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The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells
Pingyuan Xie, Qi Ouyang, Lizhi Leng, Liang Hu, Dehua Cheng, Yue- qiu Tan, Guangxiu Lu, Ge Lin
PII: S1873-5061(16)30049-6 DOI: doi: 10.1016/j.scr.2016.05.011 Reference: SCR 773
To appear in: Stem Cell Research
Received date: 2 February 2016 Revised date: 11 April 2016 Accepted date: 20 May 2016
Please cite this article as: Xie, Pingyuan, Ouyang, Qi, Leng, Lizhi, Hu, Liang, Cheng, Dehua, Tan, Yueqiu, Lu, Guangxiu, Lin, Ge, The dynamic changes of X chromosome in- activation during early culture of human embryonic stem cells, Stem Cell Research (2016), doi: 10.1016/j.scr.2016.05.011
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The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells
Pingyuan Xie1,2,3,#, Qi Ouyang1,2,3,#, Lizhi Leng1,3, Liang Hu1,2,3, Dehua Cheng1,2,3,
Yueqiu Tan1,2,3, Guangxiu Lu2,3, Ge Lin1,2,3*
1Institute of Reproductive & Stem Cell Engineering, school of basic medical science,
Central South University, Changsha 410078, China
2National Engineering and Research Center of Human Stem Cell, Changsha 410078,
China
3Key Laboratory of Stem Cells and Reproductive Engineering, Ministry of Health,
Changsha 410078, China
#Co-first author
*Correspondence to: Ge Lin (Email: [email protected]); Telephone:
86-731-82355100.ext 8478; FAX: 86-731-84497661
ACCEPTED MANUSCRIPT
1 ACCEPTED MANUSCRIPT
Abstract: X chromosome inactivation (XCI) is required for dosage compensation of
X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells
(hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown.
Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20-P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability eventACCEPTED during early culture, which MANUSCRIPT is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity. Abbreviations: ihESCs: initial human embryonic stem cells ehESCs: early human embryonic stem cells 2 ACCEPTED MANUSCRIPT XCI: X chromosome inactivation Xi: inactivated chromosome Xa: activated chromosome SNP: single-nucleotide polymorphism Keywords: human embryonic stem cells, X chromosome inactivation, epigenetics, gene expression regulation, culture alteration 1. Introduction Human embryonic stem cells (hESCs) are regarded as one of the most promising cells for the future of regenerative medicine because of their capacity for pluripotency and the ability to indefinitely self-renew [1]. However, during long-term culture, many concerns have been raised regarding genetic and epigenetic instability, which hinder the development of hESCs and their related applications [2-4]. The epigenetic alterations previously reported include aberrant X chromosome inactivation (XCI) states [5-8], ACCEPTEDDNA methylation instability MANUSCRIPT [9], aberrant silencing of DLK1-DIO3 imprinted cluster [10], and biallelic expression of imprinted genes [11]. XCI is required for dosage compensation of X-linked genes in mammals [12]. In humans, the initiation of XCI is dependent on the X inactivation center, and the X inactive specific transcript (XIST) is a critical gene in this region, which produces a non-coding RNA and coats the X chromosome in cis and triggers its inactivation [13, 14]. In addition, XCI has been associated with characteristic patterns of histone 3 ACCEPTED MANUSCRIPT modifications, such as tri-methylation of H3 Lys-27 (H3K27me3) [15]. Another characteristic associated with XCI is that the inactive X chromosome (Xi) is distinctly replicated later in S phase compared with the autosomes and the active X counterpart [16]. A growing number of studies are concerned regarding XCI states in hESCs. XCI status in long-term cultured female hESCs is promiscuous and can be divided into 3 distinct XCI states [8]. Class I female hESCs refer to the hESCs that have two active X chromosomes and retain competence to initiate random XCI upon differentiation, which is similar to mouse embryonic stem cells (mESCs). Class II female hESCs refer to hESCs that have undergone XCI and express XIST and H3K27me3 punctate enrichment in both undifferentiated and differentiated