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• W2624200931 abstract "•Hypoxia-induced DNA demethylation allows hPSC-NPCs to differentiate into astrocytes•HIF1α and Notch signal activation play a critical role in this epigenetic change•RTT patient-derived astrocytes induced under hypoxia impair neuronal development Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders. Human neural precursor cells (hNPCs) derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders. The mammalian CNS is composed mainly of three neural cell types, neurons, astrocytes, and oligodendrocytes, all of which are generated from common multipotent neural precursor cells (NPCs) (Namihira and Nakashima, 2013Namihira M. Nakashima K. Mechanisms of astrocytogenesis in the mammalian brain.Curr. Opin. Neurobiol. 2013; 23: 921-927Crossref PubMed Scopus (65) Google Scholar, Svendsen et al., 1998Svendsen C.N. ter Borg M.G. Armstrong R.J. Armstrong R.J. Rosser A.E. Chandran S. Ostenfeld T. Caldwell M.A. A new method for the rapid and long term growth of human neural precursor cells.J. Neurosci. Methods. 1998; 85: 141-152Crossref PubMed Scopus (494) Google Scholar). With recent advances in stem cell culture techniques, NPCs derived from human pluripotent stem cells (hPSCs), and embryonic and induced pluripotent stem cells (hESCs and hiPSCs), have been shown to recapitulate neural development to some extent in vitro (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15051) Google Scholar, Thomson et al., 1998Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12344) Google Scholar, Yu et al., 2007Yu J. Vodyanik M.A. Smug-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. Nie J. Jonsdottir G.A. Ruotti V. Stewart R. et al.Induced pluripotent stem cell lines derived from human somatic cells.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (8184) Google Scholar) and to serve as a model for various neurological disorders (Israel et al., 2012Israel M.A. Yuan S.H. Bardy C. Reyna S.M. Mu Y. Herrera C. Hefferan M.P. Van Gorp S. Nazor K.L. Boscolo F.S. et al.Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells.Nature. 2012; 482: 216-220Crossref PubMed Scopus (882) Google Scholar, Marchetto et al., 2011Marchetto M.C. Brennand K.J. Boyer L.F. Gage F.H. Induced pluripotent stem cells (iPSCs) and neurological disease modeling: progress and promises.Hum. Mol. Genet. 2011; 20: R109-R115Crossref PubMed Scopus (142) Google Scholar, Park et al., 2008Park I.H. Arora N. Huo H. Maherali N. Ahfeldt T. Shimamura A. Lensch M.W. Cowan C. Hochedlinger K. Daley G.Q. Disease-specific induced pluripotent stem cells.Cell. 2008; 134: 877-886Abstract Full Text Full Text PDF PubMed Scopus (1810) Google Scholar, Sanchez-Danes et al., 2012Sanchez-Danes A. Richaud-Patin Y. Carballo-Carbajal I. Jimenez-Delgado S. Caig C. Mora S. Di Guglielmo C. Ezquerra M. Patel B. Giralt A. et al.Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease.EMBO Mol. Med. 2012; 4: 380-395Crossref PubMed Scopus (438) Google Scholar). However, although human NPCs (hNPCs) derived from hPSCs differentiate efficiently into neurons, an extremely low fraction of them generate astrocytes over a period of 4 weeks after the induction of differentiation (Hu et al., 2010Hu B.Y. Weick J.P. Yu J. Ma L.X. Zhang X.Q. Thomson J.A. Zhang S.C. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.Proc. Natl. Acad. Sci. USA. 2010; 107: 4335-4340Crossref PubMed Scopus (811) Google Scholar). Recent studies have shown that hNPCs require prolonged culture (typically around 100–200 days) under sphere-forming conditions to efficiently differentiate into astrocytes (Edri et al., 2015Edri R. Yaffe Y. Ziller M.J. Mutukula N. Volkman R. David E. Jacob-Hirsch J. Malcov H. Levy C. Rechavi G. et al.Analysing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors.Nat. Commun. 2015; 6: 6500Crossref PubMed Scopus (51) Google Scholar, Kondo et al., 2013Kondo T. Asai M. Tsukita K. Kutoku Y. Ohsawa Y. Sunada Y. Imamura K. Egawa N. Yahata N. Okita K. et al.Modeling Alzheimer's disease with iPSCs reveals stress phenotypes associated with intracellular Abeta and differential drug responsiveness.Cell Stem Cell. 2013; 12: 487-496Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, Krencik et al., 2011Krencik R. Weick J.P. Liu Y. Zhang Z.J. Zhang S.C. Specification of transplantable astroglial subtypes from human pluripotent stem cells.Nat. Biotechnol. 