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a Department of Biological Sciences, University of Durham, Durham DH1 3LE, United Kingdom;
b Institute for Ageing and Health, University of Newcastle upon Tyne NE6 4BE, United Kingdom;
c Institute of Human Genetics, University of Newcastle upon Tyne NE1 3BZ, United Kingdom
Key Words. ESCs • Telomerase • Hematopoietic stem cells • Oxidative stress • Cell proliferation • Cell cycle • Apoptosis
Correspondence: Majlinda Lako, Ph.D., Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00-44-191-241-8688; Fax: 00-44-191-241-8666; e-mail: Majlinda.Lako{at}ncl.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The telomerase holoenzyme core encompasses a reverse transcriptase (TERT) that catalyzes the addition of new repeats and a structural RNA component (TR) containing the template region that binds the telomere repeats [5–6]. Telomeric DNA consists of regions composed of conserved tandem hexanucleotide repeats with a protruding G-rich overhang and is replicated by the ribonucleoprotein telomerase. Most human somatic cells show very little or no telomerase activity, and their chromosome ends shorten at each cell division, thereby limiting the replicative lifespan and leading eventually to senescence [7]. Immortalized human cells and cancer cells, however, exhibit stable telomere length upon prolonged propagation in culture and typically have detectable telomerase activity [8]. In addition to maintaining telomere length, telomerase may also prevent the loss of G-rich single-stranded overhangs that participate, together with specific telomere-binding proteins, in forming the T-loop structure [9–11]. Erosion of the telomeric overhang can be a cause or a consequence of the collapse of the T-loop structure and the uncapping of the telomere end that eventually signals DNA damage and activates the senescence pathway [10, 11].
In addition to detection in cancer cells and in artificially immortalized cells, telomerase activity has been found in various tissues with self-renewal capacity, including the basal layer of the epidermis [12], intestinal crypt cells [13], germ line cells [14], and the hematopoietic system [15–16], and at lower levels in cycling primary presenescent fibroblasts [11]. This suggests that proper maintenance of telomere length and structure is required for the self-renewal function. Supportive evidence comes from mice lacking telomerase activity which exhibit functional impairment in organs composed of highly proliferative cells in late generations [17, 18].
Telomerase knock-down experiments in human tumor cells and overexpression studies in somatic cells have identified telomerase as a "survival enzyme" that not only allows long-term unlimited growth but also improves cellular resistance against a wide variety of stressors and cytotoxic agents. In many cases, this survival function appears unrelated to maintenance of telomere length [19, 20]. Possible mechanisms include length-independent stabilization of the telomere cap [21] or induction of stress defense genes via unknown pathways [20].
Telomerase activity has also been implicated in the maintenance of stem cell function and self-renewal, especially in the hematopoietic system. Primitive hematopoietic stem cells (HSCs) are typically quiescent and display low levels of telomerase activity, which is upregulated upon mitogenic stimulation [22, 23]. Telomerase-deficient HSCs show a reduced long-term repopulating capacity and increased genomic instability compared with wild-type cells [24]. In patients with dyskeratosis congenita, mutations in different genes compromise the function of telomerase. This leads to enhanced telomere shortening and functional deficiencies in tissues such as gut, skin, and bone marrow, all of which depend on renewal from stem cells [25–27].
Current knowledge of TERT regulation and telomerase activity in normal development is limited. ESCs afford a good model system because they can be propagated indefinitely in culture and differentiated into various cell types, recapitulating many aspects of early embryonic development [28]. In the mouse, telomerase activity is important for ESC growth. Deletions leading to loss of either the telomerase RNA or its reverse transcriptase result in progressive loss of telomeres, genomic instability, aneuploidy, telomeric fusions, and eventual reduced growth rate [29, 30, 4]. Recent work has indicated that human ESCs express the TERT gene and show high levels of telomerase activity; however, upon differentiation, the levels of TERT and telomerase activity decrease with the emergence of a maturing population of cells [31]. It is not clear from these studies whether or not the down-regulation of telomerase activity is necessary for the differentiation to proceed normally. Here, we stably overexpressed Tert in murine ESCs and investigated the effects of continuous telomerase expression on cell proliferation and apoptosis, differentiation toward hematopoietic lineage, and resistance to oxidative stress.
