Functionally diversified neuronal populations have now been efficiently generated from human pluripotent stem cells (hPSCs). However, directed differentiation of hPSCs to functional astroglial subtypes remains elusive. In this study, hPSCs were successfully directed to nearly uniform populations of immature astrocytes in large quantities (>90% S100β+ and GFAP+). The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels following transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that carry distinct homeodomain transcription factors and display phenotypic differences. These human astroglial progenitors and immature astrocytes will be instrumental for studying astrocytes in brain development and function, for revealing their roles in disease processes, and for developing novel treatments for neurological disorders.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virusinduced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.A correlation between congenital exposure to the mosquitoborne and sexually transmitted Zika flavivirus (ZIKV) and the increased incidence of severe microcephaly suggests a causal relationship between ZIKV infection and neurodevelopmental abnormalities (1, 2). However, the mechanisms of infection and specifically which cell populations are vulnerable to ZIKV during the course of human brain development remain unclear. Major insights have been drawn from in vitro models of human brain development and primary mouse tissues. In the developing mouse brain, ZIKV has been shown to infect radial glia and neurons (3), whereas studies in human pluripotent stem cell (hPSC)-derived neural cells have highlighted widespread infection and apoptosis of neural progenitor cells (4,5). Because these models do not fully recapitulate the developmental events and cell types present during human brain development, these results may not faithfully represent ZIKV-induced pathology in vivo.During human brain development, radial glial cells, the neural stem cells, give rise to diverse types of neuronal and glial cells, including neurons, oligodendrocytes, and astrocytes, in a temporally controlled pattern. We reasoned that identifying cell types that are especially vulnerable to viral infection wou...

Dysfunction of basal forebrain cholinergic neurons (BFCNs) and γ-aminobutyric acid (GABA) interneurons, derived from medial ganglionic eminence (MGE), is implicated in disorders of learning and memory. Here we present a method for differentiating human embryonic stem cells (hESCs) to a nearly uniform population of NKX2.1+ MGE-like progenitor cells. After transplantation into the hippocampus of mice in which BFCNs and some GABA neurons in the medial septum had been destroyed by mu P75-saporin, human MGE-like progenitors, but not ventral spinal progenitors, produced BFCNs that synaptically connected with endogenous neurons, whereas both progenitors generated similar populations of GABA neurons. Mice transplanted with MGE-like but not spinal progenitors showed improvements in learning and memory deficits. These results suggest that progeny of the MGE-like progenitors, particularly BFCNs, contributed to learning and memory. Our findings support the prospect of using human stem cell–derived MGE-like progenitors in developing therapies for neurological disorders of learning and memory.

Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are a useful tool for understanding brain function, modulating disease processes, and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated to neuroepithelial cells with or without exogenous patterning molecules(days 0 –21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over along term expansion (days 21–90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated to functional astrocytes upon removal of mitogens and the addition of CNTF ( days > 90). This method generates robust populations of functionally diversified astrocytes with high efficiency.