states, which is similar to mouse epiblast stem cells (EpiSCs). Class III female hESCs have undergone XCI, but never express XIST and H3K27me3 punctate enrichment in both undifferentiated and differentiated states. The majority of hESCs have undergone XCI (class II and class III). Due to the lack of adequate analysis of XCI in ihESCs, we cannot confirm whether the different XCI states in long-term cultured hESCs was inherited from the ihESCs or resultedACCEPTED from the expansion process. MANUSCRIPT In this study, we evaluated the XCI status of initial derived (P5-9) and early passaged (>P20) hESCs in 18 paired hESC lines to elucidate the dynamic changes of XCI during the early culture period. Our results showed rapid conversion of the XCI states from class II to class III during the early culture, and was accompanied by the up-regulation of a subset of X-linked genes. Furthermore, we confirmed that physiological oxygen was beneficial for the stability of XCI. 4 ACCEPTED MANUSCRIPT 2. Experimental procedures 2.1 hESC culture and in vitro differentiation This research was approved by the ethical committee of the CITIC-Xiangya Reproductive & Genetic Hospital. All hESC lines were derived and cultured in our laboratory, as previously reported [17]. Briefly, hESCs were cultured on mitomycin-C-treated mouse embryonic fibroblast feeder cells. The hESCs medium contained DMEM/F12, 15% knockout serum replacement, 2 mM L-glutamine, 2 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, and 4 ng/ml of basic fibroblast growth factor (bFGF) (all from Invitrogen). The medium was changed every day and hESCs were mechanically passaged every 6-7 days. hESCs were derived and cultured at 20% oxygen except at special mention. All cells, including different passage and different sublines, were cultured in the same culture conditions, only subjected to mechanical passaging and underwent one or two freeze/thaw cycles, but without monoclonal screening (more details available in Supporting Information Table S1). The in vitro ACCEPTEDdifferentiation protocol of MANUSCRIPT hESCs, including the spontaneously in vitro differentiation and neural induction differentiation, was performed as previously described [10]. 2.2 Microarray analysis Total RNAs were isolated from undifferentiated hESCs using TRIzol (Invitrogen) according to the manufacturer's protocol. ihESC samples ranged between passages 4-9, whereas ehESCs were used at a range between 20-30 passages. mRNA expression 5 ACCEPTED MANUSCRIPT analysis was performed using Human Genome U133 Plus 2.0 Gene Chip arrays (Affymetrix). Three biological replicates were used for each data point. The heat map for selected genes was performed using MultiExperiment Viewer (Mev) in the TM4 microarray software suite. To analyze the location of differently expressed X-linked genes, we applied a X gene expression ratio between ihESCs and ehESCs, followed by physical mapping along the X chromosome using the Feb 2009 assembly from the UCSC genome browser (http://genome.ucsc.edu) [18]. The gene expression microarray data were deposited into the NCBI Gene Expression Omnibus database under accession number GSE38662. 2.3 Quantitative PCR Quantitative PCR was performed as previously described [10]. The primer sequences and annealing temperatures are provided in Table S2. Relative RNA expression was given as 2-∆CT where △Ct = Ct (target gene-28S or ACTB). Each reaction was performed in triplicate. The independent sample t-test between ihESCs and ehESCs under 5% oxygen was used to evaluate the statistical significance of mean values using SPSS 18.0. HomogeneityACCEPTED of variance wasMANUSCRIPT analyzed before the independent sample t-test. If the variance was not equal, unequal-variances t-test was used. 2.4 RNA-Fluorescence in situ hybridization (FISH) XIST RNA-FISH was performed as previously described [6]. Three 50-mer DNA probes were designed from consensus sequences of map positions 6183-6232, 6234-6284 and 6368-6417 (accession no. L04961), which are in repeat D of XIST [7]. 