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• W2009867508 abstract "•Spinal cord astrocytes can be generated from human ESCs/iPSCs using early neuralization•ESC/hiPSC-derived astrocytes are immature, but FGF1 and FGF2 induce rapid maturation•TNFα and IL-1β selectively induce a reactive phenotype in hESC/iPSC-derived astrocytes•Human astrocytes provide a model for studying glial biology and modeling disease Differentiation of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast growth factor 1 (FGF1) or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis factor alpha and interleukin-1β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro. Differentiation of astrocytes from human stem cells has significant potential for analysis of their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic or induced pluripotent stem cells with high efficiency. Remarkably, short exposure to fibroblast growth factor 1 (FGF1) or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged by both marker expression and functional analysis. In contrast, tumor necrosis factor alpha and interleukin-1β, but not FGFs, induced multiple elements of a reactive inflammatory phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro. IntroductionAstrocytes play multiple roles in the central nervous system (CNS). Many of these are critical to normal function in the healthy adult, where astrocytes act as support cells for neurons, regulating cerebral blood flow, energy reserves, and neurogenesis (Abbott, 1988Abbott N.J. Developmental neurobiology. The milieu is the message.Nature. 1988; 332: 490-491Crossref PubMed Scopus (5) Google Scholar, Allaman et al., 2011Allaman I. Bélanger M. Magistretti P.J. Astrocyte-neuron metabolic relationships: for better and for worse.Trends Neurosci. 2011; 34: 76-87Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, Lie et al., 2005Lie D.C. Colamarino S.A. Song H.J. Désiré L. Mira H. Consiglio A. Lein E.S. Jessberger S. Lansford H. Dearie A.R. Gage F.H. Wnt signalling regulates adult hippocampal neurogenesis.Nature. 2005; 437: 1370-1375Crossref PubMed Scopus (1191) Google Scholar, Okamoto et al., 2011Okamoto M. Inoue K. Iwamura H. Terashima K. Soya H. Asashima M. Kuwabara T. Reduction in paracrine Wnt3 factors during aging causes impaired adult neurogenesis.FASEB J. 2011; 25: 3570-3582Crossref PubMed Scopus (105) Google Scholar, Takano et al., 2006Takano T. Tian G.F. Peng W. Lou N. Libionka W. Han X. Nedergaard M. Astrocyte-mediated control of cerebral blood flow.Nat. Neurosci. 2006; 9: 260-267Crossref PubMed Scopus (850) Google Scholar). Moreover, their involvement in synaptic transmission and plasticity has been the object of intensive study (Bernardinelli et al., 2011Bernardinelli Y. Salmon C. Jones E.V. Farmer W.T. Stellwagen D. Murai K.K. Astrocytes display complex and localized calcium responses to single-neuron stimulation in the hippocampus.J. Neurosci. 2011; 31: 8905-8919Crossref PubMed Scopus (48) Google Scholar, Panatier et al., 2011Panatier A. Vallée J. Haber M. Murai K.K. Lacaille J.C. Robitaille R. Astrocytes are endogenous regulators of basal transmission at central synapses.Cell. 2011; 146: 785-798Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Cell culture models used to study normal astrocyte function should therefore reflect this mature, quiescent phenotype.However, astrocyte phenotypes are extremely dynamic. Reactive phenotypes can be induced by proinflammatory factors, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, and interferon gamma, in a wide variety of traumatic and pathological contexts (Gwak et al., 2012Gwak Y.S. Kang J. Unabia G.C. Hulsebosch C.E. Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats.Exp. Neurol. 2012; 234: 362-372Crossref PubMed Scopus (201) Google Scholar). Indeed, multiple classes of reactive astrocytes can be identified, depending on the nature of activating stimulus and the time elapsed postactivation (Zamanian et al., 2012Zamanian J.L. Xu L. Foo L.C. Nouri N. Zhou L. Giffard R.G. Barres B.A. Genomic analysis of reactive astrogliosis.J. Neurosci. 