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Keywords:

  • Telomerase reverse transcriptase;
  • Telomerase RNA component;
  • Telomerase;
  • Human embryonic stem cells;
  • Cell cycle;
  • In vitro differentiation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Embryonic stem cells (ESC) are a unique cell population with the ability to self-renew and differentiate into all three germ layers. Human ESC express the telomerase reverse transcriptase (TERT) gene and the telomerase RNA (TR) and show telomerase activity, but TERT, TR, and telomerase are all downregulated during the differentiation process. To examine the role of telomerase in human ESC self-renewal and differentiation, we modulated the expression of TERT. Upregulation of TERT and increased telomerase activity enhanced the proliferation and colony-forming ability of human ESC, as well as increasing the S phase of the cell cycle at the expense of a reduced G1 phase. Upregulation of TERT expression was associated with increases in CYCLIN D1 and CDC6 expression, as well as hyperphosphorylation of RB. The differentiated progeny of control ESC showed shortening of telomeric DNA as a result of loss of telomerase activity. In contrast, the differentiated cells from TERT-overexpressing ESC maintained high telomerase activity and accumulated lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Although the TERT-overexpressing human ESC are able to form teratoma composed of three germ layers in vivo, their in vitro differentiation to all primitive and embryonic lineages was suppressed. In contrast, downregulation of TERT resulted in reduced ESC proliferation, increased G1, and reduced S phase. Most importantly, downregulation of TERT caused loss of pluripotency and human ESC differentiation to extraembryonic and embryonic lineages. Our results indicate for the first time an important role for TERT in the maintenance of human ESC pluripotency, cell cycle regulation, and in vitro differentiation capacity.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

The telomerase holoenzyme adds repetitive G-rich sequences to chromosome ends to maintain the structure of chromosome ends and protect chromosomes from degradation and end-to-end fusion [1, [2]3]. The basic components required for telomerase activity are the telomerase reverse transcriptase (TERT) unit, which possesses catalytic activity, and the telomerase RNA (TR) component, which contains the template for telomere elongation.

Most tissues express TR, at various levels; however, many appear to lack telomerase activity, perhaps suggesting that TERT is the rate-limiting factor for assembly of an active telomerase complex. In most somatic cells, telomerase activity is silenced or is present at very low levels, whereas cancer-derived cell lines, germline cells, and embryonic stem cells show abundant telomerase expression. Beyond its role in telomere lengthening, it has been suggested that telomerase prevents the loss of the G-rich single-stranded overhangs that participate, together with specific telomere-binding proteins, in forming the T-loop structure [4]. Erosion of the telomeric overhang seen in senescent cells is likely to cause collapse of the T-loop structure and uncapping of the telomere end, which eventually signal DNA damage and activate the senescence pathway [5]. Consistent with this, a reduction in telomerase activity can cause erosion of the overhangs in normal human cells, whereas ectopic expression of the TERT gene maintains the overhangs at a length similar to that found in early passage cells [6].

To date, mounting evidence suggests that telomerase has additional extratelomeric roles in mediating cell survival and antiapoptotic functions against various cytotoxic stresses [7, [8], [9], [10], [11], [12]13]. In many cell lines, exogenous telomerase was shown to stimulate cell proliferation, although the mechanistic basis for these observations is not completely understood. Our data and those of others have suggested that overexpression of TERT is associated with alteration in the expression profile of growth-promoting genes and suppression of growth-inhibitory genes [14, [15]16]. It remains to be elucidated whether TERT can act as a transcription factor or as an effector of gene expression indirectly through certain signaling pathways.

Current knowledge of TERT regulation and telomerase activity derives largely from studies of neoplastic and/or immortalized cell lines. Although useful for understanding cancer models, such studies may be less relevant to normal euploid cells, and there is a need to study telomerase regulation in normal development. In this context, studies on embryonic and somatic stem cells might prove extremely useful, since they provide an in vitro model of cell differentiation, where effects of telomerase modulation can be easily assessed. For example, forced telomerase expression in human mesenchymal stem cells results in elongation of telomeres, extended life span, and enhanced differentiation potential [17]. Similarly, upregulation of Tert expression in the K5-mTert mice promotes epidermal stem cell mobilization and proliferation in the absence of changes in telomere length [18]. Induction of Tert expression in mouse skin epithelium caused the hair follicle cycle to undergo rapid transition from a resting telogen phase to an active anagen phase [19].

In mouse, telomerase activity is important for ESC growth: deletions leading to loss of either the telomerase RNA (mTr) or its reverse transcriptase (mTert) result in progressive loss of telomeres, genomic instability, aneuploidy, telomeric fusions, and eventual reduced growth rate [20, [21]22]. Data generated by our group have shown that overexpression of Tert in murine ESC enhances self-renewal and improves resistance to apoptosis, oxidative stress, and increased proliferation, suggesting that telomerase functions as a survival enzyme in ESC [14]. Our recent work has indicated that human ESC express the TERT and TR genes and show high levels of telomerase activity; however, upon differentiation, the levels of TERT, TR, and telomerase activity decrease with the emergence of a maturing population of cells [23]. Downregulation of telomerase activity with differentiation has also been reported in embryonic carcinoma cells and immortalized cells lines that have the ability to be induced to differentiate into more mature cells, and this has been linked to downregulation in TERT expression rather than posttranslational modifications. Importantly, downregulation of TERT was found to be an early and direct event of cellular differentiation and not the cell cycle [24, [25], [26]27]. What is not clear from these studies is whether the downregulation of telomerase activity and TERT is a consequence of differentiation or is necessary for the differentiation to proceed normally. Overexpression of TERT appears to block terminal differentiation of HaCaT skin keratinocytes, neuronal differentiation of NT2 cells, and late development of immortalized sheep fibroblasts but not bovine adrenocortical cells or human adult bone cells [24, [25], [26]27]. In view of these results, it is of great value to investigate whether TERT overexpression also endows human ESC with these advantageous features.

