From bloodjournal.hematologylibrary.org by guest on January 13, 2014. For personal use only.

2008 111: 1759-1766

doi:10.1182/blood-2007-09-084913

Telomeres, stem cells, and hematology

Peter M. Lansdorp

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Stem Cells in Hematology (166 articles)

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ASH 50th anniversary review

Telomeres, stem cells, and hematology

Peter M. Lansdorp1
1Terry Fox Laboratory, British Columbia Cancer Agency and University of British Columbia, Vancouver, BC

Telomeres are highly dynamic structures capped telomeres accumulate. The nant progression is facilitated by genome
that adjust the cellular response to stress chance of the latter increases as the instability resulting from uncapped telo-
and growth stimulation based on previ- average telomere length decreases. The meres. The critical role of telomeres in
ous cell divisions. This critical function is average telomere length is set and main- cell proliferation and aging is illustrated
accomplished by progressive telomere tained in cells of the germ line that typi- in patients with 50% of normal telomerase
shortening and DNA damage responses cally express high levels of telomerase. In levels resulting from a mutation in one of
activated by chromosome ends without somatic cells, the telomere length typi- the telomerase genes. Here, the role of
sufficient telomere repeats. Repair of criti- cally declines with age, posing a barrier telomeres and telomerase in human biol-
cally short telomeres by telomerase or to tumor growth but also contributing to ogy is reviewed from a personal historical
recombination is limited in most somatic loss of cells with age. Loss of (stem) cells perspective. (Blood. 2008;111:1759-1766)
cells, and apoptosis or cellular senes- via telomere attrition provides strong se-
cence is triggered when too many un- lection for abnormal cells in which malig- © 2008 by The American Society of Hematology

Introduction
Since there are many excellent comprehensive reviews on the topic The length of the telomere repeat track determines the likeli-
of telomeres, hematology, and aging,1-4 I decided that an autobio- hood that a telomere will be properly capped. A minimum number
graphic narrative might be more interesting and perhaps more of telomere repeats is required to recruit sufficient telomere binding
entertaining. Of course, my personal story is just one of many. proteins to fold the end into a properly capped structure. Most
I understand that readers of Blood may not have time or patience likely this structure consists of a T-loop in which the single-
for such trivia. Therefore, the first section, “Telomeres and stranded 3⬘ end of the chromosome folds back into double-stranded
hematology,” contains the most important scientific messages. The telomere repeats.7 So to avoid activation of a DNA damage
later sections provide a personal account of how my interest in stem response, each telomere must have a minimum length of telomere
cells led to studies of telomeres. My hope is that my personal and repeats as well as fully functional “shelterin” proteins.2 Telomeric
inevitably biased perspective will provide some insight in the DNA together with various proteins form a proper cap.
development of these important research areas over the last The third point is that telomere length is maintained and set in
2 decades. the stem cells of the germ line that express high levels of
telomerase. However, most somatic cells, including various stem
cells, are unable to maintain telomere length. As a result, most
Telomeres and hematology somatic (stem) cells show progressive telomere shortening with
each round of replication. B lymphocytes appear to be an exception
It is widely understood that telomeres represent the very ends of to this rule in that memory B cells have longer telomeres than naive
chromosomes, characterized by guanine-rich repetitive DNA and B cells.8 Elongation of telomeres in the B-cell lineage could reflect
associated proteins.2 That telomerase is a reverse transcriptase that a requirement for extensive cell divisions imposed by clonal
uses RNA to synthesize the G-rich repeats is also well known.1 The selection and affinity maturation of antibodies. Could it be that
decrease in telomere length in most cells with proliferation and B cells are more likely to form tumors than T cells as a result?9 In
with age is appreciated, but what does it all mean? Despite general, the difference in telomere biology between the cells of the
intensive efforts by a large number of research teams, there is no germ line and various somatic (stem) cells is poorly understood.
definitive answer to this question. My hope is that, by breaking One possibility is that telomeric chromatin is different, more
down the complex topic into smaller segments, parts of the answer “open” in the germ line (and perhaps the early embryo as well),
will emerge. increasing the chance of functional interactions between telomer-
Every chromosome end needs to be capped by a minimum ase and chromosome ends. Other possibilities are that functional
number of telomere repeats to prevent activation of a DNA damage telomerase levels vary between cells of the germ line and somatic
response.5,6 The simple way to think about this is that without a cells, for example, as a result of alternative splicing of hTERT
minimum number of telomere repeats any of the 92 ends of human transcripts10 or differences in the assembly or posttranslation
chromosomes will resemble the end of a broken chromosome. modifications of the enzyme complex. Perhaps telomerase levels as
Double-strand breaks are dangerous to cells and must be repaired well as telomere chromatin differ among various cell types.
prior to mitosis to prevent genome instability or cell death. So The fourth point is that if a telomere becomes uncapped in a
chromosomes must have a proper cap. somatic cell, the outlook is not necessarily bleak as at least

Submitted September 5, 2007; accepted September 17, 2007; DOI 10.1182/blood- © 2008 by The American Society of Hematology
2007-09-084913.

BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 1759

 From bloodjournal.hematologylibrary.org by guest on January 13, 2014. For personal use only.

1760 LANSDORP BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4

2 telomere salvage pathways are available. Thus, following activa- The final points relate to the pathological consequences of
tion of a DNA damage response (via pathways that remain poorly abnormalities in telomere maintenance. Mutations in genes encod-
understood), uncapped telomeres can be elongated by telomerase ing components of the telomerase enzyme including TERT, TERC,
and/or recombination. Salvage by recombination requires the and DKC1 have been implicated in an increasing number of
presence of homologous sequences and is probably less efficient disorders including dyskeratosis congenita,4,12 aplastic anemia,13
when the average telomere length is short (ie, more efficient in and pulmonary fibrosis.14,15 Such mutations result in the assembly
mice than in humans11). Whether recombination or telomerase is of dysfunctional telomerase complexes with partial activity or no
used to salvage critically short telomeres varies between cell types catalytically activity whatsoever. Typically, only one allele of these
and presumably depends on average telomere length, telomerase telomerase genes is affected, resulting in a mixture of active and
expression, and other factors. Telomerase appears to be the major inactive telomerase enzyme complexes. There is little evidence to
salvage pathway for uncapped telomeres in hematopoietic cells. It support dominant negative effects of inactive telomerase enzymes,
is possible that homologous recombination reactions involving, for and the net effect of mutations in genes encoding telomerase
example, BRCA1 and BRCA2 and the Fanconi proteins are also components is typically a reduction of up to 50% in overall
important to sustain the proliferation of hematopoietic stem cells telomerase levels.16 Dyskeratosis congenita is a bone marrow
over a normal lifetime. failure syndrome typically associated with skin pigmentation
Unfortunately, there exists a third, dangerous pathway that abnormalities, nail dystrophy, and leukoplakia. Other clinical
extinguishes the DNA damage signals derived from uncapped manifestations are diverse in nature and can include lung fibrosis,
telomeres: the fusions of 2 uncapped chromosome ends. These can liver cirrhosis, osteoporosis, and a predisposition to develop a
variety of malignancies. It is important to note that telomerase
be sister chromatids or the ends of 2 separate chromosomes.
deficiency per se does not cause pathology, but its consequences on
End-to-end fusions are dangerous as cells with intact cell cycle
telomere length do. These indirect effects can be separated into
checkpoints will be fooled to enter mitosis with dicentric chromo-
effects in the germ line (resulting in offspring with shorter
somes. The anaphase bridges that are predicted to occur 50% of the
telomeres) and somatic cells (resulting in a reduced efficiency of
time will typically induce chromosome breaks. The resulting
the telomerase-dependent telomere salvage pathway). The net
genetic instability will kill most cells but loss or gain of genetic
result of the combined effects in the germ line and somatic cells is
material can also provide a growth advantage to rare cells. Further
disease anticipation, the onset of disease occurring at earlier age in
fusions and mitotic break-fusion-bridge cycles can result in ram-
subsequent generations. The indirect relation between mutations in
pant genome instability. In this manner, telomere shortening is telomerase genes and the occurrence of a clinical phenotype has
directly implicated in genome instability. almost certainly resulted in an underestimation of the involvement
The sixth important point is that telomere salvage pathways of mutations in genes involved in telomere maintenance in various
