Identification of senescent cendothelial cells in the aged mouse brain using single-cell mRNA sequencing
The problem we’ve faced
Aging is the main risk factor of many vascular diseases including vascular cognitive impairment and dementia. The goal of this project was to investigate the mechanism of aging at the level of individual cells in the brain vasculature.
Scientific question
By aging, so called senescence cells accumulate in the brain. These cells does not lose their cellular identity, however they have functional impairment and secrete pro-inflammatory proteins damaging the surrounding microenvironment. Identification and sorting of these cells from a tissue sample is an unsolved problem since no reliable surface marker of them was identified yet.
Solution
The proposed solution for this problem was single-cell mRNA sequencing. The technology provided by 10X Genomics makes it possible to measure gene expression of thousands of cells simultaneously, originating from a freshly isolated tissue sample. Cell types including senescence cells could be identified and subset after sequencing based on their gene expression signature.
Type of Data
First, gigabytes of raw sequence data had to be converted to a gene expression count matrix. This count matrix, a single table containing the expression level of 15.000 detected genes for 4233 individual cells, was used in the downstream analysis.
First, gigabytes of raw sequence data had to be converted to a gene expression count matrix. This count matrix, a single table containing the expression level of 15.000 detected genes for 4233 individual cells, was used in the downstream analysis.
Methods for data analysis
We used a state-of-the-art analytical workflow called Seurat to handle this huge amount of data stored in the count matrix and the associated metadata. After multiple quality control steps an unsupervised graph-based clustering was used to identify cell types. The visualization of result was done with a so-called two-dimensional embedding. To identify cells undergone senescence transformation a modified gene set enrichment method was developed which scored cells based on the expression of a senescence specific gene set.
Results
In this project we successfully identified senescence microvascular endothelial cells and demonstrated the accumulation of them by aging. Since we collected transcriptome wide data on each cell, gene expression signature of pro-inflammatory changes and blood-brain-barrier disruption was also shown in the senescence cell. With this work we laid down the foundation for future studies on anti-aging senescence removal treatments, so called senolytics.
