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 human neuronal development under healthy and pathological conditions. However, multiple studies have 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 human neurons derived from iPSCs. In this study, we conducted comprehensive cellular and network level analysis of the functional development of human neurons, generated from iPSCs obtained from healthy young female peripheral blood mononuclear cells by BDNF and GDNF treatment. We provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week in vitro period using conventional whole-cell patch clamp and dynamic clamp techniques, alongside with morphometry and immunocytochemistry. 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 the fifth week of maturation, firing profiles were consistent with those of mature regular firing type of neurons. At the network level, fast glutamatergic and depolarizing GABAergic synaptic connections were abundant together with synchronized network activity from the sixth week of maturation. Alterations in the expression of GABA receptor subunits were also observed during the process of maturation. The sequence of differentiation events was consistent, providing a robust temporal framework to execute experiments at defined stages of neuronal maturation as well as to use a specific set of experiments to assess a culture's maturation. The uncovered progression of differentiation events provides a powerful tool to aid the planning and designing of targeted experiments during defined stages of neuronal maturation.