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• W2103705124 abstract "•The regional identity of PSC-derived neurons can be controlled precisely•Phenotypes between different neuronal subtypes were compared successfully•Neuronal subtype-specific phenotypes of ALS and AD were reproduced in vitro•A novel tool is offered to study subtype specificity of disease phenotypes The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs. The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs. Modeling human diseases with pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), has remarkable potential to generate new insights into understanding disease pathogenesis and to open up new avenues for effective therapies. In particular, modeling neurological diseases is of great interest given that it is difficult to obtain patient-derived neural cells or tissues because of the limited accessibility to the brain. Indeed, ESCs and iPSCs derived from patients have been used to study several neurological diseases, including amyotrophic lateral sclerosis (ALS; Dimos et al., 2008Dimos J.T. Rodolfa K.T. Niakan K.K. Weisenthal L.M. Mitsumoto H. Chung W. Croft G.F. Saphier G. Leibel R. Goland R. et al.Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.Science. 2008; 321: 1218-1221Crossref PubMed Scopus (1616) Google Scholar, Egawa et al., 2012Egawa N. Kitaoka S. Tsukita K. Naitoh M. Takahashi K. Yamamoto T. Adachi F. Kondo T. Okita K. Asaka I. et al.Drug screening for ALS using patient-specific induced pluripotent stem cells.Sci. Transl. Med. 2012; 4: 145ra104Crossref PubMed Scopus (408) Google Scholar), Alzheimer’s disease (AD; 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, 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 Aβ and differential drug responsiveness.Cell Stem Cell. 2013; 12: 487-496Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, Yagi et al., 2011Yagi T. Ito D. Okada Y. Akamatsu W. Nihei Y. Yoshizaki T. Yamanaka S. Okano H. Suzuki N. Modeling familial Alzheimer’s disease with induced pluripotent stem cells.Hum. Mol. Genet. 2011; 20: 4530-4539Crossref PubMed Scopus (451) Google Scholar), Parkinson’s disease (Devine et al., 2011Devine M.J. Ryten M. Vodicka P. Thomson A.J. Burdon T. Houlden H. Cavaleri F. Nagano M. Drummond N.J. Taanman J.-W. et al.Parkinson’s disease induced pluripotent stem cells with triplication of the α-synuclein locus.Nat. Commun. 2011; 2: 440Crossref PubMed Scopus (350) Google Scholar, Imaizumi et al., 2012Imaizumi Y. Okada Y. Akamatsu W. Koike M. Kuzumaki N. Hayakawa H. Nihira T. Kobayashi T. Ohyama M. Sato S. et al.Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue.Mol. Brain. 2012; 5: 35Crossref PubMed Scopus (282) Google Scholar, Nguyen et al., 2011Nguyen H.N. Byers B. Cord B. Shcheglovitov A. Byrne J. Gujar P. Kee K. Schüle B. Dolmetsch R.E. Langston W. et al.LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.Cell Stem Cell. 2011; 8: 267-280Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar), schizophrenia (Brennand et al., 2011Brennand K.J. Simone A. Jou J. Gelboin-Burkhart C. Tran N. Sangar S. Li Y. Mu Y. Chen G. Yu D. et al.Modelling schizophrenia using human induced pluripotent stem cells.Nature. 2011; 473: 221-225Crossref PubMed Scopus (1039) Google Scholar, Bundo et al., 2014Bundo M. Toyoshima M. Okada Y. Akamatsu W. Ueda J. Nemoto-Miyauchi T. Sunaga F. Toritsuka M. Ikawa D. Kakita A. et al.Increased l1 retrotransposition in the neuronal genome in schizophrenia.Neuron. 2014; 81: 306-313Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, Hook et al., 2014Hook V. Brennand K.J. Kim Y. Toneff T. Funkelstein L. Lee K.C. Ziegler M. Gage F.H. Human iPSC neurons display activity-dependent neurotransmitter secretion: aberrant catecholamine levels in schizophrenia neurons.Stem Cell Reports. 