Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells

Highlights

• Up to 590-day cultures of human iPSC-derived 3D cortical spheroids (hCSs)

• Transcriptional and functional characterization of astrocytes purified from hCSs

• Astrocytes within hCSs mature during in vitro differentiation

• Early- and late-stage hCS-derived astrocytes are functionally distinct

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 (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs 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.