Roles of long non coding RNAs in human embryonic stem cell pluripotency and neural differentiation 5

Figure 8.13: Western blot confirms that hnRNPA2B1 and SOX2 specifically interact with RMST To validate findings from mass spectrometry, a Western blot using the respective antibodies was performed following biotinylated RNA pulldown Figure 8.14: RNA immunoprecipitation (RIP) established in vivo binding of RMST with hnRNPA2 and SOX2 Fold enrichment relative to the isotype IgG RIP is presented, and the respective p-values are indicated (A) hnRNPA2-FLAG was ectopically expressed in ReN-VM cells, and enrichment of RMST was observed in FLAG RIP, indicating physical association (B) Enrichment of RMST in SOX2 RIP also confirmed in vivo association of SOX2 and the lncRNA   138  Since RMST physically interacts with both hnRNPA2B1 and SOX2, the question of the assembly of the “RMST complex” arose It was found that hnRNPA2 and SOX2 assembled in an RNA-independent fashion In three independent coimmunoprecipitation (co-IP) experiments, SOX2 is co-immunoprecipitated with hnRNPA2-FLAG in RNase-treated cell lysate (Figure 8.15) In accordance with this observation, Fang et al (2011) found that SOX2 forms protein-protein interactions with several heterogenous nuclear ribonucleoproteins, including hnRNPA2B1 These evidences suggest that the RMST complex is composed of the RNA-binding protein, hnRNPA2B1 binding to RMST, and SOX2 in turn associates with hnRNPA2B1 through its protein interaction domain (Figure 8.16) Figure 8.15: Co-immunoprecipitation (Co-IP) of hnRNPA2-FLAG and SOX2 in the absence of RNA Cell lysate was treated with RNase, and the co-IP of hnRNPA2 and SOX2 indicated that the two proteins could associate by protein-protein interactions, rather than through a lncRNA scaffold   139  Figure 8.16: Proposed model of the RMST complex hnRNPA2B1 is a RNAbinding protein and probably binds directly to RMST SOX2 and hnRNPA2B1 may interact via protein-protein interactions The transcription factor SOX2 may then bind to target chromatin regions to affect gene expression 8.2.5 RMST and SOX2 co-regulate a common pool of genes During neurogenesis, RMST expression was upregulated Since RMST interacts with hnRNPA2B1 and SOX2, they can be envisaged to engage in a RMST complex, in which these three components together would regulate neurogenesis Previous reports indicate a crucial role for SOX2 in neurogenesis Depletion of SOX2 in neural progenitors resulted in impaired neurogenesis in the central as well as peripheral nervous system (Cavallaro et al., 2008; Cimadamore et al., 2011; Ferri et al., 2004; Puligilla et al., 2010) Therefore, it was expected that the knockdown of SOX2 in the ReN-VM neural stem cells would result in decreased neurogenesis To establish that the protein components of the RMST complex also played a role in neuronal differentiation, siRNAs targeting hnRNPA2B1 and SOX2 were introduced into ReNVM cells, and the effect on neuronal differentiation was assayed days post transfection by immunofluorescence (Figure 8.17)   140  Figure 8.17: Knockdown of components of the RMST complex prevents neurogenesis ReN-VM neural stem cells were transfected with the indicated siRNAs TUJ1 and MAP2, both markers for neurons, were assayed by immunofluorescence days later In the non-target siRNA (si-NT) control, TUJ1+ and MAP2+ neurons were observed When expressions of RMST, hnRNPA2B1 and SOX2 were depleted, there were significantly fewer TUJ1+ cells, indicating a loss of neurogenesis The scale bar indicates 100 µm In the bottom panel, the efficiencies of knockdown are indicated As expected, knockdown of any of the components of the RMST complex resulted in the loss of neurogenesis, indicated by decrease in the number of TUJ1+ and MAP2+ cells The similarity in loss-of-function phenotype, together with RNAprotein interactions, suggested the possibility that RMST and SOX2 could be regulating a common pool of genes To this end, a microarray experiment was carried out ReN-VM cells were transfected with RMST siRNA (si-RMST), SOX2 siRNA (siSOX2) or non-target siRNA (si-NT) RNA was extracted 48 hours after transfection   141  of siRNAs Microarray expression data was normalized to the si-NT dataset, and the extent of overlap between differentially expressed genes in the si-RMST and si-SOX2 datasets was examined To summarize the microarray findings, 1171 genes were differentially detected (FDR < 0.05; absolute fold change > 1.5) in the si-RMST cells, while there were 626 differentially expressed genes in the si-SOX2 cells There was a 100% overlap between the datasets in that the 626 genes differentially expressed in siSOX2 were also differentially expressed in si-RMST Of the 1170 differentially expressed genes in si-RMST cells, 632 were upregulated while 