2011; 29: 528-534Crossref PubMed Scopus (297) Google Scholar, Williams et al., 2014Williams E.C. Zhong X. Mohamed A. Li R. Liu Y. Dong Q. Ananiev G.E. Mok J.C. Lin B.R. Lu J. et al.Mutant astrocytes differentiated from Rett syndrome patients-specific iPSCs have adverse effects on wild-type neurons.Hum. Mol. Genet. 2014; 23: 2968-2980Crossref PubMed Scopus (136) Google Scholar), thus retarding human astrocyte functional research that is relevant to neurological diseases. 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, 2009Allen N.J. Barres B.A. Glia - more than just brain glue.Nature. 2009; 457: 675-677Crossref PubMed Scopus (560) Google Scholar). Because these roles of astrocytes are very important for brain function, astrocytes are indispensable components in CNS integrity (Allen et al., 2012Allen N.J. Bennett M.L. Foo L.C. Wang G.X. Chakraborty C. Smith S.J. Barres B.A. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors.Nature. 2012; 486: 410-414Crossref PubMed Scopus (14) Google Scholar, Christopherson et al., 2005Christopherson K.S. Ullian E.M. Stokes C.C. Mullowney C.E. Hell J.W. Agah A. Lawler J. Mosher D.F. Bornstein P. Barres B.A. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.Cell. 2005; 120: 421-433Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, Hamilton and Attwell, 2010Hamilton N.B. Attwell D. Do astrocytes really exocytose neurotransmitters?.Nat. Rev. Neurosci. 2010; 11: 227-238Crossref PubMed Scopus (524) Google Scholar, Haydon and Nedergaard, 2015Haydon P.G. Nedergaard M. How do astrocytes participate in neural plasticity?.Cold Spring Harb. Perspect. Biol. 2015; 7: a020438Crossref Scopus (107) Google Scholar, Kucukdereli et al., 2011Kucukdereli H. Allen N.J. Lee A.T. Feng A. Ozlu M.I. Conatser L.M. Chakraborty C. Workman G. Weaver M. Sage E.H. et al.Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.Proc. Natl. Acad. Sci. USA. 2011; 108: E440-E449Crossref PubMed Scopus (351) Google Scholar, Molofsky et al., 2012Molofsky A.V. Krencik R. Ullian E.M. Tsai H.H. Deneen B. Richardson W.D. Barres B.A. Rowitch D.H. Astrocytes and disease: a neurodevelopmental perspective.Genes Dev. 2012; 26: 891-907Crossref PubMed Google Scholar, Ullian et al., 2001Ullian E.M. Saperstein SK Christopherson K.S. Christopherson K.S. Barres B.A. Control of synapse number by glia.Science. 2001; 291: 657-661Crossref PubMed Scopus (1060) Google Scholar, Zhang et al., 2016Zhang Y. Sloan S.A. Clarke L.E. Caneda C. Plaza C.A. Blumenthal P.D. Vogel H. Steinberg G.K. Edwards M.S. Li G. et al.Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.Neuron. 2016; 89: 37-53Abstract Full Text Full Text PDF PubMed Scopus (1131) Google Scholar). 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., 1999Amir R.E. Van den Veyver I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3815) Google Scholar, Bienvenu and Chelly, 2006Bienvenu T. Chelly J. Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized.Nat. Rev. Genet. 2006; 7: 415-426Crossref PubMed Scopus (231) Google Scholar, Tsujimura et al., 2015Tsujimura K. Irie K. Nakashima H. Egashira Y. Fukao Y. Fujiwara M. Itoh M. Uesaka M. Imamura T. Nakahata Y. et al.miR-199a Links MeCP2 with mTOR signaling and its dysregulation leads to Rett syndrome phenotypes.Cell Rep. 2015; 12: 1887-1901Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 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., 2014Williams E.C. Zhong X. Mohamed A. Li R. Liu Y. Dong Q. Ananiev G.E. Mok J.C. Lin B.R. Lu J. et al.Mutant astrocytes differentiated from Rett syndrome patients-specific iPSCs have adverse effects on wild-type neurons.Hum. Mol. Genet. 2014; 23: 2968-2980Crossref PubMed Scopus (136) Google Scholar). 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., 1997Bonni A. Sun Y. Nadal-Vicens M. Bhatt A. Frank D.A. Rozovsky I. Stahl N. Yancopoulos G.D. Greenberg M.E. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway.Science. 