Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha, China
Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health, Changsha, China
National Engineering and Research Center of Human Stem Cell, Changsha, China
Correspondence: Guangxiu Lu, M.D., Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China. Telephone: 86–731-82355100, ext. 8478; Fax: 86–731-84497661; e-mail: email@example.com; or Ge Lin, Ph.D., Institute of Reproductive & Stem Cell Engineering, Central South University, Changsha 410078, China. Telephone: 86–731-82355100, ext. 8478; Fax: 86–731-84497661; e-mail: firstname.lastname@example.org
Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20–30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. Stem Cells2014;32:391–401
Human embryonic stem cells (hESCs) have been widely studied as an invaluable cell source for regenerative medicine and as a model for studying early human development. During prolonged passaging in vitro, hESCs commonly undergo adaptive genetic and epigenetic changes, which present a major safety concern regarding future clinical applications of hESCs. The genetic alterations include gain of chromosomes or chromosome fragments and structure variants, particularly on chromosomes 12, 17, and X [1-7]. Similar alterations are commonly observed in embryonal carcinoma cells, and they could lead to increased proliferative potential or resistance to apoptosis. The epigenetic alterations include DNA methylation instability [8-10], aberrantly imprinted gene expression [11-13], and variable X chromosome inactivation status [14-17]. Many of these epigenetic changes are also features of certain types of cancer [18, 19]. Therefore, genetic and epigenetic abnormalities gained during long-term culture are regarded to confer hESCs a tendency toward neoplastic progression. It has been suggested that periodic monitoring of genetic and epigenetic integrity should be performed to guarantee the fidelity of hESCs [20, 21].
Earlier reports indicated that passaging hESCs by dissociating them into single cells via enzymatic treatment can generate aneuploidies [22, 23], while harsh freeze-thawing cycles and atmospheric O2 exposure have also been suggested to change the X chromosome inactivation state in hESCs . However, the magnitudes of these factors' effects on genetic and epigenetic integrity are poorly understood. Early derivation and culture of hESCs are critical for the original inner cell mass (ICM) to adapt to in vitro culture and to gain the self-renewal ability ; however, little information is available on whether similar genetic and epigenetic alterations might occur during the early stages of hESC culture (<30 passages). This is partially because most hESC lines distributed worldwide for research purposes are usually above 20 passages. To investigate this question, we examined gene expression changes in early hESC cultures using cryopreserved hESCs from initial cultures (<P5) and compared them with those of later passages under different oxygen concentrations. We found that silencing of an imprinted region, the DLK1-DIO3 cluster, occurs frequently in hESC lines as a result of culture under the atmospheric oxygen (20%) conditions. Furthermore, our data suggested that such silencing is carried over by differentiated progeny and may increase the antiapoptotic ability in both hESCs and their differentiated hepatocytes.
Materials and Methods
Derivation and Maintenance of hESCs
The ethical committee of the CITIC-Xiangya Reproductive & Genetic Hospital approved the derivation of hESCs and their use for research. All hESC lines were established and cultured in our laboratory, as previous reported . Briefly, hESCs were derived and cultured on a feeder layer of mitotically inactivated human embryonic fibroblasts or mouse embryonic fibroblasts (MEFs) at a density of approximately 2,500 cells per square centimeter. The hESCs medium consists of Dulbecco's modified Eagle's medium/F-12 supplemented with 15% knockout serum replacement, 2 mM nonessential amino acids, 2 mM l-glutamine, 0.1 mM ß-mercaptoethanol, and 4 ng/ml of basic fibroblast growth factor (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The medium was changed daily and hESCs were mechanically passaged every 6–7 days. Cell lines chHES8, 10, 12, 15, 18, 22, 260, 261, 263, 268, and 283 were derived under 5% oxygen, while all other cell lines were derived under 20% oxygen. Both the initial and early passage cells used in this study were only subjected to mechanical passaging and underwent one or two freeze/thaw cycles (more details available in Supporting Information Table S1).
