Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions

Human embryonic stem cells (hESCs) gradually accumulate abnormal karyotypes during long-term suboptimal culture, which hinder their application in regenerative medicine. Previous studies demonstrated that the activation of CTNNB1 might be implicated in this process. Hence, the hESC line with stably silenced CTNNB1 was established to further explore the role of CTNNB1 in the malignant transformation of hESCs. It was shown to play a vital role in the maintenance of the physiological properties of stem cells, such as proliferation, migration, differentiation, and telomere regulation. Furthermore, the malignant transformation of hESCs was induced by continuous exposure to 0.001 μg/ml mitomycin C (MMC). The results showed that CTNNB1 and its target genes, including proto-oncogenes CCND1 and C-MYC, were aberrantly upregulated in hESCs after MMC treatment. Moreover, the high expression of CTNNB1 accelerated cell transition from G0/G1 phase to the S phase and stimulated the growth of cells containing breakage-fusion-bridge (BFB) cycles. Conversely, CTNNB1 silencing inhibited these effects and triggered a survival crisis. The current data indicated that CTNNB1 is intimately associated with the physiological properties of stem cells; however, the aberrant expression of CTNNB1 is involved in the malignant transformation of hESCs, which might advance the process by facilitating telomere-related unstable cell proliferation. Thus, the aberrant CTNNB1 level might serve as a potential biomarker for detecting the malignant transformation of hESCs.


Introduction
Human embryonic stem cells (hESCs) are derived from the inner cell mass of blastocysts with the potential of unlimited self-renewal and pluripotent differentiation that makes it a candidate source of cells for regenerative medicine [1]. Numerous studies have demonstrated that the accumulated chromosomal aberrations in long-term suboptimal cultured hESCs are similar to those found in tumorigenesis and might interfere with the clinical application [2][3][4]. Consistently, our previous studies found that the human embryonic stem cell line, chHES-3, gained increasing karyotypic abnormalities and progressed toward malignancy under long-term subopti-mal culture in vitro [5,6]. We also found that trace levels of mitomycin C (MMC), a DNA-damaging agent widely used for the preparation of feeder cells to support hESC growth, remained in the culture system which might be a major cause of these abnormalities [7]. Furthermore, we demonstrated that CTNNB1 was aberrantly upregulated in karyotypically aberrant hESCs under suboptimal culture conditions. However, under optimized culture conditions, hESCs with different passages maintained normal karyotype, and the expression of CTNNB1 did not display significant changes in karyotypically normal hESCs, thereby suggesting a link between CTNNB1 and the malignant transformation of hESCs [6].
In humans, the Wnt/β-catenin signaling pathway has a pivotal function in early embryogenesis [8]. As an essential downstream mediator of this signaling, β-catenin (the protein encoded by the CTNNB1 gene) is involved in the regulation and coordination of cell renewal, cell fate specification, and cell differentiation [9]. Deletion of CTNNB1 results in a peri-implantation lethal phenotype in knockout mice, suggesting the vital role of CTNNB1 during embryogenesis. The functional studies of CTNNB1 in ESCs primarily focused on the regulatory characteristics of pluripotency and selfrenewal [10]. However, the aberrant activation or mutation in CTNNB1 is associated with several diseases as well as cancers, such as colon cancer, pancreatic cancer, lung cancer, ovarian cancer, hepatoblastoma, and thymoma [11,12]. In recent years, the key functions of CTNNB1 in tumorigenesis have been gradually revealed; it may facilitate the carcinogenic events by promoting cell proliferation and inhibiting cell apoptosis [13]. Our previous studies suggested that CTNNB1 was also aberrantly upregulated in the malignant progression of hESCs, but the role of CTNNB1 in this process remains unclear.
