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Telomere Length Maintenance in Stem Cell Populations

Telomere Length Maintenance in Stem Cell Populations

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Telomere length maintenance in stem cell populations
Nicholas D. Allen
, Duncan M. Baird
School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3, USA
School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK 
a b s t r a c ta r t i c l e i n f o
 Article history:
Received 12 December 2008Received in revised form 4 February 2009Accepted 5 February 2009Available online 12 February 2009
TelomereTelomeraseSenescenceGenome stabilityStem cell
The maintenance of telomere length is essential for upholding the integrity of the genome. There is goodevidence to suggest that telomere length maintenance in stem cell populations is important to facilitate thecell division required for tissue homeostasis. This is balanced against the requirementin long lived species forproliferative life span barriers for tumour suppression; the gradual erosion of telomeres provides one suchbarrier. The dynamics of telomeres in stem cell populations may thus be crucial in the balance betweentumour suppression and tissue homeostasis. Here we brie
y discuss our current understanding of telomeredynamics in stem cell populations, and provide some data to indicate that telomeres in human embryonicstem cells may be more stable and less prone to large-scale stochastic telomeric deletion.© 2009 Elsevier B.V. All rights reserved.
1. The importance of telomere length in stem cell populations
The regenerative capacity of human tissues declines with age andthe incidence of cancers increases; both these processes may bedriven by a decline in stem cell function[1
5]. There has to be abalance, in long-lived organisms such as humans, between maintain-ing regenerative potential on one hand, and tumour suppression onthe other. Several studies focusing on p16
, a key mediator of replicative senescence, suggest that this may be the case. In the bonemarrow of mice, p16
levels increase with age in haematopoieticstemcells,butnotinothercelltypes[6];thiscorrelatedwithadeclineas a function of age, in the ability of these cells to reconstitute theimmune system of irradiated mice, whereas
mice hadmore stem cells and were better able to reconstitute an immunesystem. Similar observations were made in neurons from theforebrain, where again p16
levels increased with age, whichcorrelated with a decline in proliferation, this was to some extentameliorated in
mice[7]. As p16
is a tumoursuppressor, the de
ciency of protein leads to an increased frequencyofcancer[8],andyetthesedatashowthattheabsenceofp16
canslow most aspects of stem cell ageing, thus highlighting the balancebetween tumour suppression and regenerative capacity. This is likelyto result from multiple factors such as Bmi1 that promote prolifera-tion, balanced against tumour suppression for example by p16
11]. One mechanism that may contribute to
ne tune this balancemay be telomere length, whereby stem cells may need to maintaintelomeres at a length that provides suf 
cient replicative capacity fortissue homeostasis, versus the requirement to minimise telomerelength and replicative capacity as a tumour suppressive mechanism.Telomere length is an important determinant of telomericfunction, in the presence of a functional DNA damage responseshort telomeres elicit a DNA damage checkpoint, that leads to eitherreplicative senescence or apoptosis[12].Telomere-dependent repli- cative senescence represents the tumour suppressive function of telomeres[13,14]; the corollary of which may be an age-relatedaccumulation of senescent cells[15,16]. The presence of senescentcellsnotonlyreducestheproportionofmitoticallyactivecellsbutmayalso, but by virtue of exhibiting a more catabolic phenotype they canactively degrade the tissue microenvironment[17]. Interestingly eventhe presence of a small proportion of senescent cells, may render thetissue microenvironment more permissive for tumour progression[18].Theimportanceof telomerelengthfromthestandpointofageingand cancer is exempli
ed by the telomerase knock out mice. Whereafter several generations telomeres erode suf 
ciently to confer aphenotype, whereby the presence of short telomeres leads to defectsin proliferative tissues that mirror some age-related phenotypes inhumans. In addition to a shortened lifespan these include, immuno-senescence, alopecia, hair greying and intestinal atrophy andincreased rate of tumour formation[19
22]. These phenotypescould be partially rescued in the context of mutations in the DNAdamage checkpoint responses, for example the absence of p53rescued some of the proliferative defects, but this was accompaniedby an increase in the rate of tumour formation[23]. Interestingly inthe context of Terc and p21 mutations, the rescue of age relatedphenotypes was not accompanied by an increase in tumourigenesis,despite rescuing proliferative and self renewal detects in stem cellpopulations; these observations indicate that, p53/p21 dependent,telomere driven, replicative senescence was driving the age-related
Biochimica et Biophysica Acta 1792 (2009) 324
Corresponding author. Tel.: +44 29 2068 7038; fax: +44 29 2068 7343.