2012; 32: 6391-6410Crossref PubMed Scopus (1299) Google Scholar). Astrocyte activation leads to production of a wide array of mediators, including chemokines, inflammatory cytokines, and growth factors (Allaman et al., 2011Allaman I. Bélanger M. Magistretti P.J. Astrocyte-neuron metabolic relationships: for better and for worse.Trends Neurosci. 2011; 34: 76-87Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), as well as transcriptional changes of genes associated with cell-to-cell communication (e.g., connexins), cytoskeletal structure (e.g., glial fibrillary acidic protein [GFAP]), and many others (for review, see Sofroniew, 2009Sofroniew M.V. Molecular dissection of reactive astrogliosis and glial scar formation.Trends Neurosci. 2009; 32: 638-647Abstract Full Text Full Text PDF PubMed Scopus (1778) Google Scholar). It is therefore essential to precisely model the reactive state for in vitro studies of human disease.Human astrocytes have been cultured from fetal or adult postmortem CNS using expansion of neural precursors (Caldwell et al., 2001Caldwell M.A. He X. Wilkie N. Pollack S. Marshall G. Wafford K.A. Svendsen C.N. Growth factors regulate the survival and fate of cells derived from human neurospheres.Nat. Biotechnol. 2001; 19: 475-479Crossref PubMed Scopus (293) Google Scholar, Haidet-Phillips et al., 2011Haidet-Phillips A.M. Hester M.E. Miranda C.J. Meyer K. Braun L. Frakes A. Song S. Likhite S. Murtha M.J. Foust K.D. et al.Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat. Biotechnol. 2011; 29: 824-828Crossref PubMed Scopus (558) Google Scholar, Lee et al., 1993Lee S.C. Liu W. Dickson D.W. Brosnan C.F. Berman J.W. Cytokine production by human fetal microglia and astrocytes. Differential induction by lipopolysaccharide and IL-1 beta.J. Immunol. 1993; 150: 2659-2667PubMed Google Scholar, Verwer et al., 2007Verwer R.W. Sluiter A.A. Balesar R.A. Baayen J.C. Noske D.P. Dirven C.M. Wouda J. van Dam A.M. Lucassen P.J. Swaab D.F. Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin.Brain. 2007; 130: 3321-3335Crossref PubMed Scopus (88) Google Scholar), but such preparations are rare and intrinsically variable. One of the first protocols to report differentiation of human embryonic stem cells (hESCs) into astrocytes was that of 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 (289) Google Scholar. However, one practical drawback of the method is that it necessitates 6 months of culture to generate a sufficiently pure population (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 (289) Google Scholar, Krencik and Zhang, 2011Krencik R. Zhang S.C. Directed differentiation of functional astroglial subtypes from human pluripotent stem cells.Nat. Protoc. 2011; 6: 1710-1717Crossref PubMed Scopus (165) Google Scholar). Since then, other protocols starting from neural precursor cells have reported generation of astrocytes within 35–80 days (Emdad et al., 2012Emdad L. D’Souza S.L. Kothari H.P. Qadeer Z.A. Germano I.M. Efficient differentiation of human embryonic and induced pluripotent stem cells into functional astrocytes.Stem Cells Dev. 2012; 21: 404-410Crossref PubMed Scopus (108) Google Scholar, Juopperi et al., 2012Juopperi T.A. Kim W.R. Chiang C.H. Yu H. Margolis R.L. Ross C.A. Ming G.L. Song H. Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington’s disease patient cells.Mol. Brain. 2012; 5: 17Crossref PubMed Scopus (176) Google Scholar, Lafaille et al., 2012Lafaille F.G. Pessach I.M. Zhang S.Y. Ciancanelli M.J. Herman M. Abhyankar A. Ying S.W. Keros S. Goldstein P.A. Mostoslavsky G. et al.Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells.Nature. 2012; 491: 769-773Crossref PubMed Scopus (242) Google Scholar, Serio et al., 2013Serio A. Bilican B. Barmada S.J. Ando D.M. Zhao C. Siller R. Burr K. Haghi G. Story D. Nishimura A.L. et al.Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.Proc. Natl. Acad. Sci. USA. 2013; 110: 4697-4702Crossref PubMed Scopus (245) Google Scholar, Shaltouki et al., 2013Shaltouki A. Peng J. Liu Q. Rao M.S. Zeng X. Efficient generation of astrocytes from human pluripotent stem cells in defined conditions.Stem Cells. 2013; 31: 941-952Crossref PubMed Scopus (179) Google Scholar). However, by the criteria discussed below, the astrocytes generated in each case are immature and do not fully model normal astrocyte function.Astrocyte maturation occurs through a complex series of events that remain incompletely understood. There is considerable overlap between expression of different markers, and it is likely that the precise sequence of their appearance varies from one region of the CNS to another. Nevertheless, we have constructed a tentative synthesis of the appearance of known markers during maturation based on spinal cord data, where available (Figure S1). Overall, astrocyte development and maturation encompasses two phases (see Extended Results for full review, abbreviations, and citations). During the first—mainly embryonic—phase, subsets of astrocytes are generated from radial glia and progressively take on their principal functional phenotypes. Subsequently, over the first postnatal weeks in rodents, astrocytes adopt a mature, quiescent morphology and phenotype. Although all potential marker genes have not been studied in parallel in a single brain region, the sequence of appearance of markers during the first, embryonic phase is likely NF1A > GLAST > ALDH1L1 > Cx43 > S100β > CD44 > AldolaseC > GFAP. The NF1A+/GFAP+ cells generated by extant stem cell differentiation protocols (see above) likely correspond to this first, immature stage. In contrast, the second, maturational phase is associated with downregulation of GFAP, GLAST, and ALDH1L1, whereas GFAP expression persists in white matter astrocytes. In parallel, there is continued accumulation of Cx43, followed by Aqp4 and the mature astrocyte glutamate transporter 1 (GLT1). Therefore, mature gray matter astrocytes should not be expected to express high levels of GFAP, so other markers are needed to monitor their maturation and purity.To summarize, astrocytes may adopt either a quiescent state with protoplasmic morphology, characterized by low GFAP and high GLT1, or a fibrous, reactive phenotype characterized by high GFAP and low GLT1. Standard preparations of cultured GFAP+ astrocytes (McCarthy and de Vellis, 1980McCarthy K.D. de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.J. Cell Biol. 1980; 85: 890-902Crossref PubMed Scopus (3372) Google Scholar) reflect only the latter (Zamanian et al., 2012Zamanian J.L. Xu L. Foo L.C. Nouri N. Zhou L. Giffard R.G. Barres B.A. Genomic analysis of reactive astrogliosis.J. Neurosci. 2012; 32: 6391-6410Crossref PubMed Scopus (1299) Google Scholar). Therefore, a robust model of mature, quiescent astrocytes would be a significant step forward for in vitro studies of human neural function as well as disease. This is especially significant given the different effects of immature and mature astrocytes on axonal regeneration (Goldshmit et al., 2012Goldshmit Y. Sztal T.E. Jusuf P.R. Hall T.E. Nguyen-Chi M. Currie P.D. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish.J. Neurosci. 2012; 32: 7477-7492Crossref PubMed Scopus (184) Google Scholar, Tom et al., 2004Tom V.J. Doller C.M. Malouf A.T. Silver J. Astrocyte-associated fibronectin is critical for axonal regeneration in adult white matter.J. Neurosci. 