Ascorbic acid improves pluripotency of human parthenogenetic embryonic stem cells through modifying imprinted gene expression in the Dlk1-Dio3 region
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Human parthenogenetic embryonic stem cells (hpESCs) are generated from artificially activated oocytes, however, the issue of whether hpESCs have equivalent differentiation ability to human fertilized embryonic stem cells remains controversial.
hpESCs were injected into male severe combined immunodeficiency (SCID) mice and the efficiency of teratoma formation was calculated. Then the gene expression and methylation modification were detected by real time-PCR and bisulfate methods.
Comparison of five hpESCs with different differentiation abilities revealed that levels of paternal genes in the Dlk1-Dio3 region on chromosome 14 in the hpESCs with high differentiation potential are enhanced, but strictly methylated and silenced in the hpESCs with lower differentiation potential. Treatment with ascorbic acid, rescued their ability to support teratoma formation and altered the expression profiles of paternally expressed genes in hpESCs that could not form teratoma easily. No differences in the expression of other imprinting genes were evident between hpESCs with higher and lower differentiation potential, except for those in the Dlk1-Dio3 region.
The Dlk1-Dio3 imprinting gene cluster distinguishes the differentiation ability of hpESCs. Moreover, modification by ascorbic acid may facilitate application of hpESCs to clinical settings in the future by enhancing their pluripotency.
KeywordsTeratoma Germ Layer Differentiation Ability Teratoma Formation Parthenogenetic Embryo
bovine serum albumin
(Dulbecco’s) modified Eagle’s medium
differentially methylated region
human embryonic stem cells
human fertilized embryonic stem cells
human parthenogenetic embryonic stem cells
intra-cytoplasmic sperm injection
intergenic differentially methylated region
induced pluripotent stem cell
murine embryonic fibroblasts
real time polymerase chain reaction
severe combined immunodeficiency
Human embryonic stem cells (hESCs) have tremendous potential in regenerative medicine and cell therapy, as they can differentiate into cell derivatives of all three primary germ layers, endoderm, mesoderm and ectoderm. To obtain clinically applicable hESCs, parthenogenetic activation methods are preferred to the conventional method requiring oocyte fertilization. However, application of established cloned hESCs to the clinic is difficult at present because the hemagglutinating virus of Japan is used in all successful cases [1,2], whereas the hemagglutinating virus of Japan is not allowed to be used in the clinic at present. Therefore, parthenogenetic ES cells (pESCs) that were from parthenogenetic activated oocytes using an artificial method permitted in clinic settings , may be more feasible for clinical application.
pECSs have been derived from mouse , rabbit , monkey  and human sources [7-10], and their ability to differentiate into neurons and endoderm cells has been widely confirmed. Imbalance of imprinting gene expression and unique epigenetic modifications in the whole genome is reported to impair the pluripotency of pESCs. Allen and co-workers reported varying differentiation abilities of mouse pESCs, with some contributing to generation of mice after injection into tetraploid blastocysts and others failing to form specific cell tissues, particularly for skeletal muscle . Vassena et al. suggested that loss of differentiation ability is easier during serial differentiation and reprogramming processes for human pESCs (hpESCs) . Furthermore, in a study by Mai and co-workers, one of the hpESC lines failed to form derivatives from three germ layers, consistent with the suggestions of Lu et al.  and Liu et al. . Clearly, defects in differentiation ability of hpESCs limit their clinical application.
The Dlk1-Dio3 genomic regions were evolutionarily conserved in mammals. This region includes the paternally expressed genes Dlk1, Rtl1, and Dio3, and maternally expressed genes Meg3, Meg8, and antisense Rtl1. In mouse, aberrant silencing of the imprinted Dlk1-Dio3 gene cluster was observed in most induced pluripotent stem cell (iPSC) clones that contributed poorly to chimeras and failed to support the development of iPSC-derived animals [15,16]. Subsequently, Stadtfeld et al. demonstrated that ascorbic acid attenuates hypermethylation of the Dlk1-Dio3 cluster, consequently enhancing the pluripotency of iPSCs reprogrammed from differentiated B cells . Further studies by Bryce et al. suggested that aberrant DNA methylation within the Dlk1-Dio3 locus decreases the efficiency of generating mice via tetraploid complementation, supporting the contribution of this locus to the formation of low-quality iPSCs . In addition to iPSCs, activation of paternally expressed imprinted genes in mouse pESCs enhances their pluripotency, even resulting in the production of germline-competent mice . Additionally, expression profiling of the Dlk1-Dio3 cluster is correlated with pluripotency of iPSCs in pigs .
