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.

SUMMARY Low-grade astrocytomas (LGA) carry neomorphic mutations in Isocitrate Dehydrogenase (IDH), concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA and ATRX shRNA in human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo and led to a DNA methylation and transcriptional profile resembling IDH1-mutant human LGAs. The differentiation block was caused by transcriptional silencing of transcription factor SOX2, secondary to disassociation of its promoter from a putative enhancer. This occurred due to reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH-mutant LGA formation implicates impaired NSC differentiation due to repression of SOX2 as an early driver of gliomagenesis.

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