Fast-Track to Functional Neurons: Accessible and Minimalistic Neurogenin 2 Programming of Human iPSCs

Abstract

Neuronal differentiation of human induced pluripotent stem cells (hiPSCs) is a cornerstone for advancing neuroscience research and therapeutic applications. However, conventional differentiation protocols are commonly resource-intensive, technically complex, and overall time-consuming—often requiring several weeks to months to yield functional neurons. Here, a streamlined method is presented to enable fast and reliable neurogenin 2 (NGN2)-mediated neuronal differentiation by introducing the “colony-initiated differentiation” (CID) approach. The CID method yields functional neurons from a commercially available hiPSC line (BIONi010-C-13, equipped with a doxycycline (DOX)-inducible NGN2 expression cassette) in just five days. Within the first two days of CID, the colonies undergo a structural reorganization, after which the cells are replated and mature for additional 3–5 days. The neurons become functional from day five onward, demonstrated by patch clamp recordings of action potentials. Critically, CID operates entirely feeder cell- and growth factor-free. Simplicity, regular-workday compatibility, and quality of the differentiation method using a market-available hiPSC line make the method easily accessible to researchers across various fields. Thus, this work not only has implications for accelerating research in toxicity screening and drug discovery but particularly in multidisciplinary fields such as materials science and bioengineering, providing a robust platform to study neuron–material interfaces and interactions.