2004; 24: 9282-9290Crossref PubMed Scopus (152) Google Scholar). Here, we report that, using identified signaling factors, mouse or human spinal cord astrocytes generated from either ESCs or human induced pluripotent stem cells (hiPSCs) can be induced to adopt phenotypes that correspond to those of either mature or reactive astrocytes in vivo. Such defined populations of mature human astrocytes will considerably increase the resolution of studies of normal astrocyte function, whereas fully activated preparations open the door to more precise disease modeling based on patient-derived hiPSCs.ResultsAstrocytes Derived from Mouse Embryonic Stem Cells Are ImmatureBecause the development and maturation of astrocytes are better understood in rodents than in human, we first generated astrocytes from mouse embryonic stem cells (mESCs). Our overall aim was to generate astrocytes of spinal cord identity as a tool for modeling motor neuron diseases, such as amyotrophic lateral sclerosis (ALS). We therefore used an induction protocol that specifies ventral spinal progenitors (Wichterle et al., 2002Wichterle H. Lieberam I. Porter J.A. Jessell T.M. Directed differentiation of embryonic stem cells into motor neurons.Cell. 2002; 110: 385-397Abstract Full Text Full Text PDF PubMed Scopus (1390) Google Scholar) followed by neurosphere expansion, which amplifies neural progenitors and generates largely glial cells (Kuegler et al., 2012Kuegler P.B. Baumann B.A. Zimmer B. Keller S. Marx A. Kadereit S. Leist M. GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells.Glia. 2012; 60: 218-228Crossref PubMed Scopus (30) Google Scholar; Figure S1; Experimental Procedures). After 1 week, cultures of dissociated neurospheres contained cells that stained for canonical markers of developing astrocytes: GFAP, Aqp4, S100β, vimentin, and NF1A (Figures 1A and 1B ), and their spinal cord identity was confirmed by coexpression of HoxB4 (Figure 1A). The pluripotency marker Oct4 was undetectable (Figure S2C), and neurons (βIII-tubulin+), microglia (Iba1+), and oligodendrocytes (2′3′-cyclic-nucleotide 3′-phosphodiesterase [CNPase]+ or oligodendrocyte transcription factor 2 [Olig2]+) were rare or absent (Figures 1B and S1D). The mESC-derived cultures therefore contained essentially only astrocytes and glial precursors. However, they were not homogeneous, because many cells were not double stained for any given pair of antigens (Figure 1A) and less than one-third of the cells positive for vimentin and NF1A expressed the markers Aqp4, S100β, or GFAP (Figure 1B). This is comparable to the heterogeneity in cultures of neonatal astrocytes (Imura et al., 2006Imura T. Nakano I. Kornblum H.I. Sofroniew M.V. Phenotypic and functional heterogeneity of GFAP-expressing cells in vitro: differential expression of LeX/CD15 by GFAP-expressing multipotent neural stem cells and non-neurogenic astrocytes.Glia. 2006; 53: 277-293Crossref PubMed Scopus (101) Google Scholar). Our data suggested that the cultures contained developing astrocytes at different stages of maturity.Figure 1Mouse ESC-Derived Spinal Cord Astrocytes Show an Immature PhenotypeShow full caption(A) Differentiated mESC cultures contain glial cells expressing canonical astrocyte markers together with the spinal cord marker HOXB4. Insets show higher magnification of regions indicated with dotted lines. The scale bars represent 75 μm.(B) Percentages of mESC-derived glial cells expressing markers for astroglia (Aqp4, S100β, vimentin, NF1A, and GFAP), oligodendroglia (Olig2 and CNPase), neurons (β-III-tubulin), microglia (Iba1), and spinal cord identity (HOXB4). Mean ± SEM; n = 3–4 independent differentiations.(C) Western blot of mESC-derived astrocytes as compared to astrocytes derived from E12.5 mouse embryo spinal cord. GFAP protein is present in both, but ALDH1L1, GLAST, and GLT1 are only detectable in the mE12.5-derived astrocytes. α-tubulin was used as loading control.See also Figure S2.View Large Image Figure ViewerDownload (PPT)Figure S2Generation and Characterization of Mouse ESC-Derived Glial Cultures, Related to Figure 1Show full caption(A) Protocol for generation of astrocytes from mouse E12.5 spinal cord. Scale bars: 75 μm.(B) Mouse ESC-derived neurospheres contain progenitors expressing Olig2 and NF1A. Scale bar: 75 μm.