For microarray profile analysis, total RNAs were isolated from undifferentiated hESCs, ihESCs after 4–9 passages and ehESCs after 20–30 passages, using the TRIzol (Invitrogen) according to manufacturer's protocol. Three biological replicates were used for each data point. Messenger RNA (mRNA) expression analysis was conducted by CapitalBio using Human genome U133 Plus 2.0 Gene Chip arrays (Affymetrix, Santa Clara, CA, http://www.affymetrix.com), as previously reported . Gene expression intensities in numeric values were obtained using Bioconductor packages (http://www.bioconductor.org). Unsupervised clustering was performed using Cluster 3.0 and visualized by Java TreeView . The differentially expressed genes were analyzed by Significance Analysis of Microarrays software and heat maps were created using MultiExperiment Viewer (Mev) in the TM4 microarray software suite [27-29]. The new gene expression microarray data reported in this article were deposited into the NCBI Gene Expression Omnibus (GEO) database under accession number GSE38662.
Small RNA Solexa Deep Sequencing
For small RNA analysis, total RNA was prepared in the same way as for mRNA microarray analysis. The Beijing Genomics Institute (BGI) performed the small RNA sequencing using the Illumina HiSeq platform. After removing adaptor sequences, low quality tags, and contaminants, the resulting clean reads were mapped to the genome by SOAP . Known miRNAs were identified by aligning to a designated part of miRBase15.0 (http://www.mirbase.org/index.shtml), whereas SnoRNAs were identified by aligning to Rfam 9.1 (http://www.sanger.ac.uk/resources/software/) [31, 32]. Differentially expressed miRNAs and snoRNAs were identified by comparing the expression levels of known miRNAs and snoRNAs between ihESCs and ehESCs.
Analysis of Gene Expression Database from GEO
We identified 96 gene expression datasets for hESCs associated with 11 studies from the NCBI GEO database [33-43], covering cells with passages and culture conditions comparable to our samples to determine whether similar silencing of the DLK1-DIO3 cluster can be seen among hESC lines developed and used in other laboratories. The raw CEL files of these datasets were downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo) onto our local bioinformatics server for analysis using the Bioconductor R packages. These datasets were divided into two subsets based on the platform used (HG-U133 plus2 or HG-U133A Array), with each subset analyzed separately to achieve best accuracy. Data processing and normalization were performed using robust multichip average and quantile methods included in the affyPLM packages to generate the normalized numerical probe level expression values. The visualization of the expression patterns in heat maps for the selected probes and the hierarchical clustering of the samples were performed using Mev in the TM4 microarray software suite .
Bisulfite Sequencing of DLK1-DIO3 Cluster
Genomic DNA from undifferentiated hESCs was extracted using a DNeasy Tissue Kit and the bisulfite treatment of 1 µg genomic DNA was performed using an EpiTect Bisulfate Kit, according to the manufacturer's protocol (both from QIAGEN, Valencia, CA, http://www.qiagen.com). DNA was eluted with 30 µl elution buffer and 2 µl was used for polymerase chain reaction (PCR). PCR was performed using Hotstart Plus DNA polymerase (QIAGEN) under the following conditions: 95°C for 15 minutes; 35 cycles of 94°C for 30 seconds, 53°C or 57°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 10 minutes. Additional information regarding the primer sequences and PCR conditions is provided in Supporting Information Table S2. Purified PCR products were subcloned into a PMD-18-T cloning vector (TAKARA, DaLian, China, http://www.takara.com.cn/). Fifteen clones from each PCR product were examined by Sanger sequencing analysis at BGI (ShenZhen, China, http://www.genomics.cn/). The sequence data were analyzed using the biQ Analyzer software (Max Planck InstitutInformatik, Universität des Saarlandes, Germany). The data for each cell line are presented as the mean methylation degree ± SD with n = CpGs within the corresponding region.
RT-PCR and Quantitative PCR
Total RNA was isolated with TRIzol (Invitrogen) and cDNA was synthesized using 1 µg of total RNA in a 20 µl reaction by RevertAid first strand cDNA synthesis kit (Fermentas Life Sciences, Burlington, Canada, http://www.thermoscientificbio.com/fermentas/) according to the manufacturer's instructions. Real-time quantitative PCR reactions were performed in triplicate, unless otherwise specified, using 6 µl of cDNA (1:20 dilution) with the FastStart Universal SYBR Green Master with ROX (Roche, Mannheim, Germany, http://www.roche-applied-science.com/) and run on a 7500 Real-Time PCR Sequence Detection System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The product sizes, annealing temperatures, and primer sequences are provided in Supporting Information Table S2. Relative RNA expression was given as 2−ΔCt where ΔCt = Ct (target gene-28S). In some instances, the ΔCt values were further converted to relative expression levels normalized against the mean expression in the initial control. All the data were presented as means ± SD.
