Protocols for the application of human embryonic stem cell-derived neurons for aging modeling and gene manipulation

In vitro models of neuronal aging and gene manipulation in human neurons (hNeurons) are valuable tools for investigating human brain aging and diseases. Here, we present a protocol for applying human embryonic stem cell (hESC)-derived neurons to model aging and the further application of small interfering RNA (siRNA)-mediated gene silencing for functional investigations. We describe steps for neuronal differentiation and culture, siRNA transfection, and technical considerations to ensure reproducibility. Our protocol enables investigations of the molecular mechanism underlying neuronal aging and facilitates drug evaluation. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 • Generation of highly pure hESC-derived neurons • Modeling human neuronal aging via long-term culture in vitro • Achieve genetic manipulation in human neurons utilizing siRNA • Genetic or drug intervention attenuates human neuronal aging Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. In vitro models of neuronal aging and gene manipulation in human neurons (hNeurons) are valuable tools for investigating brain aging and disease. Here, we present a protocol for applying human embryonic stem cell (hESC)-derived neurons to model aging and the further application of small interfering RNA (siRNA)-mediated gene silencing for functional investigations. We describe steps for neuronal differentiation and culture, siRNA transfection, and technical considerations to ensure reproducibility. Our protocol enables investigations of the molecular mechanism underlying neuronal aging and facilitates drug evaluation.