DNA methylation alterations in iPSC- and hESC-derived neurons: potential implications for neurological disease modeling
- Laura de Boni†1, 2View ORCID ID profile,
- Gilles Gasparoni†3,
- Carolin Haubenreich†2,
- Sascha Tierling3,
- Ina Schmitt1, 4,
- Michael Peitz2, 4,
- Philipp Koch2,
- Jörn Walter3Email author,
- Ullrich Wüllner1, 4Email author and
- Oliver Brüstle2Email author
© The Author(s). 2018
Received: 31 August 2017
Accepted: 4 January 2018
Published: 29 January 2018
Genetic predisposition and epigenetic alterations are both considered to contribute to sporadic neurodegenerative diseases (NDDs) such as Parkinson’s disease (PD). Since cell reprogramming and the generation of induced pluripotent stem cells (iPSCs) are themselves associated with major epigenetic remodeling, it remains unclear to what extent iPSC-derived neurons lend themselves to model epigenetic disease-associated changes. A key question to be addressed in this context is whether iPSC-derived neurons exhibit epigenetic signatures typically observed in neurons derived from non-reprogrammed human embryonic stem cells (hESCs).
Here, we compare mature neurons derived from hESC and isogenic human iPSC generated from hESC-derived neural stem cells. Genome-wide 450 K-based DNA methylation and HT12v4 gene array expression analyses were complemented by a deep analysis of selected genes known to be involved in NDD. Our studies show that DNA methylation and gene expression patterns of isogenic hESC- and iPSC-derived neurons are markedly preserved on a genome-wide and single gene level.
Overall, iPSC-derived neurons exhibit similar DNA methylation patterns compared to isogenic hESC-derived neurons. Further studies will be required to explore whether the epigenetic patterns observed in iPSC-derived neurons correspond to those detectable in native brain neurons.
The origin and pathophysiology of sporadic neurodegenerative diseases (NDDs) such as idiopathic Parkinson’s disease (iPD) still remain enigmatic. In this regard, epigenetic alterations of genes including α-synuclein (SNCA) could contribute to the pathophysiology of this devastating disorder or the individual susceptibility .
Previously, we have identified significantly reduced DNA methylation levels of SNCA intron 1 in PD brains . In SK-N-SH cells, DNA demethylation of the SNCA intron 1—using the DNA methyltransferase inhibitor 5-Azacytidine—was associated with an increase in SNCA mRNA and α-synuclein protein expression . This is an important finding as elevated α-synuclein protein levels are directly linked to the development of PD [3, 4]. To date, these findings could not be verified in follow-up studies  and/or in native neurons derived from iPD patients compared to controls due to the inaccessibility of living disease-affected cells.
In this context, in vitro differentiated neural cells from patient-derived induced pluripotent stem cells (iPSCs) could provide a tool to study human neurons. Human iPSCs have indeed already been employed to dissect the mechanisms of several mostly inherited NDDs [6–9]. iPSC-based models of PD, mainly focusing on familial forms of the disease, displayed some interesting and relevant phenotypes . Consequently, patient-specific iPSC-derived neurons could be used to establish in vitro models for iPD allowing epigenetic and gene expression studies. Recently, a comprehensive study comprising epigenetic analyses in PD iPSC models revealed an aberrant epigenome in iPSC-derived dopaminergic neurons (DAn) of iPD and LRRK2 PD patients . These data suggest that cell-specific and presumptive disease-associated epigenetic changes can be retrieved from iPSC-derived patient-specific neurons. Nevertheless, the process of induced reprogramming is based on erasing the existing epigenetic state of the cell of origin [12, 13]. One key question emerging in the context of pluripotent stem cell-based disease modeling is whether iPSC-derived neurons display epigenetic patterns resembling those of neurons derived from non-reprogrammed human embryonic stem cells (hESCs). To address this question, we employed an isogenic stem cell system to compare DNA methylation patterns of hESC- and iPSC-derived mature neurons both generated from the same parental hESC line. Such an isogenic approach should eliminate differences due to individual genetic variation. Specifically, we assessed (i) genome-wide DNA methylation of > 485.000 CpGs in potential regulatory elements such as promoters or enhancers, (ii) gene expression of > 47.000 transcripts, and (iii) DNA methylation and gene expression changes of several NDD-related genes, in particular, SNCA.