2.5 Bisulfite sequencing 6 ACCEPTED MANUSCRIPT Bisulfite sequencing was performed as previously described [10]. Briefly, PCR was performed using Hotstart Plus DNA polymerase (QIAGEN) under the following conditions: 95°C for 15 min; 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min, followed by 72°C for 10 min. The primer sequences and annealing temperatures are provided in Table S2. Purified PCR products were subcloned into a PMD-18-T cloning vector (TAKARA). Fifteen clones from each PCR product were examined by Sanger sequencing analysis. The sequencing data were analyzed using the biQ Analyzer software (Max Planck InstitutInformatik). 2.6 Immunocytochemistry staining and DNA FISH for hESCs Immunocytochemistry staining procedures are previously described [17]. The following primary antibodies were used for hESCs and their derivatives: mouse anti-Oct4 (1:50, Santa Cruz), rabbit anti-H3K27me3 (1:800, Millipore) mouse anti-phosphoserine 2 on the C-terminal domain of RNA polymerase II (H5) (1:250, Millipore), mouse anti-β-tubulin (1:800, Sigma), and mouse anti-Pax6 (1:400, DSHB). Localization of antigens was visualized by using Alexa Fluor 488 and 594 secondary antibodies (1:1000, Invitrogen).ACCEPTED Nuclei were counterstained MANUSCRIPT with DAPI (1:1000, Sigma). DNA FISH was carried out as previously described [19]. Cells were exposed to hypotonic solution (6 mg/ml BSA+1% NaAc) for 5 min and transferred to Tween fixative (0.01 N HCL+0.01% Tween) on a clean slide. Fixed cells were hybridized with X-paint probe (Abbott Molecular Inc,Downers Grove, IL, USA). For simultaneous immunocytochemistry of RNA POL II and X chromosome FISH, immunocytochemistry was performed first, then the signals were fixed, and X 7 ACCEPTED MANUSCRIPT chromosome DNA FISH was performed. 2.7 Late replication analysis Late replication was performed by following a modification procedure [20]. hESCs were transferred to matrigel (BD) coated dishes and fed with MEF-conditioned media for 3 days. The cells were treated with 12 μg/ml Brdu (Sigma) for 10 h and 80 ng/ml colcemid (Invitrogen) for 1.5 h. Then the cells were harvested with 0.05% Trypsin / EDTA (Invitrogen). Next, we used 0.075 M KCl and 3:1 methanol : acetic acid fixative for hypotonic treatment and fixation, respectively. Cell suspension was dropped onto clean wet slides and air-dried. Then the slides were heated to 60°C for approximately 45 min in 2*SSC (0.3 M sodium chloride-0.03 M trisodium citrate) and exposed to ultraviolet light (distance of about 17 cm). At last, the slides were rinsed in running water and then stained with 1.5% phosphate-buffered Giemsa for 20 min. 2.8 XCI pattern analysis of hESCs If random XCI occurred, we can evaluate the XCI pattern using multiple polymorphic cDNA of X-ACCEPTEDlinked genes by sanger sequencingMANUSCRIPT in a population of hESCs [7]. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (QIAGEN) and RNA was extracted with TRIzol (Invitrogen). SNPs were selected for analysis along the X chromosome according to the following criteria: 1) The gene was X-linked and expressed in hESCs in moderate to high levels; 2) The gene was silenced on the Xi. PCR was performed in a 50 μl reaction volume using 1 μl DNA (~100 ng) or 2 μl cDNA converted from DNase I-treated RNA samples. Further details related to the 8 ACCEPTED MANUSCRIPT annealing temperatures, and primer sequences are provided in Table S2. PCR products were purified by using a PCR purification kit (Qiagen), and then subjected to Sanger sequencing to obtain the genotype and expression pattern via a commercial service provided by BGI. 3. Results 3.1 Gradual loss of XIST expression and H3K27me3 punctate enrichment in female hESCs during early culture To determine the XCI status of female hESCs during early culture, we first assessed the relative expression levels of XIST and H3K27me3 punctate enrichment in 18 paired female hES cell lines. High-level expression of XIST was shown in all initial passaged (P5-P9, ihESCs) female lines, and no or very low XIST expression was detected in all following early passaged (P20-P30, ehESCs) female lines by expression array, real-time PCR and RNA FISH (Figure 1A, 1B and S1A). As expected, no expression was