Author contributions: T.-L.C.: overall planning and design, collection and/or assembly of data, and data analysis and interpretation, and manuscript writing; J.T.: conception and design, collection and/or assembly of data, and data analysis and interpretation; N.T.: collection and/or assembly of data, data analysis and interpretation; G.K.: conception and design, collection and/or assembly of data, microarray data analysis and interpretation, and manuscript writing; J.C.-W.: conception and design, and financial support; S.M.G.: conception and design; M.F.P.: conception and design, data analysis and interpretation, and manuscript writing; E.W.: overall planning and design, financial support, and provision of study material or patients, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLS EXPRESS August 16, 2010.
Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions. STEM CELLS 2010;28:1782–1793
Cultured human embryonic stem cells (hESCs) exhibit properties of indefinite self-renewal and pluripotency [1–3] and therefore hold great potential for the study of early human development and use in therapeutic applications. It should be noted, however, that prolonged growth in vitro can alter the properties of hESCs. For example, hESCs cultured in serum-free media can accumulate point mutations, acquire progressive epigenetic changes over prolonged culture in vitro and develop aneuploidy [4–7]. It has been postulated that these progressive changes may be the consequence of adaptation of pluripotent stem cells to in vitro culture systems . How individual parameters dictated by particular in vitro culture methods, such as oxygen tension, medium composition, and cell harvesting and seeding methods, affect the genetic and epigenetic stability of hESCs remains unclear. The effect of culture parameters on the epigenetic state of pluripotent hESC culture is not generally considered. In fact, it is generally assumed that undifferentiated hESCs and induced pluripotent stem cells (iPSCs) do not undergo alterations in epigenetic status during in vitro expansion. However, an increasing body of evidence suggests this is not the case. For example, while examining DNA methylation patterns of various hESC lines as a function of time in culture, Allegrucci et al.  observed progressive culture-induced epigenetic changes in the hESC epigenome that mostly consisted of loss of DNA methylation. In particular, within the same BG01 cell line, switching cultures from serum-containing conditions to serum-free or feeder-free conditions lead to significant epigenetic changes regardless of passage method. Similarly, Koivisto et al.  reported that hESCs cultured in serum-free knockout serum replacement (KOSR)-containing medium display an increased growth rate compared with hESCs grown in fetal calf serum (FCS)-containing medium. Skottman et al.  subsequently performed gene expression profiling and discovered that genes involved in the regulation of transcription, RNA processing, and cell proliferation, such as SULF1  and CER1 , as well as a group of at least 50 adhesion-related genes (including integrins, laminin receptors, and TGFBR1) were differentially expressed between hESCs cultured in serum-containing medium and those in KOSR medium. Furthermore, during the process of iPSC generation, the culture of emerging iPSC clones under serum-free culture conditions is thought to either induce further epigenetic modifications that will sustain undifferentiated growth of pluripotent cells or select for iPSCs that possess such epigenetic changes . Another recent study by Pick et al.  shows that abnormal expression of imprinted genes associated with DNA demethylation occurs in different iPSC lines at various levels. The causes and molecular mechanisms of the potential culture induced epigenetic changes and the consequences of the resultant gene expression changes for hESC and iPSC behavior remain undetermined.
Here, we show that the ascorbate in the serum-free culture medium of hESCs induces expression of the tumor necrosis factor (TNFR) receptor CD30 and that CD30 expression alters hESC behavior. CD30 (TNFRSF8) was initially identified as a cell surface biomarker for Hodgkin and Reed–Sternberg (H–RS) cells in Hodgkin lymphoma [15–17]. Immunohistochemical analysis of a large range of human tumors has shown that CD30 is also overexpressed in a subset of diffuse large-cell neoplasms with anaplastic features, termed anaplastic large-cell lymphoma (ALCL) [18, 19]. Overexpression of CD30 is a characteristic of H–RS and ALCL cells and has been linked to activation of NF-κB and the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase pathway, both of which contribute to tumorigenesis and maintenance of survival of H–RS and ALCL cells [20–23]. Indeed, CD30 overexpression is thought to be essential to the pathogenesis of H-RS and ALCL [20, 23, 24]. We showed previously that aneuploid hESC lines express CD30, a biomarker for embryonal carcinoma cells, the malignant counterpart of hESCs. Expression of CD30 activates NF-κB signaling and protects hESCs from apoptosis . Several groups subsequently reported CD30 expression in diploid hESCs [26–29]. The cause and long-term biological consequences of CD30 expression in hESCs remain unknown.
