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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3255-3262
Telomerase Activity in Candidate Stem Cells From Fetal Liver and
Adult Bone Marrow
By
Jane Yui,
Choy-Pik Chiu, and
Peter M. Lansdorp
From the Terry Fox Laboratory, British Columbia Cancer Agency; the
Department of Medicine, University of British Columbia, Vancouver,
British Columbia, Canada; and the Geron Corporation, Menlo Park, CA.
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ABSTRACT |
Telomerase is a ribonucleoprotein polymerase that synthesizes
telomeric repeats onto the 3 ends of eukaryotic chromosomes. Activation of telomerase may prevent telomeric shortening and correlates with cell immortality in the germline and certain tumor cells. Candidate hematopoietic stem cells (HSC) from adult bone marrow
express low levels of telomerase, which is upregulated with
proliferation and/or differentiation. To address this issue, we
stimulated purified candidate HSC from human adult bone marrow with
stem cell factor (SCF), interleukin-3 (IL-3), and Flt3-ligand (FL). After 5 days in culture, activity was detected in total cell extracts from IL-3-, SCF + FL-, SCF + IL-3-, FL + IL-3-, and SCF + IL-3 + FL-stimulated cultures, but not from
cells cultured in SCF or FL alone. Within the CD34+
fraction of the cultured cells, significant activity was found in the
CD34+CD71+ fraction. In addition, PKH26
staining confirmed that detectable telomerase activity was present in
dividing PKH26lo cells, whereas nondividing
PKH26hi cells were telomerase negative. Because in these
experiments no distinction could be made between cycling
"candidate" stem cells that had retained or had lost self-renewal
properties, fetal liver cells with a
CD34+CD38 phenotype, highly enriched for
cycling stem cells, were also examined and found to express readily
detectable levels of telomerase activity. Given the
replication-dependent loss of telomeric DNA in hematopoietic cells,
these observations suggest that the observed telomerase activity in
candidate stem cells is either expressed in a minor subset of stem
cells or, more likely, is not sufficient to prevent telomere
shortening.
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INTRODUCTION |
THE ENDS OF all eukaryotic chromosomes
are organized into telomeres that consist of tandem arrays of G-rich
repeats and associated proteins. Telomeres protect chromosomes from
degradation, prevent end-to-end fusions, and position chromosomes
within the nucleus.1,2 In primary somatic cells such as
fibroblasts,3,4 lymphocytes,5 or hematopoietic
progenitors,6 telomeric DNA is gradually lost with each
cell division presumably because conventional DNA polymerases cannot
fully replicate the ends of linear chromosomes.7,8 Progressive shortening of chromosome ends has been suggested to act as
a mitotic clock that may contribute to cellular senescence and eventual
cell mortality of normal mammalian somatic cells.9
On the other hand, cells of the germ line, such as sperm cells, have
long telomeres of 10 to 20 kb that do not appear to shorten with aging
of the organism.4 Such long telomeric ends are assumed to
be maintained by telomerase, a ribonucleoprotein whose primary function
is to synthesize telomeric DNA, thus counteracting losses at the
chromosome termini with each round of replication. The telomerase RNA
component contains a species-specific template for synthesis of
telomeric repeats, and such RNA sequences have been cloned from
ciliates,10 yeast,11,12 mouse,13
and human.14 Recently, the gene encoding the reverse
transcriptase catalytic subunit of telomerase from several species
including humans has also been cloned.15,16 In humans,
telomerase activity is readily detectable in testes and ovaries, but
not in most somatic tissues.17,18 Telomerase activity is
also elevated in carcinomas of the ovary,19 breast,20 liver,21 lung,22
prostate,23 and in neuroblastoma24 and
hematological malignancies.25-27 In addition, the majority of immortal cell lines expresses telomerase, whereas most mortal cells
lack this activity.17 Together, these data strongly suggest that telomerase may be involved in malignant transformation and cellular immortality.
