Telomere length and proliferation potential of hematopoietic stem cells.
ABSTRACT Hematopoietic stem cells have typically been defined as pluripotent cells with self-renewal capacity. Recent studies have shown striking differences in the mean length of telomeric repeat sequences at the end of chromosomes from human hematopoietic cells at different stages of development. The most likely explanation for these observations is that hematopoietic stem cells, like all other somatic cells studied to date, lose telomeric DNA upon each cell division. In this review, limitations in the replicative potential of hematopoietic stem cells are discussed in the context of possible clinical use of such cells for transplantation and gene therapy.
- SourceAvailable from: Bipasha Bose[Show abstract] [Hide abstract]
ABSTRACT: Stem cells are the developmentally early forms of flexible cell types which can give rise to multiple cell types. Embryonic and adult stem cells are the two types of stem cells occurring in the mammalian system, which are responsible for giving rise to myriads of cell types during the development. Accordingly, harnessing the potential of stem cells is considered to have great significance for therapeutic purpose. Also, stem cells have certain features like unlimited proliferation potential, which they share closely, with the rogue cells of any mammalian system, which are cancer cells. In addition, there are stem cells, which give rise to cancer and are called cancer stem cells. In this review, we have addressed the developmental aspects of stem cells, cancer cells and cancer stem cells, their respective biological functions in maintaining an intricate balance to decide their respective contributions to the usefulness or harmfulness in the biological system.Journal of Stem Cell Research & Therapy. 02/2014; 4:173.
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ABSTRACT: Telomerase reverse transcriptase (TERT) is the protein component of telomerase and combined with an RNA molecule, telomerase RNA component, forms the telomerase enzyme responsible for telomere elongation. Telomerase is essential for maintaining telomere length from replicative attrition and thus contributes to the preservation of genome integrity. Although diverse mouse models have been developed and studied to prove the physiological roles of telomerase as a telomere- elongating enzyme, recent studies have revealed non-canonical TERT activities beyond telomeres. To gain insights into the physiological impact of extra-telomeric roles, this review revisits the strategies and phenotypes of telomerase mouse models in terms of the extra-telomeric functions of telomerase.Yonsei medical journal 01/2014; 55(1):1-8. · 0.77 Impact Factor
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ABSTRACT: Telomerase plays a key role in carcinogenesis. It is activated in most immortal cell lines and human cancers, including cutaneous melanoma (CM). Increased cell proliferation and deregulation of the cell cycle occur in human cancers. Links between telomerase activity (TA), cell proliferation, cell death and expression of cell-cycle regulators have not been extensively elucidated in CM. In this study, we investigated TA, mitotic index (MI), apoptotic index (AI), Ki-67 and nuclear positivity of cyclins D1 and A (Ki-67+N/1,000, cyclin D1+N/1,000, cyclin A+N/1,000) in 42 primary cutaneous melanomas (PCMs). TA was detected in all cases and directly correlated with MI, Ki-67+N/1,000, cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001); it was not correlated with AI. When subdividing PCMs into radial and vertical growth phase melanomas (RGPMs, VGPMs), a correlation was maintained only with MI (p < 0.005) and cyclin D1+N/1,000 (p < 0.005). Although MI and Ki-67+N/1,000 were highly correlated with cyclin D1+N/1,000 and cyclin A+N/1,000 (p < 0.001) when considering all cases together, a high correlation was found in the RGPM and VGPM groups between cyclin A+N/1,000 and Ki-67+N/1,000 only (p < 0.001), thus suggesting that cyclin A is more closely correlated with cell proliferation than cyclin D1. Our results further support the association between TA, tumor cell proliferation and cyclin D1 and A expression in PCM, though it is possible that links between TA and proliferation, on the one hand, and TA and cyclin D1 expression, on the other, might occur following various pathways. Int. J. Cancer 88:411–416, 2000. © 2000 Wiley-Liss, Inc.International Journal of Cancer 01/2000; 88(3):411-416. · 6.20 Impact Factor
The blood-forming or hematopoietic system is and has been
extensively studied for a variety of reasons. It represents the
prototype of a self-renewing biological system in that large
numbers of blood cells have to be produced daily in order to
compensate for the loss of relatively short-lived mature blood
cells. The relative ease at which blood and bone marrow
samples can be obtained and single cell suspensions prepared
has greatly facilitated in vitro and in vivo experimentation with
hematopoietic cells. Monoclonal antibodies specific for a
variety of cell surface antigens expressed on hematopoietic
cells have been produced and techniques to separate cells using
combinations of such reagents have been developed. A large
number of molecules with activity on various hematopoietic
cells are now available as are various in vitro assays to measure
functional properties of hematopoietic cells.