1997; 278: 477-483Crossref PubMed Scopus (859) Google Scholar, Nakashima et al., 1999Nakashima K. Yanagisawa M. Arakawa H. Kimura N. Hisatsune T. Kawabata M. Miyazono K. Taga T. Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300.Science. 1999; 284: 479-482Crossref PubMed Scopus (726) Google Scholar, Weible and Chan-Ling, 2007Weible 2nd, M.W. Chan-Ling T. Phenotypic characterization of neural stem cells from human fetal spinal cord: synergistic effect of LIF and BMP4 to generate astrocytes.Glia. 2007; 55: 1156-1168Crossref PubMed Scopus (44) Google Scholar). 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., 2005Fan G. Martinowich K. Chin M.H. He F. Fouse S.D. Hutnick L. Hattori D. Ge W. Shen Y. Wu H. et al.DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling.Development. 2005; 132: 3345-3356Crossref PubMed Scopus (338) Google Scholar, Takizawa et al., 2001Takizawa T. Nakashima K. Namihira M. Ochiai W. Uemura A. Yanagisawa M. Fujita N. Nakao M. Taga T. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain.Dev. Cell. 2001; 1: 749-758Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). 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., 2012Mutoh T. Sanosaka T. Ito K. Nakashima K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1alpha-notch signal interaction in the developing brain.Stem Cells. 2012; 30: 561-569Crossref PubMed Scopus (40) Google Scholar). 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., 2010Mohyeldin A. Garzon-Muvdi T. Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche.Cell Stem Cell. 2010; 7: 150-161Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, Simon and Keith, 2008Simon M.C. Keith B. The role of oxygen availability in embryonic development and stem cell function.Nat. Rev. Mol. Cell Biol. 2008; 9: 285-296Crossref PubMed Scopus (724) Google Scholar). 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., 2012Mutoh T. Sanosaka T. Ito K. Nakashima K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1alpha-notch signal interaction in the developing brain.Stem Cells. 2012; 30: 561-569Crossref PubMed Scopus (40) Google Scholar). 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. We first re-examined the differentiation tendencies of four hNPC lines established from hiPSCs (AF22 and AF24), hESCs (AF23) (Falk et al., 2012Falk A. Koch P. Kesavan J. Takashima Y. Ladewig J. Alexander M. Wiskow O. Tailor J. Trotter M. Pollard S. et al.Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.PLoS One. 2012; 7: e29597Crossref PubMed Scopus (206) Google Scholar), and human fetal brain (CB660) (Sun et al., 2008Sun Y. Pollard S. Conti L. Toselli M. Biella G. Parkin G. Willatt L. Falk A. Cattaneo E. Smith A. Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture.Mol. Cell Neurosci. 2008; 38: 245-258Crossref PubMed Scopus (177) Google Scholar) by immunocytochemistry with antibodies against the neuron and astrocyte markers tubulin β 3 class III (TUBB3) and GFAP, respectively. Whereas fetal brain-derived CB660 could efficiently differentiate into both TUBB3-positive neurons and GFAP-positive astrocytes after a 4-week differentiation period, the astrocyte population was extremely low in AF22 and AF23 (Figures 1A and 1B ). Moreover, only a small fraction of AF22 and AF23 differentiated into astrocytes even when stimulated with LIF, which activated STAT3 in these cells (Figures S1A and S1B). Interestingly, AF24 (hNPCs established from CB660-derived hiPSCs) also barely differentiated into astrocytes even in the presence of LIF (Figures 1A, 1B, S1A, and S1B). These results suggest that the capacity to differentiate into astrocytes is restricted in hNPCs if they are derived from hPSCs, regardless of the properties of the original cells. Since it has been shown that mouse mgNPCs have a limited astrocytic differentiation potential due to the hyper-methylation status in astrocytic gene promoters (Namihira et al., 2009Namihira M. Kohyama J. Semi K. Sanosaka T. Deneen B. Taga T. Nakashima K. Committed neuronal precursors confer astrocytic potential on residual neural precursor cells.Dev. Cell. 2009; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Takizawa