The hematopoietic system replenishes the loss of mature blood cells via
the recruitment of stem cells that have been defined as pluripotential
cells with self-renewal properties. We and others have shown low levels
of telomerase activity in normal bone marrow and peripheral blood
cells, both in progenitors of the myeloid and lymphoid lineages as well
as in terminally differentiated cells such as T and B
cells.26-28 Furthermore, we and others found that
"candidate" hematopoietic stem cells (HSC) upregulate telomerase activity upon stimulation in vitro.28,29 However, as the
vast majority of stem cells are quiescent during steady-state
hematopoiesis,30 the status of telomerase expression in
cycling HSC has not yet been elucidated. This is an important issue
because the self-renewal and replicative potential of the most
primitive hematopoietic cells may depend on telomerase to maintain
stable telomeres.31 In this report, we describe results of
experiments designed to address the question of telomerase expression
in cycling stem cells. For this purpose, we measured telomerase
activity in extracts from purified candidate HSC from human adult bone
marrow stimulated with different cytokines and in extracts from
CD34+CD38 candidate HSC purified from human
fetal liver. Our data indicated that telomerase activity is expressed
in most if not all cycling stem cells but is repressed in quiescent
stem cells.
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MATERIALS AND METHODS |
Purification of HSC from adult bone marrow and fetal liver.
Candidate HSC with the phenotype
CD34+CD45RAloCD71lo were obtained
from previously frozen cadaver marrow as previously
described.32 Briefly, mononuclear cells retrieved from the
interface after density separation were stained with 8G12-Cy5
(anti-CD34), 8d2-PE (anti-CD45RA), and OKT9-FITC (anti-CD71) for 30 minutes at 4°C. Cells were washed twice in Hanks' buffered saline
with 0.2% BSA (HB) and stained with 2 µg/mL of propidium iodide (PI)
before suspending in HB at a density of 5 × 106 cells/mL
for sorting. Cells were sorted on a FACStarplus (Becton
Dickinson, San Jose, CA) equipped with argon (488 nm) and helium-neon
(633 nm) lasers.
CD34+CD45RAloCD71loCD38(CD34+CD38)
cells were obtained from adult marrow and fetal liver in the 17th and
18th week of gestation according to previously described
protocols.33,34 Cells were stained with 8G12-Cy5, 8d2-FITC,
OKT9-FITC, and anti-CD38-phycoerythrin (PE; Becton Dickinson) for 30 minutes at 4°C. The washing and sorting procedures were performed as
above.
Cell culture.
Sorted candidate HSC were cultured in serum-free medium consisting of
Iscove's modified Dulbecco's medium (IMDM) supplemented with the
following reagents: bovine serum albumin (BSA) at 2%, sodium
bicarbonate at 0.1%, transferrin at 200 µg/mL, insulin at 10 µg/mL, 2-mercaptoethanol at 105 mol/L, low-density
lipoprotein (Sigma, St Louis, MO) at 40 µg/mL, and
penicillin-streptomycin at 100 U and 50 µg/mL,
respectively.32 Both stem cell factor (SCF) and Flt3-ligand
(FL) were used at a final concentration of 50 ng/mL, whereas
interleukin-3 (IL-3) was used at 20 ng/mL. All growth factors were
purchased from Pepro-tech (Rocky Hill, NJ).
Telomerase repeat amplification protocol (TRAP) assay.
Telomerase activity was measured by TRAP assay using an end-labeled
telomerase substrate (TS) primer as described.17,27,28 Briefly, cell extracts were prepared by lysing the cells in
CHAPS extraction buffer17 at a concentration
of 500 cells per µL of buffer, centrifuged at 1200g at 4°C,
and 2 µL of these extracts were used in the assay.