In Vivo and In Vitro Differentiation of hESCs
The embryoid body (EB) differentiation and teratoma assay were performed as previously described , with sufficient details also provided in Supporting Information Methods. Directed neural differentiation was performed according to Kim et al. , while the differentiation protocol for generating hepatocyte-like cells (HPLCs) was modified based on Sullivan, et al. , with details also provided in Supporting Information Methods and Supporting Information Figure S1.
Cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes and then blocked for 30 minutes with 4% normal goat serum. They were then incubated with primary antibodies diluted in blocking solution overnight at 4°C. For intracellular antigens, cells were permeabilized for 30 minutes in 0.5% Triton-X-100 before blocking. Additional details, including the antibodies used, are provided in the Supporting Information Methods.
Apoptosis Assessment by Flow Cytometry
For apoptosis assessment, hESCs were passaged onto Matrigel and cultured in MEF-conditioned medium until a confluence of 50%–70% was reached. Differentiation of HPLCs was performed as described above. hESCs and HPLCs were treated with or without Mitomycin C (1.2 µg/ml) for 6 hours. The cell apoptosis rate was measured by flow cytometry using a fluorescein isothyocyanate Annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com), and as detailed in Supporting Information Methods.
Western blotting was performed as described previously [46, 47]. In brief, cells were harvested and lysed in 1× RIPA buffer (Sigma, St. Louis, MO, http://www.sigmaaldrich.com) and protein was quantified using the Bradford reagent (BioRad, Marnes-la-Coquette, France, www.bio-rad.com). Cell lysates were loaded on SDS-polyacrylamide gels and Western blotting was performed using standard protocols with antibodies against p53 (Santa Cruz, CA, http://www.scbt.com), p53 phosphos Ser20 (Abcam, Cambridge, MA), and β-ACTIN (Sigma). After reaction with secondary antibodies, the antibody-bound proteins were detected using an ECL Western blotting kit (GE Healthcare Life Sciences, PA, www.gelifesciences.com/).
The correlation analysis in this study was performed using Pearson analysis. The independent sample t test between groups was used to evaluate the statistical significance of mean values using SPSS 18.0 for Windows. Homogeneity of variance was analyzed before the independent sample t test. If the variance was not equal, unequal-variances t test was used. The chi-square test was used to evaluate the statistical significance of methylation degree by combining all the sites. Statistical significance levels were p < .05 (denoted as *). All p-values are two-tailed test.
Silencing of the DLK1-DIO3 Gene Cluster Occurs Frequently in Early hESC Cultures
To investigate possible genetic and epigenetic changes during early hESC culture, we first performed karyotype analysis and genome-wide gene expression analysis of undifferentiated hESCs from both the initial passages (ihESCs, P4–P9) and early passages (ehESCs, P20–P30) of 12 karyotypically normal hESC lines obtained from our recently established hESC bank . All 12 cell lines were found to possess a normal karyotype during early culture (data not shown). Unsupervised clustering based on gene expression revealed that the hESC lines clustered mainly by their genetic background, rather than by passage number (Supporting Information Fig. S2). Only 0.10% ± 0.09% of the transcriptome (12–136 of the 38,500 genes/isoforms) showed significant changes (>twofold, t test p < .05) between ihESCs and ehESCs of the same line (Supporting Information Fig. S3), indicating that early culture has little impact on gene expression. Surprisingly, a common pattern of gene expression change between ihESCs and ehESCs was observed across different lines; that is, MEG3, SNORD114-3, and three other expressed sequence tags in the DLK1-DIO3 imprinted cluster were significantly downregulated in most ehESCs. All 12 ihESCs cell lines expressed high levels of these genes; however, among ehESCs cell lines, the expression of these genes was completely silenced in seven lines, and relatively high in only five lines (Fig. 1A). In contrast, the expressions of pluripotency-related genes, such as POU5F1 (also known as OCT4) and NANOG, were not significantly different between ihESCs and ehESCs (Fig. 1A).