539 were downregulated A similar trend was observed in the si-SOX2 cells, where 372 genes were upregulated, while 254 were downregulated (Table 8.1) The extent of overlap was also large (more than 60%), indicating that RMST and SOX2 possibly regulate a common pool of genes (Figure 8.18) In addition, a gene ontology (GO) analysis of the 152 genes downregulated upon both RMST and SOX2 knockdown indicated an enrichment of GO terms related to neurogenesis and neuronal function (Table 8.2) Taken together, the loss-of-function studies, coupled with whole genome gene expression analyses, indicate that RMST and SOX2 regulate a common set of target genes that are essential for neurogenesis   142  Table 8.1: Table summarizing microarray findings upon knockdown of RMST or SOX2 in neural stem cells si-RMST si-SOX2 overlap differentially expressed 1171 626 626 upregulated 632 372 331 downregulated 539 254 152 Figure 8.18: RMST and SOX2 regulate a common pool of targets The extent of overlap between differentially expressed genes in si-SOX2 and si-RMST datasets was examined by means of a microarray experiment Red circles indicate si-RMST, while blue circles indicate si-SOX2 datasets (A) Regardless of direction of fold change, the overlap between si-SOX2 and si-RMST was 100%, with 626 differentially expressed genes in si-SOX2 also misexpressed in si-RMST (B) Of these, 331 genes were upregulated in both si-RMST and si-SOX2 (C) 152 genes were downregulated in both si-RMST and si-SOX2   143  Table 8.2: Gene Ontology (GO) analysis of the 152 genes in the si-RMST and siSOX2 overlap Gene Ontology Biological Process 10 Neuron migration Regulation of Notch signaling pathway Cellular response to stress Forebrain development Neurogenesis Positive regulation of Notch signaling pathway Telencephalon development Rho protein signal transduction Brain development Oligodendrocyte differentiation Gene Ontology Term GO:0001764 GO:0008593 GO:0033554 GO:0030900 GO:0022008 GO:0045747 GO:0021537 GO:0007266 GO:0007420 GO:0048709 p-value 5.28E-06 6.55E-06 2.97E-05 2.97E-05 1.29E-04 1.46E-04 1.48E-04 1.67E-04 1.81E-04 1.89E-04 The top 10 terms, and their respective p-values are shown Gene clusters categorized into biological processes at levels 3-9 when analyzed with FatiGO 8.2.6 RMST does not regulate SOX2 expression Since RMST and SOX2 share a large set of target genes, one question that arose was whether RMST regulates SOX2 expression To this end, SOX2 expression following RMST knockdown in ReN-VM cells was measured RNA was isolated 48 hours after transfection of siRNAs Quantification by qPCR showed that upon RMST knockdown, transcript levels of SOX2 and hnRNPA2B1 remained unchanged Similarly, knockdown of SOX2 had no effect on RMST or hnRNPA2B1 expression (Figure 8.19) This demonstrates that RMST did not regulate downstream target expression by altering cellular levels of the transcription factor SOX2   144  Figure 8.19: Transcript levels of SOX2 remained unchanged following RMST knockdown Knockdown of hnRNPA2B1 (bars shown in light blue) did not alter expression levels of RMST and SOX2 significantly Similarly, knockdown of RMST (bars shown in dark blue) did not result in a change in SOX2 and hnRNPA2B1 levels in the cells 8.3 Discussion 8.3.1 RMST forms part of a complex that is required for neurogenesis In this chapter, I established that RMST interacts with hnRNPA2B1, as well as SOX2, and loss of any of these three components of the RMST complex resulted in loss of neurogenesis In accordance with literature, Sox2 induces neuronal formation, and the loss of Sox2 had been reported to cause neurodegeneration and impaired neurogenesis in the mouse brain (Ferri et al., 2004; Puligilla et al., 2010) SOX2 has also been reported to associate with hnRNPA2B1 via protein-protein interactions (Fang et al., 2011), and this interaction was also observed in this study via the co-IP experiment hnRNPA2 and hnRNPB1 (referred to as hnRNPA2B1) are spliced isoforms, and are RNA-binding proteins that regulate alternative splicing (Chen et al., 2010; Tauler et al., 2010) Therefore, apart from serving as the adaptor molecule linking RMST to   145  SOX2, it is also probable that hnRNPA2B1 may regulate the splicing of RMST into its three isoforms However, in this current study, the functions of individual isoforms are not established 8.3.2 RMST may change the binding patterns of SOX2 to chromatin From the microarray experiment described in Figure 8.17, it was evident that RMST and SOX2 share many common targets, supporting the notion that the two are part of the RMST complex that regulates genes crucial for neurogenesis There are at least two possibilities in which RMST could function in the complex First, association of RMST with SOX2 could alter binding of the transcription factor