Analysis of Primary Cilium-Bearing Human Neuroprogenitors Using Flow Cytometry

The primary cilium is a protruding organelle present on many cell types with important roles for cell signaling. Defects in primary cilium formation and function are linked to numerous pathological conditions including neurodevelopmental defects, aging and cancer. The evaluation of ciliated cells within a cell sample traditionally relies on the visual assessment of cilia in fluorescence/confocal microscopy, after immunolabeling for ciliary markers highlighting the organelle for cilium counting. This can be influenced by operator-dependent factors, notwithstanding advanced image analysis tools developed to facilitate this labor-intensive evaluation. To address these limitations, a flow cytometry approach was trialed for neuroprogenitor cells (NPCs) differentiated from human iPSCs and stained for the ciliary markers ARL13B and PERICENTRIN measured on a flow cytometer, which detected positively-labeled ciliated cells. Specific staining was confirmed by microscopy and imaging flow cytometry, demonstrating for the first time the feasibility of cilium detection with axoneme and basal body markers colocalized on a single spot on human neuroprogenitor cell surface using a scalable, objective, and quantitative modality. Flow cytometry was able to measure changes in cilium frequency in a comparative analysis of neuroprogenitors derived from ciliopathy patients and healthy controls, underlining the discriminating capacity of this streamlined approach for the study of ciliary defects in a scalable and operator-independent manner.