(C) RT-PCR shows complete downregulation of the pluripotency-associated marker Oct4 and upregulation of glial markers nestin and GFAP over the course of astrocyte differentiation. The loss of Sox10 signal in older cultures confirms the absence of oligodendrocytes.(D) Differentiated mESC cultures are almost devoid of CNPase+/Olig2+ oligodendrocytes, β-III-tubulin+ neurons and Iba1+ microglial cells. Rare examples are illustrated to demonstrate that the antibodies worked correctly. Scale bar: 75 μm.View Large Image Figure ViewerDownload (PPT)To assess their state of maturation, we used western blots to quantify levels of three markers known to be upregulated in astrocytes as they mature: GLAST and ALDH1L1, which appear at midembryogenesis, and GLT1, a postnatal marker (Figure 1C; see Extended Results). Although 4 weeks had elapsed since the mESCs were put into culture, and although GFAP was clearly present, none of these markers could be detected. This was in contrast to astrocytes generated from primary neural precursors of embryonic day 12.5 (E12.5) spinal cords using a neurosphere-based approach (Figure S2A), which expressed robust levels of GLAST, ALDH1L1, and GLT1 (Figure 1C). We therefore sought treatments through which mESC-derived spinal astrocytes could be brought to a similar degree of maturity.FGFs Promote Maturation of mESC-Derived AstrocytesIn vivo, fibroblast growth factors (FGFs) have been proposed to act as differentiation signals for astrocytes (Irmady et al., 2011Irmady K. Zechel S. Unsicker K. Fibroblast growth factor 2 regulates astrocyte differentiation in a region-specific manner in the hindbrain.Glia. 2011; 59: 708-719Crossref PubMed Scopus (20) Google Scholar, Morrow et al., 2001Morrow T. Song M.R. Ghosh A. Sequential specification of neurons and glia by developmentally regulated extracellular factors.Development. 2001; 128: 3585-3594PubMed Google Scholar) and FGF1 is strongly expressed in postnatal spinal neurons (Elde et al., 1991Elde R. Cao Y.H. Cintra A. Brelje T.C. Pelto-Huikko M. Junttila T. Fuxe K. Pettersson R.F. Hökfelt T. Prominent expression of acidic fibroblast growth factor in motor and sensory neurons.Neuron. 1991; 7: 349-364Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Addition of FGF1 to mESC-derived astrocytes from days 28–35 triggered a >4-fold increase in the number of cells expressing GLT1, with an EC50 of 5 ng/ml (Figures 2A and 2B ). Similar results were obtained using FGF2 in place of FGF1 (Figure S3). Western blotting confirmed that FGF increased overall levels of both GLT1 and GLAST (Figures 2C, 2D, and S3C) as well as other maturation markers, such as aldolase C, Cx43, and ALDH1L1 (Figures 2D and S3C). In parallel, GFAP levels were strongly downregulated (Figures 2B, 2D, S3A, and S3C). Thus, mESC-derived cultures treated with FGFs express high levels of glutamate transporters and low GFAP, a phenotype reminiscent of mature quiescent astrocytes.Figure 2FGF1 Promotes Maturation of mESC-Derived AstrocytesShow full caption(A) Effects of increasing dose of FGF1 on the relative abundance of GLT1-expressing astrocytes in mESC-derived cultures (mean ± SEM for two independent experiments, three replicates per condition). One-way ANOVA reveals an effect of treatment above 5 ng/ml. p = 0.013; F(8;9) = 5.01. The asterisk denotes p < 0.05.(B) Compared to an FBS control (left), 50 ng/ml FGF1 (right) triggers a strong increase in GLT1 staining and a nearly complete loss of GFAP immunoreactivity. The scale bar represents 75 μm.(C) Increased GLT1 expression following FGF1 treatment of astrocytes derived from two independent mESC lines (Hb9::GFP and wild-type) revealed by western blot analysis. Results are representative of three independent experiments. WT, wild-type.(D) FGF1 is sufficient to induce appearance of GLAST, CX43, and ALDH1L1 but strongly decreases GFAP expression. Results are representative of three independent experiments.