Genome-wide and single gene DNA methylation patterns of isogenic hESC- and iPSC-derived neurons are very similar
Differentially methylated CpGs (DMCGs) in iPSC-derived neurons compared to their hESC-derived counterparts (defined by a methylation difference > 20%) were analyzed in more detail; 8.680 (42.35%) of these DMCGs were hypomethylated, and 11.816 (57.65%) were hypermethylated in iPS- compared to hES-Neurons (Fig. 2b). Transcription start sites were not overrepresented in these DMCGs compared to the overall 450 K array annotation (Fig. 2c upper panels). More hypomethylated CpGs were found in CpG islands (CGI) and more hypermethylated CpGs in non-CGI DNA sequences (Fig. 2c lower panels). We also analyzed the standard deviation (> 0.2) of single DMCG in iPS-NSC (Fig. 2d) and iPS-Neurons (Fig. 2e) to identify an interclonal variation of DMCG. We identified only 3.051 and 3.264 interclonal DMCG sites with a standard deviation of > 0.2 for iPS-NSC and iPS-Neurons, respectively. We found a high correlation of variation between iPSC-derived NSC and neurons (Fig. 2d–f). To identify gene loci showing the most prominent alterations, we carried out an interindividual analysis (hES-Neurons vs. each iPSC-derived neuronal clone) applying a more stringent threshold of > 0.5 methylation difference (Additional file 1 A–C). In line with the previous results, we could identify only a few DMCGs (n = 1010 up to n = 1802) being differentially methylated comparing hES-Neurons to all three iPSC-derived neuronal clones (Additional file 1 A–C). The data indicate that (i) different iPSC neuronal clones exhibit almost the same amount of DMCG when compared to their hESC counterparts and (ii) that the differences are not biased toward certain genomic sites, keeping in mind that the distribution of CpGs on the 450 K array across the genome is not equally distributed per se. Using Fisher’s test, we could, however, identify a highly significant variation for methylation differences on the X chromosome in general (Additional file 1 D).
Consistent gene expression patterns of isogenic hESC- and iPSC-derived neurons
To compare gene expression patterns of hESC- and iPSC-derived neurons, we performed HT12v4 array experiments covering more than 47.000 transcripts. We tested the technical variation of the assay by hybridizing all the neuronal samples in duplicates. We observed high fidelity illustrated by correlation values of greater than 98% between each pair of replicates and a high interclonal correlation of hESC- and iPSC-derived neurons (Additional file 2 A–D). For all downstream comparisons, replicate 1 of each sample was used.
DNA methylation changes during differentiation of isogenic hESC- and iPSC-derived neurons
DNA methylation levels of SNCA intron 1 increased during neuronal differentiation in isogenic hESC- and iPSC-derived neurons
Impact of epigenetic alterations in NDD
There is growing evidence that epigenetic alterations might play a role in NDD [1, 22]. In particular, epigenetic modifications of dosage-sensitive genes such as SNCA, a key player in PD, could be important for the phenotypic features, individual susceptibility, and the variable course of NDD . However, the exact impact of epigenetic modifications in NDD is still unknown. To date, epigenetic studies have been hampered by the inaccessibility of disease-affected cells. Previous studies on bulk brain tissue aimed at exploring whether DNA methylation changes of the SNCA gene could contribute to the development of iPD [2, 5], but the results were not conclusive. iPSC-derived neurons could represent an attractive experimental tool to study epigenetic alterations in PD . However, the reprogramming process itself is based on a major epigenetic “reset” with the resulting iPSC exhibiting epigenetic patterns closely related to those of ESC [24, 25]. This is presumably associated with a loss of both, epigenetic somatic memory (i.e., tissue-specific epigenetic patterns of the donor cells used for iPSC generation) and age-associated epigenetic signatures [26–28]. The latter is particularly relevant as NDD typically occur in late age. A global epigenetic rearrangement during the generation of iPSC could largely eliminate epigenetic alterations resulting from prior noxious exposures. IPSC might thus not fully reflect acquired patient-specific epigenetic changes. On the other hand, epigenetic alterations are not only due to environmental impacts but also reflect changes in cis, i.e., changes in methylation patterns as a downstream result of genetic variants .