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MATERIALS AND METHODS |
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Generation of Tert-Overexpressing ESCs
Ten µg of the linearized construct pGRN190 (Geron Corp., Menlo Park, CA, http://www.geron.com) containing full-length Tert cDNA under the control of MPSV LTR (myeloproliferative sarcoma virus long terminal repeat) or control (vector only) was electroporated into 106 ESCs using an Electro Cell Manipulator (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com) and a pulse of 240 V. Cells were allowed to recover for 48 hours before puromycin was added to a final concentration of 0.6 µg/ml. Several single clones were picked and expanded, and one of these, tert-1, was selected for analysis in this manuscript. In addition, 30–40 antibiotic-resistant colonies from each electro-poration plate were pooled and expanded. One of these pooled clones, tert-2, representing Tert-transfected cells, and one pooled clone derived from the transfection with the empty vector (control ESCs) were used for further analysis.
Flow Cytometry Analysis
Embryoid bodies were disrupted by incubating them in collagenase IV (1 mg/ml; Invitrogen, Paisley, U.K., http://www.invitrogen.com) for 30 minutes at 37°C followed by 15 minutes in cell disassociation buffer (Invitrogen) for 15 minutes at 37°C. The single-cell suspension was resuspended in phosphate-buffered solution (PBS) + 2% FCS at a final density of 106 cells/ml. A total of 100 µl of this suspension were stained with phycoerythrin (PE)–conjugated antibodies to murine Flk-1 (Avas 12a1), B220 (RA3–6B2), Gr-1 (RB6–8C5), Mac-1 (M1/70), and Ter-119 (Ly-76) or their respective isotype control at a final concentration of 4 µg/ml. All antibodies were purchased from BD Biosciences (Oxford, U.K., http://www.bdbiosciences.com). Three washes in staining buffer were carried out, and the samples were analyzed using FACS Calibur (BD) using the CellQuest software. A total of 10,000 events were acquired for each sample. Cell sorting for flk-1+ cells was performed using FACS Vantage (Becton, Dickinson, Franklin Lakes, NJ, htpp://www.bdbiosciences.com).
CFU-GEMM Assay
CFU-GEMM assays were performed according to the manufacturer’s instructions (Stem Cell Technologies, Vancouver, Canada, http://www.stemcell.com). In brief, approximately 150 EBs in a total volume of 0.3 ml or 150,000 cells were added to 3 ml of MethoCult medium, yielding triplicate cultures of 1.1 ml each. The concentration of cytokines in MethoCult is as follows: interleukin-3 (IL-3; 10 ng/ml); IL-6 (10 ng/ml), stem cell factor (SCF; 50 ng/ml), erythropoietin (EPO; 3 U/ml). MethoCult containing EBs was dispensed into 35-mm dishes using a 16-g blunt-end needle. Cultures were placed in a 37°C incubator maintained at 5% CO2 and >95 % humidity. After 10–12 days in culture, the plates were assayed by counting the number of CFU-GEMM (colony-forming unit–granulocytes, erythrocytes, macrophages, and megakaryocytes), CFU-GM (colony-forming unit–granulocytes and macrophages), and BFU-E (burst-forming unit–erythroid).
Cellular Peroxide Concentrations
The conversion of the nonfluorescent substrate 2',7-dichloro-3',5-dihydrofluorescein diacetate (DCF-DA) into fluorescent 2',7-dichlorofluorescin (DCF) by intracellular peroxides was measured by flow cytometry as described [1]. Briefly, a stock solution of DCF-DA (Sigma-Aldrich Company, Dorset, U.K., http://www.sigmaaldrich.com) was prepared fresh for every experiment as a 2-mM solution in absolute ethanol. Embryoid bodies at different time points (day 0, 2, 4, and 6) were disintegrated to a suspension of single cells using trypsin (0.05%) EDTA (0.53 mM) for 10 minutes. Embryonic stem cells and embryoid bodies were collected in serum-free medium containing 80 µM DCF-DA and 5 µM verapamil (Sigma) for 30 minutes at 37°C. Cells were analyzed in a flow cytometer (Partec, Munster, Germany, http://www.partec.de) using blue excitation and the green emission channel (FL1). The median of the gated FL1 fluorescence peak was used as an estimate of the peroxide concentration.