to chromatin Preliminary evidence suggests that depletion of RMST in ReN-VM cells reduced SOX2 occupancy at some target genes (Figure 8.20), indicating that this is a plausible mechanism in which RMST may modulate neurogenesis Figure 8.20: Depletion of RMST resulted in decreased SOX2 occupancy at target genes Cells were transfected with either a non-target siRNA or RMST siRNA, and SOX2 ChIP was performed 48 hours later Fold enrichment at SOX2 targets (pDLX1, pJAG1 and pGLI2) were decreased upon RMST depletion pNECDIN was a negative control (Engelen et al., 2011)   146  The second possibility is that RMST may recruit SOX2 to specific gene loci to regulate transcription Several lncRNAs, including HOTAIR, KCNQ1OT1 and Evf2 (Bond et al., 2009; Chu et al., 2011; Mohammad et al., 2010), have been reported to recruit transcription factors or chromatin modifiers to specific regions in the genome to regulate gene expression 8.3.3 RMST may bind to proteins other than hnRNPA2B1 and SOX2 Depletion of the lncRNA RMST resulted in differential expression of more than 1000 putative target genes In contrast, depletion of SOX2 resulted in only 626 misexpressed genes (Figure 8.17A) This suggested that RMST could associate with other nuclear proteins, apart from hnRNPA2B1 and SOX2 However, from the RIP experiments in Chapter VII, it is indicated that RMST are neither bound by SUZ12, a component of the PRC2 repressive complex, nor the transcription factor REST It is likely that RMST may bind to a repressive chromatin modifier, such as DNMTs, to silence non-neuronal genes during neurogenesis More experimental validation would however, be required to provide full mechanistic insight of the role of RMST in neurogenesis 8.4 Conclusion This chapter focuses on elucidating the mechanism of RMST involvement in neuronal differentiation RMST is a conserved lncRNA that is repressed by REST During neurogenesis, when expression of REST is lost, RMST is upregulated Although RMST showed region-specific expression in the developing mouse brain, its role in neurogenesis has not been established prior to this work Though association studies, I   147  Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells Cell 113, 643-655 Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling Nat Biotechnol 27, 275-280 Chan, K.K., Zhang, J., Chia, N.Y., Chan, Y.S., Sim, H.S., Tan, K.S., Oh, S.K., Ng, H.H., and Choo, A.B (2009) KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells Stem Cells 27, 2114-2125 Chen, G., Yin, K., Shi, L., Fang, Y., Qi, Y., Li, P., Luo, J., He, B., Liu, M., and Shi, T (2011) Comparative analysis of human protein-coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq PLoS One 6, e28318 Chen, M., Zhang, J., and Manley, J.L (2010) Turning on a fuel switch of cancer: hnRNP proteins regulate alternative splicing of pyruvate kinase mRNA Cancer Res 70, 8977-8980 Chia, N.Y., Chan, Y.S., Feng, B., Lu, X., Orlov, Y.L., Moreau, D., Kumar, P., Yang, L., Jiang, J., Lau, M.S., et al (2010) A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity Nature 468, 316-320 Cho, M.S., Lee, Y.E., Kim, J.Y., Chung, S., Cho, Y.H., Kim, D.S., Kang, S.M., Lee, H., Kim, M.H., Kim, J.H., et al (2008) Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells Proc Natl Acad Sci U S A 105, 3392-3397 Chodroff, R.A., Goodstadt, L., Sirey, T.M., Oliver, P.L., Davies, K.E., Green, E.D., Molnar, Z., and Ponting, C.P (2010) Long noncoding RNA genes: conservation of sequence and brain expression among diverse amniotes Genome Biol 11, R72 Chu, C., Qu, K., Zhong, F.L., Artandi, S.E., and Chang, H.Y (2011) Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions Mol Cell 44, 667-678 Cimadamore, F., Fishwick, K., Giusto, E., Gnedeva, K., Cattarossi, G., Miller, A., Pluchino, S., Brill, L.M., Bronner-Fraser, M., and Terskikh, A.V (2011) Human ESC-derived neural crest model reveals a key role for SOX2 in sensory neurogenesis Cell Stem Cell 8, 538-551   157  Crossley, P.H., Minowada, G., MacArthur, C.A., and Martin, G.R (1996) Roles for FGF8 in the induction, initiation, and maintenance of chick limb development Cell 84, 127-136 Dinger, M.E., Amaral, P.P., Mercer, T.R., Pang, K.C., Bruce, S.J., Gardiner, B.B., Askarian-Amiri, M.E., Ru, K., Solda, G., Simons, C., et al (2008) Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation Genome Res 18, 1433-1445 Donato, R., Miljan, E.A., Hines, S.J., Aouabdi, S., Pollock, K., Patel, S., Edwards, F.A., and Sinden, J.D (2007) Differential development of neuronal physiological responsiveness in two human neural stem cell lines BMC Neurosci 8, 36 Efroni, S., Carmel, L., Schaefer, C.G., and Buetow, K.H (2008) Superposition of transcriptional behaviors determines gene state PLoS One 3, e2901 Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N.D., Tabar, V., and