Here, we report that CD30 expression is induced in hESCs grown in KOSR, but not in serum-containing culture conditions. We identify ascorbate as the only molecule in the very widely used KOSR medium that is responsible for CD30 induction. We show that ascorbate acts directly on hESCs and activates CD30 expression by triggering a dramatic loss of methylation in a CpG island in the CD30 promoter and exon I. To determine the effect of long-term activation of CD30 signaling in hESCs, we next engineered hESC lines to overexpress CD30 variant (CD30v) [30, 31], a naturally occurring splice variant that codes only for the cytoplasmic signaling domain of the protein, which is constitutively active in the absence of ligand. Our data show that such CD30v expressing hESC lines display reduced apoptosis, enhanced clonal growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. We conclude that hESC culture media containing ascorbate promotes CD30 expression through an epigenetic mechanism and that this provides a survival advantage to CD30 positive hESCs. CD30 expression and transcriptome changes mediated by CD30 may help adapt hESCs to in vitro culture.
MATERIALS AND METHODS
Cell Lines and Culture Conditions
HES2, HES3, and HES4 hESC lines were maintained in 20% FCS on mouse embryonic fibroblast feeder layers (6 × 104/cm2) with mechanical dissection every 7 days according to the protocols described [1, 2]. HES2, HES3, and HES4 were all transferred to the culture system described by Amit et al. . Collagenase was used to disaggregate colonies of hESCs for subculture. The culture medium was supplemented with 20% KOSR (Invitrogen Australia, Melbourne, Australia) and 4 ng/ml basic fibroblast growth factor (b-FGF), and feeder cells were used at a density of 2 × 104/cm2. Passage number of mechanically transferred hESCs in 20% FCS stock cultures (Px) and subsequent weekly passage number in serum free culture (Py) is indicated as Px + y. HES3 cell line was also grown in the culture system of Xu et al.  on Matrigel (BD-Biosciences)-treated surface in mouse embryonic fibroblast-conditioned medium from KOSR and 4 ng/ml b-FGF or KOSR minus ascorbate and 100 ng/ml b-FGF. MEL2 hESCs were cultured at 37°C in either a standard CO2 incubator (Sanyo Biomedical, Wood Dale/IL, USA) with 20% oxygen and 5% CO2 or a variable oxygen incubator (Binder Inc., Great River/NY, USA) with 2% oxygen with the same bulk culture conditions.
Bisulfite Sequencing of the CD30 Promoter CpG Island
DNA bisulfite reaction was performed using the MethylEasy DNA Bisulfite Modification Kit (Human Genetic Signature, North Ryde/NSW, Australia) following the manufacturers instructions. The nucleotide sequences of the primers used to amplify the CpG island region were as follows: CD30BISF 5′- GGTAGTATATTTTT TAGAGTTAGGATTATTAGTTT-3′, CD30 BISFN 5′-TTTT TTAGTGTGTTTTTTTTTGAGTTAT-3′, CD30BISR 5′CAAC TAAA CAAAAAACR AAATAAAAAATAT-3′. CD30BISF and CD30BISR were used as the first round polymerase chain reaction (PCR) primers and CD30BISFN and CD30BISR as the second round PCR primers. The PCR reaction contained 12.5 μl of PCR Master Mix (Promega), 2 μl of converted sample DNA or first round PCR product, 25 pmol each of forward and reverse primers and water to 25 μl volume. The PCR conditions were: initial denaturation at 95°C for 60 seconds, 30 cycles of denaturation at 95°C for 60 seconds, renaturation at 50°C for 2 minutes, extension at 72°C for 2 minutes, and the final extension at 72°C for 10 minutes. Products were detected by electrophoresis on a 2% agarose gel. The amplicons were then cloned into the pGEM-T easy vector (Promega Australia, Sdyney, Australia) for DNA sequencing.