While studies on the Dlk1-Dio3 imprinting domain in hESCs have mainly focused on the field of tumor promotion, limited reports to date have validated the significance of this domain gene locus. Xie et al. showed that silencing of the Dlk1-Dio3 cluster of hESCs impairs generation of their differentiated hepatocyte-like cell derivatives but does not compromise multilineage differentiation ability . Furthermore, the group of Sun revealed that the conserved miRNA cluster within the imprinted Dlk1-Dio3 region is highly expressed in mouse and rhesus macaque ESCs but rarely in hESCs . Therefore, determination of expression patterns of the Dlk1-Dio3 cluster in hpESCs and analysis of its role in pluripotency regulation of hpESCs is necessary.
In the present study, we found that the Dlk1-Dio3 gene locus in hpESCs is aberrantly expressed and methylated, which correlated with teratoma formation. Furthermore, we examined the effects of ascorbic acid on methylation and gene expression patterns in the Dlk1-Dio3 cluster, with a view to ascertaining whether this compound contributes to improvement of the differentiation ability of hpESCs.
The current study was approved by the Institutional Review Board of Peking University Third Hospital and Guangzhou Medical University Affiliated Third Hospital. The volunteers involved in the present study were informed of all details of the procedure, including sample utility and research destination, and voluntarily signed an informed consent document. All necessary consents from any participatant involved in the study, including consent to participate in the study, were obtained.
Construction and culture of human fertilized and parthenogenetic embryos
Immature oocytes were collected from patients in the ICSI cycle because of male factor infertility in the reproductive medical centers at both Peking University Third Hospital and Guangzhou Medical University Affiliated Third Hospital. All immature oocytes were cultured in commercial in vitro maturation medium (SAGE In Vitro Fertilization, Inc., San Clemente, California, U.S.), and those that expelled the first polar body were regarded as mature oocytes at the metaphase II stage. One portion of MII oocytes was subjected to intra-cytoplasmic sperm injection (ICSI) micromanipulation to construct fertilized embryos and the other portion artificially activated by treatment for five minutes with 5 μM ionomycin and for two hours with 2 mM 6- dimethylaminopyridine (6-DMAP) to form parthenogenetic embryos. All embryos were cultured in G-1 medium, followed by G-2 medium from the eight-cell to the blastocyst stages (G1 and G2 medium were all purchased from Vitrolife Inc., Goteborg, Sweden).
Derivation of hESCs from fertilized and parthenogenetic activated embryos
Expanded blastocysts were removed of zona pellucida using Tyrode’s solution, and the inner cell mass isolated via immunosurgery and placed on mitomycin C-treated murine embryonic fibroblast (MEF) feeder layers. After 7 to 10 days, the primary colony was transferred to a new feeder layer and culture continued for 10 more days. The ESC colony was dissected into four to eight pieces upon passage and implanted into new feeder layers. Two hpESCs and three human fertilized embryonic stem cell (hfESCs) lines were derived sucessfully. The former was designated as BGHPES-1 and BGHPES-2, and the latter was designated as BGHFESC-1, BGHFES-2 and BGHFES-3, which were also named FHES-CONT.
General characterization of human embryonic stem cells
Characteristics of hESCs were determined using established protocols , as follows:
Alkaline phosphatase (AP) activity
AP activity was assessed in both hfESCs and hpESCs. Cell colonies were stained with BCIP/NBT for 8 to 12 minutes and examined under an inverted microscope.
hESC colonies were removed to a glass sheet and fixed using 4% paraformaldehyde for 30 minutes. Fixed ESC colonies were permeabilized with 0.2% Triton X-100 for 30 minutes, followed by blocking in 3% BSA in PBS for 2 hours. Subsequently, colonies were incubated with 1:100 diluted primary antibodies, those specific for stem cell pluripotent markers (OCT4 and NANOG) (OCT4: 1:200, ab27985, Abcam, Cambridge, UK; NANOG: ab80892, Abcam) and stem cell surface markers (TRA-1-60) (1:200, ab16288, Abcam), and stained with the corresponding secondary antibodies, followed by treatment with 10 μg/mL propidium iodide for 30 minutes. Fluorescence was assessed under a confocal microscope (A1R, Nikon Corp., Tokyo, Japan).
hESC colonies were implanted onto matrigel without a feeder layer, and cells collected after three-days culture. Next, colonies were rinsed in PBS solution and incubated in 0.075 mol/L potassium chloride for 10 minutes at 37°C. Finally, cells were fixed with methanol/glacial acetic acid and dropped onto glass slides. Chromosome spreads were Giemsa-banded and photographed. The karyotypes of hESCs were determined every 10 passages.
