From stressed satellite cells to mouse and human gastruloids. Applications of single-cell and spatial transcriptomics

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

From stressed satellite cells to mouse and human gastruloids. Applications of single-cell and spatial transcriptomics

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

Generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones

Long-term consumption of isoflavone-enriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women : A randomized, double-blind, placebo controlled study

This trial was registered at clinicaltrials.gov as NCT00301353 and supported by the European Commission : Phytos QLRT-2000-00431 The PHYTOS investigators were : Alwine Kardinaal, Ineke Klöpping-Ketelaars, Linda Kok, Henriette Fick, Linda van den Bosch, Diane ter Doest, Susanne Westenbrink, Henry Brants, Petra van Aken, Carina Rubingh, Cor Kistemaker, Lizeth Vendrig, Truus Meijers, Fred Brouns, Paola D’Acapito, Lorenza Mistura, Valentina Di Mattei, Annalisa Corsi, Marika Ferrari, Paola D’Errigo, Silvia Valtueña, Vincenzo Toscano, Stefano Cianfarani, Mariarosa Giovagnoli, Stefania Sette, Kevin Cashman, Brigitte Chanteranne, Marie-Jeanne Davicco, Patrice Lebecque, Martine Advenier, Marion Brandolini, François Duboeuf, Adile Samaletdin, Stephane Doat, Veronique Braesco, Noelie Joqueviel, Philippe Ladroitte, and Duncan TalbotInternational audienceBACKGROUND: Osteoporosis is a major health problem. It was hypothesized that isoflavone-containing products may be a potential alternative to hormone replacement therapy for preventing bone loss during the menopausal transition. OBJECTIVE: The objective was to investigate whether the consumption of isoflavone-enriched foods for 1 y affects bone mineral density, bone metabolism, and hormonal status in early postmenopausal women. DESIGN: This was a randomized, double-blind, placebo-controlled, parallel, multicenter trial. Two hundred thirty-seven healthy early postmenopausal women [mean (+/-SD) age of 53 +/- 3 y and time since last menses of 33 +/- 15 mo] consumed isoflavone-enriched foods providing a mean daily intake of 110 mg isoflavone aglycones or control products for 1 y while continuing their habitual diet and lifestyle. Outcome measures included bone mineral density of the lumbar spine and total body, markers of bone formation and bone resorption, hormones, isoflavones in plasma and urine, safety variables, and adverse events. RESULTS: Consumption of isoflavone-enriched products did not alter bone mineral density of the lumbar spine and total body or markers of bone formation and bone resorption. Hormone concentrations did not differ between the isoflavone and control groups. Consumption of isoflavone-enriched products resulted in increased isoflavone concentrations in plasma and urine, whereas control products did not. This finding indicated good compliance with treatment. Subgroup analysis did not support an effect of equol phenotype on bone density. The intervention had no effect on a range of safety variables and reported adverse events. CONCLUSION: Consumption of foods containing 110 mg/d of soy isoflavone aglycone equivalents for 1 y did not prevent postmenopausal bone loss and did not affect bone turnover in apparently healthy early postmenopausal white women