(E) Na+-dependent L-(3H)-glutamate transport using two mESC lines differentiated into astrocytes shows an average 2-fold increase in uptake following treatment with FGF1 (bars show mean ± SEM; n = 4; p = 0.0095; F(3;3) = 1.286. The double asterisks denote p < 0.01).See also Figure S3.View Large Image Figure ViewerDownload (PPT)Figure S3FGF2 Promotes mESC-Derived Astrocyte Maturation as Efficiently as FGF1, Related to Figure 2Show full caption(A) Morphology of GLT1+ and GFAP+ mESC-derived astrocytes treated with FGF2 (50 ng/ml). Cultures are co-stained with the nuclear marker DAPI. Scale bar: 75 μm.(B) FGF2 also upregulates the late marker GLT1 (mean ± s.e.m.; n = 2 independent experiments in triplicate wells). One-way ANOVA reveals an effect of treatment F(8;9) = 6.06, p = 0.0078,. ∗p < 0.05 denotes statistically significant difference between FGF2- and FBS-treated samples (post-hoc test).(C) Like FGF1, FGF2 induces appearance of GLT1, aldolase C and ALDH1L1 but strongly decreases GFAP expression detected on Western blots. Results are representative of 3 independent experiments.(D) Na+-dependent L-(3H)-glutamate uptake by mESC-derived astrocytes is increased following treatment with FGF2.(E) Summary of biochemical and functional changes of mESC-derived astrocyte cultures treated with FBS and FGF1. (-) low or undetectable; (+) high.View Large Image Figure ViewerDownload (PPT)To determine whether this biochemical maturation resulted in functional changes, we measured glutamate uptake, a key role of mature astrocytes (Huang and Bergles, 2004Huang Y.H. Bergles D.E. Glutamate transporters bring competition to the synapse.Curr. Opin. Neurobiol. 2004; 14: 346-352Crossref PubMed Scopus (220) Google Scholar). In mESC astrocytes from two different cell lines grown in fetal bovine serum (FBS) alone, we detected significant levels of glutamate uptake that were Na+-dependent, as expected (Figure 2E; Mitani and Tanaka, 2003Mitani A. Tanaka K. Functional changes of glial glutamate transporter GLT-1 during ischemia: an in vivo study in the hippocampal CA1 of normal mice and mutant mice lacking GLT-1.J. Neurosci. 2003; 23: 7176-7182Crossref PubMed Google Scholar, Rothstein et al., 1996Rothstein J.D. Dykes-Hoberg M. Pardo C.A. Bristol L.A. Jin L. Kuncl R.W. Kanai Y. Hediger M.A. Wang Y. Schielke J.P. Welty D.F. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.Neuron. 1996; 16: 675-686Abstract Full Text Full Text PDF PubMed Scopus (2111) Google Scholar). Exposure to FGF1 for 7 days led to a 2-fold increase in glutamate transport (Figure 2E), reflecting upregulation of the transporters responsible (Figures 2B–2D). Thus, functional astrocytes can be efficiently generated from mESCs and FGF1 promotes their biochemical and functional maturation to a quiescent state.Human Spinal Cord Astrocytes Derived from hESCs and hiPSCs following Early NeuralizationWe then used the findings with mouse ESCs to generate mature human astrocytes. To accelerate astrocyte production, rather than passing through a stage of cycling neural precursors (Shaltouki et al., 2013Shaltouki A. Peng J. Liu Q. Rao M.S. Zeng X. Efficient generation of astrocytes from human pluripotent stem cells in defined conditions.Stem Cells. 2013; 31: 941-952Crossref PubMed Scopus (179) Google Scholar), we turned to early neuralization by dual inhibition of SMAD signaling, which had been shown by Chambers et al., 2009Chambers S.M. Fasano C.A. Papapetrou E.P. Tomishima M. Sadelain M. Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat. Biotechnol. 2009; 27: 275-280Crossref PubMed Scopus (2348) Google Scholar to enhance production of CNS precursors and neurons but whose effect on glial generation had not been studied. To inhibit SMAD signaling, we employed the activin receptor-like kinase 4 (ALK4)/ALK5/ALK7 inhibitor SB431542 together with LDN193189, a potent derivative of dorsomorphin that inhibits transforming growth factor β1/activin receptor-like kinase (Boulting et al., 2011Boulting G.L. Kiskinis E. Croft G.F. Amoroso M.W. Oakley D.H. Wainger B.J. Williams D.J. Kahler D.J. Yamaki M. Davidow L. et al.A functionally characterized test set of human induced pluripotent stem cells.Nat. Biotechnol. 