It is widely accepted that telomere is not only correlated to self-renewal ability and pluripotency of ESCs but also to the advanced invasive stage and poor prognosis of tumors [14][15][16]. Telomeres are composed of tandem repeats of the (TTAGGG) n DNA sequence and associated protein complexes that exert a protective effect on the chromosome ends. In normal somatic cells, the telomeres are shortened in each round of cellular division [17]. After telomere degradation reaches a critical level, uncapped telomeres induce replicative senescence or apoptosis to maintain genomic integrity [18]. Intriguingly, telomere maintenance is a key feature of human malignant cells and is required for the infinite proliferation and maintenance of other cancer hallmarks [19]. Our previous studies indicated that both abnormal shortening and elongation are associated with the tumorigenesis of hESCs, and the telomere dysfunction is responsible for complex chromosomal aberrations [20]. Accumulating evidence suggested that telomeres are crucial for cellular homeostasis and that telomere dysfunction can initiate genome instability and potentially trigger events that culminate in cancer [21]. As successive cell divisions occur, telomere dysfunction accumulates chromosomal instability and encourages the fusion of chromosome ends [22]. This break-fusion-bridge (BFB) event results in substantial chromosomal rearrangements, especially translocations and aneuploidy [23]. These processes promote malignant cellular transformation via stochastic inactivation of tumor suppressor genes and the activation of oncogenes [24].
Although these studies indicated that CTNNB1 and telomere are involved in the maintenance of stem cell characteristics and genomic stability, their correlation with the malignant transformation of hESCs remains to be elucidated [25,26]. In this study, we established a CTNNB1-deficient human embryonic stem cell line by short-hairpin RNA (shRNA) lentivirus to investigate the role of CTNNB1 in maintaining the stem cell physiological properties and malignant transformation of hESCs. The current data revealed that CTNNB1 deficiency not only suppresses the capacity of pro-liferation, migration, and differentiation of hESCs but also shortens the telomere length by reducing the telomerase activity. Further investigation indicated that the overexpression of CTNNB1 and its target genes, including protooncogenes CCND1 and C-MYC, was accompanied by severe BFB events, which drives the complex chromosomal rearrangements and consequent malignant transformation of stem cells. Moreover, telomere regulation is intimately related to this process. These results provided new insights into the complex biology of the malignant transformation of hESCs and suggested that CTNNB1 might be a potential biomarker for this process.

ITRAQ Proteomics
Analysis. ITRAQ proteomics analysis among Normal, SIMP, and COMP chHES-3 cells, as well as NTERA-2(EC) cells, was carried out as previously reported [29]. Briefly, cells were washed three times with ice-cold PBS collected by centrifugation at 1000 rpm and were suspended in 200 μl of lysis buffer (7 M urea, 1 mg/ml DNase I, 1 mM Na3VO4, and 1 mM PMSF) at 4°C. The cell lysate was subjected to intermittent sonication using a Vibra Cell™ high-intensity ultrasonic processor (Jencon, UK). The protein concentration of cleared lysates was determined by 2-D Quantification kit (Amersham Biosciences, Sweden) according to the manufacturer's instructions.
