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• W2157642912 abstract "•Purification of iPSC-derived mdDA neurons and primary embryonic mdDA neurons•Comparative gene-expression profiling and DNA methylation mapping of mdDA neurons•High similarity but also differences between primary and iPSC-derived mdDA neurons•Differences mainly in genes involved in neuron differentiation and development Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3Gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3Gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy. Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3Gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3Gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy. The field of regenerative medicine experienced a powerful impetus after the groundbreaking discovery of induced pluripotency (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (19304) Google Scholar). Numerous publications have shown that mouse as well as human induced pluripotent stem cells (iPSCs) have the potency to differentiate into various clinically relevant cell types, such as cardiomyocytes (Kuzmenkin et al., 2009Kuzmenkin A. Liang H. Xu G. Pfannkuche K. Eichhorn H. Fatima A. Luo H. Saric T. Wernig M. Jaenisch R. Hescheler J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro.FASEB J. 2009; 23: 4168-4180Crossref PubMed Scopus (106) Google Scholar, Ren et al., 2011Ren Y. Lee M.Y. Schliffke S. Paavola J. Amos P.J. Ge X. Ye M. Zhu S. Senyei G. Lum L. et al.Small molecule Wnt inhibitors enhance the efficiency of BMP-4-directed cardiac differentiation of human pluripotent stem cells.J. Mol. Cell. Cardiol. 2011; 51: 280-287Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), hepatocytes (Espejel et al., 2010Espejel S. Roll G.R. McLaughlin K.J. Lee A.Y. Zhang J.Y. Laird D.J. Okita K. Yamanaka S. Willenbring H. Induced pluripotent stem cell-derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice.J. Clin. Invest. 2010; 120: 3120-3126Crossref PubMed Scopus (153) Google Scholar), hematopoietic progenitors (Hanna et al., 2007Hanna J. Wernig M. Markoulaki S. Sun C.W. Meissner A. Cassady J.P. Beard C. Brambrink T. Wu L.C. Townes T.M. Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.Science. 2007; 318: 1920-1923Crossref PubMed Scopus (1245) Google Scholar), oligodendrocytes (Czepiel et al., 2011Czepiel M. Balasubramaniyan V. Schaafsma W. Stancic M. Mikkers H. Huisman C. Boddeke E. Copray S. Differentiation of induced pluripotent stem cells into functional oligodendrocytes.Glia. 2011; 59: 882-892Crossref PubMed Scopus (100) Google Scholar), and specific subtypes of neurons (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 (313) Google Scholar, Wernig et al., 2008Wernig M. Zhao J.-P. Pruszak J. Hedlund E. Fu D. Soldner F. Broccoli V. Constantine-Paton M. Isacson O. Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease.Proc. Natl. Acad. Sci. USA. 2008; 105: 5856-5861Crossref PubMed Scopus (1026) Google Scholar). Such in vitro-generated iPSC-derived cell types provide new possibilities for disease modeling and cell replacement strategies. In particular, the generation of autologous iPSC-derived midbrain dopaminergic (DA) neurons provides a very interesting tool to study and treat Parkinson’s disease (PD) (Roessler et al., 2013Roessler R. Boddeke E. Copray S. Induced pluripotent stem cell technology and direct conversion: new possibilities to study and treat Parkinson’s disease.Stem Cell Rev. 2013; 9: 505-513Crossref PubMed Scopus (9) Google Scholar). However, future clinical application of iPSC-derived DA neurons can only be considered realistic if the desired cell population is strictly purified and completely defined. Several groups have reported the generation of DA neurons from mouse and human iPSCs (Hargus et al., 2010Hargus G. Cooper O. Deleidi M. Levy A. Lee K. Marlow E. Yow A. Soldner F. Hockemeyer D. Hallett P.J. et al.Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.Proc. Natl. Acad. Sci. USA. 2010; 107: 15921-15926Crossref PubMed Scopus (380) Google Scholar, Swistowski et al., 2010Swistowski A. Peng J. Liu Q. Mali P. Rao M.S. Cheng L. Zeng X. Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions.Stem Cells. 2010; 28: 1893-1904Crossref PubMed Scopus (255) Google Scholar, Wernig et al., 2008Wernig M. Zhao J.