The telomerase reaction was performed in 50 µL of TRAP reaction
buffer containing 20 mmol/L tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 63 mmol/L KCl, 0.005% Tween-20, 1 mmol/L EGTA, 50 µmol/L deoxynucleotide triphosphates (Pharmacia, Uppsala,
Sweden), 0.1 µg each of the labeled TS primer, ACX
primer, U2 primer, 5 × 103 attamoles (amols) of an
internal control primer (TSU2), 2 U of Taq DNA polymerase (Boehringer
Mannheim, Laval, Quebec), and 2 µL of CHAPS extract. The primers for
this reaction are obtainable through Oncor (Gaithersburg, MD). Reaction
tubes were placed in a robocycler (Stratagene, La Jolla, CA) for 30 minutes at 30°C, followed by 27 cycles of polymerase chain reaction
(PCR) at 94°C for 30 seconds and 72°C for 30 seconds. One half of
the amplified products were resolved on a 12% polyacrylamide gel,
dried, and visualized by autoradiography using BioMax films (Kodak,
Rochester, NY) after 48 hours of exposure at room temperature. In some
cases, RNase A (Boehringer Mannheim) at 6 µg/mL was added during the telomerase reaction to confirm the specificity of the telomerase products that disappeared in the presence of RNase A.
For semiquantitative analysis of telomerase activity, the radioactive
bands were scanned by densitometer and determined using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). The signal from
individual test extract was normalized for the PCR efficiency and
compared with that generated by 1 amol of an oligonucleotide M2R8,
which contains the same sequence as the TS primer plus eight T2AG3 repeats using the following
formula:
where
LB is the blank control containing 2 µL of CHAPS lysis buffer
in lieu of cell extract.
Tracking proliferation by PKH26 staining.
The proliferative history of cells was followed using PKH26 labeling
and analysis as described.30 Sorted candidate HSC were washed once in Hanks' buffered saline without BSA. The cell pellet was
resuspended in 150 µL of dilutent C (Zynaxis Cell Science, Malvern,
PA), mixed with an equal volume of PKH26-GL (2 × 106
mol/L), and incubated for 5 minutes at room temperature. The reaction
was terminated by the addition of an equal volume of 10% BSA in
serum-free medium. Cells were washed twice and an aliquot was taken for
analysis of PKH26 staining intensity on day 0 of culture. PKH26 was
excited at 488 nm and the emission was measured with a 575/26 filter.
The remainder of the cells was put in culture under serum-free
conditions supplemented with SCF, IL-3, and FL for 8 days. After 8 days
of culture, cells were reanalyzed and sorted for PKHhi and
PKHlo cells using FACStarplus.
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RESULTS |
Telomerase activity in candidate stem cells from adult bone marrow.
Cell extracts from adult marrow candidate HSC with the phenotype
CD34+CD45RAloCD71lo were assayed
for telomerase activity by a modified version of the PCR-based TRAP
assay. In addition to the typical ladder of 6 bp repeats that
correspond to the amplified product of the TS primer, an internal
control (TSU2) is coamplified that yields a single lower band of 35 bp
(Fig 1). By normalizing the signal intensity of the telomerase ladder to that of the internal control, sample to sample variation due to PCR amplification efficiency was
minimized, thus allowing for semiquantitative analysis. Treatment with
RNase obliterated the 6-bp ladder, indicating that telomerase was
responsible for this reaction (Fig 1). This modified TRAP assay allowed
us to detect telomerase activity in cell extracts obtained from 10 to
100 cell equivalent of 293 cells, a telomerase-positive immortalized
kidney cell line.
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| Fig 1.
Telomerase activity in candidate HSC before culture. TRAP
products were generated from 2 µL of CHAPS extract (1,000 cell
equivalents) in the presence (+) or absence ( ) of RNase A. TRAP
products generated from sorted cells on day 0 from 4 different
cadaveric marrow (lanes 1 to 8); lane 9, no extract; lane 10, 1 amol
M2R8 standard. Arrow indicates the position of the 35-bp amplified
internal control.
