A knock-in strategy to study protein localization in human induced pluripotent stem cell (iPSC)-derived cortical neuron through genome editing

Abstract

The emergence of human induced pluripotent stem cells (hiPSCs) and their ability to differentiate into multiple subtypes of neuron present the attractive potential as a cellular model to study human-specific neural properties. Furthermore, the graft of hiPSC-derived neurons into the mouse brains potentially allows exploration of how synaptic connections form in vivo. To distinguish the synaptic proteins of the grafted neurons from those of the host neurons, it is desirable to specifically label the proteins of interest in hiPSC-derived neurons. Here in this proofof-concept investigation, we use CRISPR-Cas9 to create a knock-in epitope tag to study the localization of endogenous beta-actin in hiPSC-derived neurons. We have developed the differentiation protocol for the IMR90-hiPSC cell line to generate mature cortical neurons that express excitatory synaptic makers and produce miniature excitatory postsynaptic current. Through CRISPR-Cas9 mediated genome editing, the beta-actin gene was edited in hiPSC such that a hemagglutinin (HA) tag was added to the N-terminal of beta-actin. After differentiation of the edited hiPSCs into cortical neurons, the growth cones and dendritic spines were visualized by immunostaining through anti-HA antibody. The hiPSC-derived neural stem cells were also grafted into the mouse brain to visualize the grafted human neural stem cells by anti-HA immunoreactivity in vivo. Our findings suggest the feasibility of this strategy to label synaptic proteins for the study of protein localization during synaptogenesis in human iPSC-derived cortical neurons