Approximately 100 μg of proteins were reduced with 5 mM tris-carboxyethyl phosphine hydrochloride (TCEP) for 60 min at 37°C, alkylated with 10 mM methylethanethiosulfonate (MMTS) for 20 min at room temperature (RT), and then diluted 10 times with deionized water prior to the digestion with 2 μl of 0.25μg/μl sequencing grade trypsin (Promega, USA) overnight at 37°C. Peptides generated were labeled with iTRAQ reagents according to the manufacturer's protocol (Applied Biosystems/MDS SCIEX, USA). The samples were labeled with the respective tags as follows: chHES-3 2 Stem Cells International cell subsets including Normal, SIMP, and COMP chHES-3 cells were labeled with reporter tags 114, 115, and 116, respectively. And hECCs were labeled with reporter tags 117. ITRAQ-labeled tryptic peptide samples were fractionated by Isoelectric Focusing (IEF) on immobilized pH gradient (pH 3-10, 18 cm long, Amersham Biosciences, Sweden). These fractions were lyophilized in a vacuum concentrator and subjected to C-18 cleanup using a C18 Discovery DSC-18 SPE column (100 mg capacity, Supelco, USA). The cleaned fractions were then lyophilized again and stored at -20°C prior to mass spectrometric analysis. Ten microliters of sample was injected into the nano-LC−ESI−MS/MS system for each analysis. Mass spectrometry was performed using a QStar Elite Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer (ESI-Q-TOF-MS/MS, Applied Biosystems/MDS-Sciex, Canada) coupled to an online capillary liquid chromatography system (Dionex, The Netherlands). The mass spectrometer was set to perform data acquisition in the positive ion mode, with a selected mass range of 300-1800 m/z. The time of summation of MS/MS events was set to be 2 s. This refers to the amount of time allowed for the machine to accumulate MS/MS events before switching back to MS scan. The two most abundant charged peptides above a 20-count threshold were selected for MS/MS and dynamically excluded for 30 s with ±50 mDa mass tolerance. Protein identification and quantification for iTRAQ samples were carried out using the ProteinPilot software (version 2.0; Applied Biosystems/MDS-Sciex, USA). The database search was performed by setting cysteine modification by MMTS as a fixed modification. Other parameters include mass tolerance of up to 0.2 Da, maximum of one missed cleavage of trypsin, oxidation of methionine, Nterminal iTRAQ labeling, and iTRAQ labeled-lysine. Relative quantification of proteins in the case of iTRAQ is performed on the MS/MS scans and is the ratio of the areas under the peaks at 114, 115, 116, and 117 Da, which were the masses of the tags that correspond to the iTRAQ reagents used to label the samples. The statistical calculation for iTRAQbased detection and relative quantification was performed using the Paragon Algorithm 19 embedded within the Pro-teinPilot software. Following data analysis by the ProteinPilot software, the protein summary results were exported into an Excel sheet and manually inspected and processed. Briefly, for protein identification and quantitative analysis, 95% confidence was used. Protein identification must be based on at least two unique peptides, and the p values for the relative quantification by iTRAQ must be <0.05. Protein hits that do not satisfy these criteria are removed.
2.3. Stable Transfection. CTNNB1 shRNAs plasmid, pLKO.1puro-shCTNNB1 (pLKO.1 puro shRNA beta-catenin, Plasmid #18803) were from Addgene, with empty vector (pLKO.1 puro, Plasmid, #8453) as control. Viral particles were packaged in virus packaging cell line 293FT and collected according to the manufacturer's instructions, then added to mTeSR™1 medium (StemCell Technologies, Canada) with 8 ng/ml polybrene and incubated overnight; the second infection was performed on the next day. After infection, puromycin resistance hESC clones were selected in the medium containing 1 μg/ml puromycin (Sigma-Aldrich, USA) for two weeks. The positive clones were picked and expanded to establish cell lines, and stable transfection cell lines were determined by the Western blot analysis.

Western Blot
Analysis. The cells were harvested from dishes, washed twice with cold PBS, and lysed in RIPA lysis buffer (Beyotime, China) for 60 minutes on ice, followed by centrifuging at 11,000 × g for 15 min at 4°C to remove cell debris. Then, the supernatant was collected and the protein concentrations were determined by BCA protein assay (Sigma-Aldrich, USA). After the addition of 2× loading buffer, eighty micrograms of lysates were boiled at 95°C for 5 min and were separated on 10% or 12% SDS-PAGE gels. Proteins were subsequently electrotransferred to PVDF membranes (Millipore, Germany). After blocking with 5% nonfat dry milk in TBS-T containing 0.1% Tween-20 for 2 h at room temperature, the membranes were probed with anti-CTNNB1 (88 kDa) or anti-β-Actin (43 kDa) diluted 1 : 1000-1 : 2000 overnight at 4°C, followed by incubation in a 1 : 2000 dilution of secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Protein bands were detected using the ECL detection system, followed by exposure on Hyperfilm (Amersham Biosciences, Sweden). All the western immunoblots were performed at least three times. In each experiment, membranes were also probed with anti-β-Actin antibody to correct for differences in protein loading. Image analysis system (Image J for windows) has been applied to analyze the strap of Western blot.