Studer, L (2008) Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage Genes Dev 22, 152-165 Engelen, E., Akinci, U., Bryne, J.C., Hou, J., Gontan, C., Moen, M., Szumska, D., Kockx, C., van Ijcken, W., Dekkers, D.H., et al (2011) Sox2 cooperates with Chd7 to regulate genes that are mutated in human syndromes Nat Genet 43, 607-611 Engstrom, P.G., Suzuki, H., Ninomiya, N., Akalin, A., Sessa, L., Lavorgna, G., Brozzi, A., Luzi, L., Tan, S.L., Yang, L., et al (2006) Complex Loci in human and mouse genomes PLoS Genet 2, e47 Fang, X., Yoon, J.G., Li, L., Tsai, Y.S., Zheng, S., Hood, L., Goodlett, D.R., Foltz, G., and Lin, B (2011) Landscape of the SOX2 protein-protein interactome Proteomics 11, 921-934 Farazi, T.A., Juranek, S.A., and Tuschl, T (2008) The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members Development 135, 1201-1214 Feng, J., Bi, C., Clark, B.S., Mady, R., Shah, P., and Kohtz, J.D (2006) The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator Genes Dev 20, 1470-1484 Ferri, A.L., Cavallaro, M., Braida, D., Di Cristofano, A., Canta, A., Vezzani, A., Ottolenghi, S., Pandolfi, P.P., Sala, M., DeBiasi, S., et al (2004) Sox2 deficiency   158  causes neurodegeneration and impaired neurogenesis in the adult mouse brain Development 131, 3805-3819 Filipowicz, W., Bhattacharyya, S.N., and Sonenberg, N (2008) Mechanisms of posttranscriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9, 102-114 Finley, M.F., Devata, S., and Huettner, J.E (1999) BMP-4 inhibits neural differentiation of murine embryonic stem cells J Neurobiol 40, 271-287 Ginis, I., Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht-Nir, S., Amit, M., Hoke, A., Carpenter, M.K., Itskovitz-Eldor, J., et al (2004) Differences between human and mouse embryonic stem cells Dev Biol 269, 360-380 Gong, C., and Maquat, L.E (2011) lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements Nature 470, 284-288 Gotz, M., and Huttner, W.B (2005) The cell biology of neurogenesis Nat Rev Mol Cell Biol 6, 777-788 Gregg, C., and Weiss, S (2003) Generation of functional radial glial cells by embryonic and adult forebrain neural stem cells J Neurosci 23, 11587-11601 Guillemot, F (2007) Cell fate specification in the mammalian telencephalon Prog Neurobiol 83, 37-52 Gupta, R.A., Shah, N., Wang, K.C., Kim, J., Horlings, H.M., Wong, D.J., Tsai, M.C., Hung, T., Argani, P., Rinn, J.L., et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis Nature 464, 1071-1076 Guttman, M., Amit, I., Garber, M., French, C., Lin, M.F., Feldser, D., Huarte, M., Zuk, O., Carey, B.W., Cassady, J.P., et al (2009) Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals Nature 458, 223227 Guttman, M., Donaghey, J., Carey, B.W., Garber, M., Grenier, J.K., Munson, G., Young, G., Lucas, A.B., Ach, R., Bruhn, L., et al (2011) lincRNAs act in the circuitry controlling pluripotency and differentiation Nature 477, 295-300 Haubst, N., Berger, J., Radjendirane, V., Graw, J., Favor, J., Saunders, G.F., Stoykova, A., and Gotz, M (2004) Molecular dissection of Pax6 function: the   159  specific roles of the paired domain and homeodomain in brain development Development 131, 6131-6140 Hebsgaard, J.B., Nelander, J., Sabelstrom, H., Jonsson, M.E., Stott, S., and Parmar, M (2009) Dopamine neuron precursors within the developing human mesencephalon show radial glial characteristics Glia 57, 1648-1658 Heins, N., Malatesta, P., Cecconi, F., Nakafuku, M., Tucker, K.L., Hack, M.A., Chapouton, P., Barde, Y.A., and Gotz, M (2002) Glial cells generate neurons: the role of the transcription factor Pax6 Nat Neurosci 5, 308-315 Hu, W., Yuan, B., Flygare, J., and Lodish, H.F (2011) Long noncoding RNAmediated anti-apoptotic activity in murine erythroid terminal differentiation Genes Dev 25, 2573-2578 Iacovitti, L., Donaldson, A.E., Marshall, C.E., Suon, S., and Yang, M (2007) A protocol for the differentiation of human embryonic stem cells into dopaminergic neurons using only chemically defined human additives: Studies in vitro and in vivo Brain Res 1127, 19-25 Imanishi, T., Itoh, T., Suzuki, Y., O'Donovan, C., Fukuchi, S., Koyanagi, K.O., Barrero, R.A., Tamura, T., Yamaguchi-Kabata, Y., Tanino, M., et al (2004) Integrative annotation of 21,037 human genes validated by full-length cDNA clones PLoS Biol 2, e162 Ingolia, N.T., Lareau, L.F., and Weissman, J.S (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes Cell 147, 789-802 Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, X., Lun, Y., and Lemischka, I.R (2006) Dissecting self-renewal in stem cells with RNA interference Nature 442, 533-538 Jia, H., Osak, M., Bogu, G.K., Stanton, L.W., Johnson, R., and Lipovich, L (2010) Genome-wide computational identification and manual annotation of human long noncoding RNA genes RNA 16, 1478-1487 Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P., Zhong, S., and Ng, H.H (2008) A core Klf circuitry regulates self-renewal of embryonic stem cells Nat Cell Biol 10, 353-360   160  Jiang, X., Gwye, Y., McKeown, S.J., Bronner-Fraser, M., Lutzko, C., and Lawlor, E.R (2009) Isolation and characterization of neural crest stem cells derived from in vitro-differentiated human embryonic stem cells Stem Cells Dev 18, 1059-1070 Johnson, R., Teh, C.H., Jia, H., Vanisri, R.R., Pandey, T., Lu, Z.H., Buckley, N.J., Stanton, L.W., and Lipovich, L (2009) Regulation of neural macroRNAs by the transcriptional repressor REST RNA 15, 85-96 Judson, R.L., Babiarz, J.E., Venere, M., and Blelloch, R (2009) Embryonic stem cell-specific microRNAs promote induced pluripotency Nat Biotechnol 27, 459-461 Kapranov, P., Cheng, J., Dike, S., Nix, D.A., Duttagupta, R., Willingham, A.T., Stadler, P.F., Hertel, J., Hackermuller, J., Hofacker, I.L., et al (2007) RNA maps reveal new RNA classes and a possible function for pervasive transcription Science 316, 1484-1488 Katayama, S., Tomaru, Y., Kasukawa, T., Waki, K., Nakanishi, M., Nakamura, M., Nishida, H., Yap, C.C., Suzuki, M., Kawai, J., et al (2005) Antisense transcription in the mammalian transcriptome Science 309, 1564-1566 Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.I., and Sasai, Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity Neuron 28, 31-40 Khalil, A.M., Guttman, M., Huarte, M., Garber, M., Raj, A., Rivea Morales, D., Thomas, K., Presser, A., Bernstein, B.E., van Oudenaarden, A., et al (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression Proc Natl Acad Sci U S A 106, 11667-11672 Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H (2008) An extended transcriptional network for pluripotency of embryonic stem cells Cell 132, 10491061 Kim, T.K., Hemberg, M., Gray, J.M., Costa, A.M., Bear, D.M., Wu, J., Harmin, D.A., Laptewicz, M., Barbara-Haley, K., Kuersten, S., et al (2010) Widespread transcription at neuronal activity-regulated enhancers Nature 465, 182-187 Kong, L., Zhang, Y., Ye, Z.Q., Liu, X.Q., Zhao, S.Q., Wei, L., and Gao, G (2007) CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine Nucleic Acids Res 35, W345-349 Kriks, 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 (2011) Dopamine neurons derived   161  from human ES cells efficiently engraft in animal models of Parkinson's disease Nature 480, 547-551 Lamb, T.M., Knecht, A.K., Smith, W.C., Stachel, S.E., Economides, A.N., Stahl, N., Yancopolous, G.D., and Harland, R.M (1993) Neural induction by the secreted polypeptide noggin Science 262, 713-718 Le, M.T., Xie, H., Zhou, B., Chia, P.H., Rizk, P., Um, M., Udolph, G., Yang, H., Lim, B., and Lodish, H.F (2009) MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets Mol Cell Biol 29, 5290-5305 Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Tabar, V., and Studer, L (2007) Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells Nat Biotechnol 25, 1468-1475 Lee, S.M., Danielian, P.S., Fritzsch, B., and McMahon, A.P (1997) Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain Development 124, 959-969 Li, W., Sun, W., Zhang, Y., Wei, W., Ambasudhan, R., Xia, P., Talantova, M., Lin, T., Kim, J., Wang, X., et al (2011) Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors Proc Natl Acad Sci U S A 108, 8299-8304 Lin, M., Pedrosa, E., Shah, A., Hrabovsky, A., Maqbool, S., Zheng, D., and Lachman, H.M (2011) RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders PLoS One 6, e23356 Loewer, S., Cabili, M.N., Guttman, M., Loh, Y.H., Thomas, K., Park, I.H., Garber, M., Curran, M., Onder, T., Agarwal, S., et al (2010) Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stem cells Nat Genet 42, 1113-1117 Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., et al (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells Nat Genet 38, 431440 Malatesta, P., Hartfuss, E., and Gotz, M (2000) Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage Development 127, 52535263   162  Mehler, M.F., and Mattick, J.S (2006) Non-coding RNAs in the nervous system J Physiol 575, 333-341 Meola, N., Pizzo, M., Alfano, G., Surace, E.M., and Banfi, S (2012) The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina RNA 18, 111-123 Mercer, T.R., Dinger, M.E., Sunkin, S.M., Mehler, M.F., and Mattick, J.S (2008) Specific expression of long noncoding RNAs in the mouse brain Proc Natl Acad Sci U S A 105, 716-721 Mercer, T.R., Qureshi, I.A., Gokhan, S., Dinger, M.E., Li, G., Mattick, J.S., and Mehler, M.F (2010) Long noncoding RNAs in neuronal-glial fate specification and oligodendrocyte lineage maturation BMC Neurosci 11, 14 Mohammad, F., Mondal, T., Guseva, N., Pandey, G.K., and Kanduri, C (2010) Kcnq1ot1 noncoding RNA mediates transcriptional gene silencing