have a limited capacity. Thus telomerase levels are limiting in most diseases by genetic linkage analysis. Most individuals with a
somatic cells. When more than a few uncapped telomeres accumu- mutation in one of the telomerase genes have no phenotype.
late in a cell (the exact number is not known and could differ Pathology occurs when cells are lost or impaired by uncapped
among cell types as a function of the efficiency of the telomere telomeres to the point that the function of tissue or organ is
salvage pathways), the capacity of the telomere salvage pathways compromised. The primary determinant of when such a threshold is
is exceeded and cells either senesce or die by apoptosis. Both ATM reached appears to be the average telomere length.12 Cells at risks
and p53 are implicated in the DNA damage response originating are cells that must sustain proliferation over a lifetime including
from uncapped telomeres, and persistent DNA damage signals (eg, lymphocytes and stem cells of tissues with a high turnover such the
phosphorylated H2AX) originating from dysfunctional telomeres bone marrow. Factors in the environment or heritable factors that
have been found in senescent cells.5 In healthy humans, this compromise the function or survival of (stem) cells in specific
situation becomes increasingly likely for an increasing number of tissues increase the risk of telomere dysfunction in specific tissues.
cells as their average telomere length decreases as a function of Such collaborative effects perhaps explain the diverse disease
accumulated cell divisions and age. This is not necessarily a bad entities that have now been linked to telomerase deficiencies. Loss
thing as telomere attrition provides a hurdle for aspiring tumor cells of (stem) cells in tissues, perhaps combined with changes in the
and thereby acts as a tumor suppressor mechanism. Indeed, it marrow microenvironment,17 creates opportunities for abnormal
seems reasonable to assume that telomere length and telomerase cells that can respond to the growth stimulatory signals despite the
levels evolved in humans to suppress the growth of tumors prior to presence of critically short and dysfunctional telomeres. The loss of
reproduction and several decades of life. The flip side is that tight telomere function, resulting from either telomerase disorders or
control over replication by telomere attrition probably results in exhaustion of stem cell following endogenous or exogenous
accumulation of dysfunctional, senescent cells as well as signifi- damage, can thus result in aplastic and dysplastic changes as well
cant loss of cells with age. The possibility that telomere attrition as predispose for neoplastic growth. The intimate role of telomeres
plays a more important role in human aging than in the aging of in aging18 and cancer19 assures that these fascinating dynamic
rodents and other model organisms does not sit well with many of structures will continue to be the subject of intense investigations
my friends and colleagues who exclusively study model organisms. over the next decades.
However, the striking clinical consequences of a 2-fold reduction in
telomerase levels in humans (resulting from haploinsufficiency for
one of the telomerase genes) in comparison with the absence of a What to do?
phenotype for several generations in yeast, worms, plants, and
rodents that completely lack telomerase strongly suggests that One of my best childhood memories is crouching in the dunes
telomeres and telomerase play a role in controlling the proliferation behind my home in Wassenaar, the Netherlands, watching as
of somatic cells in humans that does not exist as such in most model gunpowder burned its way to a miniature rocket waiting in an
organisms including short-lived (inbred) mammals. improvised V-shaped rocket launching pad. After many failures,
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BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 TELOMERES, STEM CELLS AND HEMATOLOGY 1761

