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The loss of telomeric DNA may serve as a mitotic clock which signals cell senescence and exit from cell cycle. Telomerase, an enzyme which synthesizes telomeric repeats de novo, is required to maintain telomere lengths. In humans, significant telomerase activity has been found in cells with essentially unlimited replicative potential such as reproductive cells in ovaries and testes, immortal cell lines and cancer tissues, but not in most normal somatic cells or tissues. We have now examined telomerase expression in subpopulations of hematopoietic cells from adult human bone marrow using a sensitive polymerase chain reaction-based telomeric repeat amplification protocol. Telomerase activity was found at low levels in the highly enriched primitive hematopoietic cells (CD34+CD71loCD45RAlo) and was increased transiently when these cells were cultured in the presence of a mixture of cytokines. In contrast, the early progenitors (CD34+CD71+) expressed telomerase activity at a higher level which was subsequently downregulated in response to cytokines. Telomerase activity remained low in the more mature CD34− cells upon exposure to cytokines. Taken together, our results suggest that telomerase is expressed at a basal level in all hematopoietic cell populations examined, is induced in a primitive subset of hematopoietic progenitor cells and is downregulated upon further proliferation and differentiation of these cells. We have previously observed telomere shortening in cytokine-stimulated primitive hematopoietic cells. The low and transient activation of telomerase activity described here thus appears insufficient to maintain telomere lengths in cultured hematopoietic cells.
Telomeres, or chromosome ends, in eukaryotic organisms are characterized by tandem repeats of G-rich DNA . In human and other vertebrates, the telomeric sequence contains TTAGGG repeats . During cell replication, there is incomplete DNA synthesis at the 3′ end of chromosomes, and this end-replication problem predicts shortening of the telomeres with cell division [3,, 4]. Indeed, telomere shortening has been observed during in vivo and in vitro aging of normal human somatic cells, such as fibroblasts, keratinocytes, epithelial cells, peripheral blood leukocytes and endothelial cells [5–, 10]. These same cells undergo replicative senescence in vitro, i.e., they become senescent and withdraw from the cell cycle upon reaching the “Hayflick limit,” usually within 30-100 population doublings [11–, 13]. However, cells with essentially unlimited replicative potential such as the reproductive cells, immortal cell lines and cancer tissues have stable telomeres [7, 14–, 16]. The maintenance of telomere lengths in these cells is highly correlated with the presence of telomerase, a ribonucleoprotein enzyme which synthesizes telomeric repeats de novo [17–, 21]. These observations led to the proposal of the telomere hypothesis of cellular aging and immortalization . This hypothesis stipulates that telomere shortening serves as a mitotic clock for replicative senescence, ultimately signaling cell cycle exit, while telomerase activation is necessary for stabilizing telomere lengths, thereby maintaining replicative capacity of self-renewing cells such as cells in the reproductive tissues, tumor cells, and possibly stem cells of various tissue types [5, 14, 19].
The hematopoietic stem cells are usually defined as “self-renewing” multipotential cells which give rise to the myeloid, lymphoid and erythroid cell lineages. The stem cell compartment is small, but the “self-renewing” ability is thought to allow for the maintenance of stem cells despite the staggering daily demand for the production of >1011 mature blood cells [22, 23]. Using a combination of specific cell surface markers, a highly enriched population of the most primitive human hematopoietic progenitors can be isolated from adult bone marrow . These cells, expressing the CD34+CD71loCD45RAlo phenotype, are initially quiescent but can be stimulated to enter cell cycle and proliferate in response to cytokines . To date, there are no culture conditions known that allow these primitive hematopoietic progenitors to self-renew in vitro. The proliferation of such cells seems invariably associated with differentiation, mostly along the myeloid and erythroid pathways. In parallel, there are changes in the cell surface phenotype which are characterized by the activation of CD71 expression and the corresponding downregulation of CD34 expression .