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Low levels of telomerase activity were detected in candidate HSC from
four different marrow samples (Fig 1). The telomerase levels in the
purified candidate HSC varied from 3% to 20% of that generated by 1 amol of M2R8, an oligonucleotide with 8 T2AG3 repeats used as a quantitation standard, which translated to an equivalent of 0.06% to 0.4% of the activity of 293 cells when normalized on a per-cell basis.
Telomerase activity in candidate HSC after culture in cytokine
combinations of SCF, FL, and IL-3.
Because telomerase activity was reported to increase upon cellular
activation,29,35-37 candidate HSC were cultured in the presence of SCF, IL-3, and FL to examine potential upregulation of
telomerase activity. When the purified cells were cultured in SCF or FL
alone for 5 days, no upregulation of telomerase activity was observed,
whereas IL-3 by itself enhanced telomerase activity (Fig
2). Among combinations of two cytokines,
those containing IL-3 (SCF + IL-3 or FL + IL-3) were more effective
in upregulating telomerase activity than the combination SCF + FL,
giving rise to a twofold increase in telomerase activity
(Fig 2). Total cell extracts derived from candidate HSC cultured in the
presence of SCF + IL-3 + FL also showed enhanced telomerase activity
(Fig 2). The relatively low levels of telomerase activity did not
appear to result from inhibitory substances to the PCR reaction because the internal control was amplified as expected (Fig 2). Furthermore, mixing extracts from hematopoietic cells with those from 293 cells did
not result in any significant decrease in 293 telomerase activity, further indicating that inhibitors of telomerase were unlikely to be
present (data not shown).
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| Fig 2.
Telomerase activity in candidate HSC after 5 days of
culture. TRAP products using CHAPS extracts of total viable cells
derived by culturing purified candidate HSC from BM4 for 5 days in SCF (lanes 1 and 2), FL (lanes 3 and 4), IL-3 (lanes 5 and 6), SCF + FL (lanes 7 and 8), SCF + IL-3 (lanes 9 and 10), FL + IL-3
(lanes 11 and 12), and SCF + FL +
IL-3 (lanes 13 and 14). TRAP products were resolved on a 15%
polyacrylamide gel, dried, and exposed to film for 48 hours. The
intensity of the signals was analyzed by a densitometer using ImageQuant program and normalized to that of the internal control. Cellular extracts from BM1 and BM3 yielded similar results.
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Telomerase activity in cultured candidate HSC is restricted to
cycling cells.
Because telomerase activity was upregulated in cultures containing
IL-3, SCF + IL-3, FL + IL-3, and SCF + FL + IL-3, we next investigated
whether increased levels of telomerase activity were restricted to
cells of a particular phenotype. Flow cytometric analysis of purified
candidate HSC after 5 days of culture in the seven different cytokine
combinations revealed that in cultures with SCF or FL alone, the
majority of cells retained the
CD34+CD45RAloCD71lo phenotype (Fig
3). On the other hand, in cultures
containing IL-3, the percentage of
CD34+CD45RAloCD71lo cells decreased
to below 50% of total viable cells (Table
1), and increased numbers of
CD34,
CD34+CD45RAloCD71hi, and
CD34+CD45RAhiCD71hi cells were
observed (Fig 3, Table 1). Candidate HSC cultured for 5 days in SCF + FL + IL-3 were sorted into CD34, CD34+,
CD34+CD45RAloCD71lo,
CD34+CD45RAloCD71hi, and
CD34+CD45RAhiCD71hi cells.
Semiquantitative analysis of telomerase activity indicated that
CD34+ cells expressed fourfold higher levels than those in
the CD34 cells (Fig 4).Among the CD34+ cells, most telomerase activity resided in
the CD34+CD45RAloCD71hi and
CD34+CD45RAhiCD71hi
populations, whereas
CD34+CD45RAloCD71lo cells had
negligible telomerase activity (Fig 4).
