Multifactorial approach is needed to unravel the maturation phases of human neurons derived from induced pluripotent stem cells

Neurons derived from induced pluripotent stem cells (h-iPSC-Ns) provide an invaluable model for studying the physiological aspects of neuronal development and diseases. However, multiple studies have also demonstrated that h-iPSC-Ns exhibit a high degree of functional and epigenetic diversity. Due to the imprecise characterization and significant variation among the currently available maturation protocols, it is essential to establish a set of criteria to standardize models and accurately characterize and define the developmental properties of neurons derived from iPSCs. In this study, we conducted a comprehensive analysis of the h-iPSC-Ns via electrophysiological and microscopic techniques to follow their functional development at the cellular and network levels. This enabled us to provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week period in vitro. Specifically, we have used conventional whole-cell patch-clamp and dynamic clamp techniques, alongside morphometry, to assess the characteristics of maturing h-iPSC-Ns. Additionally, we utilized calcium imaging to monitor the progression of synaptic activity and network communication. At the single cell level, human neurons exhibited gradually decreasing membrane resistance in parallel with improved excitability by 5 weeks of maturation. Their firing profiles were consistent with those of mature regular firing type of neurons. At the network level we observed the development of abundant fast glutamatergic and depolarizing GABAergic synaptic connections together with synchronized network activity. The identified sequence of differentiation events are consistent and offers a robust framework for developing targeted experiments at varying stages of neuronal maturation. This framework allows for the use of different, age-related methodologies or a singular set of experiments for a cultures maturation.