observed in male samples. Similar to the above observation, singleACCEPTED H3K27me3 punctate MANUSCRIPT enrichment occurred in >90% of the initial passaged hESCs (≤P7) in all 18 examined cell lines. However, P20-30 hESCs from all 18 cell lines showed no H3K27me3 punctate enrichment (Figure 1C and S1B). In summary, these results indicate that the ihESCs expressed XIST and H3K27me3 punctate enrichment; however, the ehESCs lost XIST expression and H3K27me3 punctate enrichment. To assess how the ehESCs lost XIST expression and H3K27me3 punctate 9 ACCEPTED MANUSCRIPT enrichment, we first examined XIST expression by real-time PCR on every 3-4 passages from P5 to P20 in five cell lines. During this period, chHES51, 137, 175 showed gradual decreased XIST expression, but chHES26 and 45 showed dramatically reduced XIST expression from P4 to P9. All five cell lines fully lost the expression of XIST at P20 (Figure 1D). Simultaneously, we performed H3K27me3 staining and calculated the percentage of H3K27me3 punctate enrichment. Consistent with XIST expression, the percentage of H3K27me3 punctate enriched positive cells showed a slight decrease in chHES51, 137, 175 but dramatically reduced chHES26 and 45 (Figure 1C and 1E). These results suggest that loss of XIST expression and H3K27me3 punctate enrichment occur rapidly during initial passages. 3.2 XIST promoter methylation in ihESCs and ehESCs To evaluate the relationship of XIST expression and the epigenetic modification of the XIST promoter, we tested the degree of XIST promoter methylation in two hESC lines, chHES45 and chHES137. Our analysis showed approximately 50-60% methylation at the XIST promoter in ihESCs (XIST+), whereas the ehESCs (XIST-) showed >90% methylation atACCEPTED the XIST promoter (Figure MANUSCRIPT S2). These results indicate that the degree of XIST promoter methylation is correlated with the silencing of XIST expression. Our results are consistent with previous reports [7]. 3.3 X chromosome transcription activity in ihESCs and ehESCs To investigate the effect of the loss of XIST expression and H3K27me3 punctate enrichment on X chromosomes, we evaluated the two X chromosomes activity with two experiments. We tested the transcriptional activity of two X chromosomes by 10 ACCEPTED MANUSCRIPT double staining the X chromosomes with X chromosome painting probe and RNA polymerase II antibody. The results showed that the ihESCs and ehESCs both had one X without transcriptional activity (Figure 2A). We then performed a late replication assay to determine whether there is an X chromosome replicated in late S phase, as the indicator of the inactivated X chromosome. The results showed that the ihESCs and ehESCs both had a late replicated X chromosome (Figure 2B). These results indicate that both ihESCs and ehESCs exhibited one inactivated X chromosome (Xi) at the chromosomal level. To determine whether the X-linked genes were affected by the loss of XIST and H3K27me3 punctate enrichment, we performed transcriptomic analysis in 12 cell lines, including 10 female samples and 2 male samples as control (all including ihESCs and ehESCs). Three biological replicates were used for each data point. Among 1135 annotated X-linked genes, an average of 3.8% (57 genes) of X-linked genes exhibited a 1.5-fold increase of mRNA in female ehESCs compared with ihESCs. However, only 0.3% (4 genes) of X-linked genes showed 1.5-fold increase in male samplesACCEPTED and approximately 0.2-0.3% MANUSCRIPT of genes showed a 1.5-fold increase in all autosomes in both male and female samples (Figure 3A). From the more than 1.5-fold up-regulated genes, we identified 27 common differentially expressed genes that were up-regulated in more than one half of all 10 female hESC lines (Table 1). Furthermore, utilizing the gene expression ratio between ihESCs and ehESCs, we performed a heat map of the X chromosome by mapping the ratio (ehESCs/ihESCS) physically along the X chromosome. The results showed that the distribution of 11 ACCEPTED MANUSCRIPT up-regulated genes is not random along the chromosome but map primarily to the Xq22.3-23; Xq26; Xp22, whose region is not near the XIST gene (Xq13), which suggests that up-regulation starts from a region far from the XIST transcriptional start site (Figure 3B). Real-time PCR analysis confirmed four X-linked differentially expressed genes in two cell lines (Figure 3C and S3). 