Single‐Cell Analysis of L‐Myc Expressing Neural Stem Cells and Their Extracellular Vesicles Revealed Distinct Progenitor Populations With Neurogenic Potential

Neural stem cell (NSC)-based therapies offer a promising strategy to promote brain repair by delivering neurotrophic factors, supporting cell replacement, and stimulating endogenous neurogenesis following injury. While numerous studies have highlighted the protective and regenerative potential of NSCs and their extracellular vesicles (EVs), progress toward clinical translation remains hindered by limited molecular characterization of NSC lines and their EV cargo. To address this gap, we characterized two therapeutically relevant human fetal NSC lines, LMNSC01 and LMNSC02, both engineered to express the L-MYC gene, along with their corresponding EVs. LMNSC01 cells primarily differentiated into neurones with limited glial populations, whereas LMNSC02 cells gave rise to all three major neural lineages: neural, glial and oligodendrocyte progenitor cells (OPCs). scRNA-seq revealed distinct transcriptional profiles with minimal overlap between the two LMNSC lines. Using single extracellular vesicle nanoscopy, we observed that both lines released predominantly circular EVs, with LMNSC02-EVs exhibiting higher levels of tetraspanins (CD9, CD63, and CD81) and a larger average diameter than LMNSC01-EVs. Proteomic profiling revealed that LMNSC01-EVs are enriched in proteins involved in cell adhesion, migration, junction formation, and neuronal projection development, while LMNSC02-EVs are enriched in factors related to cytoplasmic translation initiation and biosynthesis. These LMNSC-EVs (collected from undifferentiated LMNSCs) demonstrated neuroprotective effects in a brain organoid model of methotrexate-induced toxicity when added to corresponding LMNSC01- or LMNSC02-derived brain organoids. LMNSC01- and LMNSC02-derived EVs restored neuronal and astrocytic populations but failed to rescue OPCs. These findings demonstrate the therapeutic potential of LMNSC-derived EVs to counter chemotherapy-induced neurotoxicity by preserving neurones and astrocytes, while highlighting the need for repeated or complementary interventions to restore oligodendrocyte populations.