Stem Cells 2003;21:654-660
www.StemCells.com
© 2003 AlphaMed Press
Telomere Length in Subpopulations of Human Hematopoietic Cells
Jessica A.G. Van Zifflea,
Gabriela M. Baerlochera,
Peter M. Lansdorpa,b
a Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada;
b Department of Medicine, University of British Columbia, Vancouver, BC, Canada
Key Words. Telomerase • Stem cells • Flow cytometry • Flow-FISH
Peter M. Lansdorp, M.D., Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3, Canada. Telephone: 604-877-6070, Ext. 3026; Fax: 604-877-0712; e-mail: plansdorp{at}bccrc.ca
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ABSTRACT
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In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34+CD38- cells was longer than in CD34+CD38+ cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34+CD38- cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.
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INTRODUCTION
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Telomeres are specialized structures at the ends of eukaryotic chromosomes. Human telomeres consist of hundreds to thousands of (T2AG3) repeats and associated telomere specific proteins [1]. In human somatic cells, the length of telomere repeats ranges from 2 to 15 kb [2], and telomere length was found to decrease with the number of cell divisions in vitro and with aging in vivo [3]. A striking correlation was furthermore found between telomere length and the in vitro proliferative capacity of normal human fibroblasts [4]. The progressive shortening of telomeres is thought to represent a mitotic clock that contributes to cellular senescence and the mortality of normal somatic cells [5]. The loss of telomeric DNA with each cell division appears to be related to DNA replication [6], processing of 5' telomeric strands [7], and oxidative damage to telomeric DNA [8]. Proliferative cells of the germline, such as germline stem cells, maintain relatively long telomeres (10 to 20 kilobase [kb]) throughout the lifetime of an organism [4]. Most likely, the processes that cause telomere shortening in such cells are balanced by de novo telomere repeat synthesis by telomerase, an essential reverse transcriptase enzyme [9–11].
It is estimated that a human adult produces on the order of 1012 blood cells per day [10]. In order to produce such large numbers of cells, hematopoietic tissue, typically in bone marrow, is actively proliferating at all times. What is not clear is the number of times hematopoietic stem cells (HSCs) in such tissues divide. Estimates of this number depend on the definition and assays of HSCs. Typical HSC assays involve regeneration and proliferation that may be uncommon in normal "steady state" hematopoiesis. Despite these important uncertainties, limitations in the proliferative potential of HSCs are believed to have serious consequences such as bone marrow failure. Several studies support the overall idea that the proliferative potential of HSCs is indeed finite. For example, granulocytes in blood samples from normal individuals show a striking age-related decline in telomere length that most likely reflects telomere shortening at the level of HSC [12]. This notion is supported by the observation that the telomere length in granulocytes from patients with aplastic anemia is significantly shorter than in age-matched controls [13]. Furthermore, candidate HSCs from adult bone marrow were found to have shorter telomeres than those derived from both fetal liver and cord blood cells [14]. Assuming that the telomere length in mature blood cells reflects the average telomere length in HSCs, these observations support the idea that telomerase levels in HSCs are insufficient to prevent overall shortening of telomere length relative to the length values observed in fetal cells.
It has been proposed that HSCs are telomerase competent; in general, mature hematopoietic cells do not express active telomerase, whereas HSCs do have some level of telomerase activity [15]. Indeed, CD34+CD38- candidate HSCs from fetal liver exhibit relatively high telomerase levels, whereas CD34+CD38- cells from adult bone marrow show lower levels of telomerase which is upregulated upon stimulation [16]. Until recently, it was unclear whether the telomerase activity measured in such cells is functionally significant as telomere length in more mature leukocytes clearly declines with age [12]. Recent studies of patients with dyskeratosis congenita (DKC) have clarified the situation. DKC is a progressive bone marrow failure syndrome that is predominantly inherited as an X-linked or an autosomal dominant (AD) disease [16]. X-linked DKC is caused by a mutation in the human homologue of yeast CBF5, dyskerin [17], whereas AD-DKC is caused by a mutation in the telomerase RNA template gene (hTERC) [18]. Cells from both X-linked and AD-DKC patients express reduced levels of telomerase activity and display marked telomere shortening. These findings clearly indicate that regulation of telomerase activity and telomere length in the HSC compartment is of critical importance for long-term maintenance of hematopoiesis. Further support for this notion comes from the observations of multiple groups that some degree of telomere shortening occurs in multiple lineages of the blood following human HSC transplantation [19–22]. Of note, such telomere shortening is most noticeable in the first year post transplant [20].
