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Updated: May 6, 2026
Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
Published on: March 5, 2013
Rapid, Growth Factor-Reduced Induction of Functional Neurons from hiPSCs
Natalie A Parker1, Oluwadamilola E Kolawole1, Zhixin Liao1
1Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, USA.
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 qRT-PCR, 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 h live imaging of neurite outgrowth.
Conclusion:
This streamlined, growth-factor-reduced workflow provides an accessible route for generating functional neurons from patient-derived hiPSCs, including in labs with limited hiPSC or neuronal culture experience.