et al., 2001Takizawa T. Nakashima K. Namihira M. Ochiai W. Uemura A. Yanagisawa M. Fujita N. Nakao M. Taga T. DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain.Dev. Cell. 2001; 1: 749-758Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar), we next examined the methylation status of the GFAP promoter as a representative gene promoter in these cells (Figure 1C). Bisulfite sequence analysis revealed a high-methylation status for the GFAP promoter in AF22, 23, and 24 but not in CB660 (Figures 1D and 1E). These methylation statuses were inversely correlated with the astrocytic differentiation ability of each cell line (Figures 1B and 1E). hNPCs with low astrocytic differentiation potential (AF22, 23, and 24) were all established from hPSCs, and had never been exposed to hypoxia during or after their establishment (Falk et al., 2012Falk A. Koch P. Kesavan J. Takashima Y. Ladewig J. Alexander M. Wiskow O. Tailor J. Trotter M. Pollard S. et al.Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.PLoS One. 2012; 7: e29597Crossref PubMed Scopus (206) Google Scholar). In contrast, CB660 hNPCs were prepared directly from a human fetal brain around gestational week 8 (Sun et al., 2008Sun Y. Pollard S. Conti L. Toselli M. Biella G. Parkin G. Willatt L. Falk A. Cattaneo E. Smith A. Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture.Mol. Cell Neurosci. 2008; 38: 245-258Crossref PubMed Scopus (177) Google Scholar), indicating that they had been under hypoxia until at least this time because embryonic tissues including brain are in hypoxic conditions (Mohyeldin et al., 2010Mohyeldin A. Garzon-Muvdi T. Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche.Cell Stem Cell. 2010; 7: 150-161Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, Simon and Keith, 2008Simon M.C. Keith B. The role of oxygen availability in embryonic development and stem cell function.Nat. Rev. Mol. Cell Biol. 2008; 9: 285-296Crossref PubMed Scopus (724) Google Scholar). Since we have previously shown that hypoxia confers astrocytic differentiation potential on mouse mgNPCs (Mutoh et al., 2012Mutoh T. Sanosaka T. Ito K. Nakashima K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1alpha-notch signal interaction in the developing brain.Stem Cells. 2012; 30: 561-569Crossref PubMed Scopus (40) Google Scholar), we speculated that the difference in astrocytic differentiation between these hNPCs is attributable to their exposure to hypoxia. Therefore, we decided to test whether hypoxic exposure enhances astrocytic differentiation of AF22-24. Since HIF1α, an oxygen sensor, has been shown to be crucial for mouse mgNPCs to acquire astrocytic differentiation ability (Mutoh et al., 2012Mutoh T. Sanosaka T. Ito K. Nakashima K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1alpha-notch signal interaction in the developing brain.Stem Cells. 2012; 30: 561-569Crossref PubMed Scopus (40) Google Scholar), we first examined HIF1α expression together with that of HIF2α in our hNPC culture (Figures 2C, 2D, and 2E ). Once induced, HIF1α expression was sustained until 28 days (Figures 2D and 2E). qRT-PCR data indicated that HIF1α expression peaked at 21 days after the onset of low-oxygen culture (Figure 2E). On the contrary, HIF2α and HIF2α expression were induced transiently, but then returned to basal levels (Figures 2D and 2E). These results are inconsistent with those of two previous studies (Forristal et al., 2009Forristal C.E. Wright K.L. Hanley N.A. Oreffo R.O.C. Houghton F.D. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.Reproduction. 2009; 139: 85-97Crossref Scopus (293) Google Scholar, Stacpoole et al., 2011Stacpoole S.R. Bilican B. Webber D.J. Luzhynskaya A. He X.L. Compston A. Karadottir R. Franklin R.J. Chandran S. Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen.Nat. Protoc. 2011; 6: 1229-1240Crossref PubMed Scopus (47) Google Scholar). However, this may be due to differences in cell types and culture conditions: Forristal et al., 2009Forristal C.E. Wright K.L. Hanley N.A. Oreffo R.O.C. Houghton F.D. Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.Reproduction. 