2014; 3: 531-538Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), epilepsy (Higurashi et al., 2013Higurashi N. Uchida T. Lossin C. Misumi Y. Okada Y. Akamatsu W. Imaizumi Y. Zhang B. Nabeshima K. Mori M.X. et al.A human Dravet syndrome model from patient induced pluripotent stem cells.Mol. Brain. 2013; 6: 19Crossref PubMed Scopus (87) Google Scholar, Jiao et al., 2013Jiao J. Yang Y. Shi Y. Chen J. Gao R. Fan Y. Yao H. Liao W. Sun X.F. Gao S. Modeling Dravet syndrome using induced pluripotent stem cells (iPSCs) and directly converted neurons.Hum. Mol. Genet. 2013; 22: 4241-4252Crossref PubMed Scopus (97) Google Scholar, Liu et al., 2013Liu Y. Lopez-Santiago L.F. Yuan Y. Jones J.M. Zhang H. O’Malley H.A. Patino G.A. O’Brien J.E. Rusconi R. Gupta A. et al.Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.Ann. Neurol. 2013; 74: 128-139Crossref PubMed Scopus (177) Google Scholar), and Rett syndrome (Andoh-Noda et al., 2015Andoh-Noda T. Akamatsu W. Miyake K. Matsumoto T. Yamaguchi R. Sanosaka T. Okada Y. Kobayashi T. Ohyama M. Nakashima K. et al.Differentiation of multipotent neural stem cells derived from Rett syndrome patients is biased toward the astrocytic lineage.Mol. Brain. 2015; 8: 31Crossref PubMed Scopus (66) Google Scholar, Marchetto et al., 2010Marchetto M.C.N. Carromeu C. Acab A. Yu D. Yeo G.W. Mu Y. Chen G. Gage F.H. Muotri A.R. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.Cell. 2010; 143: 527-539Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar). Because most neurological diseases affect one or more specific lesion area(s), PSCs were differentiated into corresponding neuronal subtypes in such studies (Imaizumi and Okano, 2014Imaizumi Y. Okano H. Modeling human neurological disorders with induced pluripotent stem cells.J. Neurochem. 2014; 129: 388-399Crossref PubMed Scopus (81) Google Scholar, Marchetto and Gage, 2012Marchetto M.C. Gage F.H. Modeling brain disease in a dish: really?.Cell Stem Cell. 2012; 10: 642-645Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Mattis and Svendsen, 2011Mattis V.B. Svendsen C.N. Induced pluripotent stem cells: a new revolution for clinical neurology?.Lancet Neurol. 2011; 10: 383-394Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, Okano and Yamanaka, 2014Okano H. Yamanaka S. iPS cell technologies: significance and applications to CNS regeneration and disease.Mol. Brain. 2014; 7: 22Crossref PubMed Scopus (179) Google Scholar). However, these approaches cannot procure the mechanism of subtype specificity of disease phenotypes; that is, why some neuronal subtypes are selectively damaged whereas others evade pathogenesis. To clarify the mechanism of subtype specificity, it is necessary to compare phenotypes between disease-susceptible and -insusceptible neuronal subtypes. Nonetheless, this approach has not been developed to date for two reasons. First, only a few neuronal subtypes can be successfully induced from PSCs. Second, each previously reported protocol to induce specific subtypes varies in its cultivation procedures, giving rise to variability between protocols in efficiency, maturity, and culture-afflicted stress and preventing a direct comparison across different neuronal subtypes. Here we addressed these problems by focusing on control of the regional identity of PSC-derived neural progenitors and neurons. The developing neural tube is subdivided into distinct regions along the anteroposterior (A-P) and dorsoventral (D-V) axes, and each region produces a specific subtype of neurons (Kiecker and Lumsden, 2012Kiecker C. Lumsden A. The role of organizers in patterning the nervous system.Annu. Rev. Neurosci. 