We have previously demonstrated that telomeres shorten in primitive human hematopoietic cells as a function of developmental stage and cytokine-induced proliferation . These data suggest that the proliferative potential of at least some of these cells may be limited and may decrease with age. Recent studies have shown that telomerase activity was detected at low levels in hematopoietic cells and peripheral blood lymphocytes [26–, 28]. Here, we have investigated the expression of telomerase in different subpopulations of normal human bone marrow hematopoietic cells before and after culturing in the presence of cytokines. Using a sensitive polymerase chain reaction (PCR)-based telomerase assay (telomeric repeat amplification protocol, or TRAP) , we found a low basal level of telomerase activity in the most primitive, quiescent hematopoietic cells with the CD34+CD71loCD45RAlo phenotype and the more mature CD34– cells. The cycling CD34+CD71+ progenitors expressed the enzyme activity at a higher level. Interestingly, when the quiescent primitive cells were stimulated with cytokines, telomerase activity was transiently induced in the culture. The presence of telomerase activity, however, was apparently unable to prevent the telomere loss observed previously in similar cultures .
Materials and Methods
Bone marrow cells were obtained from vertebral bodies of three cadaveric organ donors, ages 27 (BM1), 42 (BM2) and 15 (BM3), respectively. Mononuclear cells from previously frozen marrows were separated by Ficoll density centrifugation and sorted for various subpopulations based on CD34, CD71 and CD45RA cell surface expression by flow cytometry as described [24, 25]. Biological activities of the different sorted populations from the same frozen marrow samples have been previously demonstrated using the long-term culture initiating cell, methylcellulose and liquid culture assays . Sorted bone marrow cells were routinely kept in serum-free medium at 4°C overnight and then harvested (day 0) or cultured in EMX medium  (serum-free medium supplemented with Steel factor, interleukin 6 (IL-6), GM-CSF/IL-3 fusion protein [PIXY], M-CSF, G-CSF and erythropoietin) for different periods of time.
Telomerase activity was measured by the TRAP assay essentially as described with some modifications . Briefly, cells were lysed with CHAPS extraction buffer at a concentration of 1,000 cells/μl, centrifuged at 100,000 × g, and enzyme activity in the supernatant was assayed by the de novo addition of telomeric repeats to an end-labeled telomerase substrate oligonucleotide. The subsequent telomerase product was detected by PCR (27 cycles total: 94°C, 30 sec; 60°C, 30 sec; 72°C, 30 sec) using the end-labeled substrate primer and an anchored reverse primer which prevented product lengthening as a result of PCR amplification [N.W. Kim, unpublished data]. The amplified telomerase products were then separated by native gel electrophoresis and analyzed on a PhosphorImager (Molecular Dynamics; Sunnyvale, CA). Since telomerase is a ribonucleoprotein, the specificity of the telomerase products detected was demonstrated by their sensitivity to RNase digestion. Because the amounts of cell extracts were sometimes limited, preincubation of a larger volume of cell extracts with RNase was not always possible. Instead, for some of the TRAP assays performed in the present study, cell extracts were added to reaction mixtures already containing RNase to initiate the telomerase reaction. We have previously observed that this sometimes leaves a weak banding pattern reflecting the incomplete inactivation of telomerase by RNase.
To quantitate the relative levels of telomerase activity, the signal intensity of PCR-amplified telomerase products generated by a cell extract was compared to that by 293 cell extracts (after subtraction of background value from reactions with no cell extracts added) using the ImageQuant program (Molecular Dynamics). In general, the signal intensities of telomerase-specific products generated in the TRAP assay are linear for cell extracts with increasing amounts of telomerase activity. However, we have often observed that the assay becomes nonlinear when very high concentrations of 293 cell extracts (>100,000 cell equivalent per reaction) are analyzed. In the current study, we have determined that telomerase activity, as determined in the TRAP assay, is linear for cell extracts prepared from 10 to 1,000 293 cell equivalents. Results for hematopoietic cell extracts were based on two or more independent analyses.
Results and Discussion
Telomerase Activity in Bone Marrow Subpopulations
To examine telomerase activity in the bone marrow, total mononuclear cells from frozen bone marrow were analyzed for telomerase activity using a modified TRAP assay (see Materials and Methods). Figure 1 shows the specificity and linearity of this assay. Cell extracts from 293 cells (transformed kidney epithelial cells) gave a strong telomerase-specific ladder which was RNase-sensitive (Fig. 1A). In addition, telomerase activity, which is measured by the signal intensity of telomerase-specific products generated in the TRAP assay, is linear for extracts derived from 10 to 1,000 293 cell equivalents (Fig. 1B).