Reconstitution of KOSR Minus Ascorbate
Custom preparation of the various KOSR fractions was carried out by Millipore (Millipore Australia, North Ryde/NSW, Australia) according to  with the exception that ascorbate was omitted. For medium reconstitution, the pH of the amino acid solution was raised to about 7.0–7.4 and then the albumin solution and transferrin were added. The pH of the albumin-amino acid–transferrin mixture was adjusted to pH 7.7–7.9 and the insulin and trace elements were added. Cell culture grade water was added to give the desired volume and the solution was filter-sterilized. hESCs were cultured with KOSR minus ascorbate as per Amit et al.  with the modification that 100 ng/ml of b-FGF was used.
Standard G-banding karyotype analysis of Karyomax/colchicines-treated hESCs was carried out at different passage levels under serum-free conditions and serum-containing conditions. A total of at least 20 metaphases were evaluated.
PCR Amplification of CD30 Microsatellite Region
Genomic DNA of hESCs was isolated using QIAamp DNA Mini Kit (Qiagen Australia, Doncaster/VIC, Australia). The CD30 microsatellite (MS) was amplified from genomic DNA samples by PCR. Fifty to hundred nanograms of gnomic DNA was used as templates. The reactions contained 1.5 mM MgCl2, 1× PCR buffer, 0.2 mmol/l dNTPs, and 2.5 U AccuPrime Pfx DNA Polymerase (Invitrogen) and 25 pmol each of forward and reverse primers in 50 μl volume. The nucleotide sequences of the primers flanking the CD30 MS were as follows: the forward primer, 30MSF 5-AAG GAGCAAAGAGAAAACCCAGG-3 and the reverse primer, 30MSR 5-CCCAAGAAACGGTGAAATGTGAG-3. The reaction condition was as follows: initial denature at 94°C for 30 seconds, 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and the final extension at 72°C for 7 minutes. The PCR products were then analyzed by gel electrophoresis.
Colonies of hESCs and iPSCs were fixed in cold ethanol for 10 minutes at −20°C, air dried, and stained with (1:30) mouse monoclonal CD30 antibody Ber-H2 (Dako Australia, Campbellfield/VIC, Australia) and (1:50) mouse monoclonal antibody Oct-3/4 (C-10, Santa Cruz Biotechnology, Inc., Santa Cruz/CA, USA), followed by goat anti-mouse IgG1 conjugated to Alexa Fluor 488 and goat anti-mouse IgG2b (1:1,000) conjugated to Alexa Fluor 568 (Invitrogen). All are diluted in PBS containing 10% normal goat serum. Nuclei were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Appropriate isotype antibodies diluted at an identical final concentration as the primary antibodies were used as negative control for the experiments. Fluorescence microscope BX51 (Olympus Australia, Mt Waverley/VIC, Australia) was used to visualize the CD30/Oct-3/4 staining with image program Cell Pro 6.1.
hESC lines HES2, HES3, HES4, and MEL1 were harvested with cell dissociation buffer (Sigma-Aldrich, Castle Hill/NSW, Australia) and stained using TG30 (anti CD9) monoclonal antibody supernatant (Australian Stem Cell Centre, Melbourne/VIC, Australia) and with (1:30) CD30 (Ber-H2). Detection was carried out with Alexa Fluor 647 goat anti-mouse IgM2a (TG30) and Alexa Fluor goat anti-mouse IgG1 (CD30; 1:1,000). Appropriate isotype controls were included in all analyses as well as the addition of propidium iodide into all samples (0.5 μg/ml) for exclusion of dead cells. A BD FC500 analyzer (Beckman Coulter Australia, Gladesville/NSW, Australia) was used to measure CD30 and TG30 expression. In some experiments, flow cytometry was performed using a BD FACSVantage-Diva sorter.
For “Establishment of CD30 variant expressing hESC lines by lentiviral transduction” and “Analysis of phenotypes and transcriptome of CD30v-expressing hESCs” see Supporting Information.
