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

Daniel J Beisang,Peter B Bitterman,Colleen Forster,Jeremy Herrera,Eric Lock,Brian J Sandri,Emilian Racila,Craig A Henke,Alexey Benyumov,Adam Gilbertsen,Libang Yang,Karen Smith

doi:10.1038/s41598-020-66630-5

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.

Highlights

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
While a quantitative analysis of the co-localization of these markers cannot be conducted with the serial sectioning approach used here, we qualitatively found that SSEA4, MARCKS and CD44 co-localized to the perimeter region of the IPF fiboblastic focus
Through single-cell RNA sequencing and bioinformatics analysis of MPCs derived from control and IPF lung tissue, we found that IPF MPCs represent a heterogeneous population with the least differentiated MPCs displaying the greatest distinction from control MPCs

Summary

Introduction

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. We previously identified mesenchymal progenitor cells (MPCs) from human IPF lung tissue that serve as cells of origin for IPF fibroblasts[5] These cells exhibited canonical MPC properties including: (1) tri-lineage differentiation potential; (2) characteristic cell-surface markers; and (3) anchorage- independent colony formation. The network entropy algorithm (Single-Cell Entropy, SCENT) has been validated to accurately reflect differentiation trajectories using single-cell RNA sequencing data in an unbiased manner that is robust to sequencing coverage and drop-out rate[13] Given these properties of the SCENT algorithm (few pre-hoc assumptions, relatively unbiased, biologically validated, and biologically relevant) it represents a powerful tool for understanding heterogeneity within single-cell sequencing experiments

Methods

Results

Conclusion