Differentiation in vitro and in vivo
hESCs were differentiated into embryoid bodies (EBs) in vitro by suspension culture. After 10 to 14 days, EBs were collected, and gene expression analyzed using RT-PCR methods. Specific genes in all three embryonic germ layers, including AFP (endoderm) (1:100, Human Germ Layer Marker Kit, Chemicon, Merck Millipore Corporation. The United States, Massachusetts city), SMA(mesoderm) (1:200, Human Germ Layer Marker Kit, Chemicon) and TUBULIN (ectoderm) (1:200, Human Germ Layer Marker Kit, Chemicon), were identified using immunofluorescence, and Nf68kd (endoderm), Hbz (mesoderm) and Albumin (ectoderm) with RT-PCR. Gene primers are listed in Additional file 1.
For in vivo differentiation, hESCs were injected into the inguinal grooves of six-week-old male severe combined immunodeficiency (SCID) mice after division into 300 to 400 small colonies. After two months, tumors were examined. All tumors were fixed for four to eight hours in 4% paraformaldehyde and embedded in paraffin. Sections were subsequently stained with hematoxylin and eosin, and examined under a light microscope for the presence of tissue derived from the three germ layers. In cases where tumors did not form within the two-month period, an equivalent volume of hESC colonies was injected into the same location. After three repeats of this step, hESCs were considered non-pluripotent.
Gene expression profile analysis
Total RNA was extracted and purified using TRIzol with an established kit, and cDNA was generated from extracted RNA using reverse transcription. PCR was carried out using primers for the paternally expressed genes, Dlk1, Rtl1, and Dio3, and the maternally expressed genes, Meg3, Meg8, and antisense Rtl1 encoding miR433 and miR127. Amplified products were analyzed on a 1.5% agarose gel, visualized via ethidium bromide (Invitrogen, Life Technologies Corporation., Carlsbad, Calfornia, US) staining, and imaged using the BioImaging system (UVP, Upland, CA, USA). Ubiquitously expressed glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as a control. Quantitative real-time PCR for the tested genes was performed on an ABI 7500 (Applied Biosystems, Life Technologies Corporation., Foster, Calfornia, US) system using the TaqMan real-time PCR probe primer mixture according to a previous study by Kagami et al. .
DNA methylation analysis via bisulfite modification
Genomic DNA was extracted via proteinase K digestion and phenol/chloroform extraction , denatured with NaOH and modified using sodium bisulfite and hydroquinone. Bisulfite-treated DNA was purified using a Wizard DNA clean-up system (Promega, Madison, WI, USA), following the manufacturer’s instructions. DNA was precipitated with ethanol after treatment with NaOH and eluted into 50 μl distilled water. The final products were stored at −80°C until use.
Bisulfite treatment was performed using an EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Bisulfite-converted DNA was amplified using nested PCR. Primer sequences are shown in Additional file 1. Each 25 μl PCR reaction mixture contained 4 μl of bisulfite-treated DNA, and reactions were performed according to the method described by Kagamiet al. [26,27]. The presence of amplified products was confirmed by electrophoresis on a 1.5% agarose gel.
PCR products were retrieved and ligated into the pMD19-T Vector System (TaKaRa). After transformation via heat shock into 200 μl competent Top10 cells (Tiangen, Beijing, China), colonies were isolated and cultured. For each sample, 15 positive clones were sequenced.
Ascorbic acid supplementation during hESC culture
hpESCs were cultured in knockout (D)MEM medium supplemented with 20% KnockOut Serum Replacement (Gibco, Life Technologies Corporation., Carlsbad, Calfornia, US), 1 mM glutamine (Sigma, Sigma, St . Louis, Missouri, US), 1% non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 50 UI/ml penicillin (Sigma), and 50 UI/ml streptomycin (Sigma) mixture. At passage 21, 100 μg/ml ascorbic acid were added to the ESC culture medium until the end of the experiment. hpESCs cultured without ascorbic acid were taken as the control group.
Data were analyzed using SPSS 17.0 software. The differences between the two groups were calculated with the chi-square test, and those among multiple groups with one-way analysis of variance (ANOVA). Data were considered significantly different at P values less than 0.05.