Reverse Transcription-PCR (RT-PCR).
Total RNA was extracted using TRIzol reagents (Gibco-BRL, USA) according to the manufacturer's instructions. Two micrograms of RNA per sample was reverse-transcribed into first-strand cDNA by using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Germany). For RT-PCR assay, the thermal cycling conditions were as follows: initial denaturation at 95°C for 2 min, followed by 35 cycles of amplification (95°C for 30 s, 54-64°C for 30 s, and 72°C for 30 s) and a final extension at 72°C for 5 min. The PCR products were 3 Stem Cells International separated on a 1.5% agarose gel by electrophoresis and visualized on a UV transilluminator. Primer pairs are shown in Supplementary Table 2. Image analysis system (Image J for windows) has been applied to analyze the strap of RT-PCR.

Real-Time Quantitative PCR (RT-qPCR).
RT-qPCR amplifications were performed on the Roche LightCycler system (Roche Diagnostics, Germany) with SYBR Green I dye.
The cDNA was submitted to real-time PCR using the following primer pairs in Supplementary Table 2. The amplification conditions included an initial denaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. After each run, the cycle threshold (CT) values were provided from real-time PCR instrumentation by the LightCycler software. The analysis of the relative gene expression was performed by using the 2 -ΔΔCT method described by Livak and Schmittgen [30]. Evaluation of 2 -ΔΔCT indicates the fold change in gene expression relative to the internal standard gene GAPDH and takes into account the standard deviation. Individual CT values were based on three separate measurements. The specificity of the PCR amplification was directly verified by the melt curve analysis of the final products in the iCycler.

Karyotype
Analysis. The hESCs were cultured in mTeSR™1 medium (StemCell Technologies, Canada) for 3-5 days and then treated with a 0.06 μg/ml KaryoMAX® Col-cemid™ solution (Gibco-BRL, USA) for 2.5 h. After washing with PBS three times, the cells were incubated in Accutase (Millipore, USA) at 37°C for 2-3 min and harvested using standard procedures, followed by standard G-banding for karyotyping. At least 50 metaphase spreads were examined for each sample using an Olympus fluorescence microscope BX51 (Olympus, Japan) with LUCIA KARYOTYPE software (Lucia, Czech Republic).
2.9. Embryoid Bodies Formation. Spontaneously in vitro differentiation was conducted through embryoid body (EB) formation. hESC colonies were mechanically dissociated into small clumps and detached to grow as aggregates in suspension for 7 days to form embryoid bodies in DFSR medium without bFGF. The embryoid bodies were then transferred to a gelatin-coated six-well plate for adherent culture for 3-8 days in the same medium.

Analysis of Cell Growth in Vitro.
For cell proliferation and cytotoxicity assay, the aliquots of cell suspension containing 500 cells in 100 μl of medium were transferred into the individual well of 96-well tissue culture plates and were grown for 7 days. Every 24 h, 10 μl of Cell Counting Kit-8 (BBI Life Sciences Co., China) was added to wells, and the medium was removed after 4 h of incubation. The absorbance of each well was read with a Bio-Tek Instruments EL310 Microplate Autoreader (BioTek Instruments, USA) at 450 nm. The percentage of cell growth was calculated by the comparison of the A450 readings versus the first day of absorbance. Each experiment was performed at least three times in triplicate.
For the analysis of the cell proliferation, 1 × 10 7 ES cells were cultured for 12 h in 20 ml EdU (5-ethynyl-20-deoxyur-idine) medium and then harvested and stained by the Click-iTTM EdU Alexa Fluor 488 Cell Proliferation Assay Kit (Invitrogen, USA) in accordance with the manufacturer's protocol. Fluorescence data were collected using a FACS Calibur (Becton Dickinson, USA).