Cell replacement therapy is a promising treatment for diseases such as hematologic malignancies. However, during differentiation of ESC, the cells are subjected to telomere erosion [23]. In fact, several cases of leukemia with graft failure after hematopoietic stem cell transplant have been reported to have unusual telomere shortening [28]. In this study, we overexpressed and downregulated TERT in human ESC to investigate the impacts of telomerase modulation on self-renewal, differentiation, and viability and to explore the possibility of telomerase-based strategies of large-scale growth of ESC and induced differentiation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Culture and Differentiation of Human ESC

The human ESC lines H1 (WiCell Research Institute, Madison, WI, http://www.wicell.org) and human embryonic stem (hES)-NCL1 [29] were routinely passaged and maintained in human embryonic stem cell (hESC) media on mitotically inactivated mouse embryonic fibroblast feeder layers as described by Stojkovic et al. [29]. Differentiation was achieved by forming embryoid bodies (EBs) as described previously [29]. One to two passages prior to experiments, human ESC were transferred to Matrigel-coated plates (BD Biosciences, San Diego, http://www.bdbiosciences.com) with feeder-conditioned media, as described previously [29]. Transfection of TERT and control cDNA was carried out in the H1 cell line (passage 29) and hES-NCL1 (passage 24).

Generation of Human TERT-Overexpressing hESC Sublines

Ten micrograms of the linearized construct pTP6 and pTP6 containing full-length human telomerase reverse transcriptase (hTERT) cDNA was transfected into hESC using the Amaxa (Gaithersburg, MD, http://www.amaxa.com) nucleofector kit Cell Line L kit and program A-23. Two days after the transfection, stable clones were selected using puromycin selection (1 μg/ml) for 10 days. The surviving human ESC from each cell line were pooled together and expanded to form the TERT-overexpressing sublines, named H1 TERT and hES-NCL1 TERT, or the control sublines, named H1 control and hES-NCL1 control. All four sublines were maintained with 0.6 μg/ml puromycin to ensure the maintenance of transgene. Every 10–15 passages, quantitative reverse transcription (RT)-polymerase chain reaction (PCR), Western blot, and flow cytometry analysis were carried out to confirm TERT overexpression over time. All four sublines were maintained in culture up to 70 passages post-transfection.

Flow Cytometry Analysis of hESC

For the flow cytometry analysis, the hESC were collected using collagenase IV treatment (1 mg/ml for 5 minutes) followed by brief Accutase incubation (Chemicon, Temecula, CA, http://www.chemicon.com). Human ESC were suspended in staining buffer (phosphate-buffered saline + 5% fetal calf serum) at a concentration of 106 cells per milliliter. Intracellular staining was performed by fixing the cells for 10 minutes in 0.5% formaldehyde followed by gentle permeabilization in ice-cold 90% methanol. The permeabilized cells were washed three times in staining buffer before being stained with the primary antibody (TERT; 1:500; Rockland Immunochemicals, Gilbertsville, PA, http://www.rockland-inc.com) for 2 hours at room temperature. Three washes in staining buffer were carried out before staining with the secondary antibody, goat anti-rabbit IgG-fluorescein isothiocyanate (FITC) (final concentration, 6 μg/ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Cells were washed again three times and resuspended in staining buffer before being analyzed with a FACSCalibur instrument (BD Biosciences, San Diego, http://www.bdbiosciences.com) using the CellQuest software (BD Biosciences). For each sample, 10,000 events were acquired.

Small Interfering RNAs and Transfection

Short interfering RNAs (siRNAs) were obtained from Invitrogen. The siRNA sequences are shown in supplemental online Table 1. Transfection with a scrambled control siRNA provided by the same company was used as a negative control. Transfection of siRNA into human ESC was carried out using the high-efficiency nucleofection kit L from Amaxa and 80 pmol of siRNA (in 2 ml of medium) as outlined in the manufacturer's instructions.

Telomeric Repeat Amplification Protocol Assay

Telomeric repeat amplification protocol reactions were carried out using Telo TAGGG Telomerase PCR ELISA Plus (Roche Diagnostics, Indianapolis, http://www.roche-applied-science.com) following the manufacturer's instructions and as described previously [14].

LightCycler Real-Time PCR Analysis

Real-time PCR analysis was carried out using QuantiTect SYBR Green PCR Master Mix (Qiagen, Hilden, Germany, http://www1.qiagen.com). GAPDH for each sample was used as the internal control of these real-time analyses. The data were analyzed using the LightCycler relative quantification software, version 1.01 (Roche Diagnostics). For each gene, the control was normalized to 1, and all other values were calculated with respect to this. PCRs were carried out using the primers described in supplemental online Table 2.

Western Blotting

Lysates were electrophoresed on a 10% SDS-polyacrylamide gel electrophoresis gel and electrophoretically transferred to a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). Membranes were blocked in Tris-buffered saline with 5% milk and 0.1% Tween. The blots were probed with primary antibodies overnight and revealed with horseradish peroxidase-conjugated secondary antibodies. Full information on antibodies used and their concentrations can be found in supplemental online Appendix A.

Alkaline Phosphatase Staining

Alkaline phosphatase (AP) staining was carried out using the Alkaline Phosphatase Detection Kit following the manufacturer's instructions (Chemicon). The staining method was described previously [29].

Measurement of Cell Proliferation Using the 5-Bromo-2′-Deoxyuridine Incorporation Method

Human ESC proliferation was measured by incorporation of 5-bromo-2′-deoxyuridine (BrdU) (Roche Diagnostics) into the genomic DNA during the S phase of the cell cycle. Human ESC were incubated and processed with a BrdU Flow Kit (BD Biosciences) according to the manufacturer's protocol. Cells were stained with FITC, anti-BrdU, and 7-aminoactinomycin D. Cells from the same population that were not BrdU-labeled were used as negative control. Flow cytometry analysis was carried out using a FACSCalibur (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) and CellQuest software.

Cell Cycle Analysis

Cell cycle analysis was performed using the CycleTest Plus DNA reagent kit (Becton Dickinson) as described previously [14]. The data were analyzed using FlowJo software to generate percentages of cells in G1, S, and G2/M phases.

Reactive Oxygen Species Measurement

Cellular peroxide levels were assessed by staining with 30 μM dihydrorhodamine 123 (DHR) (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 30 minutes at 37° C and analyzed using FL3 fluorescence as described previously [30].

Telomere Length

The mean length of telomeric repeats was measured by flow cytometry following the hybridization of a fluorescently conjugated peptide nucleic acid probe (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com). A full description of the protocol is given in supplemental online Appendix A. Relative telomere length was calculated following the manufacturer's instructions and expressed as the percentage of the average telomere fluorescence per genome. Then, the relative telomere length for the control cells (1,301) was normalized to 100%, and all experimental samples were calculated with respect to this.

