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

Introduction: Human induced pluripotent stem cells (hiPSCs) can be rapidly converted into neurons via NGN2 overexpression, but many protocols require costly reagents during the initial induction phase that may limit adoption by labs without routine neuronal differenitation experience. We developed a simplified, low-cost protocol using a tetracyline-inducible (TET-on) NGN2 system in minimal media to generate cortical neurons in as little 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. The impact of adding the Notch inhibitor, DAPT, during doxycycline induction to enhance neurogenesis to was evaluated with immunocytochemistry (ICC) and RT-PCR. Following induction neurons were matured and characterized with ICC for mature neuronal markers and by multielectrode array recordings for functional network activity. Results: DAPT markedly improved conversion efficiency, reducing non-neuronal cells and increasing pan-neuronal TUJ1 expression. Resulting neurons expressed cortical markers and matured into functional glutamatergic neurons. 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.