The human DLK1-DIO3 cluster in14q32 also encodes a cluster of microRNAs and snoRNAs, including SNORD114-3 [48, 49] (Fig. 1B), some of which are not covered by the gene expression arrays we used. We performed Solexa deep sequencing for small RNAs in the ihESCs and ehESCs from five of the lines described above to determine whether these additional genes have the same pattern of expression variation as MEG3 and SNORD114-3. In cell lines chHES26, 45, 51, and 137, in which MEG3 and SNORD114-3 were expressed in ihESCs but later silenced in ehESCs, a group of miRNAs and snoRNAs from the locus were significantly downregulated in ehESCs (Fig. 1C and Supporting Information Fig. S4, Tables S3, S4). In contrast, line chHES175, in which the expression of MEG3 and SNORD114-3 was unchanged, showed no significant change in expressions of miRNAs and snoRNAs between ihESCs and ehESCs (Fig. 1C, Supporting Information Tables S3, S4). Thus, the expression pattern of miRNAs and snoRNAs in the cluster correlated with that of MEG3 and SNOD114-3 from ihESCs to ehESCs, suggesting that genes in the cluster are subjected to a common expression regulatory mechanism.
Imprinting of the DLK1-DIO3 cluster is known to be regulated by a differentially methylated intergenic region (IG-DMR) and by DMRs in the MEG3 promoter region  (Fig. 1B). To further determine whether silencing of the DLK1-DIO3 cluster in ehESCs correlates with increased DNA methylation of these DMRs, we examined the DNA methylation status in both ihESCs and ehESCs using bisulfite sequencing. In the chHES45 and chHES137 lines that underwent MEG3 and SNORD114-3 silencing in ehESCs, the DMRs in the ihESCs showed partial (∼50%–70%) methylation, while in ehESCs they showed nearly complete methylation (>95%; Fig. 1D and Supporting Information Fig. S5). However, in line chHES175, in which the expression of MEG3 and SNORD114-3 was persistent, methylation of all DMRs did not change from ihESCs to ehESCs (both at ∼50%; Fig. 1D and Supporting Information Fig. S5). In order to evaluate the imprinting pattern of the DLK1-DIO3 cluster, we performed allelic expression analysis in two single nucleotide polymorphism (SNP) sites of MEG3. We found that the two SNP sites were expressed in a monoallelic pattern in ihESCs of chHES45 and chHES137 (Supporting Information Fig. S6), but totally silenced in their ehESCs. The above results suggest that the DLK1-DIO3 cluster was normally imprinted in ihESCs but became completely silenced during early culture.
To further test whether aberrant silencing of the DLK1-DIO3 cluster is common in other cell lines, we used real-time PCR to examine the expression of MEG3 and SNORD114-3 in an additional 20 hESC lines from our cell bank. The results showed that MEG3 and SNORD114-3 were expressed at relatively high levels in ihESCs of all 20 cell lines, but they became completely silenced or exhibited more than twofold downregulation in ehESCs of 16 lines (Fig. 2A, 2B). We then retrieved gene expression datasets of the most commonly used hESC lines and several induced pluripotent stem cell (iPSC) lines from different laboratories around the world via the GEO database. We obtained 64 datasets generated using HG-U133 plus2 microarrays, and then compared the expression pattern of genes to our representative ihESCs and ehESCs of chHES26 (Fig. 2C, Supporting Information Table S5). In these datasets, which are mostly for human pluripotent cells after 20 passages, the pluripotency marker genes POU5F1 and NANOG exhibited a similar high level of expression across all samples. However, the genes in the DLK1-DIO3 cluster, except for DLK1, were only expressed in two initial-passaged iPSC samples (P6) and four hESC lines at a level similar to that of P6 ihESCs of chHES26, while the other 58 cell lines show no expression similar to the P24 ehESCs of chHES26. Similarly, in another gene expression dataset from 32 cell lines between P17 and P65, the expression of MEG3 was only detected in five cell lines between P17 and P23 (Supporting Information Fig. S7 and Table S5). These results suggested that most hESC and hiPSC lines used in laboratories worldwide have undergone DLK1-DIO3 silencing during early in vitro culture.
We further examined the expression status of MEG3 in the ICM of human blastocysts to identify the expression status of the DLK1-DIO3 cluster in the original embryonic cells in vivo. Our result showed that the ihESCs had comparable MEG3 expression levels to the ICM, indicating the activation of the DLK1-DIO3 cluster in ihESCs is a normal status inherited from embryonic cells and that the silencing of MEG3 expression in ehESCs represents an abnormal variation acquired through early culture (Supporting Information Fig. S8).
