DEVELOPMENT OF 3D IN VITRO MODEL TO STUDY MOLECULAR MECHANISMS OF SPINAL MUSCULAR ATROPHY

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease and the leading genetic cause of death during childhood. SMA is caused in the majority of the cases (up to 95%) by mutations in the Survival Motor Neuron 1 (SMN1) gene coding for the SMN protein, resulting in a progressive muscular paralysis due to lower motor neurons degeneration. A deeper knowledge of SMN biology and of its role in different organs/system in reliable models is very important for the optimization of available treatments and the development of complementary therapeutic approaches. In particular, it can be crucial to generate a human model able to recapitulate the complexity of the central nervous system (CNS) and its development. A promising tool to study SMA pathology is the three-dimensional (3D) organoid, obtained starting from induced pluripotent stem cell (iPSCs). Moreover, this model could be used to identify new therapeutic strategy. Rationale: In this project, we exploited CNS organoid technology, which is a novel stem cell-based 3D platform that has the potential to address the limitations of human existing bi-dimensional (2D) cultures improving preclinical testing. We aim to demonstrate that this approach can recapitulate some of the complexity of whole-organism biology overcoming the conventional use of 2D iPSCs-derived motor neurons. Nowadays, several therapeutic strategies have been tested in clinical trials and two compounds have been approved by FDA. Nevertheless, all these approaches are completely efficacious only if administered at pre-symptomatic stages. The generation of a new model for SMA could lead to a better knowledge of the mechanisms underlying the disorder during the development of the CNS and it might contribute to develop new or combined therapeutic options for affected patients. Methods: We obtained iPSCs from human fibroblasts of both healthy subjects and SMA type 1 patients and, using two different protocols that recapitulate the embryonal developmental steps, we generated 3D brain organoids and 3D spinal cord-like spheroids. We performed immunohistochemical and molecular analysis to confirm their differentiation state. Moreover, to verify their basal activity and their capability to response to stimuli, we performed calcium imaging and electrophysiological analysis. Results: CNS organoids derived from healthy subjects and patient have been successfully obtained, as suggested by the protein and gene expression data. In particular, brain organoids gave rise to an early cerebral cortex-like formation containing progenitor cells and more mature neural subtypes. Electrophysiological analysis demonstrated not only their basal activity, but also their ability to respond to stimuli. Concerning spinal cord-like spheroids, we used a modified protocol in order to induce neural caudalization and ventralization. This model gave us a powerful tool to investigate early motor neuron pathology and causes of degeneration. Like brain organoids, spinal cord-like spheroids have been characterized by immunohistochemistry, gene expression analysis and electrophysiological activity. Preliminary results suggested that SMA organoids and spheroids, compared to the controls, exhibited not only an alteration in the proper markers expression, but also in the electrophysiological activity. Conclusion: We successfully generated and characterized healthy and SMA CNS 3D organoids and spheroids that recapitulated human CNS development, showed disease-related features. This model can be used as an innovative in vitro system to study pathogenic mechanisms, identifying therapeutic targets and test potential therapeutic strategies.