E-mail address:
bairddm@cardiff.ac.uk(D.M. Baird).0925-4439/$
see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.bbadis.2009.02.004
Contents lists available atScienceDirect
Biochimica et Biophysica Acta
 journal homepage: www.elsevier.com/locate/bbadis
phenotypes, and furthermore, that apoptosis may be tumourprotective[24].Some of the phenotypes exhibited by the telomerase knockoutmice mirrorthose observed inindividuals withdyskeratosiscongenita (DC): a disease characterised by mutations in genes thatresult in impaired telomere length maintenance, such as componentsof the telomerase complex that results in haploinsuf 
ciency fortelomerase activity[25
27], or components of the shelterin complex[28]. DC patients exhibit reduced haematopoiesis, reduced number of haematopoietic progenitors and colony forming ability[29,30], andchromosomal instability phenotypes[31,32]. In addition, like telo-merase knockout mice, DC patients display disease anticipation,wherebytheseverityandageofonsetof thedisease,getprogressivelyworse fromonegenerationtothenext[33].Failure of telomere lengthmaintenance has been implicated in other bone marrow failuresyndromes including Fanconi anaemia, Shwachman
Diamond syn-drome, Diamond
Blackfan anaemia and aplastic anaemia[34
37].Thus clinical data together with mouse models indicate telomerelength maintenance is important in the context of human disease, theageing process and cancer; it appears likely that telomere lengthmaintenanceinthestemcellscompartmentsmayplayacrucialroleinthese processes.
2. Telomere length dynamics in human stem cell populations
Data concerning telomere length and telomerase activity in stemcells is limited. Adult stem cell populations undergo cell divisioncomparatively infrequently[38]and therefore, based on the endreplication problem, stem cells would be predicted to suffer limitedtelomere erosion, irrespective of telomerase expression. The mostextensivelystudiedstemcellsarethoseof thehaematopoieticsystem.Haematopoietic stem cells (HSCs) are known to express telomerase[39,40]yettheyappeartobesubjectedtotelomerelossasafunctionof age[41];telomeraseactivityisinsuf 
cienttomaintaintelomerelengthin HSCs and thus the proliferative potential of these cells may declinewith age. However telomerase is up regulated following immunestimulationofBandTcells[42
cienttoreducetherateof telomere erosion, and some cases result in telomere lengthening[47
49].Importantlytheupregulationoftelomeraseactivityfollowingstimulation, confers a proliferative lifespan extension compared tocells that do not express telomerase, this allows for repeated clonalexpansions in response to the antigen[48].However this is not unlimitedandultimatelytelomereerosionandthelossofproliferativepotential may underlie some aspects of immunosenescence[49,50].In the epidermis, telomerase is expressed by cells speci
cally onthe basal layer[51], yet telomere length decreases with age[52]. Spermatogonial stem cells express high levels of telomerase[53
55],this is considered to maintain telomere length in the germline forsubsequent generations. Indeed to date the male germline is the onlytissue that has shown to exhibit telomere lengthening as a function of age[56,57]. There is evidence to suggest that there is a gradient intelomere length from the stem cell compartments which decreases inmore differentiated cell populations. This was exempli
ed by thelingual mucosa, where
in situ
hybridisation revealed the longesttelomeres in the basal cells[58].Further evidence using
in situ
methods in mice also showed distinct gradients of telomere length,withthelongesttelomeresobservedinthestemcell compartmentsof the skin, small intestine, cornea, testis and brain[59]. Of particularimportance, this work included the observation that the telomerelength in the stem cell compartments appears to shorten with age[59]. Thus the evidence from humans and mice, indicates that stemcells maintain telomeres at a longer length, relative to the other cellswithin the tissue inwhich they reside. This is probablya function of acombination of telomerase activity together with a reduced cellturnover. However, with the exception of spermatogonial stem cells,some stem cell compartments have been shown to suffer telomereerosion as a function of age.Telomeraseactivityislikelytobelimitinginmostcellsthatexpresstelomerase at physiological levels. Over expression of the RNA andprotein components of telomerase individually does not result insigni
cant telomere lengthening[60]. However when both compo-nentsareoverexpressedsimultaneously,thesesuper-telomerasecellsexhibit ongoing telomere lengthening with no apparent lengthcontrol. These data imply that in cells where telomere length is stabletelomerase levels are limiting; it is the shortest telomeres in the cellsthat are preferentially lengthened[60,61]. This must require a
nebalance in the levels of telomerase activity to maintain telomerelength;therefore subtlylowerlevels, suchasmaybeobserved instemcell compartments, could lead to gradual telomere erosion. Whethertelomere length would become limiting in this situation is unclear;telomerase preferentially extends shorter telomeres[61]and thustelomerescoulderodetoanewshorter,butstablestate,asobservedinmany cancer derived cell lines[60]. Furthermore mice that arehaploinsuf 
cient for either the RNA or protein components of telomerase maintain telomeres at a shorter length but do not exhibitobvious direct telomere defects, such as telomere fusion[62,63].