Derivation and identification of hpESCs
Gene expression and methylation modifications in the Dlk1-Dio3 region
To confirm these findings, we ascertained whether the other three hpESCs have been established in Guangzhou before, named GHPES-1, -2 and -3. Among the three hpESC lines, GHPES-1 and -2 contributed to derivatives from all three germ layers in teratoma with high frequency (10/12 and 9/12, respectively), while GHPES-3 failed to form teratoma, even after ESC injection more than ten times (1/12) [See Additional file 2]. Gene expression profiling revealed a significant increase in the levels of paternally expressed genes in GHPES-1 and -2, compared with GHPES-3 [See Additional file 2]. In contrast, no differences were observed in the expression profiles of maternally expressed genes [See Additional file 2]. The proportion of DMR methylation modifications remained at the 1% to 5% level, with no significant differences among the three hpESC lines [See Additional file 3].
Ascorbic acid supplement alters the levels of paternally expressed genes in the Dlk1-Dio3 region
To confirm whether differences in paternally expressed genes in Dlk1-Dio3 contribute to the in vivo differentiation ability of hpESCs, expression profiles of 25 paternal and 20 maternal genes were further examined. Gene cluster data from paternally expressed genes indicated a close correlation between BGHPES-2 and GHPES-3 that did not form teratomas easily and significantly lower expression of Dlk1, Rtl1 and Dio3 in both lines. However, after ascorbic acid treatment, this correlation between BGHPES-2 and GHPES-3 cell lines was not evident, and the characteristics of paternally expressed genes in the Dlk1-Dio3 cluster normalized [See Additional file 9]. No significant changes in the levels of maternally expressed genes were observed [See Additional file 10].
Data from the present study suggest that the differences in gene expression and methylation modifications in the Dlk1-Dio3 region contribute to the differentiation ability of hpESCs. Ascorbic acid supplementation narrows this gap and enhances the ability of ESCs to form teratomas in vivo by promoting paternal imprinted gene expression.
hpESCs are derived in multiple laboratories, and a large proportion form teratomas after injection into SCID mice, which appears similar to findings with hfESCs [7-10]. However, two important facts should be taken into consideration. One is that some established hpESC lines fail to differentiate and form teratomas in SCID mice, and the other is that no studies to date have focused on the efficiency of teratoma formation for pESCs. In the current study, efficiency of teratoma formation was at least 70% in three of our hpESC lines, but ranged from only 8.3% to 20% in the other two lines. Based on the established criteria, these five hpESC lines are categorized as normal ESCs. However, limited differentiation ability restricts the potential clinical application of some of these cells. The differentiation ability of pESCs has been reported previously by Park and co-workers, who showed that EBs differentiated from mouse pESCs display growth retardation and only contribute to derivatives of the endodermal layer .
Several methods have been developed to improve the pluripotency of pESCs. Hikichi and co-workers established a new type of pESCs by transferring traditional ESC nuclei into oocytes and deriving these pESCs again at the blastocyst stage (NT-pES), leading to two to five times enhancement of differentiation ability, compared with the original pESCs . The same group reported development of parthenogenetic embryos to term using the serial nuclear transfer method . Epigenetic modification was evidently altered during reprogramming mediated by nuclear transfer, and aberrant methylation observed in cloned embryos and animals. In addition to the nuclear transfer method, embryo aggregation at the eight-cell stage was applied to improve imprinted gene expression in pESCs . Turovets et al. treated hpESCs with trichostatin A, a potent histone deacetylase inhibitor, facilitating differentiation into the definitive endoderm lineage . In the present study, treatment of hpESCs with ascorbic acid partly re-activated paternally imprinted gene expression and, consequently, enhanced differentiation potential by promoting teratoma formation.
Ascorbic acid is accepted as a beneficial agent for somatic reprogramming into iPSCs, which acts biologically via Jhdm1a/Jhdm1b to enhance reprogramming efficiency and maintain the imprinting status of the Dlk1-Dio3 region to sustain reprogramming fidelity . Similar to pESCs, iPSCs display impotent germ-line transmission ability via teraploid complementation. Stadtfeld et al. treated mouse iPSCs with ascorbic acid at the primary reprogramming stage, and observed increasing efficiency of mouse generation via teraploid complementation . The authors demonstrated that ascorbic acid specifically attenuates hypermethylation of maternal imprinting genes in Dlk1-Dio3 clusters, similar to our results.