3.4 XCI pattern in ihESCs and ehESCs Previous studies have suggested that long-term cultured hESCs show a nonrandom inactivated X chromosome and early hESCs at P5-P15 exhibit a random or skewed inactivated X chromosome [7, 21]. To determine the initial XCI pattern in female hESCs and whether variations occurred during early culture, we analyzed the XCI pattern of ihESCs and ehESCs in five cell lines, including chHES-26, -45, -51, -137, and -175, using five SNPs in the transcribed region. The results showed that the ihESCs demonstrated biallelic expression of all five SNPs, and the ehESCs, including different sublines, exhibited monoallelic expression in all five cell lines (Table 2) . However, the allelic gene expressed in subline-1 and subline-2 of chHES26 and chHES137 is ACCEPTEDdifferent, which suggests thatMANUSCRIPT the X-linked genes expressed in subline-1 and subline-2 were from a different parental X chromosome. These results indicate that the XCI pattern in ihESCs is random, similar to normal adult tissue. Furthermore, the XCI pattern in ehESCs is completely skewed, which further confirms that the ehESCs have a Xi. 3.5 XCI status after differentiation in ihESCs and ehESCs We further tested the ability to activate XIST upon differentiation, and found that the 12 ACCEPTED MANUSCRIPT ehESCs (XIST-) cannot activate XIST expression and can form H3K27me3 punctate enrichment upon differentiation, including differentiated embryoid (EB) and neural progeny. However, as expected, the ihESCs retained XIST expression and formed H3K27me3 punctate enrichment upon differentiation (Figure S4). 3.6 Effect of oxygen on XCI Previous work has shown that physiological oxygen (5%), instead of ambient (20%) levels, preserves the class I state of hESCs [22]. Therefore, we investigated whether physiological oxygen might be beneficial for XCI fidelity in our culture conditions. Herein, we derived five female cell lines under 5% oxygen and cultured the lines to P20, then detected the XIST expression and H3K27me3 punctate enrichment at P5 and P20. Unexpectedly, XCI occurred in all five initial hESC lines. Unlike the cell lines derived under atmosphere oxygen, XIST was stably expressed or showed little decrease at P5 and P20 (Figure 4A, Table S3), and more than 90% cells exhibited H3K27me3 punctate enrichment at P5 and P20 in all five cell lines (Figure 4B). Furthermore, we detected the XCI pattern in two of the above cell lines, the ihESCs demonstratedACCEPTED biallelic expression in ch HES277MANUSCRIPT and chHES278, however, the ehESCs showed monoallelic expression (Table 3). These results indicate that under 5% oxygen concentration the hESCs can preserve the expression of the XIST and H3K27me3 punctate enrichment but the XCI pattern is prone to change. 4. Discussion XCI is promiscuous in long-term cultured hESCs. In the present study, we traced the XCI dynamics during early cell culture. From P5 to P20, the ehESCs lost XIST 13 ACCEPTED MANUSCRIPT expression and H3K27me3 punctate enrichment, accompanied by the up-regulation of a small subset of X-linked genes; additionally, the XCI pattern converted from a random XCI pattern to a fully skewed XCI pattern. In the mouse, the female EpiSCs derived from the epiblast of a post-implantation embryo display random XCI, and the ESCs derived from ICMs display two active X chromosomes [23] . Our observations for XCI in ihESCs combined with other hESC characteristics suggest that the hESCs isolated under conventional methods was in a primed state, similar to the mEpiSC [24]. Our results also suggest that the variations of XCI occur in rapid dynamic procession. This may give interpretation of why variations of X chromosome inactivation occurs in early passages (P5-P15) of female hESCs [21]. Our ihESCs were similar to class II hESCs, and ehESCs were similar to class III hESCs. Some alterations in class