Isogenic stem cell systems
It is evident that successful and reliable in vitro modeling of epigenetic changes depends on the faithful replication of the epigenetic signatures observed in non-reprogrammed cells. The comparative assessment of epigenetic patterns in reprogrammed versus non-reprogrammed cells is impeded by the interindividual genetic variability. Isogenic systems can be used to resolve this problem. Indeed, while this manuscript was in preparation, several studies started to address the issue of reprogramming-associated epigenetic differences and their biological significance in isogenic stem cell systems [24, 30, 31]. Teichroeb et al. compared the transcriptome of isogenic hESC (male hESC line H9) and iPSC using an array-based analysis (HT-12v3) . They found a striking similarity (99.6%) of the transcriptome of isogenic hESC and iPSC . These results were supported by Mallon et al. who compared array-based DNA methylation (Illumina Meth.27) and gene expression (Agilent 4x44k One Color) patterns of isogenic hESC (male ESC-line H1) and iPSC . They found no major differences in global gene expression patterns and an accordance of DNA methylation patterns of 98–99% . Choi et al. compared isogenic iPSC and hESC derived from two male hESC lines (HUES2 and HUES3) . Employing global expression profiling by RNA sequencing, they found that only 49 genes were differentially expressed in these isogenic cell populations . DNA methylation pattern differences (reduced representation bisulphite sequencing, RRBS) accounted for only approximately 4% .
We were interested in extending these comparative studies from pluripotent stem cells to their neuronal derivatives. To that end, we compared DNA methylation patterns of reprogrammed and non-reprogrammed mature neurons derived from the same hESC line. Employing an isogenic human neural stem cell system, we found that the genome-wide DNA methylations (and gene expression profiles) in hESC- and iPSC-derived neurons are remarkably similar. Furthermore, iPSC-derived cells displayed only minor interclonal variability. These results are in line with several studies on epigenetic and gene expression patterns in isogenic pluripotent stem cell populations [24, 30, 31]. The need for an isogenic system is also underlined by the results of our comparative in silico analysis including hESC-derived neural cells from another hESC line (H9) and fibroblast-derived neuronal populations from controls, iPD, and LRRK2 PD patients [11, 15]. These data indicate that the individual genetic background (patient-derived vs. H9 ESC-derived vs. I3 ESC-derived) and differences in the neuronal differentiation protocols have a much higher impact on the methylation landscape than the reprogramming process itself.
Looking at five NDD-related candidate genes within our isogenic system, we also observed a high similarity between hESC- and iPSC-derived neural cell methylation patterns at the single base level. Furthermore, we detected a high DNA methylation similarity of hESC- and iPSC-derived neurons concerning the imprinted locus GNAS. Interestingly, we found a distinct increase of DNA methylation in SNCA intron 1 upon neuronal differentiation up to 6 weeks in vitro. ESC- and iPSC-derived neurons acquired a highly comparable pattern during neuronal differentiation. However, this differentiation-associated increase in methylation was paralleled by increased expression of SNCA. It remains currently unclear whether DNA methylation changes upon neuronal differentiation of iPSC- and hESC-derived neurons indeed influence SNCA gene expression levels.
Limitations of the current study and future prospects
The current study focused—as a proof of principle—on evaluating whether iPSC-derived neurons from one hESC-line exhibit similar DNA methylation patterns compared to isogenic hESC-derived neurons. A clear limitation of the current study is the lack of a profound DNA methylation analysis comparing pluripotent and differentiated cells, except for the SNCA locus. The current study did also not address the question whether certain CpG methylation patterns were erased or preserved during the reprogramming process which has to be determined using additional isogenic pluripotent, hESC- and iPSC-derived neural cells. Moreover, the complex issue of variation in genomic imprinting and X chromosome inactivation, too, would require more detailed analyses including, e.g., assessment of allele-specific expression, H3K27me accumulation, and XIST RNA levels, which are going beyond the scope of this study. Despite these limitations, our results suggest that the reprogramming process itself has no major impact on methylation patterns in iPSC-derived neurons. Yet, it remains to be determined how in vitro methylation patterns of hESC- and iPSC-derived neurons correlate to those of neurons derived from the native brain tissue. This is particularly relevant in the light of recent findings revealing substantial transcriptional differences between iPSC-derived and primary midbrain dopamine neurons .
iPSC-derived neurons represent a valuable tool for modeling neurological disorders in vitro. While a number of studies have shown that the epigenetic status of iPSC closely mimics that of non-reprogrammed pluripotent stem cells (i.e., hESC), it had remained largely unclear whether neurons differentiated from iPSC adopt methylation patterns similar to those of neurons derived from non-reprogrammed cells. Using an isogenic system, this study demonstrates that ESC- and iPSC-derived neurons are highly similar with respect to DNA methylation and gene expression. While further studies are required to explore how DNA methylation in iPSC-derived neurons correlates to methylation patterns in primary neurons derived from the CNS tissue, our results suggest that the reprogramming process itself does not confound methylation patterns in iPSC-derived neurons. Thus, iPSC-derived neurons should represent a suitable tool for studying the impact of genetic variants on the epigenome of neurons.