Dlk1-Dio3 is an evolutionarily conserved chromosome region located in chromosome 12 in mice and chromosome 14 in humans . Interestingly, Dlk1-Dio3 aberrations induce growth defects in both mice and humans with similar phenotypes, indicating that the biological function of Dlk1-Dio3 is analogous in these two species [35-37]. The Dlk1-Dio3 region appears critical for the pluripotency of iPSCs and tumor promotion [15,38,39]. Earlier data from two groups suggest that hypermethylation of maternally imprinted genes in Dlk1-Dio3 of mouse iPSCs induces abnormal silencing of the locus [17,40], in contrast to our finding that maternally imprinted genes are overexpressed and paternally imprinted genes are silenced in hpESCs. Another study by Henzler and co-workers confirmed that overexpression of Dlk1-Dio3 miRNA early in reprogramming reduces reprogramming efficiency , in accordance with our findings. The results indicate that aberrant expression of imprinted genes in this region induces reprogramming deficiency, regardless of higher or lower expression levels.
Re-activation of paternally expressed genes in the Dlk1-Dio3 cluster appears beneficial for regaining pESC pluripotency in mouse. Jiang et al. suggested that activation of paternally expressed imprinted genes in pESCs induces germline transmission when injected into blastocysts . Another study by Li and co-workers indicated that the culture environment in vitro induces epigenetic changes in paternally expressed imprinted genes, thereby increasing the pluripotency of pESCs . We additionally observed differential expression of specific paternally expressed genes in established hpESCs. However, normal expression of genes in the Dlk1-Dio3 cluster appears critical for pluripotency. Interestingly, Dlk1, Rtl1 and Dio3 gene levels were enhanced upon treatment of hpESCs with ascorbic acid, while IG-DMR and MEG3-DMR methylation patterns remained unchanged, indicating the involvement of other regulatory mechanisms in this modification. Hiura and co-workers demonstrated that valproic acid (VPA) enhances the pluripotency of human iPSCs without methylation modification of DMR . Thus, the DMR methylation process may not be correlated with activation of paternally expressed genes in the Dlk1-Dio3 region, supporting the contribution of other mechanisms in regulation of expression.
Menzorov et al. found that mouse ESCs can contribute into teratoma and even in chimera although their Dlk1-Dio3 imprinting control region is hypomethylated . This result is similar to our findings, which confirmed that poor methylation seems not to disturb ESCs pluripotency, but in our results, important paternally expressed genes in this imprinting region were up-regulated and positively related to ESCs differentiation ability, which indicated that methylation is not the only regulation mode for these genes in the Dlk1-Dio3 region. Non-coding RNAs may be an undelying regulator for the expression of these genes. In the Dlk1-Dio3 region, the maternally expressed genes can produce non-coding RNAs, including mir-431, mir-433, mir-127, mir-432 and mir-136. Liu et al. suggested that microRNAs in the Dlk1-Dio3 region can potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region . Regarding the fact that microRNA expression can be regulated by ascorbic acid treatment , it is possible that ascorbic acid indirectly changed the expression level of paternal genes by microRNA regulation.
Imprinting genes have shown a tremendous potential to regulate their pluripotency and multi-differentiation ability. During cell differentiation and tissue formation, it is critical that genomic demethylation and de novo methylation take place for lineage specification and to establish tissue-specific methylation patterns. Often, non-fertilized-originated human pluripotent stem cells showed abnormal epigenetic modification and imprinting gene expression, including nuclear transfer embryonic stem cells  and iPSCs ; however, hpESCs have a stable epigenetic modification mode, which is different from the dynamics and inconsistent epigenetic status in the other two kinds of pluripotent stem cells. In 2004, Kono et al. proved first that modification of H19 and IGF2 in oocytes can result in the birth of parthenogenetic mice . This indicated that H19-IGF2 was important to parthenogenesis, which suggested that manipulation of imprinting genes can reverse parthenote to fertilization-like status. In Mitalipov's study, he suggested that hypermethylation within the IGF2/H19 IC in all analyzed primate ESC lines resulted in a risk of cellular overproliferation and tumor formation . Recently, Yin et al. demonstrated that knockdown of H19 can improve mouse pESC differentiation potential in the lineages of ectoderm and mesoderm . In further study, H19-Ifg2 and Dlk1-Dio3 imprinting region co-modification may have the greatest impact on improvement of the differentiation ability of pESCs.
In conclusion, the efficiency of teratoma formation by hpESCs is improved with ascorbic acid treatment, leading to enhancement of potential differentiation ability. Ascorbic acid has beneficial effects on general human physiology and may, therefore, be safely used as a stimulatory drug to facilitate clinical application of hpESCs.
This work was supported in part by the Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Funds for general program (31371521 and 81370766), and Ph.D. Programs Foundation of Ministry of Education of China (20110001120008), and Beijing Nova Program, and the Zhujiang Science and Technology Star Project of Guangzhou (2012 J2200006) and Guangdong Province Higher Education Funding(Yq2013135 and 2013KJCX0149).
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