Silencing of DLK1-DIO3 Is Irreversible
Normal expression of the Dlk1-Dio3 gene cluster has been related to the establishment of full pluripotency in mouse iPSCs [51, 52]. We compared the differentiation capability of ihESCs and ehESCs from three lines with an initial-on-late-off MEG3 expression pattern: chHES55, 60, and 137 to determine whether aberrant silencing of the DLK1-DIO3 cluster in ehESCs affects the pluripotency of hESCs. All three lines were able to differentiate into the three germ layers both in vitro and in vivo (Supporting Information Fig. S9). This is consistent with most published studies, which report that late-culture hESCs maintain their multilineage differentiation ability .
To assess whether the silencing of the DLK1-DIO3 cluster in ehESCs could be reversed during differentiation, we analyzed the expression level of MEG3 and SNORD114-3 in differentiated progenies from ihESCs and ehESCs. Compared to hESCs, the differentiated cells in day 15 embryonic bodies (EB day15) exhibited marked changes in cellular morphology and downregulation of NANOG, but showed similar expression patterns of MEG3 and SNORD114-3, with a statistically significant linear correlation (r = 0.921 for MEG3, p < .001; r = 0.789 for SNORD114-3, p < .001) (Fig. 3A–3D and Supporting Information Fig. S10). Upon directed differentiation toward the neural lineage, we observed the same inheritance tendency in differentiated ß-tubulin-positive neural cells (Supporting Information Fig. S11A, S11B). Taken together, our results indicate that differentiation does not reverse the silencing of the DLK1-DIO3 cluster.
Early hESCs and Their Liver-Like Cell Derivatives Exhibit Attenuated Apoptosis Induced by DNA Damage
Downregulation of MEG3 in the DLK1-DIO3 cluster was previously shown to participate in hepatocarcinoma tumorigenesis, possibly by suppressing p53 to exert an antiapoptotic effect . To evaluate whether this effect occurs in ehESCs or their hepatocyte derivatives, we compared the apoptosis rate in ihESCs and ehESCs from the chHES56, 90, and 12 lines, treated with or without mitomycin C (MC). After MC-induced DNA damage, both ihESCs and ehESCs exhibited one- to two fold higher rates of apoptosis, as indicated by fluorescence-activated cell sorting analysis. However, in the ehESCs that were negative for MEG3 expression (MEG3off) from the chHES56 and chHES90 lines, the rates of MC-induced apoptosis were 20% lower than the rates observed in their ihESC counterparts with MEG3 expression (MEG3on), with a statistical significance (Fig. 4A and Supporting Information Table S6). By contrast, in the MEG3on ehESCs of the chHES12 line that was positive for MEG3 expression, the rate of MC-induced apoptosis was comparable to their MEG3on ihESC counterparts (Fig. 4A).
We further examined the effect of MEG3 silencing by differentiating both ihESCs and ehESCs from the chHES56, 90, and 12 lines into HPLCs. After a three-step induction, the cells exhibited the typical hepatocyte morphology and were positive for both the hepatocyte progenitor marker alpha fetal protein and hepatocyte nuclear factor 4 alpha (Supporting Information Fig. S1A, S1B). Similar to their original hESCs, after MC-induced DNA damage, HPLCs derived from MEG3off ehESCs exhibit significantly lower rates of apoptosis than HPLCs derived from MEG3on ihESCs (Fig. 4B and Supporting Information Table S6). Taken together, this data suggest that MEG3 silencing attenuates DNA damage-induced apoptosis in hESCs and their differentiated cells.
We next examined whether the attenuated apoptosis in MEG3off ehESCs and their differentiated derivatives were associated with suppression of the p53 pathway, as previously reported [47, 55]. Our data showed that TP53 mRNA levels were decreased by up to 30% in both control and MC-treated ehESCs from the chHES90 and chHES56 lines, which had up to 70% downregulation of MEG3 compared to the corresponding ihESCs (Fig. 4C). However, upregulation of TP53 mRNA was observed in MEG3on ehESCs from the chHES12 line, compared to the level in its ihESC. Increased p53 and phosphor-p53 protein levels were observed in all hESCs after MC treatment; however, ehESCs from MEG3off cell lines exhibited reduced phosphor-p53 levels (Fig. 4C, 4E). Likewise, HPLCs from MEG3off ehESCs inherited compromised p53 reactivity to MC-induced DNA damage in terms of mRNA and protein levels compared to its ihESC-derived counterpart (Fig. 4D, 4F). Taken together, our data suggest a positive correlation between the expression level of MEG3 and TP53 (Supporting Information Fig. S12 and Table S7), which indicates that the antiapoptotic effects observed in MEG3off ehESCs and the differentiated HPLCs may be caused, at least in part, by suppression of the canonical p53 pathway.
