From stressed satellite cells to mouse and human gastruloids

Abstract

The first part of this thesis describes how widely-used tissue dissociation protocols that are often required prior to single-cell RNA sequencing (scRNA-seq) procedures can alter the transcriptome of cells. The second part of this thesis focuses on gastruloids, aggregates of embryonic stem cells that can be used to study mammalian post-implantation development in vitro, and describes how scRNA-seq and spatial transcriptomics technologies can aid in the characterization and development of improvement versions of gastruloid models. Chapter 1 provides an introduction into embryology and stem-cell based in vitro models for embryology, with a focus on gastruloids. This chapter describes the historical context of the gastruloids model, and explains why this model is a useful addition to the toolbox of modern-day embryologists. This chapter also summarizes the advantages and current limitations of the gastruloids system. In addition, this chapter provides a brief introduction into scRNA-seq and spatial transcriptomics technologies. Chapter 2 shows that dissociation procedures that are required for many scRNA-seq experiments can induce a stress response in a subpopulation of these cells. This chapter shows that satellite cells (muscle stem cells) are particularly sensitive to such a dissociation-induced stress response. This chapter highlights that results obtained with scRNA-seq require validation with microscopy, and provides experimental and computational solutions that can be used to remove dissociation-affected subpopulations from scRNA-seq experiments. Chapter 3 provides a detailed single-cell and spatial transcriptomics-based characterization of mouse gastruloids. A detailed comparison with embryos reveals that most embryonic cell types are present in gastruloids, and shows that key markers of somitogenesis are expressed in the correct spatial location. We follow up on these observations in the second part of Chapter 3, and show with live-imaging experiments that the somitogenesis clock is active in mouse gastruloids. We then perform a small drug screening study to perturb the somitogenesis clock in gastruloids. This drug screening reveals how reduced FGF signalling induces a short-tail phenotype in embryos, and exemplifies how gastruloids, which can easily be generated in large numbers, can be used to perform large-scale drug screening procedures. Finally, this chapter describes the discovery that the addition of a small amount of Matrigel can induce the formation of somite-like structures in mouse gastruloids. Chapter 4 describes the development and characterization of the first human version of the gastruloids model. In this chapter, this new human gastruloids system is characterized and compared to mouse gastruloids with spatial transcriptomics. In addition, this chapter describes how various teratogens and inhibitors affect the development of human gastruloids, suggesting that this system can indeed be used to study how environmental factors affect human development. Chapter 5 provides a discussion of the work described in this thesis. This chapter describes alternative tissue dissociation protocols that have been developed by others that followed up on the dissociation-induced stress-response that we discovered in Chapter 2 of this thesis. In addition, Chapter 5 provides an extensive overview of the current challenges, ethical considerations and potential future applications of the (human) gastruloid field.