Researchers use single-cell RNA-Seq to unlock genetic code of diseased lung cells

A new study has determined the genetic codes of healthy and diseased human lung cells, a finding that may allow doctors to better diagnose and follow idiopathic pulmonary fibrosis (IPF) progression, as well as advance treatments for the disease.

Published in the Journal of Clinical Investigation Insight, the study, “Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis,” is believed to be the first single-cell genetic investigation uncovering four distinct epithelial lung cell types, each with specific genetic characteristics, making this a promising tool to further examine the pathogenesis of IPF.

Researchers analyzed normal and IPF human lung tissues through a technique called single-cell RNA sequencing, which allows scientists to determine the RNA sequences present in a cell, ultimately offering a “genetic blueprint” of the cell.

Single-cell RNA sequencing analysis from human IPF and normal lung epithelial cells

CD326⁺ epithelial cells were isolated from peripheral lung tissues by FACS as described in Methods, followed by single-cell isolation using Fluidigm C1 system and RNA sequencing. (A) Hierarchical clustering and principal component analysis (PCA) of 540 single cells from control (n = 3) and IPF patients (n = 6) reveals 4 major cell types (C1–C4), termed as normal AT2 (C1, green), indeterminate (C2, yellow), basal (C3, red), and club/goblet (C4, blue) cells. Single cells are colored by cluster on a 3D space. (B) Heatmap represents the expression of distinct RNAs that identify each of the 4 cell types. (C) Hierarchical clustering of all IPF and control cells using differentially expressed genes involved in epithelial proliferation (GO:0050673), “response to cytokines” (GO:0034097), and “response to growth factors” (GO:0034097) is shown. Minimum expression values were set to 0.01 TPM. Genes (n = 9,154) with specificity >0.7 and with TPM >1 in at least 10 cells in at least 6 samples were selected for hierarchical clustering using Z score–transformed expression.

While the team found several overlapping characteristics between the cells analyzed, predominant genetic markers of four different cell types were apparent. Normal lung tissue cells expressed genes linked to fully formed alveolar type 2 (AT2) lung cells, which play a vital role in normal respiration, response to oxidative stress, as well as cell protection and detoxification.

However, AT2 expression was abnormal in the remaining three cell types (IPF cells). Instead, these cells exhibited the expression of genes important for epithelial proliferation, “response to cytokines” (important signaling molecules) and “response to growth factors.” IPF basal cells showed the greatest deviation from normal epithelial lung cells, with an increase in “wound healing” genes, among other factors.

Based on their results, the authors suggested that respiratory epithelial cells undergo differential stages in IPF, and that these cells seemed to have lost the normal genetic control systems needed to ensure the development of functional, mature lung cells, such as AT2 cells.

“In the present study, we identify profound changes in epithelial cell gene expression at the single-cell level of resolution in IPF. Our findings identified the activation of a number of canonical signaling and transcriptional pathways associated with cell injury and repair in IPF and identify IPF-related epithelial cells of indeterminate differentiation,” the researchers wrote. “Our present single-cell data support the concept that an abnormal differentiation program has been initiated in the tissue microenvironment of IPF … with many respiratory epithelial cells acquiring aberrant, multilineage-like states.”

The team believes that identifying these abnormalities in gene patterns may lead to the development of new, targeted therapies and help in disease diagnosis.

“This paper identifies a number of novel targets and molecular pathways for IPF, for which there are pharmaceutical approaches,” said Jeffrey Whitsett, MD, lead investigator and co-director of the Perinatal Institute at Cincinnati Children’s Hospital, adding that airway cells “can be obtained by brushing the airway or biopsy, and marker genes can be tested to make a diagnosis or monitor treatment.”

Source – Eurekalert