When we analyzed cell extracts from 105 bone marrow cells, low levels of telomerase activity were detected in three separate donors. Figure 2A shows the representative results from two donors. The telomerase products were characterized by the presence of a typical ladder of six base repeats which were sensitive to RNase digestion. The residual bands in the RNase-treated lanes are most likely due to incomplete digestion of the telomerase enzyme (see Materials and Methods). For comparison, telomerase activity in 50 or 500 293 cell equivalents from the same analysis is also shown. Overall, the telomerase activity detected in the bone marrow was low and variable among different individuals. Since the bone marrow cells and the sorted subpopulations (below) are heterogeneous populations, accurate quantitation of telomerase activity in specific cell types is not possible. However, when compared to 293 cells and normalized to a per cell basis, the enzyme activity was present at 0.2% or less of that found in 293 cells (0.03% for BM1 and BM3; 0.2% for BM2).
To further localize the expression of telomerase activity in bone marrow, we fractionated total mononuclear cells into functionally distinct populations based on their cell surface expression of CD34 , CD71 and CD45RA . Cells with the CD34+CD71loCD45RAlo phenotype are highly enriched in the most primitive and largely quiescent stem cells, whereas the CD34+CD71+ cells are enriched in the actively cycling early progenitors. The CD34– population, on the other hand, contains the more mature hematopoietic precursors [24, 25]. Telomerase activity in these three cell populations was assayed using the TRAP assay, and representative analyses for all three donors are shown in Figure 2B. Telomerase activity was detected at low levels in the most primitive cells (lanes 2, 16, 19) and the more mature precursors (lanes 6, 17, 20). By contrast, much higher levels of the enzyme activity were present in the early progenitors (lanes 4, 15, 18). The level of telomerase activity in the early progenitors was between 20%-50% of that found in 293 cells (lanes 8-13) whereas those of the primitive cells and the more mature cells were 4% or less. In the same analysis, telomerase activity was not detectable in cell extracts obtained from exponentially growing normal human foreskin fibroblast strain, IMR90 (lane 14).
The CD34+CD71+ population can be further divided into CD45RA+ or CD45RA– subsets which are enriched in the myeloid or erythroid progenitors, respectively . Examination of telomerase activity in both of these subpopulations did not show any consistent quantitative differences, suggesting that telomerase activity did not segregate with commitment to specific lineages (data not shown). Taken together, the results above show that telomerase activity is differentially expressed in hematopoietic cell subpopulations derived from normal adult human bone marrow; namely, low levels of enzyme activity are detected in the primitive cells and the more mature cells, whereas elevated telomerase activity is present in the early progenitors. Recently, differential expression of telomerase activity in freshly isolated primitive (CD34+CD38lo/–) and committed hematopoietic progenitors (CD34+CD38+) has also been described in bone marrow and mobilized peripheral blood .
Telomerase Activity in Cultured Hematopoietic Cells
The detection of elevated levels of telomerase activity in the early progenitors led us to further examine telomerase activity in CD34 subpopulations upon cytokine stimulation. CD34+CD71loCD45RAlo, CD34+CD71+, and CD34– cells were cultured in the EMX medium containing cytokines which favored myeloid and erythroid differentiation and analyzed at three to four-day intervals over a total of 11 days (see Materials and Methods). Similar patterns of telomerase expression were observed for all three donors analyzed. Figure 3 shows the results from the TRAP assays for two donors, and Figure 4 shows the mean levels of telomerase activity in the different cultures relative to that in 293 cells (lower panels). The level of telomerase activity remained low in cultures of the more mature CD34– cells throughout the 11 days of culture. By contrast, telomerase activity was initially found at high levels in the CD34+CD71+ early progenitors but was downregulated in response to cytokines. By day 11, telomerase activity was almost undetectable in these cultures. Interestingly, the level of telomerase activity was increased transiently in the most primitive CD34+CD71loCD45RAlo cells after eight days of cytokine stimulation and declined by day 11. In some cases, telomerase induction could be detected as early as four days in culture. The mean level of telomerase activity induced in these primitive cells (4.6% of 293 cells), however, was on average lower than that detectable in uncultured early progenitors (11.8% of 293 cells). It is conceivable that the in vitro culture conditions do not reproduce the exact in vivo environment, and therefore activation and differentiation of primitive stem cells in vivo and in vitro are qualitatively and/or quantitatively different from each other.
