Optimization of Activin A-driven differentiation of neural progenitors into medium spiny neurons

Medium spiny neurons (MSNs) are the main projection neurons of the striatum and play a central role in voluntary movement regulation, emotional processing, motivation, and reward-related behaviour. Despite their functional relevance, MSNs are particularly vulnerable in several neurological disorders. Importantly, an altered striato-cortical function is a common feature of a broad spectrum of neurodevelopmental disorders (NDDs). The development of the human striatum remains poorly understood. Some developmental structures, such as the Ganglionic eminences (GEs), are indeed transient, which adds an additional layer of complexity. Although GEs are crucial structures that give rise to MSNs and multiple populations of cortical interneurons, as well as olfactory bulb interneurons, inter-species differences in the brain structure and the complexity of the human brain limit the translational relevance of the animal models. To overcome these challenges, human induced pluripotent stem cells (iPSCs)-derived cultures represent a relatively simple yet physiologically relevant tool to study MSNs differentiation. To date, several protocols of MSNs differentiation have been developed. Most of them rely on temporal and dose-dependent manipulation of key morphogens, such as SHH (Sonic Hedgehog), WNT (Wingless-related integration site), and BMPs (Bone morphogenic proteins). These steps are followed by a prolonged maturation phase, often supplemented with neurotrophic factors, cyclic AMP analogs, retinoic acid (RA), and optimization of the differentiation media, to support MSN survival and maturation. A common limitation of these protocols is that they begin with the induced pluripotent stem cells (iPSCs), thus adding complexity to the differentiation steps and requiring extended differentiation timelines, ranging from 40 to 90 days, to achieve full maturation of the MSNs. In the present thesis project, a recombinant protein, Activin A, was employed as a differentiation cue using neural progenitor cell (NPC) cultures as a starting cell population. NPCs were differentiated in a medium containing N2 and B27 supplements under three conditions: Activin A, PMA, or a combination of both. Cells were fixed after one or two weeks and labelled for the MSN markers CTIP2 and FOXP2 by immunofluorescence. Our findings revealed a marked increase in FOXP2 following two weeks of Activin A treatment. Moreover, differentiation outcomes were also influenced by media change conditions. A half media change promoted CTIP2 expression during the 1st week, whereas FOXP2 expression was significantly increased at second week compared to the first when a full media change was applied. In addition, supplementation with B27 containing Vitamin A resulted in significant changes in FOXP2 expression during the first week in both PMA- and Activin A-treated conditions. Altogether, these findings support that Activin A promotes differentiation of NPCs towards the MSN fate, thereby providing a more efficient strategy compared with the previous approaches. Furthermore, fine-tuning additional molecular cues and culture parameters may represent a powerful strategy for further optimization of MSN differentiation protocols.