Telomeric Repeat Amplification Protocol (TRAP)
TRAP reactions were carried out using the TRAPeze ELISA Telomerase Detection Kit (InterGen, Burlington, MA, http://www.intergen.com), following the manufacturer’s instructions. Several dilutions of protein extract from each sample (0.1, 1, and 10 ng) were used in the polymerase chain reaction (PCR): for each sample, three PCRs were performed. Telomerase-specific products were determined by the presence of a 6-bp incremental DNA ladder on a 12% polyacrylamide gel stained with ethidium bromide. TRAP products were quantified by a colorimetric reaction at 450 nm (using the manufacturer’s instructions) in a Titertek Multiskan plate reader (Flow Laboratories, High Wycombe, U.K.). Heat-denatured samples and the CHAPS lysis buffer (2 µ1) (InterGen) only were used as negative controls.
Microarray Analysis
Analysis was performed with the Affymetrix Mouse 430A target array (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) according to the manufacturer’s protocol with the modifications as described earlier [1]. Total RNA extracted from ESCs and EBs at day 4 using TRI zol (Invitrogen) was used. The whole analysis was carried out twice using samples from two independent experiments. Using MAS5 software provided by Affymetrix, the identification of candidate genes was based on "change p-values," which are calculated by comparing 11 individual probe pairs per gene between baseline and experimental arrays. To further increase the statistical significance, we only selected candidate genes for which the "change p-value" was below .004 in both experiments. This improves the p value to at least .000016 (= .004 x .004), which was more than sufficient to directly verify the expression changes by a different experimental approach, such as reverse transcription (RT)-PCR. It is important to note that the interpretation of our data is based on the results of both the array data and RT-PCR. The Gene Ontogeny mining tool (Affymetrix) was used to identify, among significantly changed genes, the ones with functions in stress defense, cell proliferation, and hematopoietic differentiation. The changes in expression levels of these genes were confirmed by RT-PCR.
RT-PCR Analysis
About 500 EBs were selected each day, and total RNA was extracted using TRIzol and reverse transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega Corp., Madison, WI, http://promega.com) and random oligonucleotide hexamers following the manufacturer’s instructions. The amount of cDNA in each sample was normalized using Gapdh as control. Primers and reaction conditions for all genes are given in Table 1
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Apoptosis Assay
Apoptosis in ESCs and EBs was induced by treatment with the protein kinase C inhibitor bisindolylmaleimide II [32] and measured using the annexin V-FITC apoptosis detection kit (BD Biosciences-Pharmingen) in accordance with the manufacturer’s instructions.
Cell-Proliferation Assay
Cells were harvested by trypsinization, and live cells were counted using Trypan Blue (Sigma) exclusion. Some 50,000 live cells were added to each well of a 12-well gelatine-coated plate (Iwaki; Bibby Sterilin, Staffordshire, U.K., http://www.bibby-sterilin.co.uk) containing 1 ml of murine CGR8 medium (detailed above) and LIF (10 ng/ml). Cells were permitted to grow for 3 days, and cell numbers were estimated using the Trypan Blue exclusion method. The number of population doublings was estimated as PD = ln (n2/n1)/ln2, where n1 was the seeded cell amount, and n2 was the obtained cell amount.
Cell-Cycle Analysis
Cell-cycle analysis was performed using the CycleTest Plus DNA reagent kit (Becton, Dickinson). Cells were first treated with serum-free medium for 12 hours and left to recover for 48 hours in serum-supplemented medium. Cells were harvested by trypsinization and counted (hemocytometer). Some 500,000 cells were fixed, permeabilized, and stained in accordance with the manufacturer’s instructions, and the sample was analyzed by flow cytometry (Becton, Dickinson FACS Vantage) measuring FL2 area versus total counts. The data were analyzed using ModFit 3 (Verity Software House, Topsham, ME, http://www.vsh.com) to generate percentages of cells in the G0/G1, S, and G2/M phases.