the rocket would fizz off into the sky and disappear out of sight. Ah, best murine stem-cell purification procedures did not yet yield
the sizzle, the sparks, the whoosh! This childhood experience has “pure” stem cells in the late 1980s, and my own work at the time
of course nothing to do with telomeres or hematology, but I was was primarily focused on the human system. Apart from differ-
asked to provide a historic perspective. Sure enough I will get to ences in telomere biology (discussed later) it is probably true that
stem cells and telomeres if I continue tracing my footsteps. the frequency of stem cells in human bone marrow is several orders
Like most teenagers, I did not have a clear picture of what to do of magnitude lower than in the mouse. Jan Abkowitz and her
with my life. I subscribe to the motto “when in doubt follow your collaborators in Seattle have made compelling arguments (Shep-
nose but keep your options open.” The many different career herd et al34,35) in support of the idea that the absolute number of
options as an MD appealed to me. Nearing the end of my medical stem cells and the number of times they divide are very similar in
training, my main interest switched from psychiatry (having all mammals. If this is correct, the estimated 104 to 105 stem cells in
learned that sympathetic ears alone accomplish little) to internal the mouse are distributed over approximately 2 liters of bone
medicine and immunology. At the time, it seemed reasonable to do marrow in humans. Based on this estimate, the frequency of stem
“research,” although I was largely ignorant about the nature of such cells in adult human bone marrow is expected to be less than 1 per
activities. After unproductive exercises in immunology, I decided 107 nucleated bone marrow cells. No wonder attempts to purify,
to determine whether I could reproduce the work of Köhler and genetically modify, or expand human adult stem cells have run into
Milstein and make monoclonal antibodies.20 After many trials and hard times. It seems very possible that the low frequency of actual
errors, I eventually succeeded. How exciting it was to direct the stem cells in human bone marrow (and “mobilized” peripheral
growth, selection, and expansion of antibody-producing cells. In blood) represents a major, underappreciated challenge for both
reproducing this published work, I had learned 2 important lessons: basic studies and clinical applications.
anything worthwhile can be reproduced and “practice makes To avoid variables in the quality and quantity of human blood
perfect.” This was a turning point for me because it gave me the and bone marrow samples available for my research, I decided to
courage to choose a career in science. work with previously frozen aliquots of bone marrow cells from
My main focus initially was technical and, after further work on cadaver organ donors. This was a great advance because it avoided
assays to screen for monoclonal antibodies against cell-surface biologic variation among samples and allowed systematic work on
antigens,21 hybridoma growth factor22,23 (which turned out to be stem-cell purification, assays, and culture. Based on the work of
IL-624,25), and the discovery of tetrameric antibody complexes,26 it Mike Dexter et al who had developed culture systems for murine
became time for me to decide on a specific research area. I selected hematopoietic cells,36 we developed a surrogate assay for human
blood cell formation and hematopoietic stem cells, encouraged by stem cells using limiting dilution strategies based on the their
the work of Stuart Schlossman and colleagues in Boston, who had presumed ability to initiate long-term cultures.37 Other approaches
shown that monoclonal antibodies are powerful tools to distinguish to measure human stem cells were explored by John Dick in
between morphologically similar cells in the immune system Toronto, who transplanted human stem cells into immune-deficient
(Kung et al27). Inspired by the seminal work of Donald Metcalf,28 animals.38 Both assays have been very useful in human stem-cell
I believed the hematopoietic system was waiting to be “dissected” research. However, doubts remain about the efficiency, reproduc-
by monoclonal antibodies. By chance and the efforts of Allen ibility, and cell types measured in either assay relative to the cells
Eaves, I ended up pursuing such studies in Vancouver, a truly that sustain normal hematopoiesis or the cells that repopulate bone
spectacular place, secluded at the very border of the “civilized” marrow upon transplantation.
world, yet a great place to pursue science, especially in the Terry Another area of interest was the development of tissue culture
Fox laboratory at the BC Cancer Research Center with colleagues medium for human hematopoietic cells.39 Most tissue culture
such as Connie Eaves, Keith Humphries, and Gerry Krystal. media were developed and optimized for various immortal cell
One of the first antibodies I produced in Vancouver was 8G12 lines more than 50 years ago, and it seemed reasonable to assume
specific for CD34.29 Curt Civin had shown that CD34 is a useful that primary human hematopoietic cells could have different
marker for stem and progenitor cells (Strauss et al30), and our requirements. My work in this area was inspired by Norman
antibody, also called HPCA-2, was found to be very useful to Iscove, who had developed a superior serum-free medium for the
enumerate and study CD34⫹ cells. We also started exploring growth of B cells and hybridomas (Iscove and Melchers40). In view
various applications of tetrameric antibody complexes,26 including of the doubts about which cells to study and the unknown growth
applications for cell separation,31 which are now widely used. factor requirements of primary hematopoietic (stem) cells, our
progress was limited. Nevertheless, the recipe we developed
(containing high levels of transferrin and low-density lipoproteins)
is still used and probably provides a good starting point for further
Hematopoietic stem cells: where to start? work in this important area.39,41
With “in-house” monoclonal antibodies and cell separation tech-
niques at hand, we embarked in the late 1980s on activities that
remain very popular to this day: purify stem cells as best as possible Developmental changes in stem-cell
and study the behavior of such cells in culture in response to function?
endless culture variables. The goal was to identify culture condi-
tions that would allow meaningful “expansion” of stem cells in While the work on monoclonal antibodies, culture systems, tissue
vitro for clinical applications. This strategy was based on compel- culture media, and cell separation techniques represented signifi-
ling direct and indirect evidence supporting the “self-renewal” of cant technical advances, it did not serve the main objective of my
stem cells in vivo. For example, in the mouse it is possible to isolate studies: to purify human stem cells and cultivate such cells to
a single stem cell, transplant it into a lethally irradiated recipient, obtain a clinical meaningful numeric expansion in culture. On the
observe donor cell bone marrow reconstitution, and harvest many contrary, the whole exercise seemed tedious and trivial when it
stem cells after a few weeks.32 Despite claims to the contrary,33 the became apparent that the maintenance of CD34⫹ cells in serum-
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1762 LANSDORP BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4

