The Application of Genome-editing to Study the Pathophysiological Mechanism of Neurodegeneration and Aging in Human Induced Pluripotent Stem Cells (iPSC)

The arrival of human induced pluripotent stem cells (hiPSCs) and their ability to differentiate into multiple subtypes of neurons have opened new avenues in studying cellular properties of human neurons. hiPSCs are easily accessible as they can be directly induced from somatic cells of individuals at different ages, hence providing a pre-clinical cellular model to study neuronal dysfunction in human brain disorders, in particular, neurodegeneration in the elderly people. However, the process of the lengthy development, maturation, and aging of human brain are very difficult to model in vitro. And one way to address this is to label the iPSC-derived neurons with epitope tags by genome editing, followed by grafting into the brains of aged mice and study the effect of aging on human neuron. The recent advance in genome editing through CRISPR-Cas9 has greatly facilitated the generation of disease-related mutations in hiPSCs. Here we use CRISPRCas9 to create a knock-in epitope tag in hiPSC-derived cortical neurons. We have developed the differentiation protocol for the iPSC line IMR90 to generate mature cortical neurons that expressed pre- and postsynaptic makers of excitatory synapses and produced miniature excitatory postsynaptic current. In this proof-of-concept investigation, the β-actin gene was edited in hiPSC through CRISPR-Cas9 mediated genome editing, such that a hemagglutinin (HA) tag was added to the N-terminal of -actin. After differentiation of the genome-edited hiPSCs into cortical neurons, the growth cones and dendritic spines, cellular compartments that are involved in synaptogenesis, were visualized by immunostaining through anti-HA antibody. Furthermore, after transplantation, the grafted tagged hiPSC-derived neural stem cells protrude branches in the host brain. Our findings suggest the feasibility of this knock-in strategy to label synaptic proteins for the study of neuronal morphology in human iPSC-derived cortical neurons in the brains of aged mice and animal model of neurodegeneration in vivo.