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| Fig 3.
Phenotypic analysis of candidate HSC after culture.
Candidate HSC were cultured for 5 days in (A) SCF, (B) FL,
(C) IL-3, (D) SCF + FL, (E) SCF + IL-3, (F) FL + IL-3,
and (G) SCF + FL + IL-3. After 5 days, cells were
stained with antibodies against CD34, CD45RA, and CD71. Profiles
shown were from events gated for low PI, low SCC, and high
expression of CD34. In (G) boxes I, II, and III represent the windows
used to sort for
CD34+CD45RAloCD71lo cells,
CD34+CD45RAloCD71hi cells, and
CD34+CD45RAhiCD71hi cells,
respectively. The same windows were applied to Fig 4 and Table 1.
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| Fig 4.
Telomerase activity in subpopulations of cells present
after 5 days in cultures of purified candidate HSC from adult marrow. See also Fig 3G. (A) CHAPS extracts from BM4-derived cells (lanes 1 to
10): CD34 fraction (lanes 1 and 2), CD34+
fraction (lanes 3 and 4),
CD34+CD45RAloCD71lo fraction
(lanes 5 and 6),
CD34+CD45RAloCD71hi fraction
(lanes 7 and 8), and
CD34+CD45RAhiCD71hi cells and
(lanes 9 and 10); CHAPS extracts from BM1-derived cells (lanes 11 to
14): CD34 fraction (lanes 11 and 12) and
CD34+ fraction (lanes 13 and 14). All extracts were
generated from 1,000 cells.
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To further investigate the relationship between telomerase activity and
cell proliferation in hematopoietic progenitors, we used PKH26 to track
cell divisions at the level of single cells. Sorted candidate HSC were
labeled with PKH26, a fluorescent dye that stably incorporates into the
lipid bilayer and is diluted among the daughter cells with each
successive cell division. After 8 days of culture in SCF + FL + IL-3,
cells were sorted into PKHhi and PKHlo
fractions (Fig 5A). Telomerase activity in
PKHlo cells was 5 to 10 times higher than the ones in
PKHhi cells (Fig 5B), indicating that viable cells that
remained quiescent in cytokine-stimulated cultures did not express
detectable levels of telomerase.
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| Fig 5.
PKH26 staining of purified candidate HSC before and after
culture in SCF + IL-3 + FL. (A) PKH26 was incorporated into the lipid bilayer of purified candidate HSC at day 0. On day 8, PKH26 intensity was analyzed again and cells were sorted into
PKHlo (gate1) and PKHhigh fractions (gate2) for
TRAP. The cells in the PKHlo fraction have undergone
several rounds of division, resulting in diminished dye fluorescence.
(B) Telomerase activity in PKHhi and PKHlo
cells. PKHhi and PKHlo cells were sorted from
BM1 after 8 days of culturing SCC in SCF + FL + IL-3.
TRAP assay was performed on CHAPS extracts equivalent to 1,000 cells in
the absence () and presence (+) of RNase
A.
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Telomerase activity in fetal liver
CD34+CD38 cells.
Although telomerase activity was upregulated in 5-day cultures of adult
marrow candidate HSC stimulated by SCF, IL-3, and FL, the activity was
found to reside predominantly in the proliferating cells containing
mainly committed progenitors. To address the question of whether
cycling stem cells express telomerase activity, we examined telomerase
expression in CD34+CD38 candidate HSC from
fetal liver. Higher telomerase activity was detected in the fetal liver
CD34+CD38 cells than those from adult bone
marrow (Fig 6). Pooled data from three
different samples showed that the activity in fetal liver
CD34+CD38 cells ranged from 0.5% to 1.5%
of that found in 293 cells, whereas in adult bone marrow the activity
was at most 0.3% of that present in 293 cells. These results suggest
that either all or a subfraction of cycling fetal liver stem cells
express telomerase activity.
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| Fig 6.
