Rapid, Growth Factor-Reduced Induction of Functional Neurons from hiPSCs

Introduction: Human induced pluripotent stem cells (hiPSCs) can be rapidly converted into neurons via Neurogenin-2 (NGN2) overexpression, but many protocols require costly reagents during the initial induction phase that may limit adoption by labs without routine neuronal differentiation experience. We developed a simplified, low-cost protocol using a tetracycline-inducible (TET-on) NGN2 system in minimal media to generate cortical neurons in as few as 6 days. Methods: KOLF2.1J hiPSCs were stably transfected with a TET-on NGN2 cassette using the nonviral PiggyBac system and induced with doxycycline in Essential 6 media with or without the Notch inhibitor DAPT. Neurogenesis was evaluated with immunocytochemistry (ICC) and RT-qPCR, and cultures matured in defined conditions were characterized by multielectrode array (MEA) recordings to assess functional maturation. Results: DAPT markedly improved hiPSC-to-neuron conversion efficiency, and yielded glutamatergic neurons expressing cortical markers. MEA recordings showed spontaneous activity by day 14 and synchronous network firing by day 35. Secondary PB transfection enabled Td-Tomato labelling of KOLF2.1J:pB-TO-NGN2 hiPSCs, allowing 24-hour live imaging of neurite outgrowth. Conclusion: This streamlined, growth-factor-free workflow provides an accessible route for generating functional neurons from patient-derived hiPSCs, including in labs with limited hiPSC or neuronal culture experience.