Isolation of high yield and quality RNA from human precision-cut lung slices for RNA-sequencing and computational integration with larger patient cohorts

Abstract

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology and disease, as well as for screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology and disease as compared to PCLS derived from animals (e.g. clinical heterogeneity), but access to human tissue is limited. A number of different experimental readouts have been established for use with PCLS, but obtaining high yield and quality RNA for downstream gene expression analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for non-biased and high through-put analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis (IPF). We show that the RNA isolated using this method is of sufficient quality for both RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS was comparable to data generated from native tissue and could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data sets. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS derived from distal lung tissue, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease, such as IPF.

Modeling of Aberrant Epithelial Reprogramming in Idiopathic Pulmonary Fibrosis using Human Induced Pluripotent Stem Cell-derived Alveolar Organoids

“…After observing signatures of epithelial reprogramming induced by the FC stimulation based on expression of individual markers, we were curious to explore changes in phenotypes associated with cell types. For this, we used computational deconvolution with Bisque (46,47). For the analysis, we used a public single-cell RNA-seq dataset (GEO Accession: GSE135893) as a reference filtered to include lung epithelial cells from nonfibrotic controls and IPF donors (10) (Fig.…”

Targeting Alveolar Repair in Idiopathic Pulmonary Fibrosis

“…How relevant either the PCLS approach, or even TGF-beta stimulation alone on PCLS, might be in predicting clinical efficacy remains unknown. However, several omics-based approaches have recently been optimized for PCLS and will most likely accelerate the characterization of this ex vivo model (156)(157)(158)(159). In addition, other small molecules in Phase I and Phase II (e.g.…”