14 (Mmp14), transforming growth factor beta-1 (Tgfb1), platelet-derived growth factor receptor alpha (Pdgfra) and beta (Pdgfrb). The linked pattern of gene expression is apparent for Col18a1 and Mmp14 as well as Fn1 and Tgfb1. Cluster 1 expression of the lung matrix genes is high, but variable within the Cluster 1 subpopulations. All timepoints are combined in the analysis. In the first panel, the cluster boundaries are shown for clarity.

Figure 8

The tSNE map of the combined single-cell transcription of genes classified as angiogenesis genes. The map is color-coded to reflect relative gene expression. The genes areH platelet endothelial cell adhesion molecule 1 (Pecam1), endoglin (Eng), vascular endothelial growth factor A (Vegfa), fibroblast growth factor 1 (Fgf1), fms-related tyrosine kinase receptor 1 (Flt1) and 4 (Flt4), naturetic peptide receptor 1 (Npr1) and sphingosine kinase 1 (Sphk1). The linked pattern of gene expression is apparent for Pecam1 and Eng as well as Vegfa, Flt4, Npr1, and Sphk1. Almost all subpopulations of Cluster 1 demonstrate high expression of angiogenesis genes. All timepoints are combined in the analysis. In the first panel, the cluster boundaries are shown for clarity.