KOSR Upregulates CD30 in Diploid hESCs
We previously reported that the cell surface receptor CD30 (TNFRSF8) is highly expressed in karyotypically abnormal hESCs . Here, we show that hESC lines freshly established from CD30 negative standard (serum-containing medium) cultures display a progressive increase in CD30 expression on their expansion in KOSR medium. This is detected between passage 3 and 15, so that after 15 weekly passages in each of the three hESC lines examined approximately 70%–80% of hESCs-expressed CD30 when analyzed by flow cytometry (Fig. 1A). In all lines cultured in KOSR medium (only HES3 shown), a subset of cells begin to display the characteristic plasma membrane labeling of CD30 when analyzed by fluorescence microscopy (Fig. 1B) and increased CD30 mRNA expression (Fig. 1C). Importantly, after extended passages in KOSR, all three lines display abundant CD30 expression, but were overtly karyotypically normal (Supporting Information Fig. 1). In agreement with our previously published data , no CD30 expression was detected in the parental hESC lines after 15 weekly passages under standard 20% FCS culture conditions, in contrast to KOSR-cultured hESCs (Fig. 1D).
To distinguish between KOSR upregulation of CD30 versus FCS repression of CD30, we cultured hESCs with either 20% FCS, 20% KOSR, 10% FCS plus 10% KOSR, or 10% KOSR plus 20% FCS culture medium for 4 weeks. Subsequent analysis of CD30 expression in these hESC cultures clearly demonstrated that CD30 upregulation was in response to KOSR, not the absence of FCS (Fig. 1E). KOSR induction of CD30 was consistent across the five hESC lines tested (HES2, HES3, HES4, MEL1, and MEL2 [not shown]), and was independent of b-FGF (Supporting Information Fig. 2A) or the batch of KOSR (Supporting Information Fig. 2B). These experiments unequivocally demonstrate that the KOSR medium is responsible for CD30 expression and excludes the possibility that FCS represses the expression of CD30 or that CD30 immunoreactivity is unmasked on switching to serum-free cultures. In this respect, it is important to note that the epitope of the Ber-H2 CD30 antibody used throughout this study was previously reported to be unaffected by the thioredoxin-mediated redox modulation of the CD30 protein .
KOSR Induction of CD30 Is Due To Ascorbate
To determine which component of KOSR is responsible for CD30 induction, we retroengineered the medium using the KOSR patent data  and tested nine individual fractions of compounds that together make up KOSR. Only one component of one of the KOSR fractions, namely ascorbate (50 μg/ml) induced CD30 (Fig. 2A). Indeed, omission of ascorbate from complete KOSR prevented the induction of CD30 in HES2 or HES3 over 15 and 23 weeks in serum-free culture conditions, respectively (Fig. 2B). hESC proliferation in KOSR minus ascorbate was reduced such that population doubling time was increased twofold (data not shown), but this could be overcome by increasing the b-FGF concentration to 100 ng/ml. Complete KOSR (with either 4 or 100 ng/ml b-FGF) induced >90% CD30 expression in these hESC lines over the same time period (Fig. 2B). We therefore conclude that ascorbate is the sole component in KOSR responsible for CD30 induction in hESC, having excluded harvesting technique, b-FGF addition, KOSR batch or reduced feeder density.
Ascorbate induction of CD30 expression in hESCs seems not to be reversible by ascorbate-free culture. For example, MEL3 hESCs, originally established in KOSR, remained uniformly CD30 positive for more than 14 weekly passages after transfer to standard FCS conditions (Supporting Information Fig. 3A). We next verified that ascorbate-mediated CD30 induction occurs through direct interaction with hESCs and not through a possible paracrine mechanism via the mouse embryonic fibroblast (MEF) feeder layer by showing that under feeder-free conditions, ascorbate also induces CD30 expression (Supporting Information Fig. 3B). We further tested multiple hESC media that are used for serum-free and feeder-free hESC culture (Vitro-HES and mTeSR1 medium) for their CD30 inducing ability. As predicted by the fact that these media, like KOSR, also contain 50 μg/ml ascorbate, both media were found to induce CD30 expression in hESCs (Supporting Information Fig. 3C). We also show that CD30 expression is induced by ascorbate concentrations as low as 5 μg/ml and as high as 500 μg/ml, albeit with different kinetics of CD30 induction (Supporting Information Fig. 4). We next investigated CD30 expression in human iPSCs that are derived in KOSR medium that contains ascorbate. Indeed, CD30 is highly expressed in the iPSC line ES4CL1  cultured in KOSR (Supporting Information Fig. 5), suggesting that ascorbate may have a similar CD30 inducing effect on human induced pluripotent cells as on hESCs.
