Micropatterned and stretchable human neuromuscular organoids to study Duchenne Muscular Dystrophy

Recent advances in bioengineering allow to develop human neuromuscular in vitro models that more closely resemble the physiological three-dimensional (3D) cellular environment and structure of neuronal-coupled skeletal muscle. Neuromuscular organoids (NMOs) derived from human induced pluripotent stem cells (hiPSCs) have been used as in vitro models to investigate the neuromuscular system. However, organoids do not fully recapitulate the physiological 3D organization of the native skeletal muscle tissue. Cell culture substrates with a defined microgrooved surface topography have been used to control myogenic cell differentiation and spatial organization. In this thesis we aimed to drive the self-assembly behavior of hiPSCs during their differentiation in NMOs using an engineered micropatterned stretchable substrate to generate functional patterned-NMOs (p-NMOs). Under static culture conditions, we confirmed that microgrooves impose myogenic and neural cells organization along the longitudinal direction of the patterned substrate. However, we showed that initial cell density, as well as cell embedding, influence cell behavior and organization, leading in some case to a partial or complete loss of the patterned orientation. Duchenne muscular dystrophy (DMD) is a rare genetic neuromuscular disease that causes progressive skeletal muscle loss and weakness. To assess whether the p-NMO model could be used to study such neuromuscular disorder, hiPSCs derived from one DMD patient were used. Interestingly, DMD p-NMO exhibited some specific DMD phenotypes, in comparison with healthy donor derived p-NMO. Indeed, upon stimulation with neuronal and muscular specific neurotransmitters, we observed altered functional intracellular calcium responses, revealing altered calcium flux in the DMD p-NMO. Moreover, gene expression analysis showed altered expression of the muscle stem cell determinant transcription factor PAX7 and a higher dispersion degree of myogenic cells in DMD samples. In the investigation of p-NMO functionality the presence of clustered acetylcholine receptors reached by neural projections were observed in healthy p-NMO, while this aspect was rarer in DMD samples. Although deeper analyses are needed to optimize the model, the overall preliminary results suggest that p-NMO represents a promising three-dimensional in vitro model for studying human neuromuscular mechanisms and related diseases. Finally, with the aim to apply dynamic culture conditions to p-NMOs, a novel home-made soft-tissue specific bioreactor was produced and characterized to analyze the effect of different physiological stretching protocols (passive and cyclic uniaxial deformations) for future applications on both healthy and DMD p-NMOs.