Mapping tissue immunity in the human urinary bladder across lifespan

The urinary bladder is a hollow spherical organ situated in the lower abdomen. It forms part of the urinary tract, which is responsible for expelling waste and surplus water from the body. Urine, containing waste and excess fluid, is produced by the kidneys, and transported to the bladder where it is stored prior to voiding. This means that the bladder is in relatively close proximity to the external environment, and may be attacked by bugs (microbes) coming in from body surfaces, including microbes originating in the gut. We know that all organs, including the bladder, contain some resident defence (immune) cells that help to fight off microbes. Despite this, infection of the bladder is common, affecting 1 in 2 women in their lifetime and 1 in 20 men. Susceptibility to bladder infection increases with age, as do other bladder diseases, like inflammation (that can give symptoms such as pain or incontinence) and cancer. Despite the fact that immune cells play an important role in all of these conditions, we currently don't understand the details of how bladder defence is set-up, how this goes wrong or changes with age, and how this differs between men and women. Our research project aims to address this knowledge gap. Our overall aim is to produce a map or atlas of all the immune cells in the human bladder across lifespan, in men and women.

We will take two experimental approaches:

The first is to take a piece of human bladder, and to mash it up so that we can analyse individual cells, including immune cells. Different cells have different functions and identification marks because of differences in their genetic material (called genes), and differences in how their genetic code is translated into the working parts of cells, called proteins. Every cell contains thousands of proteins, but these are hard to measure at scale in a single cell. However, we can measure the molecule that acts as an intermediate between genes and proteins - this is called messenger RNA or mRNA. In the past 10 years, technology has advanced such that we can measure thousands of mRNA molecules in a single cell, this is what we plan to do with the mashed bladder samples.
We already have ethical permissions in place to take human bladder samples across lifespan, including from embryos (from the Human Development Biology Resource at Newcastle University), and adults (from organ donors in Cambridge that have consented for their tissues to be used for research).

Our second experimental approach is to take a piece of bladder and keep it intact so that the relationship of the cells in space is maintained. We will then make slices of the piece of bladder so that they are thin enough to be visualised under a microscope. We will use the mRNA information from our first experiment to identify protein markers that can be visualised by the microscope using probes that are fluorescently labelled and we can also map mRNA molecules in space. This will allow us to investigate the position of cells relative to each other and to determine whether the position of cells differs between men and women, and in the adult and developing bladder.

Making a complete map of cells in the bladder across lifespan will provide a critical reference atlas that will help researchers in the future to understand which cells become abnormal in different bladder diseases and how it might be possible to prevent unhelpful age-related changes.

The urinary bladder contains a network of cells, including resident immune cells, which interact to maintain organ homeostasis, defence and repair. A comprehensive understanding of the cellular networks in the normal human bladder across lifespan, and how these differ between male and female bladders, is currently lacking, and will provide an important reference dataset from which to investigate disease-associated perturbations. This project aims to address this need by:

1. Generating single-cell molecular profiles of female and male pre-natal, young and old adult human bladders. We will perform droplet-based sequencing (using the 10x Genomics platform) on single cell suspensions obtained from n=6 dissociated tissue samples per group (36 samples in total). We will profile unselected bladder cells (our pilot data shows that we get an excellent representation of immune cells), and will use CITEseq antibodies to delineate cell protein expression to enhance immune cell characterisation.

2. Analysing the scRNAseq dataset generated in (1) to identify temporal changes in:
a. Immune functionality of urothelial cells, stromal cells and endothelial cells
b. Resident-immune cell number and phenotype.
c. Resident-immune cell interactome using receptor-ligand expression analysis
d. Resident-immune cell function by assessing geneset and pathway enrichment.
Age and sex-associated differences identified in the analyses performed in 2a-d will be validated using confocal imaging, flow cytometry, and ex vivo stimulation of bladder samples or cells.

3. Ascertaining the precise spatial relationships of transcriptionally characterized cells within the bladder in females and males across lifespan. We will use:
a. High dimensional (25-plex) immune-relevant protein-marker imaging (using an optimised iterative strip-stain protocol).
b. Spatial transcriptomics using the Visium platform (10x Genomics).
c. Validate findings made in Parts 1 and 2 using confocal imaging.