-P. Pruszak J. Hedlund E. Fu D. Soldner F. Broccoli V. Constantine-Paton M. Isacson O. Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease.Proc. Natl. Acad. Sci. USA. 2008; 105: 5856-5861Crossref PubMed Scopus (1026) Google Scholar). In these studies, iPSC-derived neurons displayed expression of crucial DA markers and exhibited typical neuronal electrophysiological properties. Furthermore, these iPSC-derived DA neurons could functionally integrate into a rat PD model upon transplantation. In principle, these results indicated that a DA neuronal population could be obtained from iPSCs and present important midbrain DA neuronal characteristics. However, genome-wide studies comparing the genetic and epigenetic features of iPSC-derived DA neurons versus primary DA neurons are currently lacking. Since reprogramming of somatic cells to iPSCs resets their identity back to an embryonic stage, iPSC-derived differentiated neurons should be considered freshly formed “embryonic” neurons. Accordingly, a relevant comprehensive comparison of iPSC-derived DA neurons can only be done with freshly formed embryonic and perinatal primary mesodiencephalic DA (mdDA) neurons. We generated iPSC lines from Pitx3Gfp/+ knockin mouse embryonic fibroblasts. PITX3 is a highly specific mdDA neuron marker that is required for DA neuron differentiation in the substantia nigra (Jacobs et al., 2009Jacobs F.M. van Erp S. van der Linden A.J. von Oerthel L. Burbach J.P. Smidt M.P. Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression.Development. 2009; 136: 531-540Crossref PubMed Scopus (131) Google Scholar, Jacobs et al., 2011Jacobs F.M. Veenvliet J.V. Almirza W.H. Hoekstra E.J. von Oerthel L. van der Linden A.J. Neijts R. Koerkamp M.G. van Leenen D. Holstege F.C. et al.Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons.Development. 2011; 138: 5213-5222Crossref PubMed Scopus (53) Google Scholar, Smidt et al., 2004Smidt M.P. Smits S.M. Bouwmeester H. Hamers F.P. van der Linden A.J. Hellemons A.J. Graw J. Burbach J.P. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3.Development. 2004; 131: 1145-1155Crossref PubMed Scopus (272) Google Scholar). Specific PITX3-associated GFP expression allowed us to strictly identify and purify DA neurons from either iPSCs or the ventral midbrain at specific developmental stages by fluorescence-activated cell sorting (FACS). We then subjected these mdDA neurons to genome-wide gene-expression analysis comparing iPSC-derived DA neurons and primary isolated mdDA neurons. Induction of pluripotency in somatic cells is considered an epigenetic process that entails, among other events, a large series of changes in DNA methylation patterns (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 (746) Google Scholar, Maherali et al., 2007Maherali N. Sridharan R. Xie W. Utikal J. Eminli S. Arnold K. Stadtfeld M. Yachechko R. Tchieu J. Jaenisch R. et al.Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell. 2007; 1: 55-70Abstract Full Text Full Text PDF PubMed Scopus (1425) Google Scholar, Nishino et al., 2011Nishino K. Toyoda M. Yamazaki-Inoue M. Fukawatase Y. Chikazawa E. Sakaguchi H. Akutsu H. Umezawa A. DNA methylation dynamics in human induced pluripotent stem cells over time.PLoS Genet. 2011; 7: e1002085Crossref PubMed Scopus (235) Google Scholar, Ohi et al., 2011Ohi Y. Qin H. Hong C. Blouin L. Polo J.M. Guo T. Qi Z. Downey S.L. Manos P.D. Rossi D.J. et al.Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells.Nat. Cell Biol. 2011; 13: 541-549Crossref PubMed Scopus (460) Google Scholar). Furthermore, iPSC differentiation into neural progenitor cells and subsequently into a specific neuronal subtype depends on properly established de novo DNA methylation (Lee et al., 2010Lee S.-H. Jeyapalan J.N. Appleby V. Mohamed Noor D.A. Sottile V. Scotting P.J. Dynamic methylation and expression of Oct4 in early neural stem cells.J. Anat. 2010; 217: 203-213Crossref PubMed Scopus (21) Google Scholar, Watanabe et al., 2006Watanabe D. Uchiyama K. Hanaoka K. Transition of mouse de novo methyltransferases expression from Dnmt3b to Dnmt3a during neural progenitor cell development.Neuroscience. 