Abstract
Human induced pluripotent stem cell-derived (iPSC) neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer’s, and Autism Spectrum Disorder. In situ characterization of the spatial-temporal evolution of cell state in 3D culture and subsequent 2D dissociated culture models based on protein expression levels and localizations is essential to understanding neural cell differentiation, disease state phenotypes, and sample-to-sample variability. Here, we apply PRobe-based Imaging for Sequential Multiplexing (PRISM) to facilitate multiplexed imaging with facile, rapid exchange of imaging probes to analyze iPSC-derived cortical and motor neuron cultures that are relevant to psychiatric and neurodegenerative disease models, using over ten protein targets. Our approach permits analysis of cell differentiation, cell composition, and functional marker expression in complex stem-cell derived neural cultures. Furthermore, our approach is amenable to automation, offering in principle the ability to scale-up to dozens of protein targets and samples.
Highlights
Human induced pluripotent stem cell-derived neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer’s, and Autism Spectrum Disorder
Such work can benefit from a complementary IF method that is not constrained by limitations inherent to traditional antibody-based imaging
We used DNA-conjugated PRobe-based Imaging for Sequential Multiplexing (PRISM) antibodies to evaluate complex cortical neurons (CN) and Motor neurons (MN) cultures derived from stem cell lines in dissociated 2D cultures
Summary
Human induced pluripotent stem cell-derived (iPSC) neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer’s, and Autism Spectrum Disorder. Compared to standard antibody stripping procedures[21,22,23], PRISM offers non-destructive imaging probe exchange, cycling fluorescent imaging strands within several minutes permitting multiple rounds of imaging data acquisition[24,25,26] from the same culture These features allow for the use of large panels of markers, providing higher content datasets. 2D cultures can be used for high-throughput characterization of stem cell-derived neurons, which lends itself well to automation assays We hypothesize that this technique could be adapted to 3D organoid sections, and as these retain the higher order structure of cellular organization, especially in neural cultures, this may be critical for disease modeling in vitro. Because PRISM is a direct analog to IF/ ICC imaging, our approach complements assays in model culture systems currently used to evaluate stem cell differentiation, compound and drug candidate screening, tissue engineering, and potentially in vitro disease diagnostics development
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