2012; 35: 347-367Crossref PubMed Scopus (115) Google Scholar). Namely, neuronal subtype specification in the neural tube is determined in a region-specific manner. If the regional identity of PSC-derived neurons could be regulated at will based on the same protocol, then any desired subtypes could be induced with the same efficiency and under the same culture conditions. A protocol permitting such regulation would enable the reproduction of disease phenotypes in any given brain region and also the comparison of phenotypes between different neuronal subtypes. In this study, we report a robust method to control the regional identity of PSC-derived neural progenitors and neurons based on a single protocol. To do so, Wnt, retinoic acid (RA), and sonic hedgehog (Shh) signaling were modulated to manipulate the A-P and D-V identities of neural progenitors. Regional identity was maintained throughout neural differentiation, generating a variety of neuronal subtypes, including cortical projection neurons, cortical interneurons, midbrain dopaminergic neurons, hindbrain serotonergic neurons, spinal cord sensory interneurons, and spinal cord motor neurons. Finally, we compared these neuronal subtypes and detected ventral spinal cord-specific neurite swellings in ALS iPSCs and forebrain-specific accumulation of phosphorylated tau (p-tau) in AD iPSCs. Therefore, this culture system could be a useful tool to approach the mechanism of subtype specificity of neurological disease phenotypes. Because Wnt, RA, and Shh signaling are involved in the formation of the A-P and D-V axes during neural development (Briscoe and Ericson, 2001Briscoe J. Ericson J. Specification of neuronal fates in the ventral neural tube.Curr. Opin. Neurobiol. 2001; 11: 43-49Crossref PubMed Scopus (429) Google Scholar, Marshall et al., 1992Marshall H. Nonchev S. Sham M.H. Muchamore I. Lumsden A. Krumlauf R. Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity.Nature. 1992; 360: 737-741Crossref PubMed Scopus (404) Google Scholar, Nordström et al., 2002Nordström U. Jessell T.M. Edlund T. Progressive induction of caudal neural character by graded Wnt signaling.Nat. Neurosci. 2002; 5: 525-532Crossref PubMed Scopus (211) Google Scholar), we explored the possibility that these patterning signaling molecules might have analogous effects in neural cells differentiated from PSCs. We hypothesized that the A-P identity (ranging from the telencephalon to the spinal cord) can be controlled by regulating Wnt and RA signaling and that the D-V identity can be controlled similarly by regulating Shh signaling (Figure 1A). To evaluate this hypothesis, we induced neural progenitors derived from ESCs (KhES-1; Suemori et al., 2006Suemori H. Yasuchika K. Hasegawa K. Fujioka T. Tsuneyoshi N. Nakatsuji N. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage.Biochem. Biophys. Res. Commun. 2006; 345: 926-932Crossref PubMed Scopus (279) Google Scholar) by using the neurosphere culture system (modified from Chaddah et al., 2012Chaddah R. Arntfield M. Runciman S. Clarke L. van der Kooy D. Clonal neural stem cells from human embryonic stem cell colonies.J. Neurosci. 2012; 32: 7771-7781Crossref PubMed Scopus (37) Google Scholar) and treated the cells with modulators of these patterning signaling pathways during neurosphere formation (Figure 1B). The modulators included the porcupine inhibitor IWP-2 (a Wnt antagonist; Chen et al., 2009Chen B. Dodge M.E. Tang W. Lu J. Ma Z. Fan C.-W. Wei S. Hao W. Kilgore J. Williams N.S. et al.Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.Nat. Chem. Biol. 2009; 5: 100-107Crossref PubMed Scopus (1106) Google Scholar), the glycogen synthase kinase (GSK) 3β inhibitor CHIR99021 (a Wnt agonist, referred to hereafter as CHIR; Ring et al., 2003Ring D.B. Johnson K.W. Henriksen E.J. Nuss J.M. Goff D. Kinnick T.R. Ma S.T. Reeder J.W. Samuels I. Slabiak T. et al.Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo.Diabetes. 