Cells of the human intestinal tract undergo dynamic changes from development to adulthood and in response to environmental stimuli. Recent advances in high-throughput profiling of cells using transcriptomic approaches have vastly expanded the catalogue of cells that are found across the human body. Nonetheless, a holistic view of intestinal cell diversity across distinct intestinal regions (space) and multiple life stages (time) is still lacking. The work presented in this thesis is focused on building a reference of human intestinal cell types using novel genomic approaches including single-cell RNA sequencing (scRNA- seq) and spatial transcriptomics. Importantly, we use this data to gain new insights into intestinal cell organisation and function during in utero development, homeostasis, and in rare and complex diseases.

Chapter 1 introduces the advances in single-cell genomic approaches within the last decade and contrasts them with previous technologies used for cataloguing cells. Following an overview of single-cell technologies, the intestinal spatial architecture, as well as key cell types (e.g. epithelium, enteric neurons, stromal and immune cells) required for the proper intestinal function, will be summarized. As an intestinal surface area, innervation and immunity are established during in utero development, this chapter will also outline the principles of relevant developmental events, namely villus formation, enteric nervous system development, and lymphoid structure formation.

Chapter 2 outlines the materials and methods used for the generation and analysis of single-cell and spatial data that will be discussed in the following results chapters.

Chapter 3 focuses on deciphering the key cell players in the process of human villus formation. This chapter outlines the single-cell profiles of human embryonic and early fetal gut tissue and explores the interactions between epithelial and mesenchymal cells. In addition, these cell type profiles and cell networks are compared and contrasted with cells found in the children diagnosed with chronic inflammation of the small intestines or Crohn’s disease. This analysis reveals similarities in epithelial cell changes during development and regeneration.

Chapter 4 is focused on an in-depth analysis of epithelial and neuronal lineage composition and developmental relationships across different intestinal regions and life stages. In this chapter, an integrated view of the gut is presented, encompassing cells from in utero development, childhood and adulthood, and up to 11 intestinal regions. We resolve regional differences of BEST4+ absorptive cells, implicate IgG sensing as a novel function of intestinal tuft cells and resolve differentiation of enteroendocrine cell subsets. Analysis of the epithelial cells is followed by resolving the developing enteric nervous system at the single-cell resolution and showing the patterned expression of Hirschsprung’s disease-associated genes.

Chapter 5 is focused on the development of local intestinal immunity. In this chapter, the three key cell types that orchestrate lymph node and gut-associated lymphoid tissue formation in humans are described. The comparisons of these subsets with cells expanded in Crohn’s disease patients reveal re-initiation of this cellular program during inflammation to recruit and retain immune cells to the site of damage.

Lastly, Chapter 6 summarises the insights gained through single-cell analysis of intestinal cell types and discusses these results in the light of recent literature on development and inflammation.