Telomere Length
The mean length of telomeric repeats was measured by flow cytometry following the hybridization of a fluorescently conjugated peptide nucleic acid (PNA) probe [33]. Briefly, cells were harvested by trypsinization, washed with PBS with 0.1 % bovine serum albumin (BSA), and counted. A total of 3 x 105 cells (in triplicate) were resuspended in a hybridization mix comprising formamide (70% aqueous solution; deionized), 20 mM Tris, pH 7.0, 1% (w/v) BSA, and fluorescein isothiocyanate (FITC)–conjugated PNA telomeric probe (PerSeptive Biosystems, Framingham MA) to a final concentration of 0.3 µg /ml. The mixture was denatured (at 800°C for 10 minutes), then allowed to hybridize at room temperature (2 hours in the dark). The cells were collected by centrifugation (at 3,000 rpm for 5 minutes) and washed twice with wash buffer (hybridization buffer plus and 0.1% [v/v] Tween 20). A further wash was performed using PBS with 0.1% (w/v) BSA and 0.1% Tween 20. The cells were resuspended in 300 µ1 PBS with 0.1% (w/v) BSA plus RNase A at a final concentration of 10 µg/ml. Propidium iodide was added at a final concentration of 0.06 µg/ml, the sample was incubated 2–4 hours at room temperature, then analyzed by flow cytometry measuring FL1 fluorescence intensity versus total counts. In addition, the length of telomeric restriction fragments was measured by pulsed field gel electrophoresis followed by in-gel hybridization, as described [34–36; also see below].
Overhang Length
The length of single-stranded terminal overhangs in telomeres was measured by nondenaturing in-gel hybridization, as described [34–36]. Briefly, cells were embedded in 0.65% low-melting agarose plugs at a density of 107 cells/ml before deproteination by proteinase K treatment. The DNA was digested until completion with HinfI (60 U per plug; Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com) at 37°C. Plugs were analyzed in a 1.0% agarose gel by pulsed field gel electrophoresis (CHEF-DRIII-SYSTEM; Bio-Rad) at 5.5 V/cm for 17 hours with a switching time of 2 to 10 seconds in 0.5x TBE (Tris-borate-EDTA: 4.5 mM Tris, 4.5 mM boric acid, 0.1 mM EDTA, pH 8.0). The gel was dried at room temperature without DNA denaturation and hybridized with 32P-
-ATP end-labeled (CCCTAA)4 at 37°C for 16 hours using high-stringency hybridization buffer (Amersham Biosciences, Piscataway, NJ, http://www.amershambiosciences.com). Gels were washed twice each in 2x standard saline citrate (SSC) and 0.2x SSC, the first wash at room temperature and the next three at 37°C. for 30 minutes each and then exposed overnight in a phosphoimager (Storm 820, Molecular Dynamics, Amersham). The gels were then denatured and rehybridized with the same probe at 43°C. Lanes were scanned, and the ratios of signal intensities from native versus denatured gel lanes were taken as a measure of the amount of single-stranded telomeric DNA (overhangs).
Karyotype Analysis of ESCs
Chromosome preparations were made using standard cytogenetics techniques and a 16-hour colcemid/BrdU mitotic arrest step. The karyotype of ESCs was determined by the standard G-banding procedure.
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RESULTS |
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). Although tert-1 ESC and tert-2 ESC clones over-expressed Tert mRNA at different levels, the telomerase activity levels were similar to each other. Most probably, an 11-fold over-expression of Tert as found in tert-2 ESCs is already sufficient to saturate the formation of active telomerase complexes. During differentiation, telomerase activity decreased in control ESCs but was maintained in the Tert-transfected cells (Fig. 1B).
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). Observation of 30 metaphases for tert-1 ESCs and tert-2 ESCs showed no deviations from the normal karyotype at PD130. Increasing aneuploidy was observed in metaphases obtained at PD190 and PD300 for both wild-type and Tert-transfected ESCs. It is well known that murine ESCs in prolonged culture become severely aneuploid [37]. Chi-square analysis (p < .01) revealed that Tert overexpression did not increase the severity or frequency of these aberrations.
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; supplementary online Fig. 2).
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). This appeared to be due to faster transit of Tert-transfected cells through the G2 and M phases of the cell cycle and, accordingly, higher fractions of cells in S phase (Fig. 3B). The doubling time for tert-1 ESCs is 16.6 hours and for tert-2 is 16.3 hours, compared with the control clone which is 23.2 hours. In view of this result, it is possible that the G2/M checkpoint might have been activated. It is equally possible that the cells simply transit G2/M phase more rapidly because of their intact telomeres.