2.11. Cell Cycle Analysis by Flow Cytometry. For cell cycle analysis, 1 × 10 6 cells were harvested, washed twice with cold PBS buffer, and fixed with 70% cold ethanol. After incubation at 4°C overnight, cells were washed with PBS, resuspended in FACS buffer containing RNase A (0.2 μg/ml) and propidium iodide (20 μg/ml, Sigma-Aldrich, USA), and incubated at 37°C for 30 min. The stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson, USA) with excitation at 488 nm and the emission recorded 675 nm long pass (FL4, mitoxantrone) filters, and the data were analyzed by the ModFIT/LT software.
2.12. Wound Healing Assay. Cell migration was detected by wound healing assay, which is a measure of the speed of the collective motion of the cells. Plate 5 × 10 5 cells in a plasticbottomed 6-well dish. Mitomycin C (10 μg/ml) was incubated for 2 h to arrest mitosis when the cells were fused to 90%. To simulate wounding, scratch a confluent monolayer with a pipette tip for a gap size of 0.5 mm, rinse the wound with DPBS to remove debris, and replace it with a serum-free medium for continuous culture. Record the wound healing images at 0 h, 18 h until the gap is approximately half-closed. Image analysis system (Image J for windows) has been applied to analyze the scratch area at each time point and calculate the cell mobility.
2.13. Telomerase Activity. Quantitative measurement of telomerase activity was performed using the TRAPeze Telomerase Detection Kit (Millipore, USA) according to the manufacturer's instruction. In brief, after the addition of 100-200 μl CHAPS buffer, the cells were lysed on ice for 30 min and then centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected, and the protein concentrations were determined by standard procedures (BCA protein assay, Sigma-Aldrich, USA). In each reaction, a volume of 0.33 μg protein equivalent was added to a 48 μl reaction solution consisting of TRAP buffer, dNTP Mix, TS primer, RP primer mix, and 2 U Hotstart Taq polymerase (Qiagen, USA). After incubated at RT for 30 min and stop at 80°C for 10 min, telomerase activity was determined by real-time quantification of the PCR products using a Roche Lightcycler II.
2.14. Telomere Length Quantitative FISH. For Q-FISH analyses on metaphase, cells were grown for 3-4 days after passage and stalled at metaphase by incubation with 0.1 mg/l colcemid for 1.5 h. After that, cells were collected, treated in 0.56% KCl buffer for 10 min, and fixed 3 times in fixation buffer (methanol to acetic acid, 3 : 1). Metaphase spreads were prepared by dropping fixed cells onto cleared glass slides. The Q-FISH procedure was performed as previously described [31]. Cell line LY-S and LY-R were employed to generate the calibration curve in every experiment. Fluorescence images were captured on fixed exposure timing and 4 Stem Cells International insensitivity, and telomere length was determined by measuring the telomere fluorescence intensity, which was evaluated by using the Tel-TELO program.

Quantitative Proteomics Showed Upregulated Expression of CTNNB1 Proteins in a Malignant Transformed Stem Cell
Line. Our previous studies reported that the chHES-3 cell line underwent karyotypic changes from simple to complex and experienced dysregulation of self-renewal pathway and dysfunction of related oncogenes during long-term suboptimal culture, leading to malignant transformation (Yang et al. [5]). Hence, chHES-3 is a specific cell model for the investigation of the mechanism underlying stem cell malignant transformation. Significantly, the intracellular Wnt signaling pathway was preferentially activated in malignant transformed chHES-3 cells, especially the expression of CTNNB1 was aberrantly upregulated, which might be associated with this process [6]. To further investigate the differential expression of CTNNB1 during the chHES-3 malignant transformation, Normal (chHES-3 cells retained a normal karyotype), SIMP (chHES-3 with a simple duplication karyotype), and COMP (chHES-3 with the accumulation of more chromosomal abnormalities) chHES-3, as well as human embryonal carcinoma cell (hECCs) line NTERA-2 cl.D1 (EC) were analyzed by quantitative proteomics. Four unique reporter ions (m/z = 114-117) were used to label the Normal, SIMP, COMP chHES-3 cells, and EC cells, respectively. Consistent with a previous study [6], the MS/MS spectra and iTRAQ ratios of the peptides from β-catenin showed a high expression of CTNNB1 in karyotypically aberrant chHES-3 ( Figure 1).

