Alsafadi, H. N. | bioRxiv Science

bioRxiv2022

Simultaneous isolation of proximal and distal lung progenitor cells from individual mice using a 3D printed guide reduces proximal cell contamination of distal lung epithelial cell isolations

The respiratory epithelium consists of multiple, functionally distinct cell-types and is maintained by regionally-specific progenitor populations which repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung epithelial cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D-printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice which give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, classic manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal progenitors with distal cells which altered the transcriptional landscape of distal organoid cultures. Thus, cell isolation with the 3DLD generates reproducible distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays. HighlightsO_LI3DLD reproducibly separates lung lobes and extrapulmonary airways (bronchi/trachea) C_LIO_LI3DLD cell isolation yields consistent isolation of distal epithelial cells (DECs) C_LIO_LIContamination of proximal cells in classic DEC isolations may alter in vitro results C_LIO_LI3DLD allows for simultaneous isolation of proximal and DECs from single animals C_LI eTOC blurbAlsafadi et al. describes a new method for simultaneous isolation of lung epithelial proximal and distal progenitors using the aid of a 3D printed device (3DLD). Both isolated cell types differentiate in multiple in vitro assays. The 3DLD guide minimized contamination of proximal cells in distal cell isolations whose presence can alter the transcriptional landscape of distal epithelial organoids.

cell biology↗

bioRxiv2020

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

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.

molecular biology↗