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). Frequencies of both spontaneous cell death and inhibitor-induced apoptosis were higher in day 6 wild-type EBs than in wild-type ESCs. Overexpression of Tert protected day-6 EBs from spontaneous apoptosis. However, bisindolylma-leimide II induced close to 100% apoptosis in day-6 EBs, and this could not be rescued by Tert overexpression (Fig. 4B).
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, and Prdx2) were similar during the differentiation of Tert-overexpressing and control ESCs (Fig. 5B
). In contrast, the expression of 10 out of these 15 candidate genes (Mortalin, Hspa1b, Bmi1, Gpx2, Sod2, Txnip, Ercc4, Hspa1a, Tgr, and Gsta3) was maintained by forced telomerase expression in day 4 EB cells while downregulated in control day 4 EBs (Fig. 5B). Together with the functional data shown above, this suggests that telomerase overexpression can maintain stress defense capacities during differentiation at levels more similar to undifferentiated ESCs.
To investigate whether continued Tert expression influences the efficiency of differentiation toward hematopoietic lineages, we measured the hematopoietic commitment of wild-type and Tert-overexpressing EBs. First, we estimated the total frequencies of colony-forming cells (CFCs) within preparations of EBs at day-0 to day-6 stages of the EB differentiation protocol by progenitor colony assays (CFU-GEMM). Formation of myeloid cell colonies was significantly more frequent from Tert-overexpressing EBs from day 2 onward (Fig. 6A). We then compared the abilities of day-3.5 wild-type and tert-2 EBs to differentiate along different hematopoietic lineages. Day-3.5 EBs were disrupted to single cells, and the ability to form colonies was measured under combinations of growth factors and cytokines that stimulate the differentiation toward myeloid and erythroid lineages. This showed that Tert-2 EBs gave rise to significantly higher numbers of mixed colonies (CFU-GEMM) and colonies derived from more committed progenitors (CFU-GM and BFU-E) than wild-type EBs (Fig. 6B; supplementary online Fig. 1).
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). We then sorted 10,000 Flk-1+ cells from day-3.5 EBs derived from wild-type and Tert-transfected cells and subjected them to CFU-GEMM assays. We obtained higher numbers of colonies from Tert-transfected cells (Fig. 6D), suggesting that overexpression of Tert results in an increase in the number of hematopoietic progenitor cells obtained during differentiation of ESCs. This is also supported by staining of day 6 EBs with myeloid (Gr-1, Mac-1), erythroid (Ter-119), and lymphoid lineage markers (B220). All of these markers were expressed in a higher fraction of tert-2 EBs than in wild-type EBs (Table 3).
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). Differential expression was confirmed for all of these genes by RT-PCR. Moreover, RT-PCR also confirmed that the differential expression pattern that existed between wild-type and Tert-overexpressing ESCs was maintained during differentiation into EBs at least until day 4 (Fig. 7).
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). Two genes with roles in detoxification of peroxidation products (Aldh3AI, Mgst1) were found to be upregulated as a result of Tert overexpression. This corresponds well with the lower peroxide levels in Tert-over-expressing cells (Fig. 5A). Together with the upregulation of an additional DNA damage repair gene (Rad51), this finding is well in accordance with the downregulation of the two important cell-cycle checkpoint arrest genes p21 and Gadd45a. p21 is known to be upregulated in telomere-dependent cellular senescence [41], and Gadd45 was also shown to be upregulated in response to telomere dysfunction before [42]. Finally, we observed upregulation of Nov, a gene involved in regulation of differentiation, and of c-myb, a key hematopoietic transcription factor that is involved in regulation of proliferation and commitment within the hematopoietic hierarchy [43–45]. c-myb expression is even more increased in day 4 EBs derived from the differentiation of Tert-transfected ESCs (Fig. 7). This could be indicative of an increased proliferation of hematopoietic progenitor cells within the EBs compared with those derived from control ESCs. |
DISCUSSION |
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We found that Tert-overexpressing ESCs grow faster and are more resistant to apoptosis than are wild-type ESCs. Likewise, differentiated progeny with high levels of telomerase activity show lower levels of spontaneous apoptosis; slower accumulation of peroxides; and importantly, significantly higher numbers of cells able to differentiate along hematopoietic lineages.
