Validazione di un nuovo protocollo per differenziare neuroni spinosi medi striatali da cellule progenitrici neurali da piccole molecole

Medium spiny neurons (MSNs) constitute approximately 90-95% of striatal cells and represent the primary integrative and output component of the striatum. Due to their extensive involvement in cortico-striatal circuits, MSNs play an essential role in coordinating motor, cognitive and behavioural processes. Therefore, alterations in MSNs function and striatal network organization have been strongly linked to a broad spectrum of neurological conditions. Growing evidence indicates that dysfunction within the striato-cortical tracts is a key feature of several neurodevelopmental disorders (NDDs), which are characterized by impairments in social interaction, cognition, reward processing and attention. Despite their functional importance, the molecular and developmental mechanisms underlying MSNs specification and maturation remain partially understood. MSN populations originate from transient embryonic regions known as ganglionic eminences (GEs), which also give rise to diverse interneuron populations across multiple brain regions. The transient nature of these structures, their complex developmental dynamics and interspecies differences in brain composition, present major limitations for accurately modelling human striatal development using conventional experimental systems. Efforts to recapitulate MSNs development in vitro have largely relied on human stem cell-based systems, aiming to mimic key aspects of striatal maturation and patterning. These approaches rely on the temporal and dose-dependent modulation of key developmental signalling pathways, such as Sonic Hedgehog (SHH), WNT (Wingless-related integration site), and BMP (Bone Morphogenic Proteins)-related mechanisms, often implemented through small molecule-mediated modulation to achieve ventral telencephalic identity. However, these strategies often require prolonged differentiation timelines and complex culture conditions and remain limited by the generation of heterogeneous neuronal populations with variable differentiation efficiency. In this study, Activin A was investigated as a potential regulator of MSNs development, using human derived small molecule neuronal progenitor cells (smNPCs) cultured in N2B27 media as starting cell population. An initial one-week differentiation was performed to identify the most suitable conditions for striatal MSNs maturation. smNPCs were exposed to increasing concentrations of Activin A, in the presence or absence of RB5, a cell penetrating peptide that potentiate ERK signalling pathway, crucial for cell survival, proliferation and differentiation. Early differentiation outcomes were assessed using the MSN-early markers CTIP2 and FOXP2. Among the tested concentrations, treatment with 100ng/mL Activin A in the absence of RB5 resulted in the highest FOXP2 expression, suggesting enhanced early MSNs specification. This optimal condition was subsequently applied in a four-week differentiation protocol, to evaluate neuronal maturation over time. Prolonged Activin A exposure promoted progressive maturation, with the highest DARPP-32 expression detected at week 4. In addition, morphological observations revealed enhanced neurite outgrowth under this long-term Activin A treatment condition. Collectively, these findings suggest that Activin A is a promising regulator of MSNs differentiation from human smNPCs, promoting sustained neuronal maturation, supporting the development of in vitro models for studying human striatal development, disease mechanisms, and potential drug discovery approaches for neurological disorders.