Generation and molecular characterization of mouse embryonic stem cells derived Trunk-Like Structures

One of the greatest mysteries in developmental biology is how the orchestrated process of cellular differentiation guides a single cell to form a functional organism comprised of complex tissues and organs. A detailed understanding of these events in mammals is obscured by the inaccessibility of embryonic development in utero. In recent years, community efforts have led to the development of stem cell-based models of embryogenesis, able to mimic some cellular and molecular aspects of the early stages of development in vitro. Despite these advances, such models lack proper embryo-like morphogenesis necessary for tissue formation and organ development. To overcome this limitation, I optimized and characterized a protocol to precisely model the morphogenetic changes that occur during gastrulation and early organogenesis. Within only five days, the protocol produces “Trunk-Like Structures (TLS),” self-organized synthetic embryos originating from mouse pluripotent stem cell aggregates treated with chemical signaling molecules (gastruloids) and embedded in an extra-cellular-matrix (ECM) to mirror the in utero environment. TLS morphologically and molecularly resemble in vivo development, displaying a high level of trunk-tissue organization that includes the presence of somites (building blocks of future bones, muscles, and cartilage), a neural tube (future spinal cord), and a gut tube (future gastrointestinal tract). To test TLS similarities to the in vivo embryo, I integrated different single-cell technologies and confirmed the high complexity of these synthetic embryos. Specifically, I observed that TLS progress from their stem cell origin through cell type maturation, producing relevant subtypes of the developing trunk. Unlike in vivo development, the TLS platform allows rapid and tunable genetic and chemical perturbations with the benefits of uninterrupted and continuous observation. As a proof of principle, I utilized CRISPR/Cas9 based genetic ablation to recapitulate a well-studied mutant phenotype. Next, I influenced TLS developmental trajectories by targeted chemical modulation, resulting in the overproduction of somites, a phenotype never observed before in vivo. Finally, I investigated the molecular mechanism responsible for the enhanced tissue morphogenesis observed in TLS. I found that cells’ ability to organize into complex tissues requires the presence of the extra-cellular-matrix, a specific feature we implemented in our protocol. Trunk-Like Structures provide a scalable, tractable, and reproducible platform to study normal and aberrant embryonic development in vitro at an unprecedented spatiotemporal resolution