2006; 142: 727-737Crossref PubMed Scopus (82) Google Scholar). Therefore, it is essential not only to evaluate gene expression but also to compare the methylome of iPSC-derived DA neurons with their primary counterparts. In order to obtain a comprehensive profile of the functionally most relevant DNA methylation sites in iPSC-derived DA neurons versus primary mdDA neurons, we performed a genome-wide analysis of CpG island (CGI) methylation using reduced representation bisulfite sequencing (RRBS) (Meissner et al., 2005Meissner A. Gnirke A. Bell G.W. Ramsahoye B. Lander E.S. Jaenisch R. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis.Nucleic Acids Res. 2005; 33: 5868-5877Crossref PubMed Scopus (826) Google Scholar, Smallwood et al., 2011Smallwood S.A. Tomizawa S.-I. Krueger F. Ruf N. Carli N. Segonds-Pichon A. Sato S. Hata K. Andrews S.R. Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos.Nat. Genet. 2011; 43: 811-814Crossref PubMed Scopus (506) Google Scholar) on genomic DNA samples isolated from purified neuronal populations. iPSCs were generated from embryonic fibroblasts of Pitx3Gfp/+ transgenic mice and characterized (Figure S1 available online). DA-specific differentiation was performed according to previously described protocols (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 (2477) Google Scholar, Kawasaki et al., 2000Kawasaki H. Mizuseki K. Nishikawa S. Kaneko S. Kuwana Y. Nakanishi S. Nishikawa S.I. Sasai Y. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.Neuron. 2000; 28: 31-40Abstract Full Text Full Text PDF PubMed Scopus (1120) Google Scholar) and resulted in microtubule-associated protein 2 (MAP2)- and tyrosine hydroxylase (TH)-positive PITX3-expressing neurons that exhibited the typical morphology of mature DA neurons (Figures 1A and 1B ). A comparative FACS profile showed an absence of GFP-expressing cells within the undifferentiated iPSC population, whereas the DA-differentiated population contained a distinguishable subgroup of GFP-expressing cells (Figure 1C). Upon lineage-specific differentiation, iPSC-derived DA neurons were able to secrete dopamine (Figure 1D), and patch-clamp recordings revealed bona fide electrophysiological properties of the iPSC-derived mdDA neurons (Figures 1E–1G). PITX3-GFP-sorted cells functionally integrated into 6OHDA-lesioned rat brains after intrastriatal implantation, causing a reduction in amphetamine-induced rotation behavior (Figure S2). Our data show successful differentiation of iPSCs toward functional PITX3-expressing mdDA neurons that could be purified from an undefined iPSC-derived cell population. Next, we set out to compare the global gene-expression profile of the iPSC-derived DA neurons with that of the primary mdDA neurons. We performed a genome-wide comparative gene-expression analysis with iPSC-derived PITX3-GFP-positive cells and primary isolated mdDA neurons at several developmental stages (embryonic day 12.5 [E12.5] to postnatal day 0 [P0]; see Figure 2A for experimental scheme). FACS sorting with an efficiency of 98% allowed purification of iPSC-derived PITX3-GFP-positive cells and primary PITX3-GFP-positive mdDA neurons (Figure 2B). Telencephalic brain homogenate served as the negative control for primary cells (Figure 2B), and undifferentiated iPSCs served as the negative control for iPSC-derived GFP-positive cells (as shown in Figure 1C). Correlation of the genome-wide expression profiles of Pitx3Gfp/+ mdDA neurons at different developmental stages (Figure 2C) revealed the highest similarities between embryonic mdDA neurons (r = 0.92–0.98). In comparison, the gene-expression profile of iPSC-derived DA neurons was less correlated (highest correlation [r = 0.66] with E14.5 mdDA neurons); however, the weakest correlation was found between iPSC-derived DA neurons and P0 mdDA neurons (r = 0.55). We performed Gene Ontology (GO) term analyses to annotate genes that were differentially expressed in iPSC-derived DA neurons in comparison with primary embryonic mdDA neurons. We found that the genes that were most downregulated in iPSC-derived DA neurons were associated with GO terms such as nervous system development, neuron differentiation, and neurogenesis (Figure 2D). We analyzed the expression of an mdDA-specific subset of genes in more detail and found high similarities between iPSC-derived DA neurons and most primary mdDA neurons (Figure 2E). Hierarchical clustering revealed several mdDA-specific genes equally up- or downregulated in primary isolated and iPSC-derived DA neurons. Sample cluster analysis