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

Abstract

Background: 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 biomarker readout from every model that is developed. One option is to use a highly multiplexed biomarker imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ at single-cell resolution. New Method: This paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs. Results: 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. Comparison with existing methods: Compared to standard immunocytochemistry, CODEX oligo-conjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing. Conclusion: 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 biomarkers in the same samples. CODEX is a powerful technique that complements other single-cell omics technologies for in-depth phenotype analysis.

Challenges and Opportunities for Immunoprofiling Using a Spatial High-Plex Technology: The NanoString GeoMx® Digital Spatial Profiler

“…In addition to its protein biomarker assays, the platform will soon be enabled with RNA detection using the new PhenoCycler-Fusion system ( 102 ), which could be compared in the future to a new nanoString technology called “CosMx” that integrates protein analysis with RNA and also offers the possibility of single-cell resolution ( 103 , 104 ). From a histotechnical point of view, one of the disadvantages of the PhenoCycler compared to the DSP is the challenge to process tissue section and slide preparation, because this assay uses a coverslip of a limited size (22 × 22 mm) ( 105 ).…”