Human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) constitute a powerful platform to study human microglial biology in health and disease. Here, we present a protocol for microglial differentiation and efficient gene delivery to these cells using adeno-associated virus (AAV). We describe steps for differentiation, viral transduction, and validation using flow cytometry and fluorescence imaging. Finally, to demonstrate the utility of this approach, we outline procedures to record and analyze calcium activity in hiMG using the genetically encoded sensor GCaMP8s. For complete details on the use and execution of this protocol, please refer to Fruhwürth et al. 1 and Heiss et al. 2 • Steps for differentiation of human iPSC-derived microglia (hiMG) • Instructions for efficient gene delivery to hiMG using adeno-associated virus • Guidance on how to perform calcium imaging in hiMG using a genetically encoded sensor Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) constitute a powerful platform to study human microglial biology in health and disease. Here, we present a protocol for microglial differentiation and for delivering genes to these cells using adeno-associated virus (AAV). We describe steps for differentiation, viral transduction, and validation using flow cytometry and fluorescence imaging. Finally, to demonstrate the utility of this approach, we outline procedures to record and analyze calcium activity in hiMG using the genetically encoded sensor GCaMP8s.
Heiss et al. (Mon,) studied this question.