The presence of mar mosaicism in source HFs, presumably clonal hiPSCs and hiPSC NPCs, indicates that these lines will not be readily amenable to functional evaluation via in vitro modeling using current methods, as one cannot distinguish 9p24.1 and control cells in live cultures. Standard phenotypic comparisons in these variably mosaic lines would be expected to yield large inter-experiment and intra-individual variation, requiring comparisons of increased numbers of cells and/or biological replicates to reach statistically significant conclusions. While phenotypic assays could theoretically be combined with stringent molecular techniques to resolve the 9p24.1 status of individual neural cells, combining post hoc FISH with many standard hiPSC-based assays of neuronal function (dendritic branching, synaptic imaging, multi-electrode array [MEA], electrophysiology, neurotransmitter release) and/or global transcriptomic, proteomic, or epigenetic approaches would likely prove practically difficult. FISH sample processing methodologies may be technically difficult to pair with some sensitive synaptic staining protocols, many commercial MEA plates are not amenable to imaging, and post hoc FISH would dramatically reduce the throughput of dendritic tracing or electrophysiological comparisons, approaches already limited by the small number of cells
