Here using hNPCs derived from RTT patient iPSCs as

Here, using hNPCs derived from RTT patient iPSCs as an example, we have shown that our method of hypoxia-mediated rapid astrocytic differentiation of hNPCs can be applied to functional analysis of patient-derived astrocytes. We revealed that MECP2 MT astrocytes diminish the soma size, neurite outgrowth, and synaptic formation of neurons. As well as in RTT, astrocyte dysfunction has also been implicated in many other neurological diseases such as Alexander disease, which is characterized by genetic mutations in GFAP (Hagemann et al., 2013). Several types of transgenic mice with mutations in GFAP have been established. However, not all of the human pathological phenotypes of astrocytes can be reproduced in mice, necessitating research using human astrocytes, and this holds true for other astrocyte-mediated neurological disorders. Our hypoxic culture greatly accelerates the production of patient-relevant astrocytes, bringing us one step closer to better understanding the mechanisms underlying these disorders and also paving the way for development of strategies for their treatment.
Experimental Procedures
Author Contributions
Acknowledgments We appreciate the technical assistance from The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences. We have greatly benefited from discussions with J. Kohyama, S. Katada, and T. Imamura. We thank A. Smith for providing the human NPCs (CB660, AF22-24), and I. Smith for editing the manuscript. This work was supported by grants from the Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (AMED-CREST), JSPS KAKENHI (15K14452), MEXT KAKENHI (16H06527), and the Uehara Memorial Foundation to K.N.
Introduction Astrocytes are a major component of the human CNS. The versatile and heterogeneous nature of astrocytes allows them to perform multiple essential functions during brain development and later in neuronal soluble guanylate cyclase and synaptic transmission. Astrocytes are essential for the establishment and plasticity of neural circuits since they participate in the formation of synapses, in the phagocytic removal of unwanted synapses, and in neurotransmitter recycling (Khakh and Sofroniew, 2015). Astrocytes are located close to blood vessels and participate in the formation of the blood-brain barrier and in the regulation of blood flow. They regulate extracellular ion concentrations and provide energy and substrates to neurons (Sofroniew and Vinters, 2010). In addition to these sustaining functions, astrocytes participate in the response to injury and disease by becoming reactive. Astrocyte reactivity is a graded and complex response that includes changes in morphology, gene expression, and inflammation (Anderson et al., 2014; Colombo and Farina, 2016; Pekny and Pekna, 2014; Sofroniew, 2009, 2015). Morphological changes range from hypertrophy and polarization toward the site of injury to the formation of glial scars. Genomic studies using two injury mouse models, ischemic stroke and neuroinflammation, revealed extensive modifications in the transcriptome profiles of reactive astrocytes with a common gene component and gene changes specific to each stimulus (Zamanian et al., 2012). As a result, the role of astrocyte dysfunction and neuroinflammation in multiple CNS pathologies is attracting attention (Pekny et al., 2015). During embryonic development, radial glial cells generate progenitors that differentiate into neurons in the early embryonic phase and into glial cells in the late embryonic and postnatal periods. This switch from neurogenesis to gliogenesis involves epigenetic changes and Notch signaling (Fan, 2005; Morrison et al., 2000; Takizawa et al., 2001). Notch activates the JAK/STAT3 (Janus kinase/signal transducer and activator of transcription 3) pathway, which promotes astrogliogenesis (Kamakura et al., 2004). In mice, astrocyte progenitors migrate radially from the germinal ventricular zone and occupy a stable territory throughout the life of the animal (Tsai et al., 2012). Thus, morphological and functional astrocyte diversity is determined during development by the regional patterning of the precursors (Hochstim et al., 2008; Tsai et al., 2012). Data obtained from in vitro culture astrocytes purified from human and rat brains showed that human astrocytes occupy an extended territory and display a superior total arborization length (Zhang et al., 2016). Indeed, significant differences exist between human and rodent astrocytes (Han et al., 2013; Oberheim et al., 2006, 2009; Zhang et al., 2016) that may be relevant for functional studies of astrocytes in the context of human neurological disorders.