Oxygen at 5% Is Required to Maintain Persistent Expression of the DLK1-DIO3 Cluster
In searching for the causes for aberrant silencing of the DLK1-DIO3 cluster, we noticed that, among the 32 lines analyzed, six cell lines with persistent MEG3 expression were derived under 5% oxygen before shifting to atmospheric oxygen conditions for prolonged culture (Figs. 1A, 2A). From the GEO database, the lines WIBR1, 2, and 3, which were derived under 5% oxygen, also expressed high levels of MEG3 and SNORD114-3 (Fig. 2C) . This led us to speculate that the oxygen concentration might be a critical factor in maintaining normal expression of the DLK1-DIO3 cluster during culture, especially during the initial derivation and expansion stages. To test this hypothesis, we derived five new hESC lines from human blastocysts under 5% oxygen conditions, and divided them at P5 for subsequent culture either in 5% or 20% oxygen until P20 (Supporting Information Fig. S13). As expected, MEG3 and SNORD114-3 were stably expressed in culture under continuous 5% oxygen in all five lines, including those that were cultured to P20 under the same conditions. However, those lines that were transferred to 20% oxygen at P5 and cultured to P20 showed 1.5–3.2-fold downregulation of MEG3 and SNORD114-3 (Fig. 5A). Furthermore, we analyzed the methylation level in ihESCs and ehESCs (including samples under 5% oxygen and 20% oxygen). We found that the level of DNA methylation was approximately 50%–60% for both IG-DMR and MEG3 promoter DMRs in ihESCs and ehESCs under 5% oxygen, which is similar to the DNA methylation level of normal imprinting; however, the ehESCs under 20% oxygen showed significantly higher methylation level, approximately 70%, consistent with the downregulation of MEG3 and SNORD114-3 (Fig. 5B and Supporting Information Fig. S14). To exclude the possibility that loss of imprinting may also occur during early culture, we analyzed the allelic expression of MEG3 in ihESCs and ehESCs cultured under both 5% and 20% oxygen. The results indicated that MEG3 was expressed monoallelically in ihESCs and ehESCs (including samples under 5% oxygen and 20% oxygen) (Supporting Information Fig. S15). This data indicated that low oxygen levels during initial culture are important to maintain DLK1-DIO3 cluster expression during late culture, even when the culture is shifted to atmospheric oxygen conditions. To further test whether late exposure of ihESCs derived under 20% oxygen to 5% oxygen could recover the expression of the DLK1-DIO3 cluster, we transferred P5 ihESCs from four lines derived under 20% oxygen to 5% oxygen, and cultured the cells until P20. Out data showed that late exposure to 5% oxygen could not recover the expression of MEG3 and SNORD114-3 (Fig. 5C). Taken together, our results indicate that atmospheric oxygen is the main cause for the silencing of the DLK1-DIO3 cluster during early culture and further that an initial low oxygen concentration (5%) is essential for maintaining the normal expression of the DLK1-DIO3 cluster (Fig. 5D).
Maintenance of the epigenetic stability of hESCs is critical for their future therapeutic applications; however, the impact of early culture on hESCs has been largely ignored or underestimated. In this study, we investigated gene expression data from a large number of hESC lines generated in our own laboratory and laboratories worldwide, and identified a hallmark for the impact of early culture in most lines: the aberrant silencing of the DLK1-DIO3 imprinted region. This process seems to occur early before P20, a period for which the epigenetic instability of hESCs has been seldom addressed. Our observation of gene expression changes in the DLK1-DIO3 cluster provides new insight into the epigenetic instability during the early culture of hESCs, which may help to better characterize the genuine molecular profile of hESCs.