Reduced Differentiation Capacity in CTNNB1-Depleted
Stem Cells. CTNNB1-deficient hESC colonies were established by shRNA infection, and the CTNNB1 expression and stem cell characteristics were explored after interference (Figures 2 and 3 and Supplemental Figure 2). The silencing efficiency of CTNNB1 was measured by Western blot and immunofluorescence assays. The results showed that compared to the wild-type (WT) and empty vector group (MOCK), the expression of CTNNB1 was remarkably decreased in the shCTNNB1 group, while no significant difference was detected between the MOCK and WT groups (Figures 2(a) and 2(b)). The G-band analysis after long-term culture showed that hESCs after CTNNB1 interference retained a normal karyotype (Supplemental Figure 1(b)). Immunofluorescence and RT-PCR results indicated that the downregulation of the CTNNB1 level decreased the expression of intrinsic pluripotency factors, including OCT4, NANOG, TDGF1, TERF1, KLF4, REX1, CRIPTO, and THY-1 (Figures 2(b) and 2(c)). Interestingly, shCTNNB1-hESCs formed smaller and fewer embryonic bodies (EBs) that grew slowly 7 days after replating into the dishes, which was opposite to that of the control and WT groups under identical conditions (Figure 3(a)). In addition, immunofluorescence using antibodies against β-tubulin (ectoderm), SMA (mesoderm), and AFP (endoderm) confirmed that both the WT and MOCK groups are differentiated into three germ layers, but no positive signal was recorded in the shCTNNB1 group. Surprisingly, the majority of the shCTNNB1-hESCs persistently showed strong OCT4-positive signal even after 27 days of culture in the differentiation medium (Figure 3(a)). RT-PCR analysis showed a significantly lower expression of the three germ layer markers such as KRT17, ACTC1, RUNX1, HAND1, GATA4, GATA6, SOX17, HCG, and CDX2 in shCTNNB1-hESCs as compared to WT and MOCK groups (Figure 3(b)), suggesting a conspicuous inhibition of differentiation in hESCs after CTNNB1 interference. Taken together, these results indicated that continuous expression of pluripotency markers is exhibited in CTNNB1-interfered hESCs, and the expression of differentiation markers is suppressed.

Suppressed Proliferation and Migration Capacity in CTNNB1-Depleted Stem Cells.
To further evaluate the role of CTNNB1 in hESCs, we investigated the cell proliferation and migration capabilities of shCTNNB1-hESCs. The results indicated that in comparison with the WT and MOCK groups, the interference of CTNNB1 distinctly delayed the growth rate of hESCs (Figure 4(a) and Supplemental Figure 1(a)). The cell cycle assay was used to further investigate the effect of CTNNB1 on cell cycle distribution, the percentage of cells in the G0/G1 phase was increased markedly after CTNNB1 silencing, while a significant decrease was observed in the S phase as compared to the control groups (Figure 4(b)). Moreover, the wound healing assay in shCTNNB1-hESCs showed a significantly slower healing process as compared to the control group (Figure 4(c)). These data indicated that knocking down CTNNB1 in hESCs decreases the rate of cell proliferation, causes G0/G1 cell cycle phase arrest, and lowers the rate of cell migration, thereby implying its function in supporting the cell growth and migration.

