Constituting about of all cells in the

Constituting about 40% of all cells in the brain, astrocytes have long been classified as mere passive supporting cells that, for example, promote survival and functional synaptic formation of neurons; however, astrocytes are also essential for the phagocytic elimination of synapses, which refines neuronal circuit development (Allen and Barres, 2009). Because these roles of astrocytes are very important for oxytocin antagonist function, astrocytes are indispensable components in CNS integrity (Allen et al., 2012; Christopherson et al., 2005; Hamilton and Attwell, 2010; Haydon and Nedergaard, 2015; Kucukdereli et al., 2011; Molofsky et al., 2012; Ullian et al., 2001; Zhang et al., 2016). Therefore, astrocyte dysfunction is thought to be implicated in various neurological disorders including Rett syndrome (RTT), which is caused by methyl-CpG binding protein 2 (MECP2) mutations (Amir et al., 1999; Bienvenu and Chelly, 2006; Tsujimura et al., 2015). Mutant (MT) MECP2-expressing astrocytes derived from RTT-hiPSCs have recently been reported to have adverse effects on neuronal maturation compared with their isogenic wild-type (WT) MECP2-expressing astrocytes (Williams et al., 2014). However, little progress in human astrocyte functional analysis has been made because, as noted above, differentiation of hPSC-derived hNPCs into astrocytes is a time-consuming process. The interleukin-6 family of cytokines, including leukemia inhibitory factor (LIF), are well known to efficiently induce astrocytic differentiation of late-gestational (lg)NPCs by activating the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway (Bonni et al., 1997; Nakashima et al., 1999; Weible and Chan-Ling, 2007). However, these cytokines are incapable of inducing astrocytic differentiation of mid-gestational (mg)NPCs because astrocytic genes, such as Glial fibrillary acidic protein (Gfap), are silenced by DNA methylation (Fan et al., 2005; Takizawa et al., 2001). Thus, mgNPCs have a strong tendency to differentiate into neurons rather than astrocytes. mgNPCs prepared from embryonic day 11 (E11) mouse telencephalon can be induced with moderate efficiency to differentiate into astrocytes after culturing for 4 days (nominally corresponding to E15), while astrocytic differentiation is effectively induced in lgNPCs prepared directly from E15 mouse telencephalon. We have previously shown that this weaker acquisition of astrocytic differentiation potential by mgNPCs cultured in dishes is due to the high oxygen level compared with that in vivo (Mutoh et al., 2012). The atmosphere contains 21% O2 (160 mm Hg), whereas interstitial oxygen concentration ranges from 1% to 5% (7–40 mm Hg) in mammalian tissues including the embryonic brain (Mohyeldin et al., 2010; Simon and Keith, 2008). Thus, 21% O2 (atmospheric) is actually physiologically abnormal in vivo; however, because cell cultures are generally conducted in 21% O2, and it is common to define atmospheric O2 concentration as normoxia, we refer to 21% O2 as normoxia in this study. Notably, when we cultured E11 mgNPCs for 4 days under hypoxia (2% O2), the cells differentiated efficiently into astrocytes, to a level comparable with that of E15 lgNPCs. We also revealed that demethylation of Gfap in mgNPCs is enhanced in hypoxic culture compared with that in normoxia (21%) (Mutoh et al., 2012). Given these findings, we hypothesized that the inefficient astrocytic differentiation of hPSC-derived hNPCs is due to a retarded or suspended transition from mid- to late-gestational stages of NPC development, so that hypoxia should confer astrocytic differentiation potential on hNPCs as we observed in mouse mgNPCs. We therefore cultured hPSC-derived hNPCs under hypoxic conditions and found that this is indeed the case. The hNPCs differentiated rapidly (within 4 weeks) into astrocytes, and this was inversely correlated with the methylation status of the GFAP promoter. We also show that conferral of astrocytic differentiation potential on the hNPCs is achieved by a collaboration between hypoxia-inducible factor 1α (HIF1α) and Notch signaling. Furthermore, we show that astrocytes derived from RTT-hiPSCs using our method impair aspects of neuronal development such as neurite outgrowth and synaptic formation, indicating that our protocol will accelerate investigations of the functions of neurological disorder-relevant astrocytes in vitro.