Unravelling the dynamics of 2D and 3D neuronal networks derived from human induced pluripotent stem cells

Objective. In this work, I conducted a study focused on the development of 2D and 3D in vitro human induced pluripotent stem cells (hiPSCs)-derived neurons over a long period of time, to understand the stability of the experimental model and its evolution over the time. In particular, I investigated and characterized cortical excitatory (E) and inhibitory (I) hiPSCs-derived neuronal networks using low- and high- density Micro-Electrode Arrays (MEAs). This peculiar system allowed me to investigate whether and how the E/I balance affected the in vitro development in terms of spontaneous and both electrically and chemically evoked activity. Subsequently, I increased the complexity of the in vitro model to resemble the in vivo system more closely, by exploring the role of modularity and three-dimensionality. Approach. Excitatory (E) and inhibitory (I) neurons were obtained from hiPSCs thanks to the overexpression of the transcriptional factors Neurogenin-2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), respectively. Five different configurations were realized by systematically varying the glutamatergic (E) and GABAergic (I) percentages, starting from fully excitatory (E:I 100:0) up to fully inhibitory (0:100) cultures, including heterogeneous combination of both cell types with different percentages (E:I 25:75, 50:50, 75:25). Spontaneous electrophysiological activity was recorded during the development up to 98 Days in vitro (DIV), and the evoked activity was collected during electrical stimulation and chemical modulation. The main features were extrapolated to describe the firing, bursting and network bursting activity, typical of the in vitro populations, as well as features describing the topology and the connectivity of the networks. The neuronal networks were characterized on both low- and high- density MEAs and in 2D and 3D configurations. Main results. The nominal E/I ratio of the heterogeneous neuronal networks was consistent with immunostaining and imaging quantification. The homogeneous networks behaved differently from all the heterogeneous configurations. The fully inhibitory networks lacked in the organization into bursts and exhibited a pronounced tonic firing. On the other hand, increased inhibition in the heterogeneous cultures affected the duration and the organization of the bursting and network bursting activity. The electrical stimulation was effective in producing reliable responses in both heterogeneous and homogeneous configurations. As for chemical modulation, BIC, PTX and PTZ provoked major changes in the dynamics and connectivity of the heterogeneous networks, while APV and CNQX caused drastic decreases in the activity and alterations of the functional connectivity of both homogeneous and heterogeneous configurations. Significance. Due to the intrinsic complexity of the human brain, scientific research is exploring alternative methods to investigate neural functions in reduced experimental models that preserve intrinsic brain features such as modularity, segregation, heterogeneity, and three-dimensionality. To this end, in vitro models on MEAs combined with the use of hiPSCs-derived networks opened new opportunities in the field of personalized medicine. Despite the promising premises, these cultures have not been widely characterized from an electrophysiological point of view. Thus, my PhD research project represents a step forward to provide the scientific community with a human-based in vitro system, investigating and reproducing in vitro several peculiar brain’s features such as heterogeneity, modularity, and three-dimensionality. My findings will lay the foundation for unravelling the complex relationships among different neuron types and will help us better grasping how these interactions influence network activity. This has the potential to significantly enhance the exploration of a wide range of both physiological and pathological conditions.