Apoptosis Assay

Apoptosis assay was carried out using the Annexin V-FITC apoptosis detection kit (BD Biosciences) as described previously [14].

Karyotype Analysis of hESC

Chromosome preparations were made using standard cytogenetic techniques and a 16-hour colcemid/BrdU mitotic arrest step. The karyotype of ESC was determined by the standard G-banding procedure.

Hematopoietic CFU-GEMM Assay

Hematopoietic CFU-GEMM assays were performed according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) essentially as described previously [14].

Cell Signaling Assays

Panorama antibody microarrays for cell signaling, containing 224 different antibodies spotted in duplicate on nitrocellulose-coated glass, were purchased from Sigma-Aldrich. One milligram of cell extracts prepared from TERT-overexpressing and control sublines was collected, labeled with Cy3 and Cy5, respectively, and hybridized to the slides according to manufacturer's instructions. Cy3 and Cy5 signals were read on Gene Pix Pro 4.0. More information on analysis of results is given in supplemental online Appendix A.

Tumor Formation in Severe Combined Immunodeficient Mice

All procedures involving mice were carried out in accordance with institutional guidelines. Approximately 106 hESC were injected into the testis of adult male severe combined immunodeficient (SCID) mice. After 70–90 days, mice were sacrificed, tissues were dissected, and material was analyzed by histology. A full description of the method is presented in supplemental online Appendix A.

Statistical Analysis

Two-tailed pairwise Student's t test was used to analyze results obtained from two samples with one time point. Analysis of variance (two factors with replication) was used to compare multiple samples (at several time points). The results were considered significant if p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Generation and Characterization of TERT-Overexpressing Human ESC Clones

Two human ESC lines (H1 and hES-NCL1) were stably transfected with the empty construct pTP6 or pTP6-TERT to generate two control sublines (H1 control and hES-NCL1 control) and two TERT-overexpressing sublines (H1 TERT and hES-NCL1 TERT), respectively. Real-time RT-PCR (Fig. 1A) and Western blotting (Fig. 1B) were carried out to confirm overexpression of full-length TERT cDNA. To ensure maintenance of TERT overexpression, all the sublines were maintained in the presence of 0.6 μg/ml puromycin and analyzed every 10–15 passages by real-time RT-PCR and Western blot or flow cytometry analysis. This showed maintenance of TERT overexpression over time in culture (Fig. 1C). Real-time RT-PCR analysis showed that overexpression of TERT did not result in changes in TR expression (supplemental online Fig. 1). Overexpression of TERT resulted in the generation of colonies that showed the typical tightly packed human ESC morphology with large nuclei and visible nucleoli (Fig. 1D). Although H1 TERT and hES-NCL1 TERT ESC sublines overexpressed TERT mRNA at different levels, the telomerase activity levels were similar to each other (Fig. 1E). Most probably, a 10-fold overexpression of TERT, as found in hES-NCL1 TERT subline, is already sufficient to saturate the formation of active telomerase complexes. An alternate possibility is that human TR or other components of the telomerase holoenzyme became limiting. This could perhaps explain why the H1 and hES-NCL1 TERT sublines showed increases in telomerase activity of only 30% and 26%, respectively, compared with the controls (Fig. 1E). Identical results were obtained when different amounts of cellular extracts were used for measurement of telomerase activity (data not shown), confirming the validity of the results and lack of any inhibitors which can be detrimental for this type of assay.

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Figure Figure 1.. Characterization of TERT-overexpressing human ESC sublines. (A): Real-time reverse transcription-polymerase chain reaction for expression of TERT in H1 and hES-NCL1 sublines. The data represent the mean ± SEM from three independent experiments. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. (B): Western blotting showing TERT overexpression in H1 and hES-NCL1 ESC sublines. GAPDH was used as a loading control. (C): Flow cytometry analysis for expression of TERT in H1 control and H1 TERT sublines at passage 40 (11 passages after transfection) and passage 50 (21 passages after transfection). (D): Microphotograph of the center of a human ESC colony taken from the H1 TERT subline showing typical ESC morphology. (E): Assessment of telomerase activity in TERT-overexpressing and control ESC sublines. The data represent the mean ± SEM from three independent experiments. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. (F): Assessment of telomere length in TERT and control human ESC sublines and during the 3-week differentiation by flow-fluorescence in situ hybridization. The data are expressed as relative telomere length in relation to the control cell line (1,301), which used as positive control and normalized to 100%. The data represent the mean ± SEM from three independent experiments. (G): Intracellular peroxide levels were analyzed using 2′,7′-dichlorofluorescin diacetate staining. All data are from four independent experiments and represent mean + SEM. (H): Karyotype analysis in hES-NCL1 TERT-overexpressing human ESC subline at passage 60. Thirty metaphases were analyzed. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hES, human embryonic stem; TERT, telomerase reverse transcriptase.

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In a recent study, we showed that telomerase activity and telomere length measured by metaphase fluorescence in situ hybridization (FISH) decreased during differentiation of human ESC [23]. Using flow-FISH, we were able to confirm a significant decrease in telomere length during the 3-week differentiation time course of control sublines (p < .05; Fig. 1F). Most importantly, a significant difference was observed between the TERT-overexpressing and control sublines (p < .01; Fig. 1F), since the TERT-overexpressing sublines did not show the telomere length decrease observed upon differentiation of the controls.

Human ESC have highly efficient antioxidant defense mechanisms, but this protective capacity is progressively lost during differentiation into embryoid bodies [23]. To investigate whether continued expression of TERT would interfere with the loss of antioxidant defense during differentiation, we measured intracellular levels of peroxides by DHR staining. Differentiation of control subline into embryoid bodies resulted in 25% increase in cellular DHR fluorescence at day 20, in accordance with our earlier results [23]. In contrast, peroxide levels decreased by 25% during differentiation of TERT-overexpressing human ESC (Fig. 1G).

To investigate whether continuous overexpression of TERT in human ESC contributes to gross genomic instability, we carried out G banding on TERT and control sublines at 30 and 60 passages after transfection (Fig. 1H). Observation of 20 metaphases for each subline showed no deviations from the normal karyotype.