2011; 29: 279-286Crossref PubMed Scopus (367) Google Scholar, Kriks et al., 2011Kriks S. Shim J.W. Piao J. Ganat Y.M. Wakeman D.R. Xie Z. Carrillo-Reid L. Auyeung G. Antonacci C. Buch A. et al.Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease.Nature. 2011; 480: 547-551Crossref PubMed Scopus (1272) Google Scholar, Yu et al., 2008Yu P.B. Deng D.Y. Lai C.S. Hong C.C. Cuny G.D. Bouxsein M.L. Hong D.W. McManus P.M. Katagiri T. Sachidanandan C. et al.BMP type I receptor inhibition reduces heterotopic [corrected] ossification.Nat. Med. 2008; 14: 1363-1369Crossref PubMed Scopus (495) Google Scholar).We used one hESC line (R1; James et al., 2006James D. Noggle S.A. Swigut T. Brivanlou A.H. Contribution of human embryonic stem cells to mouse blastocysts.Dev. Biol. 2006; 295: 90-102Crossref PubMed Scopus (127) Google Scholar) and one hiPSC line (18c; Bock et al., 2011Bock C. Kiskinis E. Verstappen G. Gu H. Boulting G. Smith Z.D. Ziller M. Croft G.F. Amoroso M.W. Oakley D.H. et al.Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.Cell. 2011; 144: 439-452Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, Boulting et al., 2011Boulting G.L. Kiskinis E. Croft G.F. Amoroso M.W. Oakley D.H. Wainger B.J. Williams D.J. Kahler D.J. Yamaki M. Davidow L. et al.A functionally characterized test set of human induced pluripotent stem cells.Nat. Biotechnol. 2011; 29: 279-286Crossref PubMed Scopus (367) Google Scholar) derived from a healthy volunteer, which we have shown to have a strong propensity for neural differentiation. When SB431542 (10 μM) and LDN193189 (0.2 μM) were added to cultures from 1 to 5 days in vitro (DIV) (Figure 3A), 70%–80% of cells in the culture at 10 DIV were PAX6+ Oct4− neural progenitors (data not shown). To direct the cells toward a caudal ventral identity, retinoic acid (RA) and sonic hedgehog cysteine mutated (SHH-C) were added, as indicated, and neurotrophic factors were provided to support neuronal survival (Figure 3A). By 40 DIV, as with earlier protocols (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 (796) Google Scholar, Karumbayaram et al., 2009Karumbayaram S. Novitch B.G. Patterson M. Umbach J.A. Richter L. Lindgren A. Conway A.E. Clark A.T. Goldman S.A. Plath K. et al.Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.Stem Cells. 2009; 27: 806-811Crossref PubMed Scopus (311) Google Scholar, Li et al., 2005Li X.J. Du Z.W. Zarnowska E.D. Pankratz M. Hansen L.O. Pearce R.A. Zhang S.C. Specification of motoneurons from human embryonic stem cells.Nat. Biotechnol. 2005; 23: 215-221Crossref PubMed Scopus (629) Google Scholar, Shaltouki et al., 2013Shaltouki A. Peng J. Liu Q. Rao M.S. Zeng X. Efficient generation of astrocytes from human pluripotent stem cells in defined conditions.Stem Cells. 2013; 31: 941-952Crossref PubMed Scopus (179) Google Scholar), the cultures were enriched for markers of ventral neuronal populations (OLIG2, HB9, ISL1/ISL2, NKX2.2, and NKX6.1; data not shown). Critically for our studies, they showed spinal cord (HOXB4; >90%) but not midbrain (OTX2; <2%) identity (Figure 3B). Early neuralization did not, therefore, perturb the patterning of these neural precursors.Figure 3Rapid Generation of Spinal Cord Astrocytes from hESCs and hiPSCs following Early NeuralizationShow full caption(A) Treatments used for efficient generation of hESC- and hiPSC-derived astrocytes. P, passag" @default.
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• W2009867508 date "2013-09-01" @default.
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• W2009867508 title "Human Stem Cell-Derived Spinal Cord Astrocytes with Defined Mature or Reactive Phenotypes" @default.
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