3. Telomere instability 
Telomeres shorten primarily as a consequence of gradual end-replication losses with ongoing cell division[64,65]. However moredetailed analysis of telomere dynamics in human cells has indicatedthe existence of additional mechanisms that generate sporadic large-scale changes in telomere length[66]. In normal human cells, raretelomeres have been observed that lack signals from
uorescentlylabelled telomere repeat containing probes; these may arise asconsequence of sporadic large-scale telomeric deletion events[67
69]. Using an experimentally transformed cell line carrying atagged chromosome end, Murnane et al. observed changes intelomere length which appeared to have resulted from large-scaletelomere deletion events[70]; other types of events included thedeletion of the entire telomere and adjacent DNA coupled withhealing of the end, possibly by de novo telomere addition, orchromosomal fusion events[71]. Interestingly these events occur incells that express telomerase and can maintain telomere length forextended periods in culture, yet they can still suffer telomericinstability. Single molecule PCR analysis of telomere length hasshown that in the absence of telomerase, gradual telomere erosionresultsinadecreaseinthemeanandanincreaseinthevarianceofthedistribution[65,72].These data are consistent with the telomere dynamics predicted as a consequence of end-replication losses,together with a putative C-strand resection[64,65]. However, super-imposed on the gradual erosion of the bulk of the telomere lengthdistribution, were large-scale, apparently sporadic, telomere deletionevents. These events have been observed both in the presence orabsenceoftelomeraseandcanresultintelomerescontaininglessthan20TTAGGGrepeats[72,73].Theoccurrenceofthesedeletedtelomeresis sporadic, and they do not accumulate with ongoing cell division.Thisindicatesthat,eitherthecellinwhichthedeletioneventoccurredexitedthecellcycle,oralternativelythedeletedtelomerewasnotlongenough to confer telomeric function and was subjected to furtherprocessing. Such telomeres could be repaired to full length orsubjected to telomere fusion. Indeed fusion analysis has revealedthat telomeres that had suffered a deletion event are subjected tofusion[74]. Importantly in normal cells that contain a complement of telomeres that are long and fully functional, rare fusion events aredetected that involve very short telomeres. These events areconsistent with the concept that normal cells can be subjected tostochastic telomeric deletion, and these deleted telomeres canundergo fusion. This implies that cells within normal tissues
in vivo
may be subjected to telomeric deletion events, that can lead, viaanaphase bridging, breakage and fusion events, to large-scale,potentially oncogenic mutation. This cell intrinsic mutational load
N.D. Allen, D.M. Baird / Biochimica et Biophysica Acta 1792 (2009) 324
may be particularly signi
cant in the context of cell cycle checkpointmutations,whereatelomerethathadsufferedadeletionevent,canbesubjected to fusion, rather than causing the cell to exit the cell cycle.Indeedthere areclonalpatchesofkeratinocyteswithintheepidermis,that exhibit p53 mutations[75
77]and thus short dysfunctionaltelomeres in these cells may exhibit a greater propensity to undergofusion than in keratinocytes with functional p53. These data indicatethat it will be informative to understand the dynamics of telomerelengthinstemcellpopulations.Tothisend,wehavesomepreliminarydata to suggest that at least some stem cell populations mayexhibit agreater degree of telomere stability compared to other cell types thathave been analysed.Fig. 1shows an example of this data, wherehuman embryonic stem (hES) cells have been subjected to a largescale single telomere length analysis. At the 17p and XpYp telomeresthe mean telomere length at both ends was broadly similar at 13.0and12.8 kb respectively, this is within the range observed in the malegermline[56]. A bimodal distribution was apparent at the XpYptelomere, which is consistent with the presence of two telomericalleles that are maintained at different lengths of 6.9 and 15 kb; atelomere length differential of 8.1 kb. These data are consistent withthe concept that the telomere length setting of speci
c alleles, in thepresence of telomerase, may be determined by
acting elements orallele speci
c epigenetic modi
cations[73,78,79]. Telomere length of the hES was heterogeneous with telomeres ranging from 3.0 kb toover25kb. However it was strikingthatwe could notobserve a singletelomere out of a total sample of 753 molecules that was less than3.0 kb (Fig. 1). In
