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 oligoconjugated 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.
Competing Interest Statement
Jasmine Singh, Cassandra Hempel, Nadya Nikulina, and Oliver Braubach are employees of Akoya Biosciences. All other authors declare no conflict of interest.
Footnotes
Declaration of interest, JS, CH, NN, and OB are employees of Akoya Biosciences. All other authors declare no conflict of interest.
https://www.akoyabio.com/wp-content/uploads/2021/01/CODEX-User-Manual.pdf
Abbreviations
- iPSC
- induced pluripotent stem cells
- CODEX
- CO-Detection by indexing
- DIV
- days in vitro
- ICC
- immunocytochemistry
- NPCs
- neural progenitor cells
- KO-DMEM
- knockout Dulbecco’s modified Eagle’s medium
- PBS
- phosphate-buffered saline
- THZ
- Thiazovivin
- RT
- room temperature
- LGE
- lateral ganglionic eminence
- MSN
- medium-sized spiny neurons
- KSR
- Knockout serum replacement
- PenStrep
- penicillin, streptomycin
- NEAA
- non-essential amino acids
- Vit. A
- vitamin A
- FBS
- fetal bovine serum
- Ngn2
- neurogenin 2
- NT3
- neurotrophin 3
- cDNA
- complementary DNA
- NC
- negative control
- ddH2O
- double-distilled water
- PFA
- paraformaldehyde
- MAV
- Multiplex Analysis Viewer
- BSA
- bovine serum albumin
- PLL
- poly-L-lysine
- BS3
- bis(sulfosuccinimidyl)suberate