2003; 52: 588-595Crossref PubMed Scopus (393) Google Scholar), RA, Shh protein, and the Smo receptor agonist purmorphamine (a Shh agonist; Sinha and Chen, 2006Sinha S. Chen J.K. Purmorphamine activates the Hedgehog pathway by targeting Smoothened.Nat. Chem. Biol. 2006; 2: 29-30Crossref PubMed Scopus (293) Google Scholar). Although the diameter of the neurospheres was increased by treatment with CHIR and RA, each of the experimental conditions was able to generate neurospheres (Figures S1A and S1B). Moreover, the patterning factors did not influence the number of neurospheres generated (Figure S1C), and the neural progenitor marker NESTIN was expressed similarly under all conditions (Figure S1D). These results indicate that neural induction is unaffected by Wnt, RA, or Shh signaling. We next examined the expression of A-P markers (Figure 2A) in ESC-derived neurospheres by qRT-PCR (Figure 2B). IWP-2-treated neurospheres expressed high levels of the forebrain markers FOXG1 and SIX3 (Oliver et al., 1995Oliver G. Mailhos A. Wehr R. Copeland N.G. Jenkins N.A. Gruss P. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development.Development. 1995; 121: 4045-4055Crossref PubMed Google Scholar, Xuan et al., 1995Xuan S. Baptista C.A. Balas G. Tao W. Soares V.C. Lai E. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.Neuron. 1995; 14: 1141-1152Abstract Full Text PDF PubMed Scopus (462) Google Scholar). The expression levels of the forebrain/midbrain markers OTX1 and OTX2 (Simeone et al., 1992Simeone A. Acampora D. Gulisano M. Stornaiuolo A. Boncinelli E. Nested expression domains of four homeobox genes in developing rostral brain.Nature. 1992; 358: 687-690Crossref PubMed Scopus (667) Google Scholar) were highest in untreated neurospheres. The low expression level of OTX1 in IWP-2-treated neurospheres agrees with the observation that this gene is not expressed in the anterior-most region of the forebrain (Shimamura et al., 1997Shimamura K. Martinez S. Puelles L. Rubenstein J.L. Patterns of gene expression in the neural plate and neural tube subdivide the embryonic forebrain into transverse and longitudinal domains.Dev. Neurosci. 1997; 19: 88-96Crossref PubMed Scopus (89) Google Scholar). EN1, which is expressed in the midbrain and the anterior hindbrain (Hanks et al., 1995Hanks M. Wurst W. Anson-Cartwright L. Auerbach A.B. Joyner A.L. Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2.Science. 1995; 269: 679-682Crossref PubMed Scopus (358) Google Scholar), was highly expressed in neurospheres treated with CHIR at concentrations of 0.5 and 1 μM. HOXB4 and HOXC4, markers of the posterior hindbrain and the spinal cord (Hunt et al., 1991Hunt P. Gulisano M. Cook M. Sham M.H. Faiella A. Wilkinson D. Boncinelli E. Krumlauf R. A distinct Hox code for the branchial region of the vertebrate head.Nature. 1991; 353: 861-864Crossref PubMed Scopus (425) Google Scholar), were expressed primarily in cultures exposed to 3 μM CHIR, and their expression levels were enhanced further by the addition of RA. These results indicate that the posteriorization of neural progenitors is driven by activation of the Wnt and RA signaling pathways. The regulation of marker expression correlating with the A-P axis was also confirmed by immunocytochemical analysis for the FOXG1, OTX1, and HOXB4 proteins (Figure 2C). Furthermore, we confirmed the robustness of our protocol by using the 201B7 and 253G1 iPSC lines (Nakagawa et al., 2008Nakagawa M. Koyanagi M. Tanabe K. Takahashi K. Ichisaka T. Aoi T. Okita K. Mochiduki Y. Takizawa N. Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.Nat. Biotechnol. 