2009; 139: 85-97Crossref Scopus (293) Google Scholar performed experiments using hESCs in the maintenance condition, and Stacpoole et al., 2011Stacpoole S.R. Bilican B. Webber D.J. Luzhynskaya A. He X.L. Compston A. Karadottir R. Franklin R.J. Chandran S. Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen.Nat. Protoc. 2011; 6: 1229-1240Crossref PubMed Scopus (47) Google Scholar did so using hESC-derived NPCs, which were maintained in aggregation form, in spinal motor neuron- and midbrain dopaminergic neuron-inducing conditions, whereas we maintained and differentiated hPCS-derived hNPCs in monolayer. Furthermore, cultures in both of those studies but not in ours contained basic fibroblast growth factor (bFGF), which has a variety of effects on cell behavior. We therefore presume that these discrepancies explain the difference in HIF1α and HIF2α expression between the previous experiments and ours, although we cannot completely exclude other possibilities. When AF22–24 were cultured under hypoxia, we observed a dramatic induction of GFAP- (Figures 2A and 2B for AF22-24) and aquaporin-4 (AQP4)-positive astrocytes (Figure S2A for AF22) after 28 days of differentiation. In addition to GFAP and AQP4 mRNA expression, we also confirmed the upregulation of expression of another astrocyte-specific gene, aldehyde dehydrogenase 1 family member L1 (ALDH1L1), in AF22 under hypoxia (Figure S2B). Consistent with this observation, DNA methylation in the GFAP promoter, including the STAT3 binding site, was greatly reduced in AF22 under hypoxia compared with normoxia after 28 days of differentiation (Figures 2C and 2F). Moreover, mRNA expression of nuclear factor IA (NFIA) and hairy and enhancer of split 5 (HES5), downstream targets of Notch signaling (Mutoh et al., 2012Mutoh T. Sanosaka T. Ito K. Nakashima K. Oxygen levels epigenetically regulate fate switching of neural precursor cells via hypoxia-inducible factor 1alpha-notch signal interaction in the developing brain.Stem Cells. 2012; 30: 561-569Crossref PubMed Scopus (40) Google Scholar), was upregulated under the hypoxic condition (Figure S2C). Since Notch signaling is known to be important for mouse mgNPCs to acquire astrocytic differentiation potential by reducing DNA methylation levels in the promoters of astrocyte-specific genes (Namihira et al., 2009Namihira M. Kohyama J. Semi K. Sanosaka T. Deneen B. Taga T. Nakashima K. Committed neuronal precursors confer astrocytic potential on residual neural precursor cells.Dev. Cell. 2009; 16: 245-255Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), it seemed likely that Notch signaling would also contribute to demethylation of the GFAP promoter in the hNPCs under hypoxia. We could detect the expression of at least one astrocyte-inducing factor, LIF, in AF22 in the differentiated condition (Figure S2D), suggesting that these cells are in an environment where they can express GFAP once the gene promoter has become demethylated. Since we have focused on astrocytic differentiation of hPSC-derived NPCs in this study, we did not extensively investigate their differentiation into oligodendrocytes, which are generated at late-gestational stages in addition to astrocytes. However, although we must await detailed investigations in the future, we did find that the expression of oligodendrocyte-related genes, such as platelet-derived growth factor receptor β (PDGFRβ), chondroitin sulfate proteoglycan 4 (CSPG4, also known as NG2), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNP), and proteolipid protein 1 (PLP1), was increased in AF22 under hypoxia compared with normoxia 28 days after the initiation of differentiation (Figure S2E), implying that our low-oxygen culture can likewise be applied to monitoring the induction of oligodendrocytic differentiation of hPSC-derived NPCs. Accumulating evidence suggests that aggregation (or sphere) culture of hNPCs can improve the generation of astrocytes from the cells (Imamura et al., 2015Imamura Y. Mukohara T. Shimono Y. Funakoshi Y. Chayahara N. Toyoda M. Kiyota N. Takao S. Kono S. Nakatsura T. et al.Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer.Oncol. Rep. 2015; 33: 1837-1843Crossref PubMed Scopus (452) Google Scholar, Lancaster et al., 2013Lancaster M.A. Renner M. Martin C.A. Wenzel D. Bicknell L.S. Hurles M.E. Homfray T. Penninger J.M. Jackson A.P. Knoblich J.A. Cerebral organoids model human brain development and microcephaly.Nature. 2013; 501: 373-379Crossref PubMed Scopus (2805) Google Scholar, Taguchi et al., 2014Taguchi A. Kaku Y. Ohmori T. Sharmin S. Ogawa M. Sasaki H. Nishinakamura R. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells.Cell Stem Cell. 2014; 14: 53-67Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, Volkner et al., 2016Volkner M. Zschatzsch M. Rostovskaya M. Overall R.W. Busskamp V. Anastassiadis K. Karl M.O. Retinal organoids from pluripotent stem cells efficiently recapitulate retinogenesis.Stem Cell Rep. 2016; 6: 525-538Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Taking this and our findings above into consideration, we suspected that the reason why aggregation culture can enhance astrocytic differentiation of NPCs is that the cells inside the aggregation are in a hypoxic microenvironment even though the culture itself is conducted at atmospheric oxygen level. To test this, we cultured AF22, which had never or scarcely been cultured in aggregation, for 15 days as spheres under either normoxic (21% O2) or hyperoxic (30% O2) in undifferentiated conditions, and then induced their differentiation for 28 days in monolayer culture (Figure 3A). We assessed oxygen levels in the aggregates using a chemical reagent, pimonidazole. Pimonidazole is reductively activated in hypoxic cells and forms stable" @default.
• W2624200931 created "2017-06-15" @default.
• W2624200931 creator A5011156831 @default.
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• W2624200931 date "2017-06-01" @default.
• W2624200931 modified "2023-10-01" @default.
• W2624200931 title "Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells" @default.
• W2624200931 cites W1559742681 @default.
• W2624200931 cites W1605270587 @default.
• W2624200931 cites W1964137734 @default.
• W2624200931 cites W1965425054 @default.
• W2624200931 cites W1972782503 @default.
• W2624200931 cites W1978127185 @default.
• W2624200931 cites W1980324695 @default.
• W2624200931 cites W1983149006 @default.
• W2624200931 cites W1993852989 @default.
• W2624200931 cites W2012935976 @default.
• W2624200931 cites W2016736810 @default.
• W2624200931 cites W2018286352 @default.
• W2624200931 cites W2019974998 @default.
• W2624200931 cites W2026147918 @default.
• W2624200931 cites W2031611126 @default.
• W2624200931 cites W2037988109 @default.
• W2624200931 cites W2039730754 @default.
• W2624200931 cites W2051531582 @default.
• W2624200931 cites W2052272562 @default.
• W2624200931 cites W2055835375 @default.
• W2624200931 cites W2067196568 @default.
• W2624200931 cites W2068407791 @default.
• W2624200931 cites W2068684244 @default.
• W2624200931 cites W2069611371 @default.
• W2624200931 cites W2084037323 @default.
• W2624200931 cites W2086943348 @default.
• W2624200931 cites W2088714934 @default.
• W2624200931 cites W2091898207 @default.
• W2624200931 cites W2098622411 @default.
• W2624200931 cites W2104551938 @default.
• W2624200931 cites W2116228354 @default.
• W2624200931 cites W2117058176 @default.
• W2624200931 cites W2118159405 @default.
• W2624200931 cites W2123292451 @default.
• W2624200931 cites W2126823476 @default.
• W2624200931 cites W2130419298 @default.
• W2624200931 cites W2130525167 @default.
• W2624200931 cites W2138574694 @default.
• W2624200931 cites W2138977668 @default.
• W2624200931 cites W2141351251 @default.
• W2624200931 cites W2142046859 @default.
• W2624200931 cites W2143008483 @default.
• W2624200931 cites W2144750363 @default.
• W2624200931 cites W2146890924 @default.
• W2624200931 cites W2155228504 @default.
• W2624200931 cites W2155734183 @default.
• W2624200931 cites W2156035345 @default.
• W2624200931 cites W2157645703 @default.
• W2624200931 cites W2161071327 @default.
• W2624200931 cites W2191292005 @default.
• W2624200931 cites W2319627288 @default.
• W2624200931 cites W2948665150 @default.
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