Overexpression of TERT Results in Enhancement of Human ESC Pluripotency and Suppression of the In Vitro Differentiation Capacity

To investigate the effects of continuous TERT overexpression on human ESC pluripotency, we carried out AP staining analysis and observed significantly higher numbers of AP+ in TERT-overexpressing sublines compared with the controls (Fig. 2A). Human ESC from each subline were also injected into the testis of SCID mice. One day prior to injection, a small sample of cells from all sublines was analyzed by real-time RT-PCR (data not shown) and flow cytometry to ensure TERT overexpression (Fig. 1C). Injection of TERT and control ESC sublines into the testis of SCID mice resulted in formation of teratoma. Although similar numbers of ESC were injected from each subline, the sizes of tumor were very different, with the TERT teratomas being much larger than the ones resulting from the control sublines (Fig. 2B). In both cases, teratomas were composed of cells from all three germ layers, indicating the pluripotent nature of the cells (Fig. 2C). The identity of cells derived from ectoderm (Fig. 2D) within the tumors was confirmed by immunohistochemical staining with Nestin, epithelial keratin, neurofilament 200, and glial fibrillary acidic protein. Staining with smooth muscle actin was used to confirm presence of cells deriving from mesoderm, and caudal-related homeobox transcription factor antibody (CDX2), shown to play an important role in intestinal development, differentiation, and homeostasis in adults, was used to confirm cells derived from endoderm (Fig. 2D). An additional endodermal marker, α-fetoprotein, was used to identify cells of endodermal origin (Fig. 2D).

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Figure Figure 2.. Assessment of pluripotency and differentiation capability of TERT-overexpressing human ESC sublines. (A): Assessment of pluripotency in TERT-overexpressing and control ESC sublines by alkaline phosphatase staining assays. The data represent the mean ± SEM from three independent experiments. (B): Low-magnification examination of teratoma formed by hES-NCL1 TERT and hES-NCL1 control cells. Teratomas formed by hES-NCL1 TERT (top) were clearly seen to be larger than those formed by control cells (bottom). Sections shown are from the middle section of each teratoma, where the greatest cross-sectional area was observed. Histological staining: Weigert's hematoxylin. Scale bar = 1 mm. (C): Histological analysis of teratomas formed from grafted colonies of hES-NCL1 TERT in SCID mice. (Ci): Low-power light micrograph showing heterogeneous structure within the body of the teratoma and the presence of a diverse range of different tissue types. (Cii): c and sm. (Ciii): b. (Civ): Large ng with connecting nf. (Cv): Section through primitive in with accompanying submucosal m. (Cvi): ne. (Cvii): Low-power light micrograph showing gut-like structure. (Cviii): Kidney g and associated tb. Histological staining: Weigert's hematoxylin (Ci–Civ, Di–Div) and hematoxylin and eosin (Cv–Cvi, Dv–Dvi). Scale bars = 400 μm (Ci, Cvii), 160 μm (Cii, Ciii, Cv), and 80 μm (Civ, Cvi, Cviii). (D): Immunohistochemical analysis of teratomas formed from grafted colonies of hES-NCL1 TERT in SCID mice. hES-NCL1 TERT cells were found to be pluripotent, and teratomas formed were composed of cell types from all three germ layers. (Di): α-Fetoprotein (endoderm). (Dii): Nestin-positive cells (ectoderm). (Diii): Smooth muscle actin-positive cells (mesoderm). (Div): CDX2-positive cells (endoderm). (Dv): Epithelial keratin. (Dvi): NF200. (Dvii): GFAP. (Dviii): MAP2. (Dix): Proliferative populations of cells were shown by expression of a marker for cell proliferation Ki67. Antibody-positive regions are shown in red-purple. All negative controls showed minimal levels of background staining. (Dx): Image corresponding to (Ci, Di). Scale bars = 160 μm (Di, Div, Dvi), 80 μm (Dii, Diii), and 40 μm (Dv, Dvii). Abbreviations: AFP, α-fetoprotein; b, bone; c, cartilage; GFAP, glial fibrillary acidic protein; gm, glomeruli; hES, human embryonic stem; in, intestine; m, muscle layer; MAP2, microtubule-associated protein 2; ne, neuroepithelium; nf, nerve fibers; NF200, 200-kDa neurofilament; ng, neural ganglion; sm, smooth muscle; SMA, smooth muscle actin; tb, tubules; TERT, telomerase reverse transcriptase.

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Several recent reports have shown that overexpression of TERT does suppress terminal differentiation of HaCat skin keratocytes and NT2 embryonic carcinoma cells [24, 25]. To investigate this more closely in our system, we induced differentiation of human ESC using the EB method and quantitated in a series of time points expression of the neuroepithelial marker (PAX6; Fig. 3A), the mesodermal marker (BRACHYURY; Fig. 3B), the trophectodermal marker (CDX2; Fig. 3C), primitive endodermal markers (IHH and GATA6; data not shown), and the primitive ectoderm marker (FGF5; data not shown). In all cases, we noticed that TERT-overexpressing human ESC sublines were more resistant to differentiation (p < .05) compared with the controls, although not completely refractory (Fig. 3A–3C). This was further confirmed by OCT4 real-time RT-PCR analysis (Fig. 3D), which showed less drastic reduction of this pluripotency marker in TERT sublines compared with controls (p < .05). EBs at day 10 of differentiation from each subline were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Although this analysis is not quantitative, we were able to notice neural rosettes at high frequency in EBs made from TERT-overexpressing sublines. Their presence was much more reduced in the control sublines (Fig. 3E). Since neural rosettes are thought to be rich in neural progenitors, it is tempting to speculate that overexpression of TERT perhaps leads to higher proliferation and maintenance of progenitors but is inhibitory to their terminal differentiation, although further experimental proof is needed to confirm the validity of this hypothesis.

