Comparative imaging analyses for morphometric and functional evaluation of human neuromuscular organoids

We are in the era of three-dimensional in vitro modeling of complex multicellular systems, able to mimic human tissue and organs in culture. In particular, organoids are defined as a collection of organ-specific cell types derived from stem cells, self-organized in a way comparable to the in vivo tissues. Skeletal muscle is an anatomical tissue of utmost importance for the human body, and can be considered a biomechanical device capable of generating voluntary movement and force via myofiber contraction, upon neural stimulation. It was recently shown that human neuromuscular organoids (NMOs) can be derived from pluripotent stem cells, including induced pluripotent stem cells, opening new perspective for in vitro modeling of the human neuromuscular system, in a patient-specific fashion. This has opened new perspective for the studies of disorders that are associated to both skeletal muscle and nervous system, including neuromuscular disorders and muscular dystrophies. One of the current challenges in the study of NMOs functionality and multicellular organization, resides in their intrinsic 3D complex organization. To tackle this issue, recent works have seen the development of software for bio-image analysis with increased levels of complexity, that aim to quantify relevant patho-physiologic responses of such human organoids. The advancement of bio-image analysis is very promising, both for the rise of new organoid-oriented analysis tools but also for the optimization of pre-existing techniques, valuable for NMOs analysis. In this thesis, we compared different methodologies used to quantify morphometric and biological activity of different samples, recorded via live imaging, with the aim of boosting analysis efficiency and reproducibility.