The Mechanism for Ascorbate-Induced CD30 Expression in hESCs Is Epigenetic
We next investigated potential molecular mechanisms responsible for ascorbate-induced CD30 expression. First, we tested whether ascorbate directly affects CD30 promoter activity in transient CD30 promoter-luciferase reporter assays. As shown in Figure 3A, addition of ascorbate to hESCs transiently transfected with a CD30 promoter luciferase reporter construct does not cause significant upregulation of CD30 promoter activity. In Hodgkin lymphoma and ALCL, where CD30 is used as a diagnostic marker, increased CD30 expression has been linked to modulation of a microsatellite repeat in the CD30 promoter  as well as with increased expression of AP-1, a critical transcription factor for CD30 expression in Hodgkin lymphoma . We could, however, neither detect any modulation of microsattelite repeat length between CD30 positive and CD30 negative hESCs by PCR (Fig. 3B) and DNA sequencing (data not shown) nor detect a difference in AP-1 (Jun B) expression between CD30 positive and CD30 negative hESCs (Fig. 3C). We next investigated the possibility that ascorbate induces CD30 expression via an epigenetic mechanism. (CD30+ or CD30−) hESCs cultured in either KOSR or KOSR minus ascorbate were sorted for expression of the cell surface pluripotency marker TG30/CD9 and CD30 expression using flow cytometry. 43 CpG sites in the core promoter region of the CD30 gene (−249 to +509) of CD9+CD30− and CD9+CD30+ hESCs were bisulfite sequenced and compared. KOSR-cultured CD9+CD30+ hESCs showed clear demethylation of these 43 CpG sites, whereas CD30 negative undifferentiated hESCs show almost uniform methylation (Fig. 3D). These results suggest that ascorbate upregulates CD30 in hESCs by the demethylation of the CpG island in the promoter region.
Changes of Oxygen Levels in Culture Affects Ascorbate-Mediated CD30 Induction
Ascorbate is an antioxidant and a cofactor for several enzymes, including ascorbate oxidase (Fig. 4A). To determine whether oxygen is involved in ascorbate-mediated CD30 induction in hESCs, we cultured hESCs in KOSR medium under 20% and 2% oxygen. Our data show that culturing hESCs in 2% oxygen enhances ascorbate-mediated CD30 induction (Fig. 4B, 4C), suggesting that a redox-based mechanism may be involved in CD30 induction. Further research is needed to determine whether other antioxidants like N-Acetyl-L-cysteine  or Trolox [40, 41] have a similar effect on CD30 induction.
Generation of CD30v Expressing hESC Lines
Most laboratories have adopted serum-free culture of hESCs as the method of choice and nearly all human iPSC generation involves culture of reprogrammed cells in KOSR. As our data show that the ascorbate in KOSR is responsible for CD30 expression in hESCs and that mTeSR1 and VitroHES also induce CD30 expression, it is likely that the majority of hESCs and iPSCs studied worldwide express CD30. Does this, however, affect the behavior of hESCs? To better understand the downstream consequences of CD30 expression for hESC biology, we generated hESC lines that overexpress CD30 variant (CD30v), a CD30 splice variant that codes for the cytoplasmic signaling domain of CD30 and that is constitutively active in the absence of ligand [30, 31]. These lines were next cultured under FCS culture conditions, thus, preventing expression of endogenous CD30. We chose this approach, rather than overexpressing full-length CD30 because it simplifies the interpretation of the effect of CD30 signaling on hESC as it circumvents issues such the shedding of the CD30 ligand binding ectodomain [42, 43] and ligand availability. We employed the promoter of elongation factor-1α for constitutive overexpression of either CD30v-IRES-GFP (IRES, internal ribosome entry site; GFP, green fluorescent protein) or the GFP control. Transduced hESC cultures were analyzed weekly by fluorescence microscopy and only GFP-positive cells were transferred for 6–8 weeks by mechanical dissection until all CD30v-IRES-GFP and GFP-transduced hESCs (HES2, HES3, and HES4, only HES2 and HES4 shown) were almost uniformly GFP positive (Fig. 5A). Endogenous CD30 expression remained undetectable, as expected of hESCs cultured under FCS containing conditions (results not shown). Importantly, all GFP and CD30v expressing lines displayed a normal karyotype (Supporting Information Fig. 6).
