A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells

Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.