broblast cell cultures sporadic telomere deletionresults in telomeres of less than 10 TTAGGG repeats at a frequency of 9×10
;thesearetelomeresthathavebeenshowntoundergofusion[74]. Compared to these cells, the paucity of stochastic telomericdeletion events in our sample of human embryonic stem cells issigni
cant (
0.001, Chi sq.). These data indicate the possibility thathumanembryonicstemcellsmayexhibitenhancedtelomerestability.Consistent with this view, whilst some human ES cells displaykaryotypic changes following prolonged periods in culture, thepredominantaberrationsareaneuploidy,speci
callygainsofchromo-somes17,12andX,withlessevidenceofnon-reciprocaltranslocationsthat occur as a consequence of telomere dysfunction[80]. Themechanisms underlying telomere deletion events is not clear[81],but may include mechanisms such as T-loop recombination[68,82],unequal sister chromatid exchange[83],replication slippage, or resolution of stalled replication forks at sites of unrepaired damageor DNA structures, such as G-quartets, that cannot be replicated[84].Thus the mechanism by which human embryonic stem cells mayexhibit enhanced telomeric stability is also not clear, however in thiscontext it is pertinent to note that these cells exhibit enhancedgenomic maintenance in humans[85], and lower levels of reactiveoxygenspeciesinmice[86,87].DNAdamage,includingthatgeneratedby oxidative stress, may provide an obstacle to telomeric replication,the resolution of which could initiate large-scale changes in telomerelength. Adult stem cells may not exhibit the same levels of genomicstability compared to embryonic stem cells; it will therefore beinformative to understand how levels of telomeric stability differbetween these cell types.
4. Chromatin structure and genomic organisation in stem cells
If the apparent stability of telomeres in hES cells, and indeed otherstem cell types, is borne out by further analysis, the mechanistic basisof telomeric stability would warrant investigation. In addition to theenhanced levels of DNA repair one could speculate that telomericstability could be related to differences in chromatin distributionsand/or differences in nuclear organisation. The organisation of lociwithinthenucleuscan regulate transcription,withdifferentcelltypesdisplaying distinct nuclear organisation patterns[88,89]. Stem cellsare no exception, as they display a nuclear architecture, such that locirequired for the maintenance of pluripotency, display speci
c nuclearlocations; for example NANOG on 12p has a more central location inES cells compared to differentiated cells, or 6p that maintains itschromosome position but the OCT4 locus on that chromosome isoutsideof itsusualposition[90].Itisknownfromstudiesinyeastthatthe tethering of telomeres at the nuclear periphery is important toregulate transcription of telomere associated genes such as themating-type loci[91], which may be mediated by regulating thetelomeric chromatin[91]. It also appears that embryonic stem cellsexhibitadistinctchromatinstructure,thatisimportantforcontrollingthe expression pro
les required to maintain pluripotency[92
95]. EScells are uniquely characterised by a prevalence of a bivalentchromatin state, in which many developmentally regulated genessimultaneously possess both activating and silencing epigeneticchromatinmodi
cations[96].Whetherthesedifferencesinchromatinextend to the telomeres is not clear. It is becoming apparent thatthe chromatin status of telomeres, which exhibit epigenetic marksof compacted heterochromatin, can control telomeric stability[78,79,97,98]. For example in mice the subtelomeric regions areheavily methylated, but mammalian telomere repeats lack the CpGsites for methylation. Interestingly the knockout of DNA methyltrans-ferases results in large-scale changes in telomere length, that may beassociated with increased recombinational activity including sisterchromatid exchange[98].Thus chromatin structure contributes to telomeric stability, and speci
c chromatin patterns in stem cells mayconfer enhanced telomere length stability.In summary, telomere length in stem cell populations is animportant determinant of the replicative potential that is required fortissue homeostasis, yet telomere length may decline as a function of 
Fig. 1.
Telomere length analysis of the human embryonic stem cell line, HUES9. Largescale analysis of telomere length at the XpYp and 17p telomeres using single telomerelength analysis (STELA). Telomere length statistics are shown below.326
N.D. Allen, D.M. Baird / Biochimica et Biophysica Acta 1792 (2009) 324

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