The hematopoietic system has been subdivided into a
hierarchy of three distinct populations (Till et al., 1964;
Metcalf, 1984). In this model the most mature cells are mor-
phologically identifiable as belonging to a particular lineage
and have very limited proliferative potential. The cells in this
most mature compartment are derived from committed prog-
enitor cells with a higher but still finite proliferative potential.
Committed progenitor cells in turn are produced by a popula-
tion of multipotential hematopoietic stem cells with self-
renewal potential, i.e. the capacity to give rise to more cells
with indistinguishable properties and developmental potential.
Self-renewal of stem cells is believed to be essential for main-
tenance of hematopoiesis over time.
Studies with cultured hematopoietic cells have shown that the
formation of hematopoietic colonies as well as the proliferation
and survival of various hematopoietic cells typically requires
the presence of ‘hematopoietic growth factors’, many of which
have been cloned over the last decade (Metcalf, 1989, 1993).
Many investigators have interpreted these observations as
evidence that the behavior of hematopoietic cells is ultimately
controlled by extracellular signals. This notion, together with
the possibility of purifying various hematopoietic cells with
unprecedented precision has led to frantic research efforts over
the last five years to achieve clinical useful manipulation of
blood-cell production in vitro. Two sets of observations cast
doubt about the feasibility of some of these perceived applica-
tions in the short term. First, there are a number of observations
indicating that extracellular factors do not appear to control
lineage commitment and self-renewal but instead seem to
permit exhibition of a predetermined cellular proliferative and
differentiation potential (Ogawa, 1989; Lansdorp et al., 1993;
Mayani et al., 1993). These findings suggest that the decisions
that ultimately determine the fate of hematopoietic stem cells
are not dictated by the environment but instead are controlled
by currently ill-defined, intrinsic genetic mechanisms with a
developmental component. A second hurdle to meaningful in
vitro stem cell ‘expansion’ is the observation that hematopoi-
etic cells, including purified hematopoietic stem cells, appear to
loose telomeric DNA with each cell division and with age
(Vaziri et al., 1994). This Commentary is focused on the role
of telomeres and telomerase in normal and abnormal
hematopoietic cells. As telomerase activity has so far not been
found in any somatic tissues (Counter et al., 1994), the findings
in the hematopoietic system appear to be applicable to other
self-renewing somatic tissues as well. More extensive and
general reviews on telomeres and telomerase have been
published elsewhere (Blackburn, 1991, 1992; Harley, 1991).
STRUCTURE AND FUNCTION OF TELOMERES
Without extremely reliable mechanisms to duplicate and
Telomere length and proliferation potential of hematopoietic stem cells
Peter M. Lansdorp
Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3
Journal of Cell Science 108, 1-6 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
Hematopoietic stem cells have typically been defined as
pluripotent cells with self-renewal capacity. Recent studies
have shown striking differences in the mean length of
telomeric repeat sequences at the end of chromosomes from
human hematopoietic cells at different stages of develop-
ment. The most likely explanation for these observations is
that hematopoietic stem cells, like all other somatic cells
studied to date, lose telomeric DNA upon each cell division.
In this review, limitations in the replicative potential of
hematopoietic stem cells are discussed in the context of
possible clinical use of such cells for transplantation and
Key words: telomeric DNA, proliferative potential, self-renewal,
hematopoietic stem cell
segregate complete copies of the genome into daughter cells,
life in any form could not exist. It seems certain that given this
importance, many factors and pathways involved in securing
the fidelity of gene duplication and chromosome segregation
remain to be uncovered. Recently, the role of telomeres in
some of these processes has received increased attention.