tion and expansion of stem cells be developmentally controlled?

Because our findings greatly complicated simple notions about
“stem cells” and “self-renewal,” they remain as unpopular today as
they were 15 years ago. A sobering lesson for aspiring scientists:
you can be right and make exciting advances but whether your
work is accepted or acknowledged depends largely on social
networks in science and is often independent of its quality or merit.
The news is not all bad: most gems are eventually rediscovered.
The good news for the investigator is that lack of recognition has
advantages in terms of freedom to pursue novel research directions
and time not wasted on making appearances.
Because the observed functional differences among hematopoi-
etic cells at different stages of development were obtained using
conditions that were identical, we proposed that the functional
differences were intrinsic to cells at different stages of develop-
ment.44 The very low frequency of actual stem cells in adult bone
marrow mentioned above provides a caveat: it is possible that the
cell populations purified from fetal liver and cord blood were far
more enriched for actual stem cells than the candidate stem cells
purified from human bone marrow. However, our data seemed to
indicate that the behavior of stem cells could be primarily
determined by unknown factors with a developmental component.
If correct, dreams of stem-cell therapies starting with very few
purified stem cells had run into a major roadblock. The idea of
embarking on a quest to unravel the molecular mechanism that
control stem-cell behavior and expansion at various stages of
development furthermore did not appeal to me. I was aware of the
limited progress in understanding the more accessible and better
documented developmental clock regulating the expression of
globin genes and the idea of similar studies with rare primary cells
and unknown genes was unattractive. This was my mindset in early
1993 when I met Houmayoun Vaziri, who was interested in a
Figure 1. Developmental changes and telomere loss in hematopoietic cells.
position as a graduate student in my laboratory.
(A) Ontogeny-related differences in the production of CD34⫹ cells in culture.
(Reproduced from Lansdorp et al42 with permission.) Candidate stem cells with a
CD34⫹CD45RAlowCD71low phenotype were purified from fetal liver (wk12), umbilical
cord blood, and adult bone marrow and 104 sorted cells were cultured in serum-free A mitotic clock in stem cells?
culture medium supplemented with IL-6, IL-3, steel factor, and erythropoietin as
described.39 At the indicated time interval, the total number of nucleated cells (light
blue squares) and CD34⫹ cells (dark blue squares) present in the cultures was
Vaziri asked me if I had looked at telomere length as a variable that
calculated from cell counts and the percentage of viable CD34⫹ cells measured by could be related to the developmental changes in stem-cell function
flow cytometry. All CD34⫹ cells from bone marrow cultures and fractions of the we observed. He showed me data on telomere length in human
CD34⫹ cells from cord blood and fetal liver cultures were sorted and used for lymphocytes that he had just published together with Cal Harley
continuation of the cultures. (B) Loss of telomeric DNA in human hematopoietic cells
upon proliferation in vivo and in vitro (reproduced from Vaziri et al43 with permission and Richard Allsopp (Vaziri et al45). Looking at the smears of
from the National Academy of Sciences of the United States). DNA samples from total terminal restriction fragments (TRFs) obtained using Southern
nucleated cells from 2 different donors for each tissue before and after culture at analysis, I remember being distinctly unimpressed. Nevertheless,
increasing time points were subjected to terminal restriction fragment (TRF) size
Southern blot analysis as described elsewhere.43 Cultures were initiated with highly
the idea that fetal and adult stem cells could have differences in
enriched stem cells as described in panel A. (C) Quantitative analysis of the TRF data DNA that could be measured intrigued me and we decided to
shown in panel B. The first time point (blue square with circle) represents the TRF collaborate. One problem was that Southern analysis requires a lot
value in nucleated cells from each tissue before purification. The mean and standard
of DNA (extracted from a million cells or more) and we could not
error of 2 independent TRF measurements (different gels) for each DNA sample
purified from total nucleated cells at each time point are shown. The total number of purify that many human candidate stem cells. Instead, we decided
cells was used to calculate population doublings. The loss of telomeric DNA in these to analyze the telomere length in the millions of cells that were
cultures varied between 19 and 54 base pairs per population doubling. For details see readily produced in our serum-free cultures initiated with a few
Vaziri et al.43
thousand purified candidate stem cells. The results of the blinded
TRF analysis were shocking to me: the average telomere length in
free long-term cultures was the result of sequential recruitment of the cultured cells followed a very clear pattern: fetal liver longer
CD34⫹ cells that were initially quiescent.39 Frustrated by our than cord blood, and cord blood longer than adult bone marrow
inability to obtain any meaningful numeric expansion in culture (Figure 1B). Furthermore, the telomere length showed a steady and
with purified candidate stem cells from adult human bone marrow, progressive decline in culture (Figure 1C), in line with the idea that
we decided to explore the behavior of cells with the same every cell division was accompanied by a loss of measurable
phenotype purified from human cord blood and fetal liver. To our number, estimated at 30 to 100 base pairs, of telomere repeats.43 To
great surprise, the ability to produce CD34⫹ cells was hundreds to convince ourselves that our observations were not related to
thousands of times better in cultures initiated with purified cells suboptimal culture conditions, we put our magnetic cell separation
from cord blood and fetal liver (Figure 1A).42 Could the prolifera- technique to work and we purified several million human CD34⫹
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BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 TELOMERES, STEM CELLS AND HEMATOLOGY 1763