revealed the strongest gene-expression correlation between iPSC-derived mdDA neurons and embryonic primary neurons, whereas postnatal-stage primary mdDA neurons clustered separately. Several key DA genes, such as Otx2, FoxA1, FoxA2, Nr4a2 (Nurr1), Lmx1a, and Lmx1b were similarly expressed in iPSC-derived mdDA neurons and in embryonic mdDA neurons. Other DA genes, such as En1 and En2, showed expression levels in iPSC-derived mdDA neurons comparable to those in postnatal mdDA neurons. We extended our gene-expression profiling to additional mdDA-specific transcription factors (Chakrabarty et al., 2012Chakrabarty K. Von Oerthel L. Hellemons A. Clotman F. Espana A. Groot Koerkamp M. Holstege F.C. Pasterkamp R.J. Smidt M.P. Genome wide expression profiling of the mesodiencephalic region identifies novel factors involved in early and late dopaminergic development.Biol. Open. 2012; 1: 693-704Crossref PubMed Scopus (35) Google Scholar), axonal guidance factors, and ion channels (Figure S3). While we observed moderate correlations between iPSC-derived mdDA neurons and primary DA neurons for transcription factors and axonal guidance factors, we found a high correlation for expression levels of ion channels, with the strongest overall correlation (r = 0.82) between E12.5 mdDA neurons and iPSC-derived DA neurons. In view of the origin of iPSC-derived DA neurons, we also analyzed the transcript profile for pluripotency genes and fibroblast-related genes, visualized by dendrograms (Figure 2F). The gene expression of a set of pluripotency markers was subjected to cluster analysis, which showed similar transcript levels (e.g., Nanog, Oct4 (Pou5f1), Zfp42, and Nr0b1 [also known as Dax1]) in primary and iPSC-derived cell types (Figure 2F). This result not only substantiates a successful transition away from a pluripotent state but also indicates appropriate silencing of pluripotency genes upon in vitro differentiation. However, a similar cluster analysis performed on a subset of fibroblast-specific markers revealed differential expression in primary and iPSC-derived neurons, suggesting remnants of a still active fibroblast gene program in iPSC-derived DA neurons. In summary, comparative gene-expression profiling of purified iPSC-derived DA neurons and embryonic mdDA neurons revealed a clear correlation, but less similarity was found between iPSC-derived DA neurons and P0 DA neurons. Downregulated genes in iPSC-derived DA neurons were mainly associated with biological functions such as nervous system development, neurogenesis, and neuron differentiation. These findings prompted us to investigate the nature of this downregulation and to extend our gene-expression analysis by performing in-depth epigenetic profiling focused on DNA methylation. First, in order to validate our genome-wide expression profiling data, we performed quantitative PCR (qPCR) experiments for a set of selected key DA genes (Pitx3, Nurr1, Dlk1, En1, and En2), Sox9, and Desmin. The expression levels of these particular genes appeared to be in line with the results obtained by microarray analysis, and indicate that DA-specific marker expression in iPSC-derived DA neurons most closely resembles that of terminally differentiated-stage mdDA neurons (Figure 3A). As far as the expression of Pitx3, Nurr1, En1, and En2 was concerned, the highest similarity was found between iPSC-derived DA and E16.5 mdDA neurons (Figure 3B). Next, we set out to compare the DNA methylation profiles associated with the selected key DA gene expression of iPSC-derived DA and primary mdDA neurons. DNA methylation is a major player in epigenetic regulation of gene expression, and strong methylation of promoters is generally associated with gene silencing. RRBS allowed us to analyze specific CpG methylation at nucleotide resolution on a genome-wide scale. RRBS provides coverage preferentially of CpG-rich regions, such as CGIs. CGIs are predominantly associated with promoter regions surrounding the transcription start site (TSS). We identified and analyzed CGIs and their methylation status for the aforementioned key mdDA factors and found a striking correlation between CGI methylation levels and gene expression (Figure 3C). Pitx3, the mdDA marker we used to purify both cell types, revealed equally low methylation in the CGI around exon 1. Active expression of Pitx3 