Decreased Telomerase Activity and Shortened Telomere
Length in CTNNB1-Depleted Stem Cells. In hESCs, telomere regulation is one of the crucial mechanisms that maintain stem cell characteristics. Also, Wnt/β-catenin signaling is reported to play a major role in regulating telomerase reverse 5 Stem Cells International transcriptase (Tert) expression and telomere length in mESCs [32]. Herein, we compared the telomerase activity and telomere length in CTNNB1-depleted hESCs, WT, and MOCK groups. The TRAPeze Telomerase Detection Kit was used to evaluate the telomerase activity, and lower telomerase activity was detected in CTNNB1-depleted hESCs than in the WT and MOCK groups (Figure 4(d)). Furthermore, a short telomere length (about 2.2 Kb shortening) was detected in CTNNB1-depleted hESCs by quantitative fluorescence in situ hybridization (Q-FISH) (Figure 4(e)). These results suggested a positive role of CTNNB1 in the maintenance of telomeres of hESCs.

CTNNB1 Is Also Required for the Survival and
Proliferation of Cells with Genomic Instability. To further understand the role of CTNNB1 during the malignant transformation of hESCs, cells were continuously exposed to 0.001 μg/ml MMC for 10 passages to activate the malignant transformation of hESCs. RT-qPCR revealed that the expression level of CTNNB1 and its target genes, including proto-oncogenes CCND1, C-MYC, MDM2, and BCL-2, was upregulated, while that of the apoptosis-related molecule, P21, was downregulated ( Figure 5(a)). Next, we treated CTNNB1-silenced hESCs with MMC in order to evaluate the cellular response after CTNNB1 knockdown. Cell cycle analysis demonstrated that the overexpression of CTNNB1 was accompanied by a decreased population in the G0/G1 phase and a significantly increased population in the S phase. Conversely, the population in the G2/M phase in shCTNNB1-hESCs treated with MMC was increased, and that in the S phase was decreased ( Figure 5(b)). Furthermore, severe BFB events were triggered in shCTNNB1-hESCs as compared to the control groups after MMC treatment ( Figure 5(c)). The control groups showed superior proliferation ability as compared to shCTNNB1-hESCs and died within one week after the MMC treatment ( Figure 5(d)).

Discussion
Although hESCs are promising seed cells for regenerative medicine, the accumulated chromosomal abnormalities during prolonged culture is a major bottleneck that hinders its clinical application. Previous studies have reported that malignantly transformed genetically unstable hESCs harbor the classical stem cell surface markers similar to normal hESCs, and therefore, it is difficult to selectively remove   (a) 9 Stem Cells International unhealthy hESCs from healthy cultures [33]. Our previous studies demonstrated that the overexpression of CTNNB1 was accompanied by increased karyotype complexity during the malignant transformation, indicating a putative correlation between CTNNB1 and the malignant transformation of hESCs [5,6]. Therefore, the present study aimed to investigate the effects of CTNNB1 in the malignant transformation of hESCs, which is conducive to the safety assessment of the clinical application of hESCs.
In mammals, CTNNB1 controls cell proliferation and cell fate decisions before and after birth and plays a crucial role at multiple stages of embryonic development [34]. The current results also indicated that in normal physiological conditions, CTNNB1 played an essential role in maintaining cell proliferation, migration, and differentiation capacities of hESCs; it also participates in cell cycle and telomere regulation. Since the Wnt/β-catenin signaling pathway is one of the integral structural components of the crucial nuclear effector, the hyperactive Wnt/β-catenin signaling was involved in a wide variety of diseases, and hence, the imbalance and dysregulated overexpression of CTNNB1 is related to several human cancers [12,35]. Consistently, our previous results suggested that CTNNB1 was aberrantly upregulated accompanied by chromosomal aberrations in transformed hESCs [5,6]. These Telomere length 12 18 24 Telomere length (Kb) 30     11 Stem Cells International findings indicated that CTNNB1 plays an essential role in the maintenance of the physiological functions of hESCs; however, the dysregulation of CTNNB1 might be related to the malignant transformation of hESCs.