Figure 2. Fluorescence in situ hybridization to

detect telomere repeats. Quantitative in situ hybridiza-
tion (Q-FISH) using Cy3-labeled (CCCTAA)3 peptide
nucleic acid probes on metaphase chromosomes from
a human lymphocyte (A) and an embryonic fibroblast
from a late-generation telomerase RNA KO mouse (B).
Note that the fluorescence intensity is very similar for
both sister chromatids at individual chromosome ends
but very heterogeneous between ends. The arrows in
panel B point to pseudo metacentric chromosomes
resulting from the fusion of 2 acrocentric mouse chromo-
somes. Note the lack of telomere repeats at the
junction of the 2 fused chromosomes.

CD38⫺ cells directly from more than 2 ⫻ 1010 organ donor bone trials and errors, I finally managed to obtain reproducible results
marrow cells. The telomere length in those purified cells was longer with human chromosome preparations. The key innovations in the
than in CD34⫹ CD38⫹ cells but shorter than in the differentiated technique that is now known as quantitative FISH (Q-FISH)53 were
cells from fetal liver.43 Taken together, these results supported the the use of high concentrations (70%) of formamide in the
idea that cell divisions are indeed “counted” in hematopoietic stem hybridization as well as the wash solutions, the use of low ionic
cells by progressive telomere loss. The immediate implication of strength to enable quantitative hybridization, and the inclusion of
this conclusion was that the self-renewal and proliferation of stem blocking protein to prevent nonspecific binding of the directly
cells could eventually be limited by progressive telomere loss. It labeled, hydrophobic PNA probes specific for (TTAGGG)n repeats.
was time for me to learn more about telomeres.

Telomere length analysis by FISH? Chromosome-specific differences in telomere

length
In 1994, the telomere field was very small and spearheaded by a
number of exceptional scientists including Elizabeth Blackburn, Q-FISH analysis of metaphase chromosomes from normal human
Ginger Zakian, Titia de Lange, and Carol Greider. In 1985, Carol cells revealed that every chromosome end carries a detectable
Greider and Liz Blackburn had published their pioneering work on number of telomere repeats and that the length of telomere repeats
telomerase in the unicellular organism Tetrahymena,46 and a year at individual chromosome ends is strikingly heterogeneous (Figure
later Howard Cooke, based on his finding that sperm DNA had 2A). Immediate questions that arose were the following: How does
longer telomeres than nucleated blood cells, suggested that telomer- the variation at individual ends relate to the progressive loss of
ase levels in stem cells could be insufficient to maintain telomere telomere repeats in cells with each round of cell division? Are
length (Cooke and Smith47). Further support for this idea was length differences chromosome specific? Could the presence of
obtained a few years later,48,49 and subsequent studies showed that long telomeres support repair of short telomeres in the absence of
the telomere length in fibroblasts predicted their replicative life telomerase? Does telomerase play a role in generating telomere
span50 and that telomere shortening in immortal tumor cells is length diversity? To start addressing some of these questions, I was
prevented because they express high levels of telomerase.51 For keen to apply Q-FISH to cells from telomerase RNA (Terc)
these studies, telomere length was invariably measured using TRF knockout (KO) mice that were rumored to have no phenotype
length analysis by Southern analysis. My first venture into the whatsoever. When we obtained cells from such mice, we found that
telomere field, after collaborating with Houmayoun Vaziri, was a telomeres decreased in length by around 5 kb with each subsequent
quest to develop novel tools to measure telomere length at generation, and we readily observed chromosome fusions indica-
individual telomeres and in individual cells. This quest was driven tive of loss of telomere function in generation 6 (Figure 2B), the
by necessity. If telomeres were indeed important parameters in the last (infertile) generation.54 Because this telomere length pheno-
biology of stem cells (I was now convinced of this), then better type had been completely missed by conventional Southern
tools to study telomere length would be required. I was intrigued by analysis, the general interest in our Q-FISH technique greatly
the possibility of using fluorescence in situ hybridization (FISH) increased.
for such measurements, although I had no knowledge of FISH The telomere knockout mouse has been instrumental in elucidat-
techniques. This was an advantage as several FISH experts whom I ing the critical role of telomerase in telomere maintenance in
consulted invariably told me that to measure repetitive DNA with a mammals and has highlighted the critical role for telomeres in
length of less than 10 kb in a quantitative way using FISH was maintaining genome stability. The findings in this mouse model
impossible. However, encouraged by Ger van den Engh and argue against a role of telomeres as a developmental clock:
Michael Egholm, I was hoping that advances could be made using subsequent generations of mice with progressively shorter telo-
newly discovered peptide nucleic acid (PNA) probes,52 and meres develop normally. However, the mouse model has proven to
I decided to give PNA a try during a sabbatical stay in the be somewhat misleading for human disease in that the conse-
Netherlands with easy access to the outstanding “FISH” laboratory quences of partial telomerase deficiency in humans appear to be
of Hans Tanke and Ton Raap at Leiden University. After many much more severe than that of complete lack of telomerase in mice.
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1764 LANSDORP BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4