III ehESCs were similar to what is observed in cancer cells, including loss of XIST expression, H3K27me3 punctate enrichment and extremely skewed XCI, which have been reported in several human cancers, including breast, lung, esophageal and germ cell tumors [25-30]. These reports suggest that XCI abnormalitiesACCEPTED may contribute to the pathogenesis MANUSCRIPT of cancer. In our ehESCs, we found at least 7/27 common up-regulated genes in cancer. For example, RAP2C, a member of the Ras family, is involved in regulating cellular proliferation, differentiation and apoptosis [31]. Pirin (PIR) may play a role in melanoma progression and migration [32]. Glypican 3 (GPC3) is highly expressed in hepatocellular carcinoma but not in normal tissue [33]. Heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) is up-regulated in human pancreatic cancer (PC), and silencing of HS6ST2 inhibits progression of PC 14 ACCEPTED MANUSCRIPT through the inhibition of notch signaling [34]. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is associated with cancer progression, including increased proliferation and tumor growth promotion [35]. Proteasome 26S subunit (PSMD10) is up-regulated in various malignancies, including hepatocellular carcinoma, pancreatic cancer and breast cancer [36, 37]. Therefore, these genes may promote the tumorigenicity and metastasis of some malignancies. Among these genes, up-regulation of PSMD10 and GPC3 has been reported in XIST+ hESCs [7]. As a result, whether the transition of XCI states from class II to class III is correlated with the development of malignancy is unclear, and we cannot rule out the influence on hESCs and their derivatives. Simultaneously, we should evaluate the XCI states and X-linked gene expression before using hESCs in regenerating medicine and as an X-linked disease model. Previous reports have suggested that XIST binds to the gene-rich, open chromatin regions and interacts with XCI-related proteins to move genes subject to X inactivation into the proximity of the compacted/repeat-rich region of the Xi, which ACCEPTED MANUSCRIPT would prevent access of the transcription factors to regulatory regions of genes, resulting in silencing. When the cells lose the expression of XIST [38], we speculate that the XIST-coated X-chromosome silent compartment is disrupted and some genes that are not located near XIST are exposed to active gene domains and are re-expressed. The fully skewed XCI pattern in our early hESCs was similar to what has been 15 ACCEPTED MANUSCRIPT reported in established hESC lines [7]; however, whether the skewed XCI pattern represented an imprinted XCI, such as what occurred in preimplantation murine development, was unclear. We addressed this question by analyzing the XCI pattern of different sublines from the same hESC line, and found that different sublines expressed X-linked genes from different parental origin X chromosomes, and combined the random XCI pattern of ihESCs, which supports the notion that the fully skewed XCI pattern is the result of clonal selection without relationship to the parental original. In addition, the hESC lines under 5% oxygen processed the transition from random to fully skewed XCI, which further confirmed the selection was not the result of atmospheric oxygen. However, the factors that determine the clonal selection remain unclear. Previous research suggests that oxygen concentration is a critical requirement to derive female hESCs with two activated X chromosomes and cellular stress, such as atmosphere oxygen exposure and harsh freeze-thaw cycles, can induce irreversible XCI [22]. In our culturing condition that was under 5% oxygen, the hESCs maintained theACCEPTED expression of XCI MANUSCRIPT markers, XIST and H3K27me3 punctate enrichment, but the XCI pattern was converted from random to skewed. It is possible that the reason why we failed to derive female hESCs with two active X chromosomes may be the different culture conditions. For example, 1000 UI of human LIF was added in the previous conditions and 5% oxygen working cabinets were used in their culture [22]. A previous study hinted