HESCs (line I3) were maintained and differentiated to rosette-forming neural stem cells (NSCs) according to standard protocols and differentiated to neurons as described previously (Additional file 9) . iPSCs were obtained and validated according to established protocols. In brief, NSCs were transduced with lentiviral TetON-system in order to generate a stable inducible cell line harboring the reprogramming factors OCT4 and KLF4 (pLVXTP-Tet-On (Clontech), FUW-OCT4, and FUW-KLF4 (Addgene)). Cells were induced to reprogram by the addition of 1 μg/ml doxycycline (Sigma-Aldrich) and cultured on irradiated mouse embryonic fibroblasts in Knockout Dulbecco’s modified Eagle’s medium containing 20% serum replacement, 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, and 4 ng/ml FGF2. Doxycycline was withdrawn upon colony formation, and doxycycline-independent colonies were mechanically isolated and further propagated. iPSC lines were differentiated to NSC and cultured as described . Terminal differentiation of NSC was performed on Geltrex-coated dishes (Life technologies) in DMEM/F12 and Neurobasal (B27 supplement 1:50, penicillin/streptomycin 1:100, cAMP 300 ng/ml, and 1.6 g/l glucose) mixed at a 1:1 ratio. Neurons were cultured for 2, 4, and 6 weeks, and media were changed every second day (Additional file 9).
Extraction of DNA and RNA
Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
cDNA synthesis and semiquantitative PCR
One microliter of total RNA was used for cDNA synthesis with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories) according to the manufacturer’s instructions. A final concentration of 150 ng/μl of cDNA was used for real-time PCR with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Possible contamination with genomic DNA was tested using samples processed without reverse transcriptase. Gene expression levels were normalized to human β-actin using the following primers (final concentration 10 μM): SNCA forward 5′-cgacgacagtgtggtgtaaag-3′ and SNCA reverse 5′-aaatgttggaggagcagtgg-3′.
Bisulphite treatment and single CpG analysis
Bisulphite treatment of genomic DNA was performed with the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s protocol. Amplicons were generated using region-specific primers (final concentration 10 μM each) with the recommended adaptors (Additional file 6). Purified PCR products were pooled in an equimolar ratio and sequenced after cluster formation on a MiSeq instrument benchtop sequencer with the sequencing-by-synthesis technology  according to the manufacturer’s protocol. Runs were set for “Generate FASTQ only” workflow in Illumina Experiment Manager. ESC and iPSC were analyzed as described elsewhere .
Infinium HumanMethylation450 BeadChip
Genome-scale DNA methylation profiles were generated using Illumina’s Infinium HumanMethylation450 Beadchip assay (Illumina). The assay allows the determination of DNA methylation levels at > 450.000 CpG sites covering promoters and putative regulatory domains of all designable RefSeq genes. The Infinium Methylation Assay was performed according to the manufacturer’s instructions. The fluorescently stained chips were imaged using an Illumina HiScan scanner.
Infinium HT12v4 BeadChip
Genome-wide gene expression profiles were generated using Illumina’s Infinium HT12v4 BeadChip assay (Illumina). The assay allows the determination of gene expression of > 47.000 probes covering designable RefSeq genes. The Infinium Expression Assay was performed according to the manufacturer’s instructions. The scanning was performed using an Illumina HiScan. Data from these images were loaded into Illumina’s GenomeStudio software suite (GS) and exported as a GS export file.
Genome-wide methylation raw data were pre-processed using R statistics software suites minfi and RnBeads  to extract the data, subtract the background, and to normalize the data using internal controls present on the chips. Only CpGs with a detection p value < 0.01 in all samples were included (n = 484,932 of 485,577). All samples were analyzed as individual samples (n = 1). For genome-wide expression analysis, raw data was background subtracted and quantile normalized using R and the lumi software suite. Only probes with a detection p value < 0.01 in all samples were included (n = 20,586 of 39,050). All NSCs were analyzed as individual samples (n = 1); neuronal samples were analyzed in duplicates (n = 2). Further statistical analysis was performed with R statistics software or SPSS version 22. p values were based on a (paired) two-sided t test. Associations between variables (expression and DNA methylation) were measured by Pearson’s rank correlation.
For amplicon-based single gene CpG methylation analysis, sequencing reads obtained from the Illumina MiSeq were processed and assigned to the reference sequence and respective sample using specific tags in the universal portion of the adaptors. Methylation level and patterns were assessed using multiple sequence alignment using BiQ Analyzer HT . All samples were analyzed as individual samples (n = 1) excluding all reads with a maximum fraction of unrecognized CpG sites of 10%. Data are shown as mean ± SD.