Comparison of telomerase activity in freshly isolated
CD34+CD38 cells from fetal liver and adult
marrow. TRAP assay performed on CHAPS extracts equivalent to 1,000 cells in the absence () and presence (+) of RNase A. CD34+CD38 cells from adult marrow (lanes 1 and 2) and fetal liver (lanes 3 and 4). Lysis buffer (LB) served as the
negative control (lane 5), whereas 293 cells at the equivalent of 20 cells (lane 6) and 10 cells (lane 7) served as the positive controls.
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DISCUSSION |
The ability to induce or enhance telomerase activity may be important
in maintaining the replicative potential of normal stem cells found in
self-regenerating tissues such as those of the hematopoietic
system.28,29 The studies reported here were aimed to
investigate telomerase expression in the most primitive hematopoietic cells in humans. We confirmed our previous results regarding the low
level of telomerase activity in freshly isolated "candidate" HSC
with the CD34+CD45RAloCD71lo
phenotype from adult marrow.28 These sorted cells are
highly enriched (several hundred-fold when compared with unpurified
cells) in long-term culture-initiating cells (LTC-IC), arguably the
best in vitro assay for human stem cells.38,39
Unfortunately, most bone marrow cells with a
CD34+CD45RAloCD71lo phenotype are
unable to initiate long-term cultures, and rare committed progenitors
from the adult bone marrow also share this phenotype.32 The
latter, which are actively proliferating, could contribute partially or
completely to the low but readily detectable levels of telomerase in
purified candidate HSC before culture.
In the present study, we found that the level of telomerase activity in
CD34+CD45RAloCD71lo cells sorted
from cytokine-stimulated cultures was reduced as compared with that in
freshly isolated cells with the same phenotype. On the other hand,
telomerase activity was increased in cultured candidate HSC
concomitantly with the upregulation of CD45RA and CD71
expression. By sorting various subpopulations after
stimulation with SCF, FL, and IL-3 for 5 days, we found that
telomerase activity was mainly confined to
CD34+CD45RAloCD71hi and
CD34+CD45RAhiCD71hi cells, which
are known to be enriched in cycling progenitors committed to
differentiate into the erythroid and myeloid lineages, respectively.32 In addition, tracking cellular division by
PKH fluorescence confirmed that telomerase activity was confined to cells that had proliferated in culture. Cells with a
CD34+CD45RAloCD71lo phenotype
present after 5 days in cytokine culture could represent a population
that failed to respond to SCF, FL, and IL-3 stimulation and remained
quiescent or exited from the cell cycle. In both cases, telomerase
activity is expected to be low in view of the data describing
upregulation of telomerase activity upon entry into the cell
cycle.35-37,40
Because cycling stem cells are extremely rare in the adult bone
marrow,30,33 and because our culture conditions are unable to induce the selective self-renewal of adult bone marrow candidate stem cells, we next examined telomerase expression in
CD34+CD38 cells from fetal liver. It has
been shown that the proliferative potential of hematopoietic cells
changes during ontogeny and that candidate HSC from fetal liver contain
a very high proportion of cycling cells as compared with candidate HSC
from bone marrow.33 Our finding that
CD34+CD38 fetal liver cells express readily
detectable levels of telomerase activity strongly suggests that such
cycling candidate HSC express telomerase activity. In view of the loss
of telomerase DNA in hematopoietic cells with age,6 this
observation can be explained by assuming that the measurable telomerase
activity is not preventing the overall telomere shortening in candidate
HSC.31 Alternatively, telomerase could be expressed in only
a proportion of the cells. If the latter hypothesis is correct, then
identification and selective expansion of such telomerase positive
clones could be useful in transplantation and gene transfer protocols
because their progeny would possibly maintain a high proliferative
potential. To address this issue, more information about telomerase
expression in single CD34+CD38 cells from
fetal liver and the in vivo of the telomerase levels detected by
telomerase assays is urgently needed. Antibodies to the telomerase
reverse transcriptase protein15,16 could possibly be used
to address this issue.