How can continuous expression of telomerase cause these beneficial effects? We show here that shortening of telomeric single-stranded overhangs occurs during differentiation of wild-type ESCs but is compensated for in Tert-overexpressing cells. Loss of telomeric overhangs is expected to destabilize telomeric loops, the structures that seal or "cap" the ends of chromosomes [55]. Uncapped telomeres trigger apoptosis [56], or cell senescence [57], depending on the cellular context and, especially, the integrity of the cell cycle checkpoint machinery. In intact human fibroblasts, uncapped telomeres are recognized as substrates for signaling kinases ATM and, to a lesser extent, ATR and DNA-PK, which induce the formation of DNA damage foci. That, in turn, activates the effector kinases Chk1, Chk2, and p53 and finally arrests cell proliferation via activation of p21 [58, 59].
Different models have been suggested regarding the role of telomeric overhangs for the formation of telomeric DNA damage foci and activation of proliferation arrest [10, 11, 21, 60, 61]. In any of these models, stabilization of overhangs by telomerase, as shown here (Fig. 2), would counteract the formation of a DNA damage signal. Thus, we conclude that stabilization of telomeric ends by telomerase contributes to the improved growth, survival, and differentiation of mouse ESCs.
There is also the possibility that active telomerase may enhance DNA repair and improve cellular stress defense by nontelomeric pathways. For instance, mice with a knockout of telomerase RNA are more sensitive to ionizing radiation [62, 63], and this might not fully be explained by their shorter telomeres. Importantly, Sharma et al. [20] found improved repair of radiation-induced double strand breaks and cisplatinum-dependent crosslinks in fibroblasts after transfection with hTERT and observed transcriptional upregulation of a number of genes, including some genes with functions in DNA damage repair and stress protection. Inhibition of telomerase also changed transcription of a large number of genes [64]. We found that important stress defense genes and, specifically, antioxidant defense genes were downregulated during ESC differentiation [1] in parallel with the loss of telomerase activity [2]. This downregulation could be blocked by maintenance of telomerase activity (Fig. 5B), and additional genes with roles in cell cycle progression, stress defense, and DNA damage repair were upregulated in Tert-expressing cells (Fig. 7). This is in accordance with other studies showing that hTERT expression supports proliferation of human cells by stimulating transcription of several growth-promoting factors, downregulation of p21, induction of hyperphosphorylation of Rb, and activation of E2F [65, 66]. It is not clear at present how direct the effect of Tert overexpression on gene transcription is. It is possible that telomerase participates in signaling cascades that can directly induce transcription of genes that are important in cell-cycle progression. For instance, it has been suggested that Tert can induce structural modifications in chromatin [20]. It is also known that telomerase activation is involved in the Akt kinase pathway, which is a major response pathway to cellular oxidative stress [67, 68].
We expect that the improvement in antioxidant and general stress defense during ESC differentiation as a result of continuing telomerase activity is likely to minimize the accumulation of genomic damage during both in vitro and in vivo expansion of these cells, thus making them better candidates for cell-replacement therapies. This might be particularly relevant in the treatment of hematologic disorders since overexpression of Tert seems to increase the potential for differentiation toward hematopoietic lineages. Our in vitro assays suggested that this is due to an increase in numbers of hematopoietic stem or progenitor cells induced during the differentiation of ESCs and to better survival and net growth of hematopoietic progenitor cells. This is in accordance with recent data showing that enhanced telomerase expression in human bone marrow stromal cells results in increased osteogenic differentiation by maintaining the stem cell pool during ex vivo expansion [52, 53]. However, our in vitro data need to be confirmed by in vivo work into myeloablated recipients.
In conclusion, we have established that maintenance of telomerase activity during differentiation of ESCs confers several advantages on the differentiated cells, such as enhanced proliferation, resistance to apoptosis and oxidative stress, and improved differentiation toward hematopoietic lineages by expansion of the progenitor population. These cells did not show an increase in karyotypic instability. However, it is well established that overexpression of Tert in differentiated cells increases tumor formation in both wild-type and p53 knockout mice and induces a premalignant phenotype in mesenchymal stem cells [50, 69, 70]. Thus, it will be necessary to extend this work using highly controlled transient upregulation of TERT. In summary, this work has supports emerging evidence that Tert has additional physiological roles in addition to maintaining telomere length, and deciphering these mechanisms merits further investigations.
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ACKNOWLEDGMENTS |
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