Human Lung Cell Atlas Project: genetic regulation of foetal and healthy adult lung cell states in 3D spatial resolution

Cells are the functional units of our bodies. Different diseases are caused by changes in the types of cells in our bodies, or by changes in cell behaviour (cell state). The Human Cell Atlas is an international collaboration to characterise all cell types and states of the human body to understand how the healthy body functions. It will provide a reference map to identify cell changes that are associated with disease. We propose to study cell types and cell states in the healthy human lung to contribute to understanding of changes that occur in lung cancer and degenerative diseases, like pulmonary fibrosis and chronic obstructive pulmonary disease. Importantly, we will study the developing (foetal) human lung alongside the healthy adult lung. This will allow us to make comparisons between immature and mature cell states and, in the long-term, provide information that can be used as the basis for regenerative medicine.

Cell type and cell state are defined by the different genes expressed in each cell. Single cell RNA sequencing technology, scRNA-seq for short, makes it possible to determine exactly which genes are active in any cell for thousands of cells in parallel. Using scRNA-seq will allow us to identify and characterise cells in the developing and adult human lung. We will build on our recent work in the adult lung that has identified an entirely new cell type, the ionocyte, to understand how different cells cooperate to build and maintain lung function.

Firstly, we will carry out scRNA-seq in distinct spatial locations to identify all cell types of the developing lung. By looking at individual cells it becomes possible to identify characteristics that are associated with each cell. For example, whether it is still immature or has already acquired the characteristics of a more mature cell type. This makes scRNA-seq a very powerful tool to study human development. We will be asking how the human lungs develop during weeks 6-20 post-conception. We will compare our results from the foetus to how cell types and states are maintained in the adult human lung, leveraging funding that we have already obtained to study the adult tissue.

Secondly, we will use a new single cell technology (single cell ATAC-seq) which will allow us to identify regions of DNA that control which genes are turned on or off in each cell. These experiments will help us to better define cell states and provide information about how these states are controlled. In the future, this knowledge will be used to find ways of modifying cell states to develop new treatments for disease.

An atlas is not just a list, but also provides spatial information in the sense of a map. In the third part of our proposal we will place the identified cell types back into their spatial context. This is of particular importance for studying development and adult stem or progenitor cells, where the presence of neighbouring cells strongly influences behaviour. We will use spatial transcriptomics which allows the activity of all genes to be studied in space, but currently not at the resolution of individual cells. We will also use a technique called HCR, coupled with high performance microscopy, which measures fewer genes, but at single cell resolution. By combining these two techniques we will build a 3D map of the lung.

Our work will generate large and complex data-sets that need to be integrated computationally. At the Wellcome Genome Campus we have world-leading computational expertise and infrastructure. We (Meyer) are already working in close collaboration with the Human Cell Atlas to maximise the benefits of this project for international biomedical research.

This work will lay the foundations for a better understanding of many lung diseases. It will help us to interpret results from genetic studies of lung disease and impact on regenerative medicine and therapeutics development by revealing pathways controlling lung development and maintenance.

We will contribute to the Human Cell Atlas for the developing and adult lung by defining lung cell types, their chromatin states and spatial organisation. This will allow us to analyze cell trajectories, and genetic regulatory elements predicted to establish and maintain cell identities, in the developing versus adult lungs. We will also delineate how early immunity is acquired in the lung. Furthermore, analysis in 3D context will inform future studies of morphogenesis and stem cell regulation. The data obtained will provide a baseline for comparison with disease situations and for functional studies of lung development, maintenance and disease.

We will use scRNA-seq of trachea, large airway and future alveoli from 6-20 post-conception weeks to define the cell types present in the developing lung. These data will be compared to similar regions of the adult, leveraging funding that we (Meyer) have already obtained to profile the adult lung.

Using a new cost-effective, high-throughput experimental platform for scATAC-seq we will study chromatin organisation in the same regions of developing and healthy adult lungs. These data will be integrated with the scRNA-seq for a "multi-omics" approach to analysing cell trajectories. These data will also be used to analyse the large publically-available data-sets of genetic variants associated with lung function and disease to produce improved predictions of the cell types and developmental stages at which these variants function.

To localise the identified cell states within the lung, we will use a combination of spatial transcriptomics (genome-wide, but resolution of 30-100 cells) and hybridisation chain reaction (highly sensitive detection of a limited number of transcripts at single cell resolution).

We (Meyer) are already working with the HCA Data Coordination Platform (DCP) at EBI and the Hinxton Single Cell Portal and will continue to do so to maximise the benefits of this project for biomedical research.

1. Impact for human health.
We will profile cell states in the healthy human adult lung and the developing embryonic/foetal lung which will be incorporated into the wider HCA project, allowing rapid international access by the research community. Our data will provide a baseline for comparison with disease states, allowing disease-associated changes in cell states to be clearly identified. It will also contribute to the interpretation of the many completed, and ongoing, studies of genetic variants which contribute to susceptibility to lung disease, providing a cellular and gene regulatory framework in which to interpret the data. Moreover, the information that we will generate on pathways involved in lung development and maintenance will contribute to ongoing academic and commercial efforts in therapeutic lung regeneration.

2. Impact on capacity-building of a scientifically literate workforce.
This project will contribute to the production of a scientific-literate workforce with skills in cell biology/imaging and computational analysis of complex biological data sets. Involvement with the HCA is an excellent opportunity for these researchers to gain exposure to a large-scale, international, collaborative project. Additionally, the University of Cambridge and Sanger Institute offer numerous formal training opportunities in leadership, mentoring and science communication that we will take advantage of. These will all be important impacts for all staff involved and will be achieved within the life-time of the award.

3. Impact on the wider public.
The public is interested in science and research into health issues. Engaging with the wider public about the HCA project, and the lung atlas in particular, will promote public understanding of the nature of scientific research and contribute to the national conversation on the need for informed decision making around scientific issues.