that human induced pluripotent stem cells derived on a LIF expressing SNL feeder shows two active X 16 ACCEPTED MANUSCRIPT chromosomes, which suggests that LIF may be an important factor to maintain two active X chromosomes [38]. Some procedures in our conditions, including passage and culture change were carried out in atmospheric oxygen, whether the temporary exposure would lead to the XCI alterations need further study. In subsequent research, we will further improve our culture conditions to preserve XCI in a stable state. In conclusion, our study showed that XCI alteration was a frequent epigenetic reprogramming event, however, the atmosphere was not the only influencing factor. Further studies need to be directed at investigating the influence of XCI alteration on the function of various differentiated cell types and find other critical factors for the stability of XCI. Acknowledgments This work was supported by grants from the National Basic Research Program of China (973 program, 2011CB964901 and 2012CB944901), and the National Natural Science Foundation of China (81222007 and 31101053). We thank the IVF team of the ReproductiveACCEPTED & Genetic Hospital of CITICMANUSCRIPT-Xiangya for their assistance. We thank the Postdoctoral Research Station of Central South University for their support. Conflict of interest The authors declare no conflict of interest in this paper. 17 ACCEPTED MANUSCRIPT References [1] J.A. Thomson, J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, J.M. Jones, Embryonic stem cell lines derived from human blastocysts, Science, 282 (1998) 1145-1147. [2] C. Spits, I. Mateizel, M. Geens, A. Mertzanidou, C. Staessen, Y. Vandeskelde, J. Van der Elst, I. Liebaers, K. Sermon, Recurrent chromosomal abnormalities in human embryonic stem cells, Nat Biotechnol, 26 (2008) 1361-1363. [3] J.S. Draper, K. 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Gradual loss of XIST expression and H3K27me3 punctate enrichment in female hESCs during early culture. (A): Heat map showing the relative expression levels of selected transcripts in initial-passage hESCs (ihESCs) and early-passage and hESCs (ehESCs). The colors indicate low (3green) to high (11red) absolute log2 expression level of genes. The two rows of XIST are two representative probes for XIST. (B): Expression of XIST in eight paired ihESCs (black) and ehESCs (grey) lines by RT-PCR. (C): Representative immunostaining of chHES51 at different passages with antibodies against H3K27me3 (green) and Oct4 (red). DAPI stained nuclei in blue. chHES51-P5 showed nearly 100% H3K27me3 positive punctate enrichment, chHES51-P9 showed 32.6% H3K27me3 positive punctate enrichment, and chHES51-P20 showed no H3K27me3 positive punctate enrichment. Scale bar = 50 μm. (D): Relative expression level of XIST in passages from P5-P20 in five female hESCs lines. (E): The percentage of H3K27me3 positive punctate enrichment in different passages from P5-P20 in five female hESCs (n > 1000). Bars = mean ± SD (two biologicalACCEPTED replications). MANUSCRIPT Figure 2. Both the ihESCs and the ehESCs showed a Xi. (A): Representative immunostaining for RNA pol II (green) followed by DNA FISH (red) for X chromosomes in the ihESCs (up) and ehESCs (bottom) of chHES51 cells. The arrow indicates Xi or its position. Please note that this active polymerase is excluded from the region surrounding the inactive X chromosome. Magnification × 1000. (B): Late 21 ACCEPTED MANUSCRIPT replication assays by R banding analysis in paired chHES51. Please note the inactivated X (the red arrowhead) showing high BrdU incorporation (late replicated) and the active X chromosome (the black arrowhead) show no BrdU incorporation (early replicated). Figure 3. A small subset of X-linked genes is up-regulated in ehESCs. (A): Percentage of each chromosomes-linked genes (probes) that are up-regulated more than 1.5-fold between ehESCs and ihESCs in female (46,XX) and male (46,XY) hESCs. The percentage of X-linked genes in the female is much higher than that of autosomes and the X chromosome in males. (B): Heat map of X-linked genes expression ratio. The expression