Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Our understanding of gliogenesis remains incomplete and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient, chemically-defined astrocyte differentiation strategy that overcomes current limitations. Remarkably, this gliogenesis-promoting approach bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like molecular signatures. Modulation of several cell signaling pathways including NOTCH and JAK/STAT in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to astrocyte differentiation and absence of neurogenesis. The functional and morphological identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g., electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic cell culture platform and used to model Alexander disease. In conclusion, our findings uncover unexpected plasticity in neural lineage progression that can be exploited to manufacture large numbers of hPSC-derived RGCs and astrocytes for basic neuroscience research, drug development, and regenerative medicine.