Figure 4 shows the growth kinetics in the EMX medium of the various bone marrow subpopulations from all three donors. The increase in cell number (upper panels) and the level of telomerase activity relative to that found in 293 cells (lower panels) are expressed as a function of time in culture. It is evident that the level of telomerase activity in cultured CD34– cells (right) remained low despite the exponential increase in total cell number with time. Similarly, for cells with the CD34+CD71loCD45RAlo(left) or CD34+CD71+ (middle) phenotype, the up- or downregulation of telomerase activity did not correlate with cell proliferation.
When the primitive CD34+CD71loCD45RAlo cells are stimulated with IL-3, IL-6, Steel factor and erythropoietin, expression of CD34 has been shown to downregulate while the expression of CD71 is upregulated . Consistent with this, we observed a transient appearance of a CD34+CD71+ population in cultured primitive cells, while the proportion of CD34+CD71+ cells gradually declined in cultures of the early progenitor cells (data not shown). Since telomerase activity in both of these cultures paralleled CD34+CD71+ expression, the transient increase in telomerase activity is very likely due to dilution of the more telomerase positive CD34+CD71+ cells by the subsequent expansion of late progenitors that are CD34–.
In summary, the results shown here demonstrated that telomerase activity was expressed at a low basal level in the most primitive hematopoietic stem cells which were largely quiescent . Upon activation by cytokines to grow and differentiate in culture, telomerase activity became induced but only transiently. This was consistent with the higher levels of telomerase activity detected in the early progenitor cell population isolated directly from bone marrow. Downregulation of telomerase activity with further growth and differentiation in both cell populations indicates that proliferation per se does not maintain enzyme activity in hematopoietic cells. Whether telomerase activation in the primitive hematopoietic cells is related to their commitment to differentiation and/or functional “self-renewal” is currently under investigation.
We have previously shown that the telomeres of the most primitive hematopoietic cells gradually shorten upon stimulation with the same cytokine mixture used in this study . The presence of telomerase activity in hematopoietic subpopulations is therefore insufficient to overcome the net telomere loss in the total population. It is possible that a threshold level of telomerase activity may be required to maintain telomere lengths. Currently available technology does not allow us to determine whether telomerase expression in these cells functioned to slow the rate of telomere loss. There is also a formal possibility that telomerase activity detected in cell extracts does not correlate with functional activity in intact cells. For example, telomere-binding proteins [31–, 33] or chromatin structures might regulate the accessibility of telomeres to the telomerase enzyme. Lastly, telomerase activity might be expressed in only a small subpopulation of cells (e.g., CD34+CD71+), and stabilization of telomere length in these cells could be masked when the cultures are analyzed as a whole. If there exists a subpopulation of cells which maintains stable telomeres and does not differentiate, we would predict that it has an unlimited replicative capacity. Given that there are no in vitro growth conditions in which primitive hematopoietic cells can selectively self-renew, the presence of such a population would not be easily identified. Ultimately, the use of single cell assays for telomerase activity and more sensitive techniques for telomere length measurements will help to resolve this issue.
There is accumulating evidence that hematopoietic stem cells have a finite, albeit extensive, replicative capacity [22, 23, 34]. The expansion potential of adult hematopoietic stem cells is also reduced when compared to those from neonates or fetuses . Thus, in clinical applications where hematopoietic stem cells are placed under excessive proliferative demand such as repeated cycles of chemotherapy or hematopoietic transplantations, there may be exhaustion of their replicative potential. Our observations of weak and transient expression of telomerase and gradual telomere loss in the early hematopoietic progenitors provide a molecular mechanism to account for their extensive yet limited replicative capacity.
We thank Karen Prowse for critically reading the manuscript, Mike Strong and Ted Rigley (NorthWest Tissue Center, Seattle, WA) for making cells available for these studies, and Peter Ho for expert technical assistance.
This research was supported in part by Grant AI-29524 (to P.M.L.) and Grant AG09383A (to C.B.H.) from the National Institutes of Health and the Allied Signal Award for Biomedical Research on Aging (to C.B.H.). H.V. is a recipient of a student fellowship of the Medical Research Council of Canada.