The expression status of the DLK1-DIO3 cluster in hESCs is poorly characterized. Based on the analysis of gene expression from late-cultured hESCs, it was previously concluded that expression of the DLK1-DIO3 region was regulated in a species-specific manner and is silenced in all human pluripotent stem cells. In two recent studies [33, 56], MEG3 was shown to be not expressed in hESC and iPSC lines, and this silencing was regarded as a normal phenotype, while the expression of MEG3 was considered as an aberrant epigenetic change seen only in a few cell lines. However, our results suggest the opposite model, in which DLK1-DIO3 is normally active in human pluripotent stem cells, and that silencing of this cluster is an aberrant change resulting from inappropriate culture conditions, particularly the use of atmospheric oxygen level. Furthermore, the expression status of this cluster can be used as an indicator for the epigenetic stability of early cultures.
The DLK1-DIO3 cluster is an evolutionary conserved imprinted region in mammals and altering the dosage of the imprinted genes in this cluster causes a range of phenotypes from growth deficiencies and developmental defects in the embryo and placenta to defects in adult metabolism and brain function observed in human and the mouse [57, 58]. Thus, the biological consequences of the DLK1-DIO3 cluster silencing in early hESC cultures deserve extensive evaluation, particularly because of our limited understanding of the function of the noncoding RNAs in this region. Although loss of DLK1-DIO3 expression does not seem to compromise the multilineage differentiation capacity of the hESCs, the positive correlation between downregulation of MEG3 and resistance to DNA damage-induced apoptosis in both ehESCs and their derived HPLCs raises a serious safety issue. Aberrant silencing of MEG3 has been observed in several types of cancers, for example, in nonfunctioning pituitary tumors and hepatocellular carcinoma, and further evidence suggested the silencing contributes to tumorigenesis [59, 60]. Therefore, it is possible that aberrant silencing of MEG3 may lead to increased accumulation of genetic abnormalities and might prime the differentiated progeny toward malignant transformation. Although the tumorigenicity of ehESCs, especially their differentiated derivatives, needs to be further evaluated, our observations coincide with previous findings that culture-adapted genetic and epigenetic alterations usually lead to increased antiapoptotic or proliferation advantages [5-7]. For this reason, we suggest that the active expression of the DLK1-DIO3 cluster should be included as a quality control marker for hESCs. Furthermore, MEG3 and other small RNAs in this cluster were suggested to regulate other neighboring imprinted protein-coding genes and further study will be needed to evaluate whether the silencing will affect other imprinted genes in hESCs and their progeny .
Physiological oxygen is necessary for maintaining two active X chromosomes by reducing cellular stress during the initial derivation and early culture of hESCs . Physiological oxygen also seems to be required for maintaining a normal expression profile for the DLK1-DIO3 cluster. However, it remains to be confirmed whether a similar mechanism is involved in the two processes. Nevertheless, our study provides new evidence that a nonphysiological culture environment, even just over a short period of time, can have a long-term impact on epigenetic stability in hESCs, possibly conferring aberrant functional consequences on both the undifferentiated and differentiated progeny. Therefore, optimization of culture conditions for hESCs should be implemented from the very beginning of their derivation and culture.
We identified in this study for the first time a hallmark epigenetic abnormal alteration event during initial culture in most hESC lines, which is irreversible and transmissible into differentiated progeny and seems to contribute to an antiapoptotic property. Further we found that culturing under atmosphere oxygen level (20%) is the cause of this change, and keeping culture under the physiological oxygen level (5%) is required to prevent this alteration. Our study provides new insight into the importance of early culture conditions on maintaining the epigenetic instability of hESCs, and may help to better characterize the genuine molecular profile of hESCs. Further studies need to be directed at investigating the influence of DLK1-DIO3 cluster silencing on the function of various differentiated cell types.
We thank the IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance. This work was supported by grants from the National Basic Research Program of China (973 program 2011CB964900 and 2012CB944901), the National Natural Science Foundation of China (81222007), and the Ministry of Science and Technology of China (863 program 2011AA020113 and 2006AA02A102). We also thank Dr. William Held for his critical review of the manuscript.
P.X.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; Y.S., Q.O., L.H., Q.T. X.Z., B.X., D.Y., Y.P., and T.L.: collection and/or assembly of data, data analysis and interpretation; P.L.: data analysis and interpretation and manuscript writing; G.L. and G.L.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript. P.X and Y.S contributed equally to this article.
Disclosure of Potential Conflicts of Interest
The authors indicate no potential conflicts of interest.