in both cell types might be due to this permissive DNA methylation state. CGIs in gene bodies of Nurr1, En1, and En2 appeared to be more strongly methylated in iPSC-derived DA neurons than in primary mdDA neurons. This specific DNA methylation state in iPSC-derived DA neurons may underlie the somewhat lower relative levels of expression of these key mdDA factors in iPSC-DA neurons in comparison with the primary mdDA neurons. Interestingly, Nurr1 showed the highest methylation in a CGI around exon 5, which may point to an alternative promoter for the expression of Nurr1. Similarly, increased methylation was observed for En1 and En2, in particular at CGIs around exon 1. Additional key mdDA transcription factors, such as Otx2, Lmx1a, Lmx1b, FoxA1, FoxA2, and the dopamine receptor Drd2, were analyzed and their CGI methylation status was examined (Figure S4). Overall, increased CGI methylation in iPSC-DA neurons appeared to be at intermediate levels (around 50%) in comparison with native mdDA neurons. These differences in DNA methylation patterns and, accordingly, the reduction in DA gene-expression levels may have subtle, as yet uncharacterized effects on the proper functionality of iPSC-derived DA neurons. In order to obtain a global view of DNA methylation, we next set out to compare total CpG methylation and CGI methylation in primary mdDA neurons and iPSC-derived mdDA neurons. To determine whether DA neurons derived from iPSCs recapitulate the DNA methylation landscape of their in vivo counterparts isolated from the ventral midbrain, we performed a genome-wide analysis of 5mC (Figure 4). We obtained information on 844,812 CpGs (≥5 read depth) for iPSC-DA neurons and mdDA neurons, with a bisulfite conversion efficiency of >98% as assessed by non-CpG methylation. Of these, 72% corresponded to CGIs in iPSC-DA neurons and mdDA neurons. Global methylation levels outside of CGIs were similar in iPSC-DA and mdDA neurons, and comparable to those observed in somatic tissues in general (Illingworth et al., 2008Illingworth R. Kerr A. Desousa D. Jørgensen H. Ellis P. Stalker J. Jackson D. Clee C. Plumb R. Rogers J. et al.A novel CpG island set identifies tissue-specific methylation at developmental gene loci.PLoS Biol. 2008; 6: e22Crossref PubMed Scopus (502) Google Scholar, Illingworth et al., 2010Illingworth R.S. Gruenewald-Schneider U. Webb S. Kerr A.R. James K.D. Turner D.J. Smith C. Harrison D.J. Andrews R. Bird A.P. Orphan CpG islands identify numerous conserved promoters in the mammalian genome.PLoS Genet. 2010; 6: e1001134Crossref PubMed Scopus (389) Google Scholar; Figures 4A and 4B). Similarly, the CpG methylation levels of iPSC-DA neurons and mdDA neurons were highly correlated (r = 0.85; Figures 4C and 4D). On the other hand, in a CGI context, iPSC-DA neurons were relatively hypermethylated in comparison with mdDA neurons (Figure 4B), with the relative proportion of CpGs presenting intermediate levels of methylation (i.e., 40%–60%; Figures 4B and 4C). By plotting the percentage of CGI methylation in mdDA neurons against iPSC-DA neurons, we found a population of more than 2,000 genes with relative CGI-associated hypermethylation in iPSC-DA neurons (Figure 4D). As described above, this was also observed in the methylation levels of individual CGIs, with CGIs unmethylated in mdDA neurons presenting intermediate levels of methylation in iPSC-DA neurons (Figure 3C). Since CGI methylation is essentially bimodal (i.e., either methylated or unmethylated), this reflects a degree of epigenetic heterogeneity within the population of iPSC-derived DA neurons. To assess whether there is an impact on gene expression in this distinctly hypermethylated population, we analyzed the function of the associated genes and compared expression levels in developing mdDA neurons and iPSC-DA neurons. Although gene expression in general did not seem to be reduced in iPSC-derived DA neurons, a significant proportion of hypermethylated CGIs was observed (Figures 4D and S5). The most hypermethylated subset of genes (Figure S5C) was subjected to GO analysis (Figure 5A). Interestingly, the most significantly enriched biological functions, such as neuron differentiation and neuron development, corresponded with the most enriched GO terms found in our reduced-gene-expression analysis (see Figure 2D). We then analyzed