Taken together, telomere length data in leukocytes from normal individuals, patients with DKC, and transplant recipients strongly suggest that the telomerase level in HSCs is very important but insufficient to maintain the telomere length in HSCs. A prediction from this notion is that the most primitive HSCs will have the longest telomeres and that committed progenitors and mature progeny will have shorter telomeres than HSCs. In order to test this hypothesis, we analyzed the telomere length in purified populations from adult cadaver bone marrow cells using a newly developed fluorescent in situ hybridization (flow-FISH) technique.
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MATERIALS AND METHODS
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Cells
Previously frozen bone marrow cells from vertebrae bodies of organ donors were processed and used as previously described [14]. Cells were resuspended in Hank’s buffered salt solution containing 5% fetal calf serum and 0.1% sodium azide (HFN) at a concentration of 5 x 106 cells/ml.
Flow Cytometry and Cell Sorting
Cells were labeled with monoclonal antibodies against CD34 (HPCA-2-fluorescein isothiocyanate [FITC], Becton Dickinson and Company [BD]; San Jose, CA; http://www.bd.com) and CD38 (HB7-PE, BD) as described [14]. Labeling of side population (SP) cells was performed as described [23]. After labeling, cells were resuspended in HFN containing 2 µg/ml propidium iodide. CD34+CD38-, CD34+CD38+ and SP cells were sorted on a BD FACSVantage cell sorter equipped with an enterprise laser generating both UV and 488 nm excitation lines. Cells in selected windows were sorted and used for flow-FISH analysis.
Telomere Length Analysis by Flow-FISH and Flow Cytometry
The average length of telomere repeats at chromosome ends in individual peripheral blood leukocytes was measured by flow-FISH [24] with important modifications [25, 26]. Briefly, bone marrow-derived mononuclear cells of each individual were hybridized with or without 0.3 µg/ml telomere specific FITC conjugated (C3TA2)3 PNA probe (kindly provided by Applied Biosystems; Bedford, MA; http://www.appliedbiosystems.com), washed and counterstained with 0.01 µg/ml LDS 751 (Exciton Chemical Co. Inc.; Dayton, Ohio). The fluorescence in FL1 for individual cell types was acquired on a FACSCalibur (BD) and analyzed with CellQuest or CellQuestPro (BD). To convert the specific fluorescence (fluorescence measured in cells hybridized with the FITC-labeled telomere PNA probe minus the autofluorescence of unstained cells) into kb of telomere repeats, we processed and analyzed an internal standard bovine thymocyte with a known telomere length simultaneously with each sample [26]. Since the telomere length of the bovine thymocytes is known, the telomere length of the donor bone marrow population can be calculated from the ratio of the mean specific fluorescence of the bone marrow population to the mean specific fluorescence of the population of bovine thymocytes. The latter can be separated from the bone marrow cells by selection of appropriate light scatter and fluorescence properties [26].
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RESULTS
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The telomere length in mature hematopoietic cells and purified candidate HSCs was measured by flow-FISH. Cells were sorted from cadaver bone marrow samples from seven human organ donors (Table 1
). Telomere length in the CD34+CD38- and CD34+CD38+ compartments of all seven donors was analyzed. In addition, two of the seven donors were analyzed for telomere length in the SP compartment (Table 1). In order to obtain sufficient viable cells for telomere length analysis, the frozen marrows were separated by density gradient centrifugation to remove dead cells. Cells were additionally processed by lineage depletion to purify SP cells, resulting in a cell suspension highly enriched for CD34+ cells (increasing the relative percentage SP cells from ~0.05% in whole bone marrow to over 2% in lineage depleted marrow).