To validate CD30v expression in the transduced hESCs, mRNA and protein levels were determined by quantitative real-time PCR and western blotting. CD30v mRNA expression was over 100-fold higher in CD30v-transduced hESC lines, when compared with the control CD30-negative hESC line (Fig. 5B). Western analysis detected CD30v protein in transduced hESCs, but not in control hESCs (Fig. 5C). To determine whether CD30v expressed in the transduced hESC lines is biologically active, an in vitro NF-κB luciferase reporter assay was performed. Indeed, CD30v expressing hESCs displayed increased NF-κB transcriptional activity (Fig. 5D). This increase is comparable with that observed in hESC that endogenously express full-length CD30 . Furthermore, we also observed that phospho-ERK1/2 is upregulated in CD30v expressing hESCs (Supporting Information Fig. 7). Activation of NF-κB activity and increased P-ERK expression was previously observed in H–RS and ALCL cells [20, 23, 44] that express CD30.
CD30v Reduces Spontaneous Apoptosis of hESCs
Our group has previously reported that transient expression CD30 or CD30v in hESCs leads to inhibition of apoptosis . This earlier observation was confirmed with the stable CD30v-expressing hESC lines established in this study. Wild-type, GFP control, and CD30v-expressing hESCs maintained in serum-containing conditions in the absence of ascorbate were expanded in serum-free conditions for 7 days and spontaneous apoptosis was quantified by flow cytometry using the terminal deoxynucleotidyl transferase dUTP nick end labeling. Our data show that, while GFP control HES2 and HES3 cells display 26% and 14% spontaneous apoptosis, respectively, CD30v-expressing HES2 and HES3 cells display 12.3% and 6.5% spontaneous apoptosis over 48 hours (Fig. 6A), respectively. These data indicate that CD30v-transduced hESC lines are more resistant to cell death under in vitro culture conditions.
CD30v Increases Single-Cell Plating Efficiency of hESCs
hESCs undergo a process of adaptation in serum-free culture conditions that is accompanied by a progressively increased ability to seed both less cells at each split, and smaller clumps of hESCs [4, 8, 45–47]. As this process of adaptation is also accompanied by progressively increased CD30 expression, we investigated the cloning efficiency of CD30v expressing hESCs that had never been in serum-free medium culture conditions. All hESCs examined were first expanded into serum-free culture conditions for 7 days. Then these cells were harvested and dissociated into single cells and sorted into 96-well tissue-culture plates at 100 cells per well coated with Matrigel. After 7 days, wells with surviving colonies (Fig. 6B) were scored. The relative cloning efficiency was compared between wild-type, GFP control, and CD30v-transduced hESCs. HES2 CD30v and HES3 CD30v expressing lines show a sixfold and threefold increase in single-cell plating efficiencies as compared with their GFP-transduced counterparts (Fig. 6C). No significant difference between GFP and wild-type hESCs was observed. Our data clearly demonstrate that CD30 signaling enhances single-cell dissociated hESC survival.
Transcriptome Analysis of CD30v Expressing hESC
To understand how CD30 signaling affects the transcriptome of hESCs, microarray analysis was profiled on Illumina Sentrix6-V2 gene expression microarrays. To prevent endogenous CD30 expression, CD30v-expressing and GFP control hESCs cultured in FCS were harvested and undifferentiated hESCs were isolated by fluorescence activated cell sorting (FACS), using the pluripotency markers TG30 and GCTM2 (Fig. 7A). RNA was isolated from TG30+/GCTM2+ CD30v and GFP control hESCs and used for array analysis. Three experimental replicates displayed very consistent gene changes, as shown by the similar heat maps (Fig. 7B). A twofold difference in expression levels and a B-statistic greater than zero were the criteria used to identify significant changes. CD30v-expressing HES2 hESCs showed upregulation of 136 genes and downregulation of 243 genes, over the GFP control line (Supporting Information Tables S1, S2). Ingenuity analysis of the cohort of upregulated and downregulated genes in CD30v-expressing hESCs shows the majority of CD30-mediated gene expression changes are associated with cell signaling, lipid metabolism, and tissue development. (Supporting Information Table S3).
