SummaryThe diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.

Melanoma cells can switch between a melanocytic and mesenchymal-like state. Scattered evidence indicates that additional, intermediate state(s) may exist. To search for such states and decipher their underlying gene regulatory network (GRN), we studied ten melanoma cultures by single-cell RNA-seq, and 26 additional cultures by bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states, and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed its intermediate migratory phenotype. By time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Jointly, these analyses indicate that an intermediate state exists and is driven by a distinct and stable "mixed" GRN rather than being a symbiotic, heterogeneous mix of cells.

While genetics highlight the role of microglia in Alzheimer's disease (AD), one third of putative AD-risk genes lack adequate mouse orthologs. Here, we successfully engraft human microglia derived from embryonic stem cells in the mouse brain. The cells recapitulate transcriptionally human primary microglia ex vivo and show expression of human specific AD-risk genes. Oligomeric Amyloid-β induces a divergent response in human vs. mouse microglia. This model can be used to study the role of microglia in neurological diseases.