In our study we found that in day-5 cultures, CD34+ cells
expressed higher telomerase activity than CD34 cells.
One possible explanation for this observation is that as
CD34+ differentiate, their telomerase activity is
downregulated together with their proliferative potential. Similarly,
freshly isolated CD34 cells from adult marrow also have
lower telomerase activity compared with
CD34+71+ cells, and when the latter were placed
in culture over a period of 10 days, their telomerase activity
declined.28 One recent report also shows that leukemic cell
lines lose telomerase activity when induced to
differentiate.40,41
In a couple of transgenic mouse models, the reactivation of telomerase
activity was correlated with tumorigenesis.42,43 However,
the role of telomerase in normal somatic cells is still largely
unknown. Telomerase activity is expressed in germline cells and is
required to maintain telomere length and preserve the unlimited
proliferative potential of these cells.44 In the hematopoietic system and skin epidermis,45,46 two other
examples of self-renewing tissues, low levels of telomerase activity
have been found. Possibly this activity could extend the proliferative potential of the stem cells in these tissues to a certain extent but
not sufficient to confer immortality as telomeric DNA is still lost
upon replication.47 In a similar manner, peripheral blood lymphocytes express low telomerase activity that is upregulated upon
activation.29,35-37 Although the levels of telomerase
activity once again appear insufficient to override telomeric decline, the enzyme activity could reduce the loss of telomeric DNA to allow
repeated clonal expansion of immune cells upon antigenic stimulation.29
In a recent study, telomerase activity in murine hematopoietic
"candidate" stem cells and various progenitors was assayed on a
single-cell basis and found to be associated with "self-renewal" potential, with lower levels in committed progenitors than in their
pluripotent precursors.48 In contrast, we and others have consistently detected higher telomerase levels in committed progenitors relative to those observed in "candidate" stem cells using human hematopoietic tissues27-29 and this study. This discrepancy
suggests that telomerase expression is regulated differently in murine versus human hematopoietic cells, a phenomenon that was previously observed with other cell types from these two species.43,49
Because to date telomere length measurements are typically based on
bulk DNA analysis using Southern hybridization, subtle changes in
telomeric length on individual chromosomes could escape detection. This
notion was recently confirmed in studies of cells from telomerase RNA
knockout mice.44 On the basis of the distribution of
telomere length in individual chromosomes of cultured hematopoietic cells using fluorescent in situ hybridization analysis, we proposed that telomerase in these cells may preferentially act on short telomeres to maintain a minimum number of repeats.50 If
this is the case, the question of what telomere parameter, if any, is
restricting the proliferative potential of adult hematopoietic cells
becomes more pertinent. Possibly the low levels of telomerase that we
measured are able to maintain the length of some but not many short
telomeres. In this model, telomerase allows for a limited extension of
the replicative lifespan of HSC. Studies in this general area using in
situ hybridization to measure telomere length on individual chromosomes
of clonally propagated hematopoietic cells in combination with assays
of telomerase activity and protein expression should further clarify
the role of telomerase in hematopoietic cells.
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FOOTNOTES |
Submitted November 6, 1996;
accepted December 21, 1997.
Supported by National Institutes of Health Grant Nos. AI29524 and
GM56162 and by a grant from Geron Corporation.
Address reprint requests to Peter M. Lansdorp, MD, PhD, Terry Fox
Laboratory, British Columbia Cancer Agency, 601 W 10th Ave, Vancouver,
BC, V5Z 1L3 Canada.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Dr Nam Woo Kim (Geron Corperation) for generously sharing
details on the modified TRAP assay before publication. Dr Mark Zijlmans
is thanked for providing the fetal liver cells and Gayle Thornbury and
Wieslawa Dragowska are thanked for their expertise in cell sorting.
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Abstract
Introduction
Abstract