Further analysis shows that 30 of the 136 upregulated genes are zinc finger (ZF) proteins belonging to the Krueppel C2H2-type ZF protein family. Almost all of these upregulated ZF proteins (27 of 30) are located together on chromosome 19 (Supporting Information Table S4) and were previously found to be upregulated in seminomas [48, 49]. Interestingly, Hittmair et al.  showed that a subpopulation of seminomas express CD30 and that CD30 expression in seminomas might indicate their upcoming transformation to embryonal carcinoma. Collectively, these data support the observed cellular survival phenotype of CD30 expressing hESCs and suggest a novel link between CD30 and altered lipid signaling as well as the potential involvement of a specific set of ZF transcription factors with preneoplastic behavior of hESC.
CD30v Does Not Affect Spontaneous Differentiation
Previously, we showed that hESCs that endogenously express CD30 display a preponderance of immature neural progenitor-like cells . To determine whether CD30v signaling has a direct impact on hESC differentiation, we performed teratoma assays with the HES2-GFP, HES4-GFP and HES2-CD30v, and HES4-CD30v overexpressing lines. Our data (Supporting Information Fig. 8A) show that all lines are able to generate noninvasive teratomas with representatives of all three germ layers. To further investigate if CD30-mediated signaling affects the kinetics of spontaneous differentiation, we quantified Nanog and Oct4 mRNA expression in embryoid bodies derived from HES2-GFP, HES4-GFP and HES2-CD30v, and HES4-CD30v overexpressing lines at 0, 6, and 12 days, respectively. These data (Supporting Information Fig. 8B) show that downregulation of Nanog and Oct4 occurs independently of CD30 status.
We previously reported that the surface antigen CD30 is expressed in aneuploid hESCs . The expression of CD30 correlated with activated NF-κB and increased resistance to apoptosis. Subsequently, several reports show that expression of CD30 is not restricted to aneuploid hESCs [26–29]. Our data demonstrating that CD30 expression is induced by the serum-free KOSR culture medium and that ascorbate is the specific compound in this medium responsible for CD30 expression now provide a rationale for these observations. In our previous study relating CD30 expression to karyotypic abnormalities, we carried out experiments in serum-containing medium that does not contain ascorbate and that consequently does not induce CD30 expression, and this thus allowed us to correlate CD30 expression with karyotypic abnormalities in hESC. The implication of our present data is that hESCs cultured in ascorbate-containing medium will express CD30. This was indeed confirmed in several commercially available hESC culture media (VitroHES, mTeSR1). Recently, Harrison et al.  reported that two sublines (H7.s14 and H7.s6) of the H7 hESC cell line when cultured in KOSR were CD30 negative when analyzed by FACS. This apparent discrepancy with our data may be related to intrinsic cell line differences. Alternatively, it is possible that these two particular H7 sublines derived from original CD30-expressing H7 hESCs acquired further epigenetic changes that lead to remethylation of the CD30 locus and downregulated CD30 expression. In our laboratory, every cell line cultured in KOSR expresses CD30 (HES2, HES3, HES4, MEL1, and MEL2). We and others have observed that CD30 expression is quickly downregulated on differentiation [25, 27, 28]. Therefore, CD30 has been put forward as a novel potential pluripotent stem cell marker (Drukker M, oral presentation at the Annual Meeting of International Society for Stem Cell Research 2006 and Lagarkova et al. ). Although CD30 is indeed rapidly downregulated on differentiation, its expression is clearly due to the ascorbate in the KOSR culture medium. Furthermore, CD30 is clearly not required for undifferentiated hESC growth in FCS and CD30-negative hESC can be expanded for more than 17 weeks in KOSR without ascorbate. Therefore, CD30 can not be regarded as a true stem cell marker.