Telomeres are the physical ends of eukaryotic chromosomes
and contain both DNA and protein (Blackburn, 1991). Various
functional properties of telomeres were recently reviewed
(Blackburn, 1994). The DNA component of telomeres consist
of (TTAGGG)nin all vertebrates including humans. Telomeres
distinguish intact from broken chromosomes (Sandell and
Zakian, 1993). This distinction is important because the ends
of broken chromosomes (but not intact telomeres) indirectly
induce cell cycle arrest and chromosome repair before cells can
proceed to cell division(s). Telomeres furthermore appear to
stabilize chromosome ends, in that telomeres (but not broken
chromosomes) are protected from nuclear degradation and
end-to-end fusion. Telomere proteins are likely to be involved
in the positioning of telomeres and chromosomes within the
nucleus and are important for the spatial structure of telomeres
and chromosome segregation (Giraldo and Rhodes, 1994;
Chikashige et al., 1994). Finally, telomeres appear to play an
important role in the replication of the very end of chromoso-
TELOMERES AND THE ‘END REPLICATION
Two characteristics of the DNA polymerases involved in dupli-
cation of DNA prior to cell division are: (1) the requirement
of label RNA primers to initiate DNA synthesis; and (2)
synthesis of new DNA in the 5′r3′ direction only. That these
characteristics pose a problem for the complete replication of
the 3′ end of linear chromosomes was realized by Olovnikov
(1971, 1973) and Watson (1972), who described it as the ‘end
replication problem’ (Fig. 1). The effect of the end replication
problem on DNA duplication at chromosome ends is illustrated
in Fig. 2. From this figure, it can be understood that the 3′ ends
of all linear chromosomes are expected to shorten progres-
sively with each round of DNA duplication (Levy et al., 1992).
It now appears that such chromosome shortening can indeed
be observed in all human somatic cells studied to date,
including primitive hematopoietic cells from adult bone
marrow (Vaziri et al., 1994).
TELOMERE LENGTH AND REPLICATIVE
A number of studies have documented that the length of
telomeric (TTAGGG)n repeats in human cells decreases with
in vitro and in vivo cell divisions (Harley et al., 1990; Hastie
et al., 1990; Lindsey et al., 1991; Counter et al., 1992; Allsopp
et al., 1992; Vaziri et al., 1993). In these studies, telomeric
length showed considerable variation between individuals and
between different tissues. Studies with cultured fibroblasts
have indicated that telomere length is a better predictor of
replicative capacity than the actual age of the fibroblast donor
(Allsopp et al., 1992). On the basis of these studies, it appears
that loss of telomeric DNA (and resulting cell cycle exit
signals) may adequately explain the observation by Hayflick
and Moorhead that normal human fibroblast become senescent
after a fixed number of doublings in vitro (Harley, 1991). This
possibility has been expanded by Harley into the ‘telomere
hypothesis of cellular aging’ (Harley, 1991; Harley et al.,
1992). Several recent observations are in agreement with pre-
dictions of this theory and it appears that the possible impli-
cations of telomere biology could be wide-spread indeed. Not
only could measurements of telomere length be used to
estimate the proliferative potential of cells or, by repeating
measurements over time, the in vivo turn-over rate of cells and
tissues, but such measurements should also be valuable in
studies of aging and associated disorders. More information on
P. M. Lansdorp
Fig. 1. Schematic illustration of the end replication problem.
Somewhere along the 3′ terminus of single-stranded DNA a specific
RNA polymerase will synthesize a short stretch of nucleotides that is
complementary to the DNA template. This RNA serves as a primer
for DNA polymerase, capable of forming DNA from the template in
the 5′ to 3′ direction of the newly formed strand only. Hydrolysis of
the RNA primer and untranslated nucleotides upstream of the 5′ end
of the primer results in a region of untranslated DNA at the 3′ end of
the DNA template.
Fig. 2. The effect of the end replication problem on DNA duplication
at chromosome ends. Newly synthesized DNA is indicated by the
hatched bars. Arrows indicate regions of untranslated DNA at the 3′
end of the original DNA strands (top) after a complete round of
replication (bottom) (see also Fig. 1).
3Telomere length in stem cells
the role of telomeres in cellular aging may furthermore lead to
meaningful predictions of organ failure with implications in
diverse areas such as gerontology and preventive medicine.