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Figure Figure 3.. Assessment of the differentiation capability of TERT-overexpressing human ESC clones. (A–D): Real-time reverse transcription-polymerase chain reaction analysis for the expression of PAX6, BRACHYURY, CDX2, and OCT4 during the differentiation of TERT-overexpressing and control sublines for 21 days in suspension culture. The data represent the mean ± SEM from two independent experiments carried out in each human ESC subline. cDNA taken from day 7 EBs from H9 human ESC was used as positive control (A–C) and normalized to 1, and all values were calculated with respect to this. In (D), the control sublines at day 0 were normalized to 1, and all other values were calculated with respect to this. Statistical analysis was carried out using two-factor analysis of variance with replication. (E): EBs at day 10 from both H1 TERT and H1 control sublines were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Neural rosettes are indicated by white arrows. (F): Total number of hematopoietic colonies obtained after 14 days of differentiation of TERT and control human embryonic stem cell (hESC) sublines. The data represent the mean ± SEM from two independent experiments carried out in triplicate in each hESC subline. (G): Graphical representation of colony types formed during hematopoietic differentiation of TERT and control hESC sublines. The data represent the mean ± SEM from two independent experiments carried out in triplicate in each hESC subline. Abbreviations: CFC, colony forming cells; CFU-E, colony-forming unit-erythrocyte; CFU-G, colony-forming unit-granulocyte; CFU-GEMM, colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-GM, colony-forming unit-granulocyte macrophage; CFU-M, colony-forming unit-macrophage; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hES, human embryonic stem; TERT, telomerase reverse transcriptase.

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We also tested the hematopoietic activity of day 14 EBs made from control and TERT sublines and noticed that this was significantly reduced as a consequence of TERT overexpression (Fig. 3F; similar reductions were obtained for days 7 and 21 of differentiation), thus corroborating our data on suppression of differentiation. It is of interest to note that during differentiation of control EBs, the majority of morphologically scored colonies showed a differentiated phenotype of granulocytic, macrophage, or erythrocyte lineages (Fig. 3G). Twenty percent of the colonies were derived from a bipotential progenitor (granulocyte-macrophage [CFU-GM]), and no mixed colonies (granulocyte-erythrocyte-macrophage-megakaryocyte [CFU-GEMM]) were observed. However, during the differentiation of TERT-overexpressing sublines, we observed the presence of mixed colonies (CFU-GEMM, 12.5%), as well as a higher percentage of CFU-GM (37.5%), which seems to have occurred at the expense of a reduction in erythrocytic colonies and granulocytic colonies. One possible explanation for this is a higher survival of the more primitive hematopoietic progenitors arising in the culture or suppression of their differentiation to more mature lineages; however, further work is needed to prove this using the inducible TERT overexpression-based system.

Overexpression of TERT Results in Changes in Human ESC Proliferation, Survival, and Cell Cycle

We noticed that TERT-overexpressing sublines grew faster in culture; this could be due to either a decrease in apoptosis, an increase in human ESC proliferation, or both. To distinguish between these possibilities, we performed Annexin V staining to detect early (7-aminoactinomycin D [7-AAD] Annexin V+) and late apoptotic (7-AAD+ Annexin V+) cells (Fig. 4A) and BrdU incorporation to detect DNA synthesizing/proliferating cells (Fig. 4B, 4C). The results showed that overexpression of TERT did not affect the rate of apoptosis, since percentages of apoptotic cells were similar to those of control clones under normal culture conditions (Fig. 4A). Addition of the nuclear factor κB inhibitor pyrrolidine dithiocarbamate [31], known to induce apoptosis, did not result in significant changes in cell death between TERT-overexpressing and control sublines (data not shown). However, overexpression of TERT caused small but significant increases in the percentage of cells incorporating BrdU (Fig. 4B, 4C) compared with the control sublines, suggesting a putative role for TERT in human ESC proliferation.

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Figure Figure 4.. Apoptosis and proliferation in TERT-overexpressing human ESC sublines. (A): Assessment of cell death in TERT-overexpressing and control human ESC sublines by flow cytometry analysis. The data represent the mean ± SEM from three independent experiments. (B): Assessment of cell proliferation by flow cytometry after 45 minutes of BrdU incorporation. The data represent the mean ± SEM from three experiments. (C): Flow cytometry images showing a higher percentage of proliferating cells (gate R2) in TERT-overexpressing sublines compared with controls. (D): Colony-forming ability of TERT-overexpressing sublines compared with controls. The data represent the mean ± SEM from three experiments. Abbreviations: 7-AAD, 7-aminoactinomycin D; BrdU, 5-bromo-2′-deoxyuridine; hES, human embryonic stem; TERT, telomerase reverse transcriptase.

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Human ESC show low survival in culture upon disassociation to single cells [32]. Since telomerase has been often described as a survival enzyme, we investigated whether overexpression of TERT conferred such advantages by gently trypsinizing TERT-overexpressing and control sublines to single cells. These were plated on mitotically inactivated feeder cells at limited dilution numbers for 10 days. AP staining was carried out to identify and facilitate the counting of human ESC colonies. This analysis showed a significant increase in the numbers of surviving human ESC that were able to form AP+ colonies (Fig. 4D).

To further characterize the role of TERT in human ESC proliferation, we carried out cell cycle analysis on TERT-overexpressing and control sublines. This showed a small but significant and highly reproducible reduction in the percentage of cells in G1 phase of the cell cycle and increases in the S phase of the cell cycle in TERT sublines compared with the controls (Fig. 5A). To investigate which cell cycle components involved in the G1/S transition were changed as a result of TERT overexpression, we carried out Western blotting in all four clones (Fig. 5B). Most interestingly, we observed in TERT sublines an increase in CYCLIN D1 expression; this has been shown by our group to be the most highly expressed cyclin in human ESC (I. Neganova, X. Zhang, S.P. Atkinson et al., manuscript in preparation). In addition, we observed a significant increase in expression of phosphorylated RB (Ser795), as well as expression of CDC6, whose transcription is controlled by E2F proteins (Fig. 5B). No changes in expression of cell cycle inhibitors p18 and p19 (p15 and p16 were absent in all four sublines) or p53 and c-MYC were observed as a result of TERT overexpression (Fig. 5B). The expression of p63, a p53-related gene, was downregulated in TERT-overexpressing sublines compared with controls (Fig. 5B). It has been reported that p63 is activated in response to TERT downregulation in cancer cells lines and might represent a mediator of p53-independent DNA damage-induced cell cycle arrest and apoptosis [33].

