Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology

Human induced pluripotent stem cell (iPSC) models have been hailed as a breakthrough for understanding disease and developing new therapeutics. The major advantage of iPSC-derived neurons is that they carry the genetic background of the donor, and as such could be more predictive for clinical translation. However, the development of these cell models is time-consuming and expensive and it is thus critical to maximize readout of markers for immunocytochemistry. One option is to use a highly multiplexed fluorescence imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ. This paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs. We differentiated human iPSCs into mixed neuronal and glial cultures on glass coverslips. We then developed and optimized a panel of 21 antibodies to phenotype iPSC-derived neuronal subtypes of cortical, dopaminergic, and striatal neurons, as well as astrocytes, and pre-and postsynaptic proteins. Compared to standard immunocytochemistry, CODEX oligo-conjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing. We show that CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology. Furthermore, we demonstrate both cellular and subcellular resolution imaging of multiplexed markers in the same sample. [Display omitted] •Demonstration of CODEX imaging in iPSC-derived neurons with subcellular resolution.•Development of workflow for fixing neurons to sustain multiple wash-stain cycles.•Demonstrating deep CODEX phenotyping for neuronal subtypes and synaptic proteins.