Transfection of the channelrhodopsin-2 gene into hESC- and iPSC-derived NSCs from a patient with ALS

hESC-derived NSCs and iPSC-derived NSCs from a patient with sporadic ALS were infected with an AAV-CAG-ChR2H134R-tdTomato-WPRE plasmid to express a mutated variant of the light-sensitive ion channel, channelrhodopsin-2H134R. AAV-CAG-hChR2H134R-tdTomato was a gift from K. Svoboda (Addgene plasmid no. 28017) (57). Adeno-associated virus (AAV) particles were also provided by Addgene. Both types of NSCs were plated in laminin-coated six-well plates at a density of 5.0 × 105 cells per well and cultured for 2 days. The cells were then incubated for 24 hours with AAV particles containing the plasmid and maintenance culture medium. The cells were cultured for 4 days, expanded for another 3 days, and sorted by fluorescence-activated cell sorting (BD Biosciences, FACSAria II) with strong expression of tdTomato. The transfection efficiency of hESC-derived NSCs and ALS-iPSC–derived NSCs was 77 and 68%, respectively. The channelrhodopsin-2 (ChR2)–NSCs were then replated in laminin-coated six-well plates. To create neurospheres, both ChR2-NSCs were dissolved using Accutase to obtain single cells.

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