2008; 26: 101-106Crossref PubMed Scopus (2207) Google Scholar, 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). In both of these iPSC lines, the expression levels of the A-P markers in the neurospheres were similar to those in ESC-derived neurospheres (Figure S2). These results suggest that the A-P identity of PSC-derived neural progenitors can be controlled by IWP-2, CHIR, and RA during neurosphere formation. In addition to the A-P identity, we next attempted to control the D-V identity by modulating Shh signaling. We hypothesized that neural progenitors derived from PSCs are dorsalized in the absence of Shh and that the activation of Shh signaling ventralizes neural progenitors with no influence on the A-P identity. To address this hypothesis, we assessed the expression levels of D-V makers in neurospheres by qRT-PCR analysis with the addition of Shh protein and purmorphamine (termed the “+Shh” group) (Figures 3A and 3B ). We used these two Shh activators simultaneously because a previous report has demonstrated that co-treatment with Shh protein and purmorphamine was greatly superior to high concentrations of either of the two alone (Maroof et al., 2013Maroof A.M. Keros S. Tyson J.A. Ying S.W. Ganat Y.M. Merkle F.T. Liu B. Goulburn A. Stanley E.G. Elefanty A.G. et al.Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells.Cell Stem Cell. 2013; 12: 559-572Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Other neurospheres received no exogenous ventralizing factors (termed the “−Shh” group). The D-V markers PAX6, PAX7, NKX2.1, and NKX2.2 are differentially expressed in vivo according to A-P and D-V positions (Figure 3A). PAX6 is expressed dorsally in the forebrain, and the expression pattern also covers both the dorsal and the ventral portions of the posterior hindbrain and the spinal cord (Ericson et al., 1997Ericson J. Rashbass P. Schedl A. Brenner-Morton S. Kawakami A. van Heyningen V. Jessell T.M. Briscoe J. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.Cell. 1997; 90: 169-180Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, Osumi et al., 1997Osumi N. Hirota A. Ohuchi H. Nakafuku M. Iimura T. Kuratani S. Fujiwara M. Noji S. Eto K. Pax-6 is involved in the specification of hindbrain motor neuron subtype.Development. 1997; 124: 2961-2972Crossref PubMed Google Scholar, Takahashi and Osumi, 2002Takahashi M. Osumi N. Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.Development. 2002; 129: 1327-1338Crossref PubMed Google Scholar, Walther and Gruss, 1991Walther C. Gruss P. Pax-6, a murine paired box gene, is expressed in the developing CNS.Development. 1991; 113: 1435-1449Crossref PubMed Google Scholar). In contrast, the midbrain and the anterior hindbrain lack PAX6 expression (with the exception of the rhombic lip) (Engelkamp et al., 1999Engelkamp D. Rashbass P. Seawright A. van Heyningen V. Role of Pax6 in development of the cerebellar system.Development. 1999; 126: 3585-3596Crossref PubMed Google Scholar, Schwarz et al., 1999Schwarz M. Alvarez-Bolado G. Dressler G. Urbánek P. Busslinger M. Gruss P. Pax2/5 and Pax6 subdivide the early neural tube into three domains.Mech. Dev. 1999; 82: 29-39Crossref PubMed Scopus (86) Google Scholar, Swanson et al., 2005Swanson D.J. Tong Y. Goldowitz D. Disruption of cerebellar granule cell development in the Pax6 mutant, Sey mouse.Brain Res. Dev. Brain Res. 2005; 160: 176-193Crossref PubMed Scopus (41) Google Scholar, Yamasaki et al., 2001Yamasaki T. Kawaji K. Ono K. Bito H. Hirano T. Osumi N. Kengaku M. Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.Development. 2001; 128: 3133-3144Crossref PubMed Google Scholar). PAX7 is expressed throughout the dorsal part of the neural tube except for the anterior forebrain, where this gene is expressed only in the small dorsal-most region (Matsunaga et al., 2001Matsunaga E. Araki I. Nakamura H. Role of Pax3/7 in the tectum regionalization.Development. 