by interacting with Dnmt1 Development 137, 2493-2499 Morizane, A., Doi, D., Kikuchi, T., Nishimura, K., and Takahashi, J (2011) Smallmolecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells J Neurosci Res 89, 117-126 Munoz-Sanjuan, I., and Brivanlou, A.H (2002) Neural induction, the default model and embryonic stem cells Nat Rev Neurosci 3, 271-280 Nakatani, T., Kumai, M., Mizuhara, E., Minaki, Y., and Ono, Y (2010) Lmx1a and Lmx1b cooperate with Foxa2 to coordinate the specification of dopaminergic neurons and control of floor plate cell differentiation in the developing mesencephalon Dev Biol 339, 101-113 Naruse, Y., Aoki, T., Kojima, T., and Mori, N (1999) Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes Proc Natl Acad Sci U S A 96, 13691-13696 Niwa, H., Miyazaki, J., and Smith, A.G (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells Nat Genet 24, 372-376 Noctor, S.C., Flint, A.C., Weissman, T.A., Dammerman, R.S., and Kriegstein, A.R (2001) Neurons derived from radial glial cells establish radial units in neocortex Nature 409, 714-720   163  Orom, U.A., Derrien, T., Beringer, M., Gumireddy, K., Gardini, A., Bussotti, G., Lai, F., Zytnicki, M., Notredame, C., Huang, Q., et al (2010) Long noncoding RNAs with enhancer-like function in human cells Cell 143, 46-58 Pandey, R.R., Mondal, T., Mohammad, F., Enroth, S., Redrup, L., Komorowski, J., Nagano, T., Mancini-Dinardo, D., and Kanduri, C (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation Mol Cell 32, 232-246 Pang, K.C., Frith, M.C., and Mattick, J.S (2006) Rapid evolution of noncoding RNAs: lack of conservation does not mean lack of function Trends Genet 22, 1-5 Pang, K.C., Stephen, S., Engstrom, P.G., Tajul-Arifin, K., Chen, W., Wahlestedt, C., Lenhard, B., Hayashizaki, Y., and Mattick, J.S (2005) RNAdb a comprehensive mammalian noncoding RNA database Nucleic Acids Res 33, D125-130 Pereira, C.F., Piccolo, F.M., Tsubouchi, T., Sauer, S., Ryan, N.K., Bruno, L., Landeira, D., Santos, J., Banito, A., Gil, J., et al (2010) ESCs require PRC2 to direct the successful reprogramming of differentiated cells toward pluripotency Cell Stem Cell 6, 547-556 Perrier, A.L., Tabar, V., Barberi, T., Rubio, M.E., Bruses, J., Topf, N., Harrison, N.L., and Studer, L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells Proc Natl Acad Sci U S A 101, 12543-12548 Poluch, S., and Juliano, S.L (2007) A normal radial glial scaffold is necessary for migration of interneurons during neocortical development Glia 55, 822-830 Pomp, O., Brokhman, I., Ziegler, L., Almog, M., Korngreen, A., Tavian, M., and Goldstein, R.S (2008) PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells Brain Res 1230, 50-60 Ponjavic, J., Oliver, P.L., Lunter, G., and Ponting, C.P (2009) Genomic and transcriptional co-localization of protein-coding and long non-coding RNA pairs in the developing brain PLoS Genet 5, e1000617 Puligilla, C., Dabdoub, A., Brenowitz, S.D., and Kelley, M.W (2010) Sox2 induces neuronal formation in the developing mammalian cochlea J Neurosci 30, 714-722 Rao, S., and Orkin, S.H (2006) Unraveling the transcriptional network controlling ES cell pluripotency Genome Biol 7, 230   164  Rapicavoli, N.A., Poth, E.M., and Blackshaw, S (2010) The long noncoding RNA RNCR2 directs mouse retinal cell specification BMC Dev Biol 10, 49 Redrup, L., Branco, M.R., Perdeaux, E.R., Krueger, C., Lewis, A., Santos, F., Nagano, T., Cobb, B.S., Fraser, P., and Reik, W (2009) The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing Development 136, 525-530 Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, L.H., Helms, J.A., Farnham, P.J., Segal, E., et al (2007) Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs Cell 129, 1311-1323 Rowitch, D.H., and Kriegstein, A.R (2010) Developmental genetics of vertebrate glial-cell specification Nature 468, 214-222 Rybak, A., Fuchs, H., Smirnova, L., Brandt, C., Pohl, E.E., Nitsch, R., and Wulczyn, F.G (2008) A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment Nat Cell Biol 10, 987-993 Salmena, L., Poliseno, L., Tay, Y., Kats, L., and Pandolfi, P.P (2011) A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? Cell 146, 353-358 Sarma, K., Levasseur, P., Aristarkhov, A., and Lee, J.T (2010) Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome Proc Natl Acad Sci U S A 107, 22196-22201 Sato, T., and Nakamura, H (2004) The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway Development 131, 4275-4285 Sheik Mohamed, J., Gaughwin, P.M., Lim, B., Robson, P., and Lipovich, L (2010) Conserved long noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic stem cells RNA 16, 324-337 Souquere, S., Beauclair, G., Harper, F., Fox, A., and Pierron, G (2010) Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies Mol Biol Cell 21, 4020-4027 