Neuromuscular Organoid Development: A Comparative Analysis of Induced Pluripotent Stem Cell Differentiation

Neuromuscular organoids (NMOs) are self-organized three-dimensional (3D) spheroids generated in vitro from human induced pluripotent stem cells (hiPSCs). Despite NMOs have been generated using different differentiation protocols, they all contain both neuronal and skeletal muscle compartments and have been used to mimic their interactions in vitro. Recent studies showed that NMOs represent an important innovation for the study of the human neuromuscular system, offering a patient-specific platform for investigating development, physiology and pathologies associated to both the skeletal muscle and the neurons. Compared to traditional bi-dimensional (2D) cell cultures, which often oversimplify cellular processes, 3D models like NMOs provide a more complex and physiologically relevant environment, promoting cellular maturation and the formation of functional neuromuscular networks that more closely resemble those found in vivo. However, NMOs still have some limitations, including variability between protocols, incomplete cell maturation, and difficulty in controlling their spheroidal morphology. In the laboratory where I conducted the internship, it has been shown that by providing specific extracellular matrix components during hiPSC differentiation, it is possible to control early-stage commitment of hiPSCs, improve maturation of the neuromuscular system model, as well as develop organoids with different morphological organization. Thus, the central objective of this thesis is to evaluate the effect that ECM exerts on early hiPSC commitment and late differentiation, with a final aim to overcome current limitations of the NMO-based in vitro models. To achieve this goal, a comparative approach along different differentiation protocols was adopted in order to understand how small-molecules and extracellular matrix components can influence the early stages of development and their subsequent cellular and functional organization. The first phase of the work focused on the early characterization of organoids, with particular attention to the identification of neuromuscular progenitors (NMPs), key cells that give rise to both the neuronal and muscular components, and mesodermal progenitors, that give rise to skeletal muscle. This analysis was essential for evaluating the efficiency of protocols in guiding hiPSCs towards specific developmental trajectory and for understanding which factors favour correct/desired cell specification. Subsequently, the NMOs generated adopting such small- and matrix-molecule based protocols were analyzed at a more advanced stage of development in terms of cellular composition, morphology and functionality. Overall, this study holds the potential to increase our knowledge on the role of extracellular matrix during the human neuromuscular system development, as well as to provide methodological advancement for a broader use of NMOs in translational studies related to the human neuromuscular system in health and disease.