the correlation of gene expression for genes involved in neuron differentiation (169 genes with a corrected p value of 2.93E-47) between all developmental stages and iPSC-derived DA neurons (Figure 5B). Correlations of neuron differentiation-associated gene expression in iPSC-derived neurons strongly decreased with increasing maturation of the embryonic mdDA neurons, with a correlation coefficient as low as 0.28 for iPSC-derived DA neurons versus P0 mdDA neurons. Of note, we found a higher correlation of gene expression between prenatal mdDA neurons and iPSC-derived neurons than between prenatal mdDA neurons and P0 mdDA neurons. After defining the biological functions of the hypermethylated subgroup of genes and establishing the correlation of expression for genes in the GO group “neuron differentiation,” we revisited the gene-expression data to visualize the actual transcript levels within a selection of the GO terms (Figures 5C–5E). We found substantial downregulation for multiple genes in three representative GO groups: “neuron development,” “axon guidance,” and “inner ear development” (nonneural GO term). Large clusters of downregulated genes were predominantly observed in P0 mdDA neurons and iPSC-derived DA neurons. Thus, we identified a subset of genes in iPSC-derived DA neurons that show intermediate hypermethylation. Many, but not all, of these genes show reduced expression levels compared with embryonic mdDA neurons. Crucially, gene annotation revealed that a large fraction of the hypermethylated genes are involved in biological functions relevant to neuron development and differentiation. In view of their potential use in cell-based therapy for patients with PD, midbrain DA neurons were one of the first cell types to be generated from iPSCs. The resemblance between iPSC-derived DA neurons and true DA neurons has been studied based on a number of mainly morphological and functional properties, as well as mdDA-specific gene sets (Hedlund et al., 2008Hedlund E. Pruszak J. Lardaro T. Ludwig W. Viñuela A. Kim K.S. Isacson O. Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson’s disease.Stem Cells. 2008; 26: 1526-1536Crossref PubMed Scopus (113) 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 (1346) Google Scholar, Wernig et al., 2008Wernig M. Zhao J.-P. Pruszak J. Hedlund E. Fu D. Soldner F. Broccoli V. Constantine-Paton M. Isacson O. Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease.Proc. Natl. Acad. Sci. USA. 2008; 105: 5856-5861Crossref PubMed Scopus (1026) Google Scholar). However, the risk of tumor growth and the heterogeneous molecular backgrounds of embryonic stem cell (ESC)- and iPSC-derived DA neurons have precluded the use of these cells for therapy in humans (Momčilović et al., 2014Momčilović O. Liu Q. Swistowski A. Russo-Tait T. Zhao Y. Rao M.S. Zeng X. Genome wide profiling of dopaminergic neurons derived from human embryonic and induced pluripotent stem cells.Stem Cells Dev. 2014; 23: 406-420Crossref PubMed Scopus (25) Google Scholar, Salti et al., 2013Salti A. Nat R. Neto S. Puschban Z. Wenning G. Dechant G. Expression of early developmental markers predicts the efficiency of embryonic stem cell differentiation into midbrain dopaminergic neurons.Stem Cells Dev. 2013; 22: 397-411Crossref PubMed Scopus (17) Google Scholar). Gaining a detailed understanding of the genetic and epigenetic signatures of ESC- and iPSC-derived DA neurons is therefore a critical step toward establishing cell-based therapy as a viable treatment for PD. In this study, we present a comparative genome-wide profile of the genetic and epigenetic features of iPSC-derived DA neurons and their primary counterparts. Although iPSC-derived DA neurons showed characteristics that are widely used to classify functional mdDA neurons (Ganat et al., 2012Ganat Y.M. Calder E.L. Kriks S. Nelander J. Tu E.Y. Jia F. Battista D. Harrison N. Parmar M. Tomishima M.J. et al.Identification of embryonic stem cell-derived midbrain dopaminergi" @default.
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• W2157642912 date "2014-04-01" @default.
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• W2157642912 title "Detailed Analysis of the Genetic and Epigenetic Signatures of iPSC-Derived Mesodiencephalic Dopaminergic Neurons" @default.
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