Single-cell RNA-sequencing of adult neural stem cells reveals novel molecular principles of stem cell activation and heterogeneity

Neural stem cells (NSCs) in the subventricular zone (SVZ) and the subgranular zone of the hippocampus continuously give rise to glial cells and neurons and are the most important source for new neurons in the adult mammalian brain1-4. It has been suggested that the NSCs in the adult brain form a heterogeneous population5,7, akin to other tissue-specific adult stem cells6. However, a molecular basis of the presumed NSC heterogeneity has been lacking. Here, we sequenced the transcriptomes of 131 individual adult NSCs from the mouse SVZ. After filtering, 117 cells were used for further analysis. We found that they consist of two distinct subgroups of about equal size, independent of the mouse's age. These subgroups were separated by molecular markers of quiescence (quiescent NSCs, qNSCs) and cell-cycle progression (active NSCs, aNSCs). We identified novel cell surface markers belonging to the tetraspanin family of transmembrane proteins for the prospective purification of qNSCs and aNSCs. The individual NSC transcriptomes were ordered along a continuous progression from qNSCs to aNSCs, with the expression of molecular pathways decreasing (G-protein-coupled receptor signaling) or increasing (MAP kinase, PI3 kinase-mTOR, Hippo, and p53 pathways) in a coordinated manner. Within the aNSCs, transcription factors for neural cell lineage specification were expressed in patterns that indicate specific lineage commitment of individual aNSC. Together, these findings imply that the switch from qNSC to aNSC is irreversible in vivo, at least in a large fraction of cells, and is accompanied by lineage commitment already at the stem-cell stage. Thus our work reveals coherent patterns of molecular heterogeneity within adult NSCs that arise from the regulation of cell-cycle activity and differentiation pathways. We also sequenced 13 bulk populations. Overall design: neural stem cells expressing the nuclear receptor Tlx were isolated via FACS and subjected for single cell RNA sequencing. The project aims to discover heterogeneity of NSCs in vivo via single cell RNA-SEQ