Su, X., Kameoka, S., Lentz, S., and Majumder, S (2004) Activation of REST/NRSF target genes in neural stem cells is sufficient to cause neuronal differentiation Mol Cell Biol 24, 8018-8025   165  Suh, M.R., Lee, Y., Kim, J.Y., Kim, S.K., Moon, S.H., Lee, J.Y., Cha, K.Y., Chung, H.M., Yoon, H.S., Moon, S.Y., et al (2004) Human embryonic stem cells express a unique set of microRNAs Dev Biol 270, 488-498 Swistowska, A.M., da Cruz, A.B., Han, Y., Swistowski, A., Liu, Y., Shin, S., Zhan, M., Rao, M.S., and Zeng, X (2010) Stage-specific role for shh in dopaminergic differentiation of human embryonic stem cells induced by stromal cells Stem Cells Dev 19, 71-82 Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131, 861-872 Takahashi, K., and Yamanaka, S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126, 663-676 Tauler, J., Zudaire, E., Liu, H., Shih, J., and Mulshine, J.L (2010) hnRNP A2/B1 modulates epithelial-mesenchymal transition in lung cancer cell lines Cancer Res 70, 7137-7147 Tay, Y., Kats, L., Salmena, L., Weiss, D., Tan, S.M., Ala, U., Karreth, F., Poliseno, L., Provero, P., Di Cunto, F., et al (2011) Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs Cell 147, 344-357 Tay, Y., Zhang, J., Thomson, A.M., Lim, B., and Rigoutsos, I (2008a) MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation Nature 455, 1124-1128 Tay, Y.M., Tam, W.L., Ang, Y.S., Gaughwin, P.M., Yang, H., Wang, W., Liu, R., George, J., Ng, H.H., Perera, R.J., et al (2008b) MicroRNA-134 modulates the differentiation of mouse embryonic stem cells, where it causes post-transcriptional attenuation of Nanog and LRH1 Stem Cells 26, 17-29 Tochitani, S., and Hayashizaki, Y (2008) Nkx2.2 antisense RNA overexpression enhanced oligodendrocytic differentiation Biochem Biophys Res Commun 372, 691696 Tripathi, V., Ellis, J.D., Shen, Z., Song, D.Y., Pan, Q., Watt, A.T., Freier, S.M., Bennett, C.F., Sharma, A., Bubulya, P.A., et al (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation Mol Cell 39, 925-938   166  Tsai, M.C., Manor, O., Wan, Y., Mosammaparast, N., Wang, J.K., Lan, F., Shi, Y., Segal, E., and Chang, H.Y (2010) Long noncoding RNA as modular scaffold of histone modification complexes Science 329, 689-693 Uhde, C.W., Vives, J., Jaeger, I., and Li, M (2010) Rmst is a novel marker for the mouse ventral mesencephalic floor plate and the anterior dorsal midline cells PLoS One 5, e8641 Valenzuela, D.M., Economides, A.N., Rojas, E., Lamb, T.M., Nunez, L., Jones, P., Lp, N.Y., Espinosa, R., 3rd, Brannan, C.I., Gilbert, D.J., et al (1995) Identification of mammalian noggin and its expression in the adult nervous system J Neurosci 15, 6077-6084 Vazin, T., Becker, K.G., Chen, J., Spivak, C.E., Lupica, C.R., Zhang, Y., Worden, L., and Freed, W.J (2009) A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells PLoS One 4, e6606 Vazin, T., Chen, J., Lee, C.T., Amable, R., and Freed, W.J (2008) Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells Stem Cells 26, 1517-1525 Walker, E., Chang, W.Y., Hunkapiller, J., Cagney, G., Garcha, K., Torchia, J., Krogan, N.J., Reiter, J.F., and Stanford, W.L (2010) Polycomb-like associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell selfrenewal and differentiation Cell Stem Cell 6, 153-166 Wang, K.C., and Chang, H.Y (2011) Molecular mechanisms of long noncoding RNAs Mol Cell 43, 904-914 Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B.R., Protacio, A., Flynn, R.A., Gupta, R.A., et al (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression Nature 472, 120-124 Wang, Z., Gerstein, M., and Snyder, M (2009) RNA-Seq: a revolutionary tool for transcriptomics Nat Rev Genet 10, 57-63 Willingham, A.T., Orth, A.P., Batalov, S., Peters, E.C., Wen, B.G., Aza-Blanc, P., Hogenesch, J.B., and Schultz, P.G (2005) A strategy for probing the function of noncoding RNAs finds a repressor of NFAT Science 309, 1570-1573   167  Wilusz, J.E., Sunwoo, H., and Spector, D.L (2009) Long noncoding RNAs: functional surprises from the RNA world Genes Dev 23, 1494-1504 Xu, N., Papagiannakopoulos, T., Pan, G., Thomson, J.A., and Kosik, K.S (2009) MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells Cell 137, 647-658 Yan, Y., Yang, D., Zarnowska, E.D., Du, Z., Werbel, B., Valliere, C., Pearce, R.A., Thomson, J.A., and Zhang, S.C (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells Stem Cells 23, 781-790 Ye, W., Shimamura, K., Rubenstein, J.L., Hynes, M.A., and Rosenthal, A (1998) FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate Cell 93, 755-766 Ying, Q.L., Stavridis, M., Griffiths, D., Li, M., and Smith, A (2003) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture Nat Biotechnol 21, 183-186 Yu, H.B., Kunarso, G., Hong, F.H., and Stanton, L.W (2009) Zfp206, Oct4, and Sox2 are integrated components of a