Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory

Abstract

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

Single-nuclei RNA-sequencing of plant tissues

“…4c, Supplementary Materials and Methods). This correlation is consistent with correlation coefficients found in previous publications (ranging from 0.7 to 0.85) 15 and therefore we found good agreement between both experiments. As shown in Supplementary Fig.…”

A CD44/Brg1 nuclear complex confers mesenchymal progenitor cells with enhanced fibrogenicity in idiopathic pulmonary fibrosis

“…Our scRNA-seq data indicate that CD44 is a marker of highly entropic IPF MPCs (10). Based on this information, we first performed IHC double staining of human IPF lung tissue to examine the distribution of CD44 expressing MPCs (double positive for SSEA4 and CD44).…”

“…As an unbiased approach to stratifying the cells, we chose transcriptional network entropy, which serves as a quantitative measure of a cells differentiated state, with higher entropy indicating a less differentiated state (11,12). Our data revealed heterogeneity among IPF MPCs without discrete subpopulations (10). The most highly entropic IPF MPCs displayed the greatest differences in their transcriptional profile compared to control MPCs.…”

“…These results indicate that IPF MPCs acquire a pathological phenotype early in their differentiation and suggest that this distinct transcriptome may underlie their fibrogenic phenotype. When examining our data for genes that best distinguished the most highly entropic IPF MPCs, we identified high CD44 expression (10).…”

Diagnosing and treating lung disease at the cellular level

“…Joint efforts through the NHLBI-supported LungMAP program ( https://lungmap.net ) ( 3 ), the Lung Cell Atlas ( 28 ) under the Human Cell Atlas program ( https://www.humancellatlas.org ), and others have utilized these single cell-based technologies to identify remarkable cell heterogeneity throughout lung development ( 9 ) and have discovered new cell types ( 21 ), specific progenitor subgroups ( 37 ), cell differentiation trajectories ( 17 ), and unique abnormal cells during disease pathogenesis ( 4 , 30 ). Recent analyses of human single-cell RNA sequencing (scRNA-seq) data sets have revealed that cell subsets that coexpress ACE2/TMPRSS2 include alveolar type II pneumocytes, absorptive enterocytes, and nasal goblet secretory cells and therefore may serve as cellular targets of SARS-CoV-2 ( 38 ).…”