Probes identified in differential methylation/expression analyses were mapped to corresponding gene IDs according to the Illumina annotation. GO enrichment analysis was performed using the GOrilla tool (http://cbl-gorilla.cs.technion.ac.il/).
Publicly available data
Published and pre-processed 450 K data from Fernandez-Santiago et al.  were downloaded from GEO accession number GSE51921. Non-publicly available data of non-dopaminergic neurons was kindly provided by R. Fernandez-Santiago. Published and pre-processed 450 K data from Kim et al.  were downloaded from GEO accession number GSE38217. For the comparison of ESC-derived and fibroblast-iPSC-derived neurons, we included all samples with a detection p value of < 0.01 and excluded all CpGs having some potential SNP bias (hES-NSC and hES-Neurons n = 4, ESC line H9, NPC line H9 and DAn line H9 n = 2, iPSC iPD and LRRK PD n = 2, iPSC control n = 3, DAn control and LRRK2 PD n = 4, DAn iPD n = 6, non-dopaminergic neurons control, LRRK2 PD and iPD n = 3). Principal component analysis was performed in R using the “prcomp” function. Obtained eigenvalues for principal components 1 and 2 were used for generation of PCA plot. The fibroblast-iPSC-derived non-dopaminergic neurons from the study of Fernández-Santiago et al. were cultured in DMEMF12/Neurobasal medium supplemented with 0.5xB27, 0.5xN2, and 2 mM GlutaMax and penicillin-streptomycin . HESC- and iPSC-derived non-dopaminergic neurons from the isogenic stem cell model were cultured with slightly higher doses of N2 and B27 (1×) and in the presence of cyclic adenosine monophosphate (cAMP, 0.1×). Dopaminergic neurons (DAn) from the study of Fernández-Santiago et al. were generated using lentiviral transduction of LIM homeobox transcription factor alpha (LMX1A), co-cultured with a mouse with mouse PA6 feeding cells overexpressing sonic hedgehog (SHH) and cultured in the presence of fibroblast growth factor 8 (FGF8) . Neurons were cultured for 30 days, and DAn exhibited a dopaminergic marker expression ranging from 44 to 67% (tyrosine hydroxylase (TH)/β-Tubulin III (TUJ1) ratio) . Dopaminergic neural precursor cells from the study of Kim et al. (ESC line H9) were generated culturing hES colonies on irradiated PA6 or MA5 cells stably over-expressing SHH and cultured in insulin/transferrin/selenium/ascorbic acid/basic fibroblast growth factor (bFGF) medium . Terminal differentiation of dopaminergic NSC into DAn was induced in the absence of bFGF but in the presence of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and cAMP . DAn were cultured for 42 days and exhibited a purity of 43% .
We thank Jasmin Kirch, Katharina Faber, and Christina Lo Porto for their assistance in MiSeq analyis. We thank Dr. R. Fernández-Santiago for the provision of the DNA methylation datasets on the non-dopaminergic neurons used in his study .
The study was supported by the BMBF project 01-KU1216F (DEEP, GG); the BONFOR program of the University Clinic of Bonn (LdB); the German Center for Neurodegenerative Diseases (Deutsches Zentrum für neurodegenerative Erkrankungen, DZNE); the German Federal Ministry of Education and Research (BMBF grant 01GNO813; CH, PK), within the framework of the e:Med research funding concept (BMBF grant # 01ZX1314A; OB); and by the BMBF/ANR through the EpiPD (Epigenomics of Parkinson’s disease) project, under the auspices of the bilateral Epigenomics of Common and Age-related Diseases Programme (grant no. 01KU1403B; UW).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
LdB contributed to the study design, cell culture, preparation and analysis of samples, data analysis, interpretation, and manuscript writing. GG contributed to the analysis of samples, data analysis, interpretation, and manuscript writing. CH contributed to the cell reprogramming, culture and differentiation, sample preparation, immunocytochemistry, and data interpretation. ST contributed to the primer design and Bi-PROF data analysis. IS contributed to the primer design. MP, PK, JW, and UW contributed to the data interpretation. OB contributed to the study design, data interpretation, and manuscript writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Approval for the derivation of pluripotent cell lines for disease and therapeutic research. Ethikkommission, Medizinische Fakultät Bonn, Universität Bonn, serial no. 275/08.
Consent for publication
The authors declare that they have no competing interests.
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