2001; 128: 4069-4077Crossref PubMed Google Scholar). NKX2.1 marks the ventral forebrain (Qiu et al., 1998Qiu M. Shimamura K. Sussel L. Chen S. Rubenstein J.L.R. Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development.Mech. Dev. 1998; 72: 77-88Crossref PubMed Scopus (133) Google Scholar), whereas NKX2.2 is expressed ventrally throughout the A-P axis (Shimamura et al., 1995Shimamura K. Hartigan D.J. Martinez S. Puelles L. Rubenstein J.L. Longitudinal organization of the anterior neural plate and neural tube.Development. 1995; 121: 3923-3933Crossref PubMed Google Scholar). Consistent with the in vivo expression patterns of these genes, PAX6 was highly expressed in neurospheres with forebrain characteristics (i.e., IWP-2-treated and untreated neurospheres) in the −Shh group as well as those with the characteristics of the posterior hindbrain and the spinal cord (i.e., CHIR3+RA-treated neurospheres). Only the latter maintained PAX6 expression after Shh activation. Meanwhile, PAX7 was upregulated in the −Shh group, except under the IWP-2-treated condition, whereas PAX7 expression was low under all conditions in the +Shh group. NKX2.1 was expressed primarily in neurospheres with forebrain characteristics (IWP-2-treated and untreated) in the +Shh group. The expression of NKX2.2 was generally high in the +Shh group. The relatively low expression level of NKX2.2 in neurospheres with posterior characteristics in the +Shh group agrees with the fact that NKX2.2 expression is initially only seen in the anterior part of the neural tube and then gradually extends to the spinal cord (Shimamura et al., 1995Shimamura K. Hartigan D.J. Martinez S. Puelles L. Rubenstein J.L. Longitudinal organization of the anterior neural plate and neural tube.Development. 1995; 121: 3923-3933Crossref PubMed Google Scholar). The control of the D-V axis shown by qRT-PCR analysis was also confirmed by immunocytochemical analysis for the PAX6, NKX2.1, and NKX2.2 proteins (Figures 3C and 3D). Moreover, D-V regulation was reproduced readily in the iPSC lines (Figure S2). Our results indicate that the D-V identity of PSC-derived neural progenitors can be controlled without perturbing the A-P identity. To further confirm the D-V control, we examined the expression of telencephalon-specific D-V markers (Figure 4A). EMX1/2 and DLX2 are confined to the dorsal and the ventral telencephalon, respectively (with the exception of small diencephalic regions) (Porteus et al., 1991Porteus M.H. Bulfone A. Ciaranello R.D. Rubenstein J.L. Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain.Neuron. 1991; 7: 221-229Abstract Full Text PDF PubMed Scopus (189) Google Scholar, Simeone et al., 1992Simeone A. Acampora D. Gulisano M. Stornaiuolo A. Boncinelli E. Nested expression domains of four homeobox genes in developing rostral brain.Nature. 1992; 358: 687-690Crossref PubMed Scopus (667) Google Scholar). Although the proneural genes NGN2 and ASCL1 are expressed in various regions of the neural tube, a D-V bias in their expression is clearly observed in the telencephalon (Fode et al., 2000Fode C. Ma Q. Casarosa S. Ang S.L. Anderson D.J. Guillemot F. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons.Genes Dev. 2000; 14: 67-80Crossref PubMed Google Scholar). EMX1/2 and NGN2 were significantly upregulated in IWP-2-treated neurospheres without Shh activation. In contrast, the expression of DLX2 and ASCL1 was increased by Shh activation. These data provide additional evidence for the D-V control of PSC-derived neural progenitors. To investigate whether the regional identity of neural progenitors was retained upon differentiation into neurons, neurospheres were subjected to a neural differentiation protocol (Figure 1B). Our protocol predominantly produced neurons rather than astrocytes or