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Table 1. Summary of cadaver marrow donor identification, sex, age, hematopoietic cells sorted, and average telomere length calculated from flow-FISH measurements as described [25]
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The telomere length difference between the CD34+CD38- and CD34+CD38+ compartments in the seven marrow donors is illustrated in Figure 1. All seven donors had longer telomere repeat length in the CD34+CD38- compartment as compared to the CD34+CD38+ compartment; the maximum telomere length difference was 2.7 kb (donor F/17).
The mean telomere length in the CD34+CD38- compartment was 8.7 ± 0.5 kb (n = 7) and the mean telomere length in the CD34+CD38+ compartment was 7.6 ± 0.4 kb (n = 7). The difference in telomere length between these two populations was statistically significant (Student’s paired t-test; p < 0.02).
The telomere length difference between SP and CD34+CD38- cells from the same donor was limited to 0.1 to 0.2 kb (Fig. 2). The SP cells from both M/17 and M/34 had longer telomeres than the CD34+CD38- cells, which in turn were longer than the CD34+CD38+ cells. However, there was no statistical difference in telomere length between SP and CD34+CD38- cells sorted from the same cadaver marrow, nor were there differences in mean telomere length comparisons (Fig. 2).
Representative plots obtained from the analysis of both CD34+CD38- and CD34+CD38+ populations from three cadaver donors (Cad 14, Cad 17, Cad 18) are shown in Figure 3. The sample populations were tightly gated in forward scatter versus side scatter to avoid the inclusion of doublets (and cells with greater than 2N DNA). This gating was refined in forward scatter versus the DNA dye LDS-751. This latter parameter was used to avoid the inclusion of cells with greater than 2N DNA content in the telomere length analysis and to identify the internal standard of bovine thymocytes. The histogram plots show the fluorescence of the FITC-labeled telomere PNA probe in the selected FSC and LDS-751 gate (Fig. 3). Events with telomere fluorescence greater than the geometric mean are observable; however, it is difficult to distinguish between a true subset of cells with greater than mean telomere length and cells in early S phase of the cell cycle.
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Figure 3. Telomere length analysis by flow-FISH. Cell populations were gated on forward scatter (FSC) versus side scatter (SSC) (left panels). The bone marrow population was discriminated from the thymocyte control cells by DNA content using LDS-751 fluorescence versus forward scatter (middle panels). The populations were narrowly gated on LDS-751 fluorescence in order to avoid inclusion of cells in S/G2/M phase of the cell cycle. Three representative samples are shown (Cad 14, Cad 17, Cad 18). The open grey peak of the histogram is the unstained sample (gated in R2 and R4), the grey peak is the stained sample (gated in R2 and R4), and the black peak is the stained bovine thymocyte control cells (R1 and R3) (unstained bovine thymocyte peak not shown). The specific fluorescence is calculated by subtracting the autofluorescence of the population (unstained peak) from the telomere fluorescence (stained peak, grey for bone marrow sample). Telomere length values in test cells were calculated relative to the known telomere length in the bovine thymocytes (as described in Materials and Methods and, more extensively, in [26]).
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DISCUSSION
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In this study, we explored the possibility that tangible differences in telomere length exist between different candidate HSC populations in humans. Flow-FISH, a reliable and accurate technique that can accommodate lower cell numbers than other available techniques to measure telomere length, was used to measure the telomere length in the purified candidate HSC populations.
In each of seven donor samples, the CD34+CD38- population had longer telomeres than the CD34+CD38+ population. This finding corroborates previous telomere length data generated by terminal restriction fragment length analysis on sorted cells from two donors [14]. Telomere length in SP cells was longer than in CD34+CD38- populations in the two donors analyzed; however, these differences were not statistically significant.
