Single-cell and spatial sequencing have revolutionised our understanding of human tissue biology in recent years. The Human Cell Atlas (HCA) initiative aims to recover and position all of the cell types in the human body. Being part of HCA, I have worked on building atlases which allow comparison across age groups for two different organs: the human thymus and skeletal muscle. Firstly, I co-led a large multinational effort to create a spatial atlas of the human thymus from fetal and paediatric stages. As a part of this effort, we expanded the census of cell types in the human thymus and mapped them spatially in the tissue. We also developed a new morphological framework (Organ Axis) which allowed us to align and compare cell type locations in fetal vs. paediatric thymus. Secondly, I led an effort to create an ageing human skeletal muscle atlas, which systematically catalogued cell types and states in young adult and elderly skeletal muscle and described age-associated changes.

In Chapter 1 I set the stage for the whole thesis by giving background on single-cell and spatial technologies as well as the research question, namely change across the lifespan. I start with a brief introduction to single-cell atlasing, the huge variety of spatial transcriptomics and proteomic technologies, focusing on Visium and IBEX. Next, I provide an overview of the prenatal and postnatal periods of development, followed by a more in depth discussion on causes of human ageing. Finally, I conclude with a description of the main events and functional changes in thymus and skeletal muscle across the lifespan.

In Chapter 2 I give an overview of the spatial human thymus cell atlas. I give a brief introduction to human thymus development, its cell type composition and function. I describe major single-cell (single-cell, CITE-seq and TCR-seq) and spatial profiling modalities (Visium and IBEX) that were applied to the thymus and outline methods for imaging data processing, annotation and cortico-medullary axis construction for the thymus. I provide an annotation and spatial mapping of T cells and supporting resident cells, including thymic epithelial cells (TECs), fibroblasts, vascular and myeloid cells. Simultaneously, I use the cortico-medullary axis to compare the spatial position of cell types in the fetal vs. paediatric thymus. I conclude by discussing the differences in cell type localisation between fetal and paediatric thymus and the importance of spatial structure for thymus function.

In Chapter 3 I provide insights from single-cell and spatial mapping of Hassall’s body, a keratinised structure in the thymus medulla, which used to be considered a degenerative epithelial structure. Firstly, I map the closest cell types to Hassall’s body and discuss the challenges of associating cell types to Hassall’s vs deep medulla. Next, I identify genes which are uniquely expressed by specific cell types in medulla, which led me to discover an underappreciated heterogeneity within mTECIII population including mucosal and skin-like subtypes. I conclude with a discussion on the putative function of Hassall’s bodies in T-cell development.

In Chapter 4 I introduce the human skeletal muscle ageing atlas. I start by summarising knowledge about the main cell types in the muscle and known ageing changes. I describe the experimental setup for the creation of the atlas and give an overview of the recovered cell types and major ageing changes that we observe. Following on from this, I describe fine-grained cell states and ageing changes that occur in the different compartments of skeletal muscle, including muscle stem cells, the myofiber itself and supporting cells of the muscle microenvironment. I conclude with a discussion on how processes in different parts of the muscle combine to cause a breakdown in muscle function over age.

In Chapter 5 I summarise the major insights from both studies and discuss their potential therapeutic uses. Next, I use my experience to provide suggestions on experimental design and challenges that need to be solved for the next generation of atlases looking to understand changes across human lifespan.