A major problem in the exploration in some of these possi-
bilities is that telomere length measurements currently require
DNA from at least 105cells and that such assays are time
consuming. Typically, DNA (extracted from 1×106to 2×106
cells) is first digested with restriction enzymes that cleave
internal but not telomeric sequences. The resulting fragments
are separated by gel electrophoresis, immobilized and
hybridized with 32P-(CCCTAA)3. Specifically bound telomere
probe results in a smear on autoradiographs that ranges
between 10 and 15 kb for ‘young’ and 5 and 10 kb for ‘old’
cells. Results of these experiments are typically expressed as
the mean telomere restriction fragment (TRF) size (in
kilobases) after quantitation using densitometry. The actual
size of telomeric repeats is shorter because the restriction
enzymes used cut 2-5 kilobases upstream from the start of the
telomeric repeats themselves (Harley, 1991). The presumed
heterogeneity in TRF length between cells within a tissue and
between individual chromosomes make alternative measure-
ments of telomere length highly desirable. Ideally, such mea-
surements should be done on individual cells. Alternatives that
are being explored are in situ hybridization and immunologi-
cal methods for quantitation of proteins that bind specifically
to telomeric repeats (Palladino et al., 1993). Potential problems
with both approaches may be the presence of sequences with
homology to telomeric repeat sequences at subtelomeric or
intra-chromosomal sites. The presence of the latter is particu-
larly a problem in inbred mice, and has prevented the use of
this animal model in studies of telomeres and cellular aging
(de Lange, 1994).
TELOMERES AND CELL CYCLE CONTROL
Most, if not all, somatic human cells lose 50-100 bp of
telomeric DNA with each successive round of cell division
(Harley et al., 1990; Allsopp et al., 1992; Vaziri et al., 1993,
1994). Because such cells ‘start’ their proliferative life at an
unidentified point in fetal life with 5-10 kb of (T2AG3)n
repeats, the replicative capacity of early fetal cells is expected
to be of the order of 50-200 doublings. Even 50 doublings rep-
resents a tremendous proliferative potential, which can, in
theory, yield up to 1015cells or approximately 1000 kg of cells.
Differentiation and cell death will almost certainly limit the
actual size of clones to a fraction of this estimate. The actual
proliferative potential of cells may furthermore be less, as the
exact length of telomeric repeats required for full telomere
function is not known. In addition, the length of telomeric
DNA repeat sequences will vary to some extent between indi-
vidual chromosomes (Moyzis et al., 1988), presumably
limiting the proliferative potential of cells to the chromosome
with the shortest length of such repeats.
As discussed above, the replicative potential of somatic cells
is expected to decrease as a function of their proliferative
history. How does critical shortening of telomeres signal cell
cycle exit and cellular senescence? Some answers to this
important question have emerged from elegant studies in yeast
(Sandell and Zakian, 1993). It was found that inducible elim-
ination of a telomere on a single chromosome resulted in arrest
of the cell cycle at the G2 checkpoint. Interestingly, many of
the yeast cells ultimately recovered from the arrest without
repairing the damaged chromosome. Such damaged chromo-
somes were much more likely to be lost upon division than
chromosomes with intact telomeres (Sandell and Zakian,
1993). From this study, it appears that loss of telomeric DNA
induces the same cellular machinery (cell cycle arrest, DNA
repair) as broken chromosomes and that intact telomeres have
important functions, by allowing cell cycle progression and
avoiding chromosome loss.
TELOMERASE AND CANCER
If somatic cells are indeed limited by the length of their
telomeric DNA to a finite replicative capacity, this could be an
important natural barrier to prevent unlimited proliferation by
malignant clones. It is currently not known whether this
mechanism indeed limits the growth of certain tumors. Fur-
thermore, given a proliferative potential of 50-100 doublings,
it can easily be envisioned how some tumors could kill their
hosts without exhausting available telomere DNA at the time
of the initial malignant transformation. Nevertheless, it now
appears that malignant human tumors may use the same
mechanism by which cells of the germ line presumably remain
immortal: expression of the enzyme telomerase (Counter et al.,
1992, 1994; de Lange, 1994). Telomerase synthesizes
telomeric repeats using an RNA template complementary to
the G-rich repeats on the 3′ telomeric chromosome end
(Greider and Blackburn, 1985; Blackburn, 1992). The human
gene(s) encoding for this remarkable RNA-dependent DNA
polymerase (a reverse transcriptase) have not yet been cloned.