oligodendrocytes (Figure S3A). We examined the expression of synapse markers and the intracellular calcium level during electrical field stimulation (Figures S3B–S3D). These results indicate that neurons derived by our protocol were functional regardless of the experimental conditions. We determined the A-P identity of neurosphere-differentiated neurons (Figure 5A). Neurons generated from IWP-2-treated neurospheres expressed high levels of the forebrain cortical neuronal marker TBR1 (Bulfone et al., 1995Bulfone A. Smiga S.M. Shimamura K. Peterson A. Puelles L. Rubenstein J.L. T-brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex.Neuron. 1995; 15: 63-78Abstract Full Text PDF PubMed Scopus (366) Google Scholar). GATA3, expressed from the diencephalon to the hindbrain (George et al., 1994George K.M. Leonard M.W. Roth M.E. Lieuw K.H. Kioussis D. Grosveld F. Engel J.D. Embryonic expression and cloning of the murine GATA-3 gene.Development. 1994; 120: 2673-2686PubMed Google Scholar), was upregulated at low CHIR concentrations (0−1 μM). LBX1, which marks dorsal neurons in the hindbrain and the spinal cord (Jagla et al., 1995Jagla K. Dollé P. Mattei M.G. Jagla T. Schuhbaur B. Dretzen G. Bellard F. Bellard M. Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes.Mech. Dev. 1995; 53: 345-356Crossref PubMed Scopus (131) Google Scholar), was highly expressed in neurons differentiated from CHIR3+RA-treated neurospheres. These results imply that neural progenitors retain their A-P identity in differentiated neuronal cultures. We next examined the D-V identity of differentiated neurons (Figures 5B–5F). In the telencephalon, TBR1+ cortical neurons are born dorsally, whereas the ventral area produces cortical interneurons, which is marked by LHX6 and LHX8 expression (Marín and Rubenstein, 2001Marín O. Rubenstein J.L. A long, remarkable journey: tangential migration in the telencephalon.Nat. Rev. Neurosci. 2001; 2: 780-790Crossref PubMed Scopus (841) Google Scholar). As expected, the expression level of TBR1 was elevated in neurons generated from IWP-2-treated neurospheres and decreased by activation of Shh signaling (Figures 5B and 5E). In contrast, LHX6 and LHX8 were significantly upregulated by Shh activation (Figure 5B). Dopaminergic and serotonergic neurons are selectively derived from the ventral portion of the midbrain and the hindbrain, respectively (Goridis and Rohrer, 2002Goridis C. Rohrer H. Specification of catecholaminergic and serotonergic neurons.Nat. Rev. Neurosci. 2002; 3: 531-541Crossref PubMed Scopus (291) Google Scholar). Although the gene expression of the dopamine-synthesizing enzyme TH did not change significantly, that of LMX1A and FOXA2, master transcriptional regulators of dopaminergic neuronal differentiation (Andersson et al., 2006Andersson E. Tryggvason U. Deng Q. Friling S. Alekseenko Z. Robert B. Perlmann T. Ericson J. Identification of intrinsic determinants of midbrain dopamine neurons.Cell. 2006; 124: 393-405Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, Ferri et al., 2007Ferri A.L.M. Lin W. Mavromatakis Y.E. Wang J.C. Sasaki H. Whitsett J.A. Ang S.L. Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron de" @default.
• W2103705124 created "2016-06-24" @default.
• W2103705124 creator A5004475963 @default.
• W2103705124 creator A5016707458 @default.
• W2103705124 creator A5019690172 @default.
• W2103705124 creator A5035479034 @default.
• W2103705124 creator A5038787228 @default.
• W2103705124 creator A5077366980 @default.
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• W2103705124 date "2015-12-01" @default.
• W2103705124 modified "2023-10-16" @default.
• W2103705124 title "Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes" @default.
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