Previously, we reported that the trace level of MMC, which is an adverse factor for malignant transformation of hESCs, causes karyotype abnormalities and carcinogenesis in long-term cultured hESCs [7]. Thus, we optimized our culture conditions by using γ-inactivated feeder cells, controlling the density of feeder cells, and passaging by manually mechanical cutting. As a result, hESCs retained a stable normal karyotype even after more than two years of cultivation under optimized conditions [6,7]. In this study, we induced the malignant transformation of hESCs by continuous exposure to 0.001 μg/ml MMC; subsequently, the cellular response was assessed. The data demonstrated that after the MMC treatment, the expression of CTNNB1 and its target genes, especially proto-oncogenes CCND1, C-MYC, MDM2, and BAX, was upregulated, while that of the apoptosisrelated molecule, P21, was downregulated. Moreover, the overexpression of CTNNB1 accelerated the cell transition from G0/G1 to S phase. Conversely, CTNNB1 depletion induced the G2/M cell cycle checkpoint arrest after MMC treatment. CCND1 is a crucial downstream target gene of the Wnt/β-catenin signaling pathway, which coordinates the cell cycle progression [36,37]. CCND1 dysregulation leads to uncontrolled cell cycle progression, and its overexpression is correlated to genomic instability, rapid cell growth, cell bypass of critical cellular checkpoints, and neoplastic growth [37,38]. In addition, pathologically active MYC and MDM2 genes drive an abnormally rapid cell cycle to dysregulate cell proliferation [39]. BAX is a proapoptotic gene and might affect cell proliferation by directly modulating the cell cycle inhibitor P21 [40,41]. Thus, our results demonstrated that elevated expression of CTNNB1 targets CCND1 upregulation and P21 downregulation, which in turn accelerates the transition of chromosomally unstable cells from G0/G1 to S phase. Conversely, CTNNB1 depleted in hESCs induced G2/M phase arrest and survival crisis after MMC treatment.
Telomere is a conserved DNA-protein complex that maintains the stability of the chromosomes. Telomeres lose about 200 nucleotides per cell division. When a telomere degradation reaches a threshold level, uncapped telomeres activate cell senescence or apoptosis by enforcing the cell cycle arrest [18,42]. Telomere dysfunction promotes tumorigenesis by inducing chromosomal instability in tumor-initiating cells, bypassing the cell cycle checkpoint, and increasing the proliferative competition of transformed cells [43,44]. As reported, hTERT promoter mutations and CTNNB1 mutations are significantly associated, and telomere and CTNNB1 have a cooperative effect in tumorigenesis [25]. In this study, the MMC treatment elevated the number of telomere BFB cycles in CTNNB1-depleted cells and significantly reduced the proliferation and survival of CTNNB1-depleted cells within 7 days. This might be due to the mismatched endto-end chromosome fusions and recombination effectuated by uncapped chromosomes leading to mitotic arrest and affecting the cellular fate in precrisis [45,46]. Conversely, cells with MMC-induced overexpression of CTNNB1 overcomes the survival crisis and contributed to uncontrolled proliferation. Moreover, CTNNB1 maintains the telomere function by regulating the telomerase activity. Taken together, the current results indicated that CTNNB1 silencing may exacerbate telomere deprotection, thereby increasing the sensitivity of cells to mutagens and promoting cell death during cell cycle arrest. Typically, the overexpression of CTNNB1 conferred a proliferative advantage, culture adaptability, and resistance to apoptosis on chromosomally unstable cells by mediating telomere maintenance, and therefore driving the malignant transformation of hESCs.
In summary, we proposed that CTNNB1 is a key factor in the early stage of malignant transformation of hESCs by regulating telomere maintenance and promoting the proliferation of chromosomally unstable cells ( Figure 6). Our data offered new insights into the complex etiology of malignant transformation of ESCs, and CTNNB1 serves as a potential prognostic safety biomarker for the application of hESCs in regenerative medicine and therapeutic target in the malignant transformation of stem cells.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declared no conflicts of interest.