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Figure Figure 5.. Changes in human ESC cell cycle distribution as a result of TERT overexpression. (A): Bar chart showing changes in cell cycle distribution as a result of TERT overexpression in H1 and hES-NCL1 sublines. The data represent the mean ± SEM from four independent experiments. An example of flow cytometry images is shown in the bottom panel. (B): Western blotting for expression of the main components involved in G1/S transition in TERT-overexpressing and control human ESC sublines. This summary is a representation of three independent experiments. GAPDH was used as loading control. HeLa cell extract was used as a positive control in Western blotting for CYCLIN D2, p16, and p15 to confirm that their absence is actually due to lack of expression in human ESC rather than technical artifacts. (C): Real-time reverse transcription-polymerase chain reaction analysis for the expression of CYCLIN D1 and CDC6 in TERT and control human ESC-overexpressing sublines. The data represent the mean ± SEM from three independent experiments. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HC, H1 control; hES, human embryonic stem; HT, H1 telomerase reverse transcriptase; NC, human embryonic stem-NCL1 control; NT, human embryonic stem-NCL1 telomerase reverse transcriptase; TERT, telomerase reverse transcriptase.

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The above results were independently confirmed by hybridization of cell extracts from TERT and control sublines to the Panorama antibody microarray (Sigma-Aldrich), which is designed to investigate several biological pathways, including cell cycle, signal transduction, apoptosis, cytoskeleton, and so on. To ensure simultaneous occurrence of changes in protein expression in both TERT sublines compared with controls, we combined the raw data obtained from the two files (obtained from two TERT sublines compared with their respective controls) into a single GeneSpring GX analysis that yields an average value for each component of the array based on confidence analysis filtration (p < .05). This analysis showed that CYCLIN D1 and CDC6 were upregulated as a result of TERT overexpression (2.27 ± 0.48-fold and 2.31 ± 0.36-fold, respectively), and p63 was downregulated (0.26 ± 0.06-fold). Taken together, these results suggest an increase in E2F-dependent transcriptional activity in TERT sublines compared with controls that could be the likely mechanism for the cell cycle changes already reported herein, although further work is needed to prove this hypothesis.

The expression of CYCLIN D1 and CDC6 was investigated by real-time RT-PCR and found to be upregulated at the transcriptional level in both TERT sublines compared with controls (Fig. 5C). These results corroborate our previous work showing upregulation of Cyclin D1 and Cdc6 as a result of Tert overexpression in murine ESC [14] and a recent report that has suggested that TERT is capable of regulating the transcription of CYCLIN D1 in human prostate epithelial cells [34].

Downregulation of TERT Results in Differentiation of Human ESC

The dominant-negative form of TERT [35] was subcloned in pTP6 vector and stably transfected into H1 and hES-NCL1 cells. After eight trials of independent transfections (four in each cell line), no stable human ESC sublines could be created (only differentiated colonies were observed after selection; data not shown), suggesting that downregulation of TERT was incompatible with human ESC self-renewal. To overcome this, we transfected short interfering RNAs for TERT (a mixture of three siRNAs directed to different coding regions of TERT), as well as a scrambled control in H1 and hES-NCL1. Reductions of 95% and 99% in the TERT expression were observed in hES-NCL1 and H1, respectively, 2 days after siRNA transfection (Fig. 6A). These results were confirmed by Western blotting (Fig. 6B). Downregulation of TERT caused a small but significant increase in the fraction of apoptotic cells compared with control (supplemental online Fig. 2).

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Figure Figure 6.. Downregulation of TERT results in human ESC differentiation. (A): Real-time reverse transcription (RT)-polymerase chain reaction (PCR) for expression of TERT in H1 and hES-NCL1 human ESC 48 hours after transfection. The data represent the mean ± SEM from three independent experiments. The value for the control was normalized to 1, and all other values were calculated with respect to this. (B): Western blotting showing TERT downregulation in H1 and hES-NCL1 human ESC. GAPDH was used as a loading control. (C): Microphotographs taken 96 hours after the transfection of the control and TERT short interfering RNAs (siRNAs). (D): Alkaline phosphatase analysis 96 hours after siRNA transfection. The data represent the mean ± SEM from three independent experiments. (E): Real-time RT-PCR analysis for the expression of OCT4 and NANOG 96 hours after siRNA transfection. The data represent the mean ± SEM from four independent experiments carried out twice in H1 and hES-NCL1. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. (F): Real-time RT-PCR analysis for the expression of various lineage markers 96 hours after the siRNA transfection. The data represent the mean ± SEM from four independent experiments carried out twice in H1 and hES-NCL1. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hES, human embryonic stem; RNAi, RNA interference; TERT, telomerase reverse transcriptase.

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The human ESC transfected with siRNA to TERT showed loss of typical ESC morphology, suggesting a loss of pluripotency (Fig. 6C). This was confirmed by AP staining, which indicated a significant reduction in the number of AP+ colonies as a result of TERT knockdown (Fig. 6D). Further evidence supporting loss of pluripotency was obtained by real-time RT-PCR analysis, which showed downregulation of specific ESC markers OCT4 and NANOG as a result of TERT downregulation (Fig. 6E). Most importantly, upregulation of CDX2 (primitive trophectoderm marker), PAX6 (neuroepithelial marker), IHH and GATA6 (primitive endoderm marker), FGF5 (primitive ectoderm marker), and BRACHYURY (mesodermal marker) was observed 4 days after TERT siRNA transfection (Fig. 6F), suggesting differentiation to various extraembryonic and embryonic lineages. Given the transient nature of transfections, it is likely that the RNAi-transfected population does not represent a homogeneous population. To look more closely at the effects of TERT downregulation in a homogeneous population, FAM-labeled siRNAs (TERT and control) were used to transfect the human ESC H1 cell line. Four days post-transfection, transfected cells were purified by fluorescence-activated cell sorting (data not shown). Real-time RT-PCR analysis indicated downregulation of NANOG and OCT4 (supplemental online Fig. 3A) and upregulation of various lineage markers in cells with reduced TERT expression (supplemental online Fig. 3B) Together, these data suggest that reduction in TERT expression results in loss of pluripotency in human ESC and induction of differentiation.

BrdU incorporation assays showed a reduction in human ESC proliferation upon TERT knockdown (Fig. 7A, 7B). This was further confirmed by cell cycle analysis, which showed a significant increase in the number of cells in G1 phase of the cell cycle and reduction in S phase in TERT RNAi-transfected cells (Fig. 7C), thus suggesting once more a role for TERT in human ESC cycle regulation. We also observed that CYCLIN D1 and CDC6 were significantly downregulated upon TERT knockdown (Fig. 7D, 7E). It is of interest to note that changes in cell proliferation and cell cycle were obvious as soon as 48 hours after siRNA transfection. In contrast, changes in cell morphology and upregulation of expression of various differentiation markers were observed 96 hours after transfection, suggesting that changes in cell cycle and proliferation precede the human ESC differentiation observed upon TERT knockdown.