The telomere length analysis of subpopulations of hematopoietic cells shown here supports the hypothesis that cells with the greatest proliferative potential have the longest telomeres. It has been demonstrated that SP cells contain a population of primitive hematopoietic progenitor cells [23]. SP cells and CD34+CD38- cells overlap in terms of cellular content; however, the SP cells are smaller, and perhaps contain a more pure population of HSC. Our data indicate that SP cells have, on average, a telomere length that is similar to the CD34+CD38- population, and we were unable to identify a clear population of cells within the SP cells that had longer telomeres than CD34+CD38- cells. These data do not, however, exclude the possibility that an elusive HSC exists within the SP cells with fetal length telomeres. However, we would have expected to see a skewed fluorescence intensity histogram if such a population represented >10% of the cells. While events that have telomere fluorescence above the mean are observable in CD34+CD38- cells (Fig. 3
), given the limitations of the LDS dye stain to allow precise discrimination of 2N versus >2N DNA content, the cells with greater telomere fluorescence could be in the S phase of the cell cycle. A DNA dye that provides more precise DNA distributions such as DAPI [3], is required in order to readdress this possibility in future studies.
Telomerase may be variably regulated depending on cellular conditions. Data from two separate studies suggest that telomerase is upregulated in response to the ex vivo expansion of hematopoietic progenitor cells [15, 16]. The data presented here suggest that the basal telomerase activity observed in the adult CD34+CD38- compartment is insufficient to prevent overall telomere shortening. That telomerase levels are nevertheless important is supported by data from DKC patients, where a reduction in telomerase activity results in compromised hematopoiesis throughout adult life and eventually leads to bone marrow failure [1, 18]. Low telomerase activity in normal HSCs may be required to maintain a limited number of short telomeres within the HSC compartment and to extend their proliferative capacity.
The great variation in telomere length between the individual donors of cells with the same surface phenotype prevents direct donor-to-donor comparisons of telomere length data from a particular cell population. This phenomenon is obvious in the seven bone marrow donors that were used in this study. Donor-to-donor variation in the telomere length highlights the need to determine the telomere length in subpopulations from the same donor in order to establish a reference point for telomere length comparisons.
While the flow-FISH technique involves analysis of single cells, it is not currently possible to confidently identify single events with longer than mean telomere length in a population. In order to reliably measure telomere length differences between cells in a given population, a subpopulation (e.g. identifiable peak or shift in a histogram of telomere fluorescence) of cells with a telomere length that is distinguishable from the major population is required. It will be interesting to further refine the stem cell populations analyzed to determine if there is indeed a phenotypically characterized population of HSCs with germline or fetal length telomeres. Unfortunately, extensive fractionation of the stem cell compartment leads to rapid decreases in cell yield. This would likely bring us to the limit of detection for measuring telomere length by flow-FISH. Ideally, methods that can measure the telomere length in viable cells [27] should be developed to further explore selection of cells on the basis of telomere length and proliferative potential. Alternatively, the telomere length in specific chromosome arms could possibly be measured in very small numbers of purified cells using a recently described polymerase chain reaction-based technique [28].
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ACKNOWLEDGMENT
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J.V.Z. was supported by an NSERC Studentship. These studies were supported by NIH grant AI29524. G.M.B. was supported by grants from the Swiss National Science Foundation (Grant number 31-53774-98) and the Bernese Cancer League.
