ABSTRACT: Neural stem cell (NSC)-based therapies can exploit the inherent pathotropism of NSCs to deliver neurotrophic factors for cell replacement therapies and to stimulate endogenous neurogenesis to permanently repair damaged brain tissue after brain injury. Despite studies on the protective/restorative functions of NSCs and their extracellular vesicles (EVs), a major impediment to advancing this approach to late-stage preclinical studies and clinical practice has been the lack of molecular characterization of NSCs and NSC-EV cargo composition. To fill this knowledge gap, we characterized therapeutically relevant human fetal NSC lines expressing the L-MYC gene (LMNSC01, LMNSC02) and their EVs using single-cell RNA sequencing (scRNAseq) and proteomic analyses. LMNSC01 cells differentiate into neurons and glial cells, including oligodendrocytes, and LMNSC02 mostly differentiate into neurons and oligodendrocyte progenitor cells (OPCs) under undirected differentiation in vitro. scRNAseq analysis indicates that LMNSC01 and LMNSC02 populations are homogenous, expressing not only NSC markers such as SOX2 and nestin, but also doublecortin (DCX), OPC marker O1, and astrocyte progenitor marker vimentin. LMNSC01 cells were enriched in biomarkers for astrocytes, excitatory neurons, glial cells, and OPCs, and LMNSC02 in inhibitory neurons, glial cells and OPCs. Using Single Extracellular VEsicle Nanoscopy, we quantified the size, shape, and content of tetraspanins (CD9, CD63, and CD81) for EVs secreted by cultured LMNSC01 and LMNSC02 cells. Both cell lines produced largely circular EVs; compared to EVs from LMNSC01 cells, those from LMNSC02 cells had on average higher detected tetraspanin content and larger diameter. We also quantified protein, RNA concentration, particle number, and batch-to batch variability for each EV. Our EV cargo global proteomics results suggest that LMNSC02 cells are enriched in processes involved in neuron development, neuron protection, synaptic transmission, and axon development, while EVs derived from LMNSC01 cells were more enriched in RNA processing. Finally, we identified neuroprotective properties of LMNSC-EVs in a brain organoid model of methotrexate-induced brain tissue toxicity. Our data suggest that LMNSC01 and LMNSC02 cell lines have distinct utilities, highlighting the need for molecular characterization to identify the cell lines and EVs with the highest therapeutic potential for various neurological conditions.