transcriptional regulatory network in embryonic stem cells J Biol Chem 284, 31327-31335 Yu, P.B., Hong, C.C., Sachidanandan, C., Babitt, J.L., Deng, D.Y., Hoyng, S.A., Lin, H.Y., Bloch, K.D., and Peterson, R.T (2008) Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism Nat Chem Biol 4, 33-41 Zeng, X., Cai, J., Chen, J., Luo, Y., You, Z.B., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., et al (2004) Dopaminergic differentiation of human embryonic stem cells Stem Cells 22, 925-940 Zhang, Y., Liu, X.S., Liu, Q.R., and Wei, L (2006) Genome-wide in silico identification and analysis of cis natural antisense transcripts (cis-NATs) in ten species Nucleic Acids Res 34, 3465-3475 Zhou, J., Su, P., Li, D., Tsang, S., Duan, E., and Wang, F (2010) High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors Stem Cells 28, 1741-1750   168  Appendices Appendix I: List of 152 genes downregulated upon RMST and SOX2 knockdown LMLN CRYM MGC42105 LOC645233 GOLSYN C2orf80 TDG LOC338758 PPEF1 UBE2L3 SMC4 LOC440585 C1orf19 CAMK2N1 TSEN15 CA14 LOC646808 LPPR5 PEG10 C9orf50 LOC646786 CEND1 CACNB2 GNPDA1 FGFBP3 ITPK1 CENPH DDIT4 PIP4K2A C8orf30A LRRC8D RAB33A CAPS C18orf51 SLAIN1 VPS24 LCLAT1 TNFAIP8L1 LOC100133298 GALNT1 CPSF2 FLJ31568 LOC387856 JAM2   MED30 DOCK10 NBPF20 E2F7 LOC282997 AFF3 NUCKS1 LOC100128221 MXRA5 LOC646345 ZNF689 PLEKHB2 RSHL3 VKORC1L1 GPM6B SAT1 STC1 SLC38A1 LOC729446 PPARGC1A NEUROG2 PLP1 NDE1 LOC442727 OSR1 FBXW8 CTHRC1 ELMOD1 BRD3 ZNF462 FLJ20021 PLA2G3 FYN PPTC7 PIK3R2 ADAM19 PCDH17 CCDC34 MTCP1 RAB13 TMTC2 FANCI ARHGEF6 KIF5C MIF4GD ENKUR SLC5A3 SLC44A1 SV2A MAPK10 LOC728475 TYSND1 FAM59A DYRK2 DNAJC30 SLC35F1 LOC389168 XPR1 EHBP1 LOC730202 DLX1 TMEM107 SIK3 BANP RECQL MRE11A FRS3 ASXL2 LOC729065 ASCL1 TOP3A KCNJ10 CLCN2 TUBB3 PRR11 SBK1 COL4A3BP TBX2 CCNY ELOVL2 ST8SIA5 LOC643995 C14orf80 DOCK7 MAP4K4 FOXO3 PCGF6 TMEM5 BEND5 PTPLAD1 QKI ZNF483 CNR1 LOC654126 RALGDS STK39 MMAB C9orf140 B4GALT5 ZNF586 C20orf100 HSPC268 RHOJ HIRIP3 MOCS1 ATP6V0E2 HEY2 KLHL13 169  Appendix II: List of 331 genes upregulated upon RMST and SOX2 knockdown CLDN1 IFI27 IL32 IFI44L VCAM1 CXCL10 MYBPC2 HERC5 EPSTI1 SAMD9 MX1 OASL GBP1 OAS2 OAS1 LOX AFAP1L2 ANGPTL4 LYSMD3 HERC6 TNFAIP2 IL15RA GXYLT2 IFI44 SLC44A3 ZNF429 DKFZP686E215 PPARA MEG3 HIST1H2AC ZNF493 RELB FBN1 ISG15 MYL9 IFIT1 TMEM229B IFIH1 LGALS9 BIRC3 ABTB1 LOC730087 LOC646731 AFTPH RAB11FIP1 MKRN3 IL15 LOC645402   LOC652741 C5orf56 IFITM1 FAM176A MATN3 PRSS35 TDRD7 BST2 ANK1 NFKB2 TAGLN MYC DDX60L N4BP2L2 OPRL1 CUL4B RARRES3 FAM5C TMEM166 OAS3 STX3 EEA1 CYP1B1 SERTAD4 LOC100129681 CLIP4 ECE1 DRAM1 RDH8 CD83 KDELR3 STAT1 DDX58 KPNA5 PLSCR4 PLAUR C19orf66 PARP12 PCTP TNKS1BP1 TTC39C CYorf15B IFIT3 UBA7 METRNL H19 NQO1 VPS4B TNS3 COL5A3 ENPP5 THBS1 DDX60 CROT TRPM4 ZNF37A PARP10 CLCN3 FAM46B ULBP2 IFI6 CARD10 WDR37 TMTC1 APOL2 ANKRD46 ROR1 NNMT LOC100129445 PGM2L1 LAMB2 QPCT CCL2 TTYH2 ZNF404 PPIC LAMB3 LOC100131091 TUFT1 CCDC19 IRAK2 GOLT1B ABCC3 C19orf12 LEPREL1 ITSN1 C17orf91 EXOC5 RSAD2 ZNFX1 EDEM1 OPTN USP18 ATP2B4 DUSP5 PRIC285 DCP2 BTN3A3 IRF9 TNPO1 CCDC126 ABCF3 GCHFR ELOVL4 ST6GALNAC6 SP110 IFI35 CHST15 TPM1 SDC4 LOC652968 C8orf58 IRF7 LYG1 EPHA2 CASP7 MBD1 GOLGA4 ZC3HAV1 ERAP2 CHPF2 IRF1 PLSCR1 C1orf109 CD46 TRIM21 NPHP3 ABCC5 C1orf133 ACSL4 PGCP HERPUD1 ALCAM FBXO32 GCA MBP PIM2 S1PR3 PRPF4B RNF24 SYTL2 CAMTA1 HLA-B XKR8 BLCAP FZD4 FAM167A 170  ZNF83 ST13 UQCC SEZ6L2 PIGZ PLCD1 CHRNA1 LOC643310 SPHAR FLNB ATF1 SEMA5B RAB11FIP2 CYP2R1 SEMA4F ENPP4 TCIRG1 TPD52L1 OGFR GAL PARP9 CNN2 GLRB VLDLR PHLDA2 MAP1LC3B LOC389386 LOC652900 LIN37 BRMS1L STAT2 RNF149 JUP OLFML2B IL18BP SPTLC1 JPH2 LPPR2 NXN CHCHD7 XRN1 LCTL TBL1XR1 PARP14 ISG20 CYR61 HLA-F SLC39A14 TNC LOC341230 SHB BTRC ATP7B   IER3 VWA1 GOLGA3 FDX1 SMAGP GALNTL1 SRPR NHLRC3 EPB41L1 PSMB8 LOC340274 TMF1 MAOB MGC4677 PCK2 LDLR INPP5A ADARB1 PLA2G15 GPR126 FAM111A EIF5A2 ZNF697 TMEM44 C10orf6 STK3 RAB4A YWHAE ZNF721 PDLIM7 RALB MIR221 C19orf28 DNAJB9 CRIM1 PHF1 SLC9A7 FAT1 FAM135A COG6 TRIM56 MPP1 ANKRD49 FER1L3 PDLIM1 GALNT10 LOC399959 ITGAV RASSF7 TNFRSF12A TYW3 PHLPP2 PCGF5 COQ10B ZNF385A PLEKHA1 TRIB1 ATG16L1 PXMP3 DICER1 ASS1 VPS36 AHNAK2 CRY2 RNF19B STK10 NAT13 C9orf3 LBH CDS2 AKAP8 COL6A1 GPR137C C6orf106 IFNGR1 IFIT2 SRXN1 TOM1L1 CGNL1 KIAA1641 LOC648740 KITLG PARD6G 171  ... established in the mouse model system, and very few human lncRNAs have been studied with such extent In this thesis, I focused on examining lncRNAs involved in the pluripotency maintenance of human embryonic. .. B., Liu, M., and Shi, T (2011) Comparative analysis of human protein -coding and noncoding RNAs between brain and 10 mixed cell lines by RNA-Seq PLoS One 6, e28318 Chen, M., Zhang, J., and Manley,... Amable, R., and Freed, W.J (2008) Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells Stem Cells 26, 151 7- 152 5 Walker, E.,