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REFERENCES
|
|---|
- Collins K, Mitchell JR. Telomerase in the human organism. Oncogene 2002;21:564–579.[CrossRef][Medline]
- Martens UM, Zijlmans JM, Poon SS et al. Short telomeres on human chromosome 17p. Nat Genet 1998;18:76–80.[CrossRef][Medline]
- Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–460.[CrossRef][Medline]
- Allsopp RC, Vaziri H, Patterson C et al. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 1992;89:10114–10118.[Abstract/Free Full Text]
- Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965;37:614–636.[CrossRef][Medline]
- Levy MZ, Allsopp RC, Futcher AB et al. Telomere end-replication problem and cell aging. J Mol Biol 1992;225:951–960.[CrossRef][Medline]
- Lingner J, Cooper JP, Cech TR. Telomerase and DNA end replication: no longer a lagging strand problem? Science 1995;269:1533–1534.[Free Full Text]
- von Zglinicki T, Saretzki G, Docke W et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 1995;220:186–193.[CrossRef][Medline]
- Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–413.[CrossRef][Medline]
- Lansdorp PM. Stem cell biology for the transfusionist. Vox Sang 1998;74(suppl 2):91–94.
- Greider CW. Telomere length regulation. Annu Rev Biochem 1996;65:337–365.[CrossRef][Medline]
- Rufer N, Brummendorf TH, Kolvraa S et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med 1999;190:157–167.[Abstract/Free Full Text]
- Brummendorf TH, Maciejewski JP, Mak J et al. Telomere length in leukocyte subpopulations of patients with aplastic anemia. Blood 2001;97:895–900.[Abstract/Free Full Text]
- Vaziri H, Dragowska W, Allsopp RC et al. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA 1994;91:9857–9860.[Abstract/Free Full Text]
- Engelhardt M, Kumar R, Albanell J et al. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997;90:182–193.[Abstract/Free Full Text]
- Yui J, Chiu CP, Lansdorp PM. Telomerase activity in candidate stem cells from fetal liver and adult bone marrow. Blood 1998;91:3255–3262.[Abstract/Free Full Text]
- Heiss NS, Knight SW, Vulliamy TJ et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet 1998;19:32–38.[Medline]
- Vulliamy T, Marrone A, Goldman F et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 2001;413:432–435.[CrossRef][Medline]
- Wynn R, Thornley I, Freedman M et al. Telomere shortening in leucocyte subsets of long-term survivors of allogeneic bone marrow transplantation. Br J Haematol 1999;105:997–1001.[CrossRef][Medline]
- Rufer N, Brummendorf TH, Chapuis B et al. Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation. Blood 2001;97:575–577.[Abstract/Free Full Text]
- Thornley I, Sutherland R, Wynn R et al. Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss. Blood 2002;99:2387–2396.[Abstract/Free Full Text]
- Awaya N, Baerlocher GM, Manley TJ et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol Blood Marrow Transplant 2002;8:597–600.[CrossRef][Medline]
- Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997;3:1337–1345.[CrossRef][Medline]
- Rufer N, Dragowska W, Thornbury G et al. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998;16:743–747.[CrossRef][Medline]
- Baerlocher GM, Mak J, Tien T et al. Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls. Cytometry 2002;47:89–99.[CrossRef][Medline]
- Baerlocher GM, Lansdorp PM. Telomere length measurements in leukocyte subsets by automated Multicolor flow-FISH. Cytometry 2003;55A:1–6.
- Maeshima K, Janssen S, Laemmli UK. Specific targeting of insect and vertebrate telomeres with pyrrole and imidazole polyamides. EMBO J 2001;20:3218–3228.[CrossRef][Medline]
- Baird DM, Rowson J, Wynford-Thomas D et al. Extensive allelic variation and ultrashort telomeres in senescent human cells. Nat Genet 2003;33:203–207.[CrossRef][Medline]
Received April 10, 2003;
accepted for publication June 17, 2003.
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Top
Abstract
Introduction
) and CD34+CD38+ (
) cell populations sorted from seven cadaver marrow donors. Mean and standard deviation of duplicate measurements. Only for M/14 and F/16 did variation in duplicates result in standard deviations larger than the symbols used. All donors had longer mean telomere length in the CD34+CD38- populations versus the CD34+CD38+ populations (p < 0.02).
), CD34+CD38- (