ratios of X-linked genes between ehESCs and ihESCs are plotted on the X chromosome. The gene expression ratio for each gene is shown in log2 scale. Figure 4. Effect of oxygen on XCI. (A): RT-PCR expression analysis of XIST in P5 ihESCs and P20 ehESCs derived and cultured under 5% oxygen. *indicates P < 0.05 by t-test; (B):ACCEPTED Representative immunostaining MANUSCRIPT of chHES277 at different passages with antibodies against H3K27me3 (red) and Oct4 (green). DAPI stained nuclei in blue. Scale bar = 50 μm. 22 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Fig. 1 23 ACCEPTED MANUSCRIPT Fig. 2 ACCEPTED MANUSCRIPT 24 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Fig. 3 25 ACCEPTED MANUSCRIPT Fig. 4 ACCEPTED MANUSCRIPT 26 ACCEPTED MANUSCRIPT Table 1 The list of X-linked genes common up-regulated at least 1.5-fold in female ehESCs Cell locatio Gene name Description line n Nosa TSC22D3 TSC22 domain family, member 3 q22.3 6 RAP2C RAP2C, member of RAS oncogene family q26.2 8 proteasome (prosome, macropain) 26S subunit, PSMD10 non-ATPase, 10 q22.3 7 PRPS1 phosphoribosyl pyrophosphate synthetase 1-like 1 q22.3 6 POLA1 polymerase (DNA directed), alpha 1, catalytic subunit p22.11 6 PIR pirin (iron-binding nuclear protein) p22.2 9 PDK3 pyruvate dehydrogenase kinase, isozyme 3 p22.11 6 PCYT1B phosphate cytidylyltransferase 1, choline, beta p22.11 6 PAK3 p21 protein (Cdc42/Rac)-activated kinase 3 q22.3 5 NXT2 nuclear transport factor 2-like export factor 2 q22.3 7 Nance-Horan syndrome (congenital cataracts and dental NHS anomalies) p22.13 5 MORC4 MORC family CW-type zinc finger 4 q22.3 5 leucine-rich repeats and calponin homology (CH) domain LRCH2 containing 2 q23 6 LOC1001308 86 /// TMEM164 q22.3 7 KCNE1L KCNE1-like q22.3 5 IGSF1 immunoglobulinACCEPTED superfamily, MANUSCRIPT member 1 q26.1 6 HTR2C 5-hydroxytryptamine (serotonin) receptor 2C q23 5 HS6ST2 heparan sulfate 6-O-sulfotransferase 2 q26.2 5 H2BFM /// H2BFXP q22.2 5 GPC4 glypican 4 q26.2 7 GPC3 glypican 3 q26.2 5 DIAPH2 diaphanous homolog 2 (Drosophila) q21.33 7 CXorf39 chromosome X open reading frame 39 q22.2 6 COL4A6 collagen, type IV, alpha 6 q22.3 5 Alport syndrome, mental retardation, midface hypoplasia AMMECR1 and elliptocytosis chromosomal region gene 1 q22.3 6 ALG13 asparagine-linked glycosylation 13 homolog (S. cerevisiae) q23 10 ACSL4 acyl-CoA synthetase long-chain family member 4 q22.3 5 a indicate the number of cell lines in which the gene up-regulated >1.5 fold changes 27 ACCEPTED MANUSCRIPT Table 2 Genomic SNP genotyping and polymorphic cDNA analysis in 5 paired cell lines Gene SNP ID Band Genotyping P4 P20 P20 name expression subline1 subline2 expression expression chHES26 LAMP2 rs12097 Xq24 [A/T] [A/T] [T] [A] WDR44 rs10521584 Xq24 [C/T] [C/T] [C] [T] MBTPS2 rs5951476 Xp22.11 [G/A] [G/A] [A] [G] chHES45 LAMP2 rs12097 Xq24 [A/T] [A/T] [A] FAAH2 rs1367830 Xp11.1 [C/T] [C/T] [C] CA5B rs1808 Xp22.2 [C/T] [C/T] [C/T] MBTPS2 rs5951476 Xp22.11 [G/A] [G/A] [A] chHES51 FAAH2 rs1367830 Xp11.1 [C/T] [C/T] [C] [C] LAMP2 rs12097 Xq24 [A/T] [A/T] [T] [T] WDR44 rs10521584 Xq24 [C/T] [C/T] [C] [T] TCEAL4 rs11010 Xq22.2 [C/T] [C/T] [C] [C] chHES137 FAAH2 rs1367830 Xp11.1 [C/T] [C/T] [C] [T] MBTPS2 rs5951476 Xp22.11 [G/A] [G/A] [G] [A] chHES175 CA5B rs1808 Xp22.2 [C/T] [C/T] [C/T] FAAH2 rs1367830 Xp11.1 [C/T] [C/T] [C] ACCEPTED MANUSCRIPT 28 ACCEPTED MANUSCRIPT Table 3 Genomic SNP genotyping and polymorphic cDNA analysis in cell lines derived 5% O2 Gene P4 P20 name SNP ID Band Genotyping expression expression chHES277 LAMP2 rs12097 Xq24 [A/T] [A/T] [T] GPC3 rs5977910 Xq26.1 [C/A] [C/A] [C/A] MBTPS2 rs5951476 Xp22.1 [G/A] [G/A] [A] GPC4 rs41537046 Xq26.1 [G/A] [G/A] [A] chHES278 LAMP2 rs12097 Xq24 [A/T] [A/T] [T] WDR44 rs10521584 Xq24 [C/T] [C] [C] GPC4 rrs41537046 Xq26.1 [G/A] [G/A] [A] ATRX rs3088074 Xq21.1 [C/G] [C/G] [C] ACCEPTED MANUSCRIPT 29 ACCEPTED MANUSCRIPT Highlights: XCI dynamic change is a frequent epigenetic instability event during early hESC culture. a subset of X-linked genes were up-regulated in ehESCs, including some cancer-related genes. 5% oxygen was beneficial for XCI fidelity. ACCEPTED MANUSCRIPT 30