Single extracellular analysis using flow cytometry for neurological disorder biomarkers

  

Poster by Houda Yasmine Ali Moussa (Hamad Bin Khalifa University) , Nimshitha Manaph (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Selma Maacha (Sidra Medicine), Kyung Chul Shin (Hamad Bin Khalifa University),, Samia M. Ltaief (Hamad Bin Khalifa University), Vijay Gupta (Hamad Bin Khalifa University), Yongfeng Tong (Hamad Bin Khalifa University), Janarthanan Ponraj (Hamad Bin Khalifa University), Salam Salloum-Asfar (Hamad Bin Khalifa University), Said Mansour (Hamad Bin Khalifa University), Fouad A. Al-Shaban (Hamad Bin Khalifa University), Hyung-Goo Kim (Hamad Bin Khalifa University), Lawrence W Stanton (Hamad Bin Khalifa University), Jean-Charles Grivel (Sidra Medicine), Sara A Abdulla (Hamad Bin Khalifa University), Abeer R. Al-Shammari (Hamad Bin Khalifa University), Yongsoo Park (Hamad Bin Khalifa University)

Background: Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility.

Objective: We aim to identify specific markers for brain-derived EVs isolated from plasma for biomarkers of neuronal disorders. 

Methods: Using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. We have also used human induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids to study neuronal EVs.

Results: Neural cell adhesion molecule (NCAM) is highly expressed and enriched in EVs released from human iPSCs-derived cortical neurons and organoids. Subpopulations of blood plasma exosomes contain NCAM, suggesting its origin from neuronal cells. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis.

Conclusion: Our data propose that NCAM-positive neuronal exosomes isolated from blood plasma can be detected and analyzed using flow cytometry. Neuronal EVs identified here will pave the way for further studies to discover biomarkers for neurological disorders.