Figure 3. Telomere length measurements using flow FISH. (A) Nonlinear decline in telomere length with age. The average telomere length in granulocytes and lymphocytes
from 392 healthy donors was calculated from the median telomere fluorescence and median autofluorescence relative to internal control cells (bovine thymocytes).61 Note that
the telomere length at any given age is highly variable, that the most rapid drop in telomere length occurs early in life, and that the rate of telomere attrition in lymphocytes
exceeds that in granulocytes. (B) Longitudinal studies of telomere length in newborn baboons point to a high turnover of hematopoietic stem cells in the first year of life.62 Note
that the 2 animals differ markedly in average telomere length and that the rate of telomere attrition drops markedly after approximately 1 to 2 years in both animals. In humans,
with a longer lifespan, this drop is expected to occur before the fourth year of life in line with previous cross-sectional observations.60 (C,D) Telomere length in individuals with
mutations in telomere genes. The telomere length in cells from healthy individuals (A) was used to plot the telomere length distribution in the normal population using a best-fit
approach (red, green, and blue curves representing expected telomere length for the indicated proportion of healthy individuals). The telomere length in lymphocytes (C) and
granulocytes (D) from patients with known mutations in telomerase genes measured in the context of several studies12-14,16,63 are shown. Each symbol represents an individual
patient diagnosed with clinical symptoms associated with a mutation in dyskerin (DKC1, black circles), hTERT (red circles), and hTERC (black squares). Some individuals are
carriers of a TERC mutation but have no clinical symptoms (blue square). The majority of individuals that carry mutations in telomerase genes display critically short telomeres,
nearly all of them below the tenth percentile of the normal distribution and a majority of these below the first percentile (typically for both cell subsets shown). Note that
individuals with early onset of disease (in the first 3 decades of life) show the most striking difference between observed and expected telomere length.

Most likely, the repair of critically short telomeres by homologous chromosomes that cannot always be obtained. So I was keen to
recombination in the absence of telomerase is more efficient when explore alternative ways to measure telomere length using our
the average telomere length is long. Murine telomeres are on PNA probes. In 1997, we started to explore in situ hybridization
average approximately 10-fold longer than in humans.11 in suspension followed by measurements of specific fluores-
Q-FISH was then used to study the length of telomere repeats cence of individual cells using flow cytometry. Major challenges
on specific human chromosomes.55 The striking heterogeneity in using this approach were keeping cells in suspension following
telomere length was not resolved by studies of specific chromo- denaturation of DNA required for hybridization and develop-
some ends. Interestingly, the average telomere length on chromo- ment of reproducible measurements while correcting for vari-
some 17p was found to be relatively short in all individuals tested, able autofluorescence. Natalie Rufer and Tim Bruemmendorf in
suggesting that this telomere will be one of the first to become my laboratory worked to develop the initial technique,59,60
uncapped upon progressive telomere shortening with proliferation which was further improved by Gabriela Baerlocher et al.61 Key
and/or age in human cells. The rate of telomere attrition at the advances were the inclusion of bovine thymocytes in every tube
inactive X chromosomes in female cells was found to exceed that as an internal reference and the use of a robotic device to
of the active X chromosome,56 indicating that differences in increase the efficiency and reproducibility of the many wash
average length of specific chromosomes could be generated during steps in the protocol. Flow FISH has revealed that the decrease
proliferation and age. Differences in chromosome-specific telo- in telomere length with age is quite different for circulating
mere length were found to be partly heritable55,57 and partly granulocytes and lymphocytes and that, at any given age,
originating stochastically in the germ line.58 Such differences could telomere length shows a marked variation among individuals
reflect differences in (sub)telomeric chromatin, repair by telomer- (Figure 3A). In collaboration with Neal Young, Rodrigo Calado,
ase or recombination, timing of replication, or combinations of Hinh Ly, Blanche Alter, Sharon Savage, Mary Armanios, and
these factors. others, we found that the “flow FISH” technique is very useful to
screen for possible involvement of telomere defects in various
disorders. Such disorders include diseases resulting from muta-
Telomere length measurements by flow FISH tions in telomerase genes such as dyskeratosis congenita,12,63
aplastic anemia,64,65 and pulmonary fibrosis.14,15 In view of the
highly significant decline in telomere length with age as well as
Q-FISH has been instrumental in the “telomere field” but the the variation in average telomere length between individuals of
technique is very time-consuming and requires metaphase the same age (Figure 3), it seems likely that these disorders
 From bloodjournal.hematologylibrary.org by guest on January 13, 2014. For personal use only.

