Single-cell transcriptomic profiling of human fetal neural stem cells isolated from the subventricular zone

Neural stem cells (NSCs) are self-renewing, multipotent cells capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside in specific brain niches: the ventricular zone (VZ), the subventricular zone (SVZ) and the subgranular zone of the dentate gyrus (SGZ). Each of these niches orchestrates finely tuned neurogenesis and gliogenesis in both the developing neocortex and the adult brain. The exact cellular composition of human NSCs (hNSCs) from the SVZ (SVZ-hNSCs) of the developing human neocortex is not yet fully understood and remains elusive. This represents a major obstacle to understanding how human neurogenesis works and limits the potential of fetal derived hNSCs in regenerative medicine applications. To address this, we performed single-cell transcriptome analysis on hNSCs, isolated from the SVZ of fetal brains of different donors, resulting from spontaneous miscarriages, and subjected to different culture passages. Our analysis revealed, in each sample, the high expression of the canonical stemness markers. Among the identified subpopulations, we observed a neural progenitor cell cluster, also expressing typical markers of the quiescent state. The remaining clusters diverged from this population along two main trajectories: one oriented toward the glial lineage and another with a more neuronal identity. Our analysis further revealed that extended in vitro culture induces a progressive transcriptional shift, characterized by the activation of differentiation programs, providing insights into the temporal dynamics of hNSCs identity. Nevertheless, despite this gradual transcriptional shift, late passage cultured hNSCs retained their stemness, as the strongly stemlike cluster persisted with a high number of cells. Overall, our findings provide a deeper characterization of hNSCs isolated from the fetal brain and demonstrate their long-term stability and safety in regenerative medicine, as they preserve their stem-like identity even after prolonged in vitro expansion.