Our data indicate that CD30 induction is not readily reversible after withdrawal of KOSR. The extensive investigations excluded the possibilities that ascorbate stimulated CD30 promoter activity, modulated the microsatellite repeat length in the CD30 promoter, or altered expression of activator protein 1 (AP-1), a critical transcription factor for CD30 expression . We next investigated epigenetic effects of ascorbate in relation to CD30 expression. We show that CD30 induction by ascorbate is strongly correlated with DNA demethylation of CD30 promoter CpG island. In agreement with this finding, 60 CpG sites in the same CpG island of the CD30 promoter were found to be demethylated in H–RS cells and ALCL cells which characteristically overexpress CD30 . At present the molecular mechanism that underlies the ascorbate-mediated demethylation of a CpG island in the CD30 regulatory region remains unclear. However, the observation that the oxygen tension affects ascorbate-induced CD30 expression suggests a potential redox-based mechanism. It has been suggested that redox misbalancing can affect DNA methylation status in CpG islands [54–56]. In this regard, it is noteworthy that CD30 is specifically redox-based functional regulated by the thio-disulfide oxidoreductase thioredoxin-1, one of the most important regulators of cellular and organismal redox homeostasis in response to redox environmental change [35, 57].
Ascorbate is regarded as an important water-soluble antioxidant in human plasma and mammalian cells , based on its property as an efficient electron donor in many biological redox reactions, and as a cofactor for enzymes such as prolyl-hydroxylases [59–61] and histone demethylases . Ascorbate (50 μg/ml) is included in the KOSR medium formulation because the inventors found that ascorbate might improve the ability of MEFs to better support mouse embryonic stem cell growth . However, a direct effect of ascorbate on hESCs, as demonstrated in this article, was not considered at that time. Ascorbate increases the efficiency of cardiomyocyte differentiation from hESCs in differentiating serum-free medium (16.7 μg/ml) , but the mechanism is not understood. Our data indicating ascorbate can bring about demethylation of CpG islands may provide a rationale for this phenomenon.
CD30 is nearly universally present on the surface of genetically unstable human embryonal carcinoma cells in vivo [64, 65], the malignant counterpart of ES cells, although the origin of these CD30-expressing cells has not been clearly defined. Its absence in hESCs cultured under serum-containing conditions indicates that CD30 is not required for pluripotency and self-renewal of hESCs. Indeed, we demonstrate that overexpression of CD30v does not affect the pluripotency of hESC, as judged by teratoma assays, and does not affect the kinetics of Nanog and Oct4 downregulation during embryoid body differentiation. We, therefore, conclude that CD30 does not significantly impact on hESC differentiation. Our data clearly show that CD30 signaling in hESCs leads to inhibition of apoptosis, and enhanced single-cell cloning efficiency The increased cloning efficiency of CD30+ hESCs reported by Harrison et al.  and Mateizel et al.  is in agreement with our data and support our conclusion that CD30 signaling is directly responsible for this phenotype. In particular, it is possible that the increased threshold to apoptosis of hESCs brought about by CD30v might contribute to the survival of cells that have accumulated genetic or epigenetic changes which would otherwise be committed to apoptosis and thereby allow the formation of precancerous lesion . A role for CD30 in cell transformation is supported by Nonaka et al.  who showed that overexpression of CD30 in rat fibroblast at levels comparable with those in H–RS cells results in NF-κB-dependent cell transformation. Inhibition of apoptosis, activation of NF-κB and enhanced clonal growth could in fact be considered beneficial for particular applications of hESCs. On the other hand, these CD30-mediated changes may also enhance the propensity to accumulate further DNA mutations and genetic instability. It remains to be determined whether CD30 expression is a deleterious adaptation to in vitro culture conditions.
We have shown that ascorbate is sufficient to induce CD30 in serum-free culture conditions through an epigenetic mechanism and that CD30 signaling enhances single-cell survival and leads to transcriptome changes related to cell signaling, lipid metabolism, and tissue development. Collectively, our data provide evidence of altered hESC behavior as a result of culture-induced epigenetic changes in hESCs following prolonged culture in ascorbate-containing media. Regardless of the sources of pluripotent stem cells, any application of human stem cells or their differentiated progeny in regenerative medicine will require the expansion of undifferentiated stem cells over prolonged periods in culture. By understanding the processes of genetic or epigenetic changes and the factors that drive selection of variant cells, it is likely that we will be able to develop culture conditions that minimize the appearance of abnormal cells, allowing “benign” adaptation of the cultured hESCs and iPSCs.
We thank the human ES cell Core facility of the Australian Stem Cell Centre for their technical support. This work was supported by the Australian Stem Cell Centre.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.