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Bioresorbable nanopatterned scaffolds functionalized with polydopamine (PDA) and graphene oxide (GO) have been shown to promote the differentiation of murine neural stem cells (mNSCs) toward neural and glial lineages. Herein, we aim to evaluate the compatibility of these scaffolds for the culture and differentiation of both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells. Our results indicate that PDA and GO scaffolds support the topographic alignment of hESCs and hiPSCs cultures, while preserving their pluripotency characteristics. Upon differentiation, PDA and GO scaffolds guide cell specification toward the neuroectoderm germ layer and the neural crest. This promotes enhanced differentiation into both neural and supportive glial cells of the central nervous system (CNS), as well as Schwann cells of the peripheral nervous system (PNS). Moreover, nanopatterned scaffolds also support the differentiation of hESCs and hiPSCs toward endothelial precursors. These findings establish a novel culture platform that enables combined differentiation pathways, potentially relevant for applications in personalized medicine and regenerative cell therapy.

ABSTRACTNeural 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.

SummaryGlioblastomas are the most common malignant brain tumors in adults; they are highly aggressive and heterogeneous and show a high degree of plasticity. Here, we show that methyltransferase-like 7B (METTL7B) is an essential regulator of lineage specification in glioblastoma, with an impact on both tumor size and invasiveness. Single-cell transcriptomic analysis of these tumors and of cerebral organoids derived from expanded potential stem cells overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cell-to-astrocyte differentiation trajectory. Mechanistically, METTL7B downregulates the expression of key neuronal differentiation players, including SALL2, via post-translational modifications of histone marks. Graphical abstract Highlights•METTL7B is highly expressed in human glioblastoma stem cells•METTL7B regulates tumor size and invasiveness in an in vivo xenograft model•METTL7B controls the neural stem cell-to-astrocyte differentiation trajectory•METTL7B regulates SALL2 expression via H3K27me3 modulation Constantinou et al. identify METTL7B as an essential regulator of lineage specification and a modulator of the expression of the transcription factor SALL2 with wide-ranging impacts on invasion and tumor growth in glioblastoma.