However, biochemical assays, using substrates of artificial
telomeric repeats, have provided clear evidence for the
existence of telomerase activity (Counter et al., 1992, 1994).
Interestingly, this enzyme activity has been found so far only
in immortal human cell lines (Counter et al., 1992) and certain
human tumors (Counter et al., 1994) but not in primary somatic
cells from normal individuals (Counter et al., 1992, 1994).
These observations suggest that telomerase could be a tumor-
specific enzyme and that non-toxic inhibitors of this enzyme
may be useful chemotherapeutic agents in the management of
patients with certain malignant tumors (Counter et al., 1994).
TELOMERES AND HEMATOPOIESIS: THE PRESENT
When DNAs from fetal liver cells, umbilical cord blood cells
and adult bone marrow cells were compared, a striking and
highly significant (P≤0.0001) difference in the mean length of
terminal restriction fragments (containing the (T2AG3)n
telomeric repeats) between the fetal/neonatal and adult tissues
was observed (Fig. 3). The observed loss of telomeric DNA in
these hematopoietic tissues appears not to be restricted to more
mature cells as purified CD34+CD38−primitive progenitor
cells from adult bone marrow were also found to have shorter
telomeres than fetal liver cells (Vaziri et al., 1994). In these
studies, there also appeared to be an age-related loss of
telomeric DNA. The measured rate of telomere loss in lym-
phocytes was calculated to reflect 0.4 stem cell doubling/year
(Vaziri et al., 1993), whereas the corresponding value was 0.25
stem cell doubling/year for (limited) adult bone marrow data
(Vaziri et al., 1994). Considerable variation in the mean
telomere length between individuals will require extension of
these studies to a larger number of samples in order to obtain
more accurate estimates of in vivo cell turn-over. Interestingly,
leukemic blast cells from a pediatric patient were found to have
significantly shorter telomeres than normal cells from the same
patient (Adamson et al., 1992). These findings are in support
of proliferation-dependent loss of telomeric DNA in cells of
hematopoietic origin. The most straightforward explanation for
these combined observations is that hematopoietic stem cells
do not express telomerase, have a very low turn-over rate in
adults and decrease their proliferative potential with each
division and, as a consequence, with age. However, the possi-
bility that minor populations of CD34+CD38−stem cells either
stop dividing at a fetal cell stage or, alternatively, express
telomerase activity and by either mechanism maintain ‘fetal
length’ telomeres throughout life has not been formally
excluded. Techniques that could be used to measure telomere
length and telomerase activity in individual cells would be
extremely useful in the further study of these possibilities.
Several observations with murine as well as human cells are
in agreement with there being a finite number of divisions of
early hematopoietic cells and some investigators have specu-
lated about the nature of such restrictions. In discussing dif-
ferences between embryonic and adult stem cells, Metcalf and
Moore (1971) calculated that embryonic (murine) stem cells
can undergo 20-80 more doublings than equivalent cells from
adult marrow (Metcalf and Moore, 1971). As one of the
possible explanations for the calculated differences, these
authors proposed that stem cells may be programmed to
undergo a fixed number of divisions. Reincke et al. (1982)
observed limitations in the proliferative capacity of normal and
irradiated stem cells in long-term bone marrow cultures. The
invariable occurrence of cellular senescence after a fixed
number of doublings was interpreted as indicating a general
biological limit to the division capacity of cells. Other reports
documenting that embryonic and fetal hematopoietic cells have
a higher in vivo (Rosendaal et al., 1979; Albright and
Makinodan, 1976) and in vitro (Nakahata and Ogawa, 1982;
Hows et al., 1992; Lansdorp et al., 1993) proliferative potential
than adult hematopoietic cells may also be in support of there
being absolute limits in replicative capacity. Similarly,
numerous observations indicating limitations in the prolifera-
tive potential of stem cells using serial transplantation experi-
ments (Metcalf and Moore, 1971; Moore, 1992) could be inter-
preted as providing support for possible telomere-related
restrictions in the proliferative potential of hematopoietic cells.
A detailed review of these studies is thought to be outside the
scope of this Commentary as the fate of primitive hematopoi-
etic cells in vitro and in vivo is undoubtedly controlled by a
variety of intrinsic and extrinsic factors, each of which may
have contributed in various degrees to these observations.
