Abstract
Human excitatory neurons programmed through neurogenin-2 (NGN2) overexpression are widely used to model brain disorders in vitro. Although growth factors (GFs) such as BDNF, GDNF, NT3 and CNTF are commonly included in differentiation protocols, their individual and combined effects on neuronal survival, morphology and function remain insufficiently characterized. Here, we systematically examined the impact of these GFs, alone or in combination, on the development and maturation of NGN2-neurons. We also compare network activity of neurons maintained in Neurobasal medium (NBM) versus BrainPhys (PB). We show that BDNF or GDNF alone were sufficient to support neuronal survival and morphological complexity, whereas functional maturation, including network activity, required CNTF. Furthermore, BP supported neuronal development and function comparable to NBM, provided appropriate supplementation. Together, our results show that CNTF in combination with either BDNF or GDNF provides the most effective support for both structural and functional maturation of NGN2-neurons derived from male induced pluripotent stem cells (iPSCs). These findings offer a better understanding of how GF supplementation shapes neuronal development and provide a framework for optimizing human neuron culture conditions in disease modeling and drug discovery.