The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/ brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

Summary The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains, and to maintain these cells in serum-free cultures. We found that human astrocytes have similar abilities to murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to existing observations in mice, we found that mature human astrocytes respond robustly to glutamate. We next performed RNA-sequencing of healthy human astrocytes along with astrocytes from epileptic and tumor foci, and compared these to human neurons, oligodendrocytes, microglia, and endothelial cells. With these profiles, we identified novel human-specific astrocyte genes, and discovered a transcriptome-wide transformation between astrocyte precursor cells and mature post-mitotic astrocytes. These data represent some of the first cell type-specific molecular profiles of the healthy and diseased human brain.

SignificanceThe brain comprises an immense number of cells and cellular connections. We describe the first, to our knowledge, single cell whole transcriptome analysis of human adult cortical samples. We have established an experimental and analytical framework with which the complexity of the human brain can be dissected on the single cell level. Using this approach, we were able to identify all major cell types of the brain and characterize subtypes of neuronal cells. We observed changes in neurons from early developmental to late differentiated stages in the adult. We found a subset of adult neurons which express major histocompatibility complex class I genes and thus are not immune privileged.

SUMMARY Glioblastoma (GBM) is the most common primary brain cancer in adults and is notoriously difficult to treat because of its diffuse nature. We performed single-cell RNA sequencing (RNA-seq) on 3,589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumor’s genome and transcriptome. We were also able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of information about tumor formation and migration.

SUMMARY There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCS) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCS at varying stages up to 20 months in vitro using immunopanning and cell sorting, and performed high-depth bulk and single cell RNA-sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.