TELOMERES AND HEMATOPOIESIS: THE FUTURE
If all hematopoietic stem cells show age- and proliferation-
dependent shortening of telomeric DNA, several notions about
the ‘self-renewal’ of stem cells and their use in transplantation
and gene therapy probably need to be revised. Culture of
purified candidate stem cells with the goal of expanding their
number would have to take ultimate telomere-related restric-
tions in their replicative potential into account. From this per-
spective, the use of fetal liver (Lansdorp et al., 1993) and cord
blood cells (Broxmeyer et al., 1992) appears to have significant
advantages over cells derived from adult peripheral blood or
bone marrow. The ±4 kb of extra telomeric repeats that cord
blood and fetal liver cells have in comparison to cells from adult
bone marrow (Fig. 3) represent an estimated extra proliferation
potential of 20-40 cell doublings (assuming loss of 100-200 bp
of telomeric DNA per cell division). Even a minimal estimated
difference of 20 doublings could produce, in theory, a 106-fold
difference in cell numbers generated from cord blood versus
adult progenitor cells. Clearly, this number compares
favourable with the 7- to 30-fold fewer CD34+progenitor cells
that can be directly harvested from cord blood compared to
adult bone marrow (Broxmeyer et al., 1992). In order to take
advantage of this theoretical superiority of cord blood cells, it
will be important to understand how their extensive prolifera-
tion potential can be selectively exploited in order to increase
the number of transplantable stem cells. At this point, it seems
unlikely that growth factors alone will be sufficient to achieve
such net expansion of cord blood stem cells. For this purpose,
studies towards determining the genetic mechanisms that ulti-
mately determine the fate of very primitive hematopoietic cells
may be more rewarding. The selective expression of certain
transcription factors in the most primitive normal hematopoi-
etic cells such as homeobox genes A2, B3 and B4 (Sauvageau
et al., 1994) may provide a starting point for such studies. If
self-renewal decisions of stem cells could be manipulated in a
meaningful way, such measures would most likely need to be
combined with techniques that allow induced extension or
maintenance of telomere length in order to be clinically useful
with cells from adult tissues. Clearly such sophisticated cellular
engineering techniques require a few breakthroughs and will
not be available in the immediate future.
In view of the likelihood that telomere length ultimately deter-
mines the replicative capacity of hematopoietic cells, the word
proliferation potential should probably be used with caution. It
now seems reasonable to distinguish cell biological or func-
tional from telomeric or genetic restrictions in the number of
P. M. Lansdorp
Fig. 3. Differences in mean length of telomere restriction fragments
(TRF) of hematopoietic cells from fetal liver (FL), cord blood (CB)
and bone marrow (BM, 4 donors aged 40, 51, 52 and 58 years).
Values of the mean and standard error of TRF measurements are
adopted from Vaziri et al. (1994).
5Telomere length in stem cells
divisions a cell can potentially undergo. Thus, hematopoietic
cells may differ in replicative capacity as determined by the
length of their telomeres and yet have similar functional pro-
It is possible that the functional proliferative potential of
primitive hematopoietic cells is somehow linked to the length
of telomeres in such cells. This notion is certainly in agreement
with the higher proliferative capacity of fetal and cord blood
cells as compared to cells with a similar phenotype purified
from adult bone marrow (Lansdorp et al., 1993). However, a
more likely explanation for this correlation is that the proba-
bility of self-renewal (reflected in the replating potential of
cells) changes with different stages of development, similar
perhaps to the well-known developmental control of hemoglo-
bin gene expression. Without conditions that allow full display
of actual genetic replicative capacity, correlation between
telomere length and functional proliferative potential are likely
to be coincidental. It could be argued that all correlations
between telomere length and replicative history/proliferative
potential of cells that have been observed to date are just that:
correlations. Although numerous observations discussed in this
Commentary are in agreement with the functional involvement
of telomere length in determining the number of divisions a
cell can undergo, direct proof of this linkage in normal cells
has not yet been provided. For this reason, expression of
endogenous or exogenous functional telomerase activity in
normal somatic cells and demonstration that this results in
extension of their proliferative potential are eagerly awaited.
This work was supported by NIH grant AI29524, and by grants
from the Medical Research Council and the National Cancer Institute
of Canada. Dr H. Mayani and C. Harley are thanked for comments
on the manuscript, which was typed by Colleen MacKinnon.
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