BLOOD, 15 FEBRUARY 2008 䡠 VOLUME 111, NUMBER 4 TELOMERES, STEM CELLS AND HEMATOLOGY 1765

represent only the tip of an iceberg and that other diseases with a Roos, Ann Rose, Sharon Savage, Hergen Spits, Guy Sauvageau,
“telomere component” will be discovered. John Schrader, Sara Selig, Salvatore Siena, Heather Sutherland,
I have come to the end of my story on telomeres, stem cells, and Hans Tanke, Leon Terstappen, Barb Trask, Houmayoun Vaziri,
hematology. A recurrent theme in my work has been that advances Neal Young, and Wim Zeijlemakers. I also want to thank the
were enabled by the development and application of new tech- following current and former technicians and trainees in my
niques to address questions raised by unexpected observations. laboratory for their contributions: Geraldine Aubert, Gabriela
This is also how we more recently became interested in the genetic Baerlocher, Agnes Baross, Tim Bruemmendorf, Liz Chavez, Iris
factors that regulate the stability of G-rich DNA66 and telomere Cheung, Wieslawa Dragowska, Ester Falconer, Maloy Ghosh, Matt
length.67 The 2 closely related helicase genes identified in these Greenwood, Prakash Hande, Mark Hills, Uwe Martens, Hector
studies are homologs of the human Fanconi J gene, and their Mayani, Vivienne Rebel, Alex Roth, Nathalie Rufer, Mike Schertzer,
precise role remains of great current interest. However, my latest Terry Thomas, Bert Wognum, Evert-Jan Uringa, Irma Vulto, and
“rocket launch” includes studies related to the possibility that sister Mark Zijlmans. Janis Abkowitz, Gerry Krystal, Olga Lansdorp, and
chromatids differ in epigenetic marks at genes that regulate Claudia Bos are thanked for critically reading drafts of the paper.
self-renewal and differentiation.68 As before, these studies require I apologize to all people whose work or contribution I forgot
development of novel tools. I am very excited about this ongoing to mention.
work and I feel extremely privileged to once again be in a position Work in my laboratory is supported by grants from the National
to experience the excitement that comes with exploring uncharted Institutes of Health (AI29524), the Canadian Institutes of Health
territory. Research (MOP38075 and GMH79042), and the National Cancer
Institute of Canada (with support from the Terry Fox Run).

Acknowledgments
I specifically want to thank Rob Aalberse, Lucien Aarden, Janis Authorship
Abkowitz, Blanche Alter, Sam Aparicio, Mary Armenios, Duncan
Baird, Michael Barnett, Graham Betton, Rodrigo Calado, Chris Conflict-of-interest disclosure: The author declares a financial
Counter, Jeff Davis, John Dick, Roeland Dirks, Bob Donahue, interest in Repeat Diagnostic, a company specializing in leukocyte
Allen Eaves, Connie Eaves, Ger van den Engh, Fred Goldman, telomere length measurements using flow FISH.
Carol Greider, Cal Harley, Lea Harrington, Phil Hieter, Richard Correspondence: Peter M. Lansdorp, Terry Fox Laboratory, BC
Hodes, Keith Humphries, Al Klingelhutz, Gerry Krystal, Andras Cancer Research Centre, 675 W 10th Ave, Vancouver, BC V5Z
Nagy, Hihn Ly, Gordon Phillips, Steven Poon, Ton Raap, Dirk 1L3; e-mail: plansdor@bccrc.ca.

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Dr Peter Lansdorp received his MD in 1976 from the Erasmus University in Rotterdam, the Netherlands. He
obtained a PhD in Immunology and Experimental Hematology in 1985 from the University of Amsterdam.
During his graduate studies he started making monoclonal antibodies and became increasingly interested in
growth factors such as IL-6 and hematopoietic stem cells. In 1985 he moved to the Terry Fox Laboratory at the
BC Cancer Agency in Vancouver, where his work on the purification and culture of human and murine hemato-
poietic stem cells led him to studies of telomere biology. Dr Lansdorp currently is a senior scientist at the
Terry Fox Laboratory and a Professor in the Division of Hematology at the Department of Medicine of the
University of British Columbia.