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Figure Figure 7.. Changes in human ESC cell cycle distribution as a result of TERT downregulation. (A): Bar chart showing changes in cell proliferation as a result of TERT downregulation in H1 and hES-NCL1 cell lines 48 hours after short interfering RNA (siRNA) transfection. The data represent the mean ± SEM from three independent experiments. (B): An example of flow cytometry images for the BrdU flow cytometry analysis 48 hours after siRNA transfection. (C): Bar chart showing changes in cell cycle distribution as a result of TERT downregulation in H1 and hES-NCL1 cell lines 48 hours after siRNA transfection. The data represent the mean ± SEM from three independent experiments. (D, E): Real-time reverse transcription-polymerase chain reaction analysis for the expression of CYCLIN D1 and CDC6, respectively, 48 hours after the siRNA transfection. The data represent the mean ± SEM from three independent experiments. The value for each control subline was normalized to 1, and all other values were calculated with respect to this. Abbreviations: 7-AAD, 7-aminoactinomycin D; BrdU, 5-bromo-2′-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hES, human embryonic stem; RNAi; RNA interference; TERT, telomerase reverse transcriptase.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Human embryonic stem cells are a unique cell population with the ability to self-renew and differentiate into cells representative of all three germ layers, which supports their potential use as a cellular source for replacement therapy [36, 37]. Our own studies have shown that human ESC express both components of telomerase complex, TERT and TR; however, these and the underlying telomerase activity are downregulated during the differentiation process, causing shortening of telomeres and decreased stress defense mechanisms in the differentiated cells [23]. Over the last 10 years, increasing evidence has suggested that TERT might have additional functions beyond its major role in telomere maintenance [38, [39], [40], [41]42]. For example, forced expression of TERT can accelerate cell proliferation [43], enhance cell protection from various cytotoxic stresses [44], and alter gene expression profiles [16]; however, the underlying mechanisms remain to be elucidated.

Our work in the murine ESC system has shown that Tert expression promotes cell proliferation, improves resistance to apoptosis, and enhances differentiation toward hematopoietic lineages [14], suggesting that Tert overexpression could be beneficial for large-scale growth and differentiation of ESC. However, these studies were conducted in the murine ESC system, and since the telomere biology is different between human and mouse, it is important to conduct similar studies in human ESC to investigate the role of TERT in human ESC self-renewal and differentiation.

In the present study, we used stable overexpression of TERT in addition to transient downregulation using small interfering RNAs. Our results showed that overexpression of TERT stimulated human ESC proliferation and increased the numbers of cells in the S phase of the cell cycle at the expense of a reduced G1, whereas TERT downregulation showed the opposite effects. At the molecular level, this was associated with increased CYCLIN D1 expression and hyperphosphorylation of RB. In somatic cells, inactivating phosphorylation of Rb by the CDK/cyclin complexes in mid- to late G1 results in liberation of E2F and other Rb-bound transcription factors, which then activate transcription of S phase genes, causing G1 to S transit. The increased CYCLIN D1 expression, together with hyperphosphorylation of RB observed in this study, is suggestive of increased CYCLIN D1/CDK activity. This would lead to increased E2F activity and enhanced G1 to S transition, which can explain the increased S phase and reduced G1 phase in TERT-overexpressing human ESC sublines. Some evidence to confirm increased E2F activity derives from upregulation of CDC6, whose transcription is known to be regulated by E2F factors [45]. This needs to be investigated further using various kinase and cell cycle duration assays and is currently the focus of the cell cycle-related studies within our group (Neganova et al., manuscript in preparation).

To date, there have been a number of studies demonstrating a link between Tert expression and cell cycle status. For example, Jagadeesh and Banerjee demonstrated upregulation of CYCLIN D1 expression in human prostate epithelial cell lines as a result of ectopic TERT expression [34]. It has also been suggested that TERT can promote cell growth in human lens epithelial cells by regulating expression of the genes mediating RB/E2F pathway [46]. Our previous work in murine ESC has also shown that ectopic Tert expression in mESC shortens the population doubling time, increases the numbers of cells in S phase, and is associated with upregulation of Cyclin D1 and Cdc6 [14]. The mechanisms for these described effects are not yet clear.

In normal somatic cells, six human telomeric proteins, TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, can form a complex called the telosome/shelterin, which determines the structure of the telomere ends and controls the generation of T loops, as well as the synthesis of telomeric DNA by telomerase [47]. In normal mammalian cells, which are usually devoid of telomerase activity, it has been observed that checkpoint proteins are on telomeres at each cell cycle and that telomere ends are transiently exposed. However, one of the components of the shelterin complex, TRF2, binds directly to telomere repeat sequences and inhibits ATM protein kinase, presumably to attenuate checkpoint signaling at telomeres [48]. The TPP1 component of shelterin can physically interact with telomerase and stimulate its activity and processivity [49, 50], whereas POT1 can inhibit telomerase activity when bound to the telomeric 3′ end [51], thus suggesting that a fine balance is likely to exist between shelterin and telomerase to control the checkpoint signaling and cell cycle progression [52]. Overexpression of TERT is likely to disturb this balance, thus altering cell cycle dynamics. This is an attractive hypothesis; however, it assumes the presence of shelterin complex and checkpoint signaling in actively dividing cells, such as human ESC, and both of these issues are unknown and remain to be investigated in this cell type. In addition, the ATM checkpoint signaling that is regulated by shelterin is mostly active at the G2/M phase of the cell cycle. Our results do suggest a faster transit of human ESC from the G1 to S phase of the cell cycle as a result of TERT overexpression, thus pointing to additional mechanisms by which TERT regulates cell cycle progression in these cells.

To investigate whether TERT plays a direct role in the transcriptional control of CYCLIN D1, we carried out chromatin immunoprecipitation assays followed by real-time PCR to quantify any enrichment of CYCLIN D1 promoter regions bound to TERT. We did not identify any positive enrichment that would suggest that TERT does not bind to the CYCLIN D1 promoter (data not shown). Direct binding of CYCLIN D1 by TERT itself may be possible but does not preclude any indirect mechanisms by which TERT and CYCLIN D1 expression might be interlinked. It has been reported that Cyclin D1 mRNA translation in murine ESC is mediated via the PI3K/p70 S6 kinase pathway, whereas its degradation is mediated by the PI3K/AKT/GSK3β pathway [53]. It is interesting to note that upon cytokine stimulation, human TERT forms physical complexes with AKT, p70S6 kinase, HSP90, and mTOR in human natural killer cells, and TERT activity is post-translationally regulated by the AKT/HSP90/p70SK6 pathway [54]. If this is true in human ESC, ectopic expression of TERT is likely to affect the amount of available AKT in the cell and the phosphorylation of its substrate GSK3β, thus changing the degradation rate of CYCLIN D1. However, this needs to be further investigated by using inhibitors of p70SK6, such as rapamycin or PI3K/AKT (LY294002), in conjunction with modulation of TERT expression and quantitation of CYCLIN D1 levels.

It is interesting to note that in both human and murine ESC, Tert is significantly downregulated during the differentiation process. Our previous work in murine ESC has suggested that downregulation of Tert by overexpression of one of its repressors, Zap3, is sufficient to slow down cell growth, reduce telomerase activity and telomere length, and cause changes in cell cycle [22]. We have shown in this study that TERT knockdown by RNA interference did result in human ESC differentiation along all tested extraembryonic and embryonic lineages, suggesting that reduction of telomerase activity was incompatible with human ESC self-renewal. This was further reinforced by stable overexpression studies, which showed that TERT-overexpressing human ESC sublines were resistant but not completely refractory to in vitro differentiation. Furthermore, these human ESC sublines showed lower spontaneous differentiation in culture, as well as higher colony-forming ability, when disassociated into single cells. The underlying mechanism for the impacts of TERT on the maintenance of human ESC pluripotency, self-renewal, and differentiation are not clear. Emerging proof suggests that TERT modulation is associated with significant changes in the transcriptome [14, 55, 56]. Therefore, one might be able to speculate that TERT may have direct or indirect involvement with the gene expression network that underlies the regulation of human ESC pluripotency and self-renewal; however, no transcriptional activity has ever been linked with TERT per se. A more attractive hypothesis would be that upregulation of TERT enhances the maintenance of pluripotent phenotype by shortening of G1 and enhancement of S phase of the cell cycle. Clearly, there are examples in the literature to suggest that lengthening of G1 phase in neuroepithelial cells does induce premature differentiation [57]. P19 murine embryonic carcinoma cells are particularly vulnerable to differentiation effects of retinoic acid in G1 phase and refractory to RA in S phase [58], suggesting that a short G1 phase and long S phase probably protect pluripotent cells from differentiation [59]. Our own work has also shown that activation of cell cycle inhibitors, such as p21 in human ESC, can cause accumulation of cells in G1 phase, resulting in human ESC differentiation (T. Maimets, I. Neganova, L. Armstrong, et al., manuscript submitted for publication). On the basis of these findings, we are more inclined to suggest that TERT has an impact on human ESC pluripotency via the cell cycle changes. Recent data show that telomere uncapping occurring in progenitor cells of TERT−/− mice does result in apoptosis in either late S phase or in G2 via a signaling cascade involving p53, thus suggesting intrinsic links between telomerase, telomere replication and capping, and S phase progression [60]. Our work has shown only a small increase in apoptosis upon TERT downregulation, suggesting that human ESC might respond differently to downregulation of TERT. It is likely that downregulation of TERT results in lengthening of G1 phase, in which human ESC are subjected to differentiation signals, thus removing them from the replicative ESC pool. Indeed, a similar mechanism has been reported to operate in murine ESC by p53 in cases of DNA damage [61]. Uncapped telomeres resulting from lack of telomerase can also be recognized as DNA damage [62]. In this context, it is possible that a p53-dependent signaling mechanism can result in ESC differentiation, as reported by our study. Although no changes in p53 expression were observed upon upregulation of TERT, this does not exclude a role for p53-mediated signaling upon TERT downregulation and needs to be investigated further.

The positive impacts of TERT expression on cell proliferation [14, 63], higher protection to oxidative stress [14, 30], and telomere length maintenance have led to the suggestion that overexpression of TERT can be beneficial for ex vivo expansion of ESC-derived cells and somatic stem cells. This has shown promising results in mesenchymal and neural progenitor cells, where ectopic TERT has led to extended expansion of these cell types in culture without impairing their differentiation potential [17, 64]. Our study has shown that overexpression of TERT does maintain telomere length and reduces the levels of intracellular peroxides accumulated in the cells during the differentiation process. Although TERT overexpression did not cause gross karyotype abnormalities, in vitro differentiation was impaired. This leads us to suggest that inducible overexpression of TERT using small molecule activators would have better practical impacts. This could be carried out once the differentiated cell type has been produced and needs to be ex vivo expanded or at the ESC stage if large numbers are required prior to differentiation.

In conclusion, our study has shown for the first time a key role for TERT in human ESC proliferation, cell cycle regulation, maintenance of pluripotent phenotype, and differentiation capacity. Our ongoing studies are focused on investigation of the underlying mechanisms of TERT actions on human ESC cycle regulation and differentiation. In addition, our investigations extend to distinguishing between the telomeric and extratelomeric effects of TERT on human ESC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank Owen Hughes for help with the reading of antibody arrays, Moira Crosier for help with EB embedding and staining, Robert Weinberg for the kind gift of the dominant-negative form of TERT cDNA, Jerome Evans for doing the karyotypic analysis, and Sun Yung and Dennis Kirk for technical assistance. This study was supported by BBSRC grant BBS/B/14779.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
SC-07-0677_Supplemental__Table_2.pdf22KSupplemental Table 2
SC-07-0677_Supplemental_Appendix_A.pdf84KSupplemental Appendix A
Sc-07-0677_Supplemental_Legends.pdf14KSupplemental Legends
SC-07-0677_Supplemental_Table_1.pdf14KSupplemental Table 1
SC-07-0677_Supplemental_Figure_1.tif311KSupplemental Figure 1
SC-07-0677_Supplemental_Figure_2.tif326KSupplemental Figure 2
SC-07-0677_supplemental_Figure_3.tif419KSupplemental Figure 3

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