Effects of Telomerase Modulation in Human Hematopoietic Progenitor Cells
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《干细胞学杂志》
a Freiburg University Medical Center, Department of Hematology/Oncology, Freiburg, Germany;
b Albert-Ludwigs-University, Department of Biology, Freiburg, Germany;
c Max-Planck Institute for Immunobiology, Hans-Spemann Laboratories, Freiburg, Germany
Key Words. Telomere length ? Telomerase ? Flow-FISH ? Cord blood ? Aging ? Stem cells
Correspondence: Dr. Uwe Martens, Freiburg University Medical Center, Department of Hematology/Oncology, Hugstetterstr. 55, D-79106 Freiburg, Germany. Telephone: 49-761-270-3401; Fax: 49-761-270-3418; e-mail: martens@ukl.uni-freiburg.de
ABSTRACT
Hematopoietic stem cells (HSCs) have a remarkable regenerative potential to replace most mature blood cells continuously throughout life. However, serial bone marrow transplantation experiments in mice associated with shortening of telomere repeats provide strong evidence that HSCs are not immortal cells but have a limited replication potential . Telomeres are the molecular caps at the ends of chromosomes that are composed of repetitive TTAGGG sequences and associated proteins . Telomeres stabilize and protect chromosome ends from end-to-end fusions, recombination, and degradation. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase, which consists of two core components, the RNA-subunit, hTR, containing the template, and a catalytic protein subunit, hTERT . In general, telomerase activity is absent in most somatic human cells, and programmed shortening of telomeres has been observed in dividing cells and with aging . In contrast, most tumor and embryonic stem cells circumvent replicative senescence by expression of hTERT, which leads to stabilization of telomeres and acquisition of an immortal phenotype .
Unlike normal somatic cells, telomerase activity has been detected at low levels in hematopoietic progenitor cells, which is upregulated in response to cytokine stimulation . However, despite telomerase activity, telomere shortening is not prevented but delayed on proliferation of hematopoietic cells . Replicative stress such as bone marrow reconstitution after autografting or allografting has been shown to result in accelerated telomere shortening . In addition, progressive telomere shortening has been reported in patients with bone marrow failure syndromes, such as aplastic anemia .
Because telomerase plays a critical role in overcoming growth limitations attributable to telomere erosion, several normal cell types, such as fibroblasts, endothelial cells, and epithelial cells, have been immortalized with telomerase without signs of malignant transformation . Such attempts may open a range of opportunities for the use of telomerase-immortalized cells in research, tissue engineering, and treatment of age-related diseases. Particularly, activation of telomere maintenance mechanisms might pose a greater potential for adult stem cells similar to advantages of embryonic cells, which are highly telomerase positive and immortal . In this study, we manipulated the endogenous telomerase activity in hematopoietic progenitor cells by ectopic transfer of hTERT as well as by a dominant-negative mutant. As a result, telomere length dynamics were only minimally influenced despite striking differences in telomerase activity. This suggests that telomerase function is more tightly regulated in HSCs compared with various other cell types. Furthermore, we provide evidence that telomerase activity is directly associated with proliferative capacity and lineage differentiation.
MATERIALS AND METHODS
Ectopic Transfer of hTERT in CD34+ CB Cells Increases the Levels of Telomerase Activity
To evaluate the function of two hTERTs containing retroviral vectors, we transduced primary human fibroblast cells that are telomerase-negative with the pBABE-hTERT-IRES-GFP and the pOS-hTERT-IRES-GFP constructs, respectively. Similar to previous reports , we found for both vectors that ectopic transfer of hTERT-induced telomerase activity, which was accompanied by substantial telomere elongation in the range of 2–2.5 kb as measured by flow-fluorescence in situ hybridization (flow-FISH; Figs. 1A and 1B). As a result, both constructs were able to immortalize human diploid fibroblasts, whereas control cells entered senescence after approximately 60 population doublings (Fig. 1C).
Figure 1. Immortalization of human diploid fibroblasts using ectopic transfer of hTERT. The HK-1 primary human fibroblast cell line was cultured as described previously . (A): Whereas untransduced (neg) and vector-transduced cells showed no detectable telomerase activity (*), fibroblasts transduced with pBABE-hTERT-IRES-GFP (dark gray bar) and with pOS-hTERT-IRES-GFP (black bar) exhibited similar levels of telomerase activity as measured by telomerase repeat amplification protocol ELISAplus assay 4 weeks after retroviral transduction. (B): Mean telomere length analyzed by flow-fluorescence in situ hybridization technique at this time point in nontransduced (white bar), vector-transduced (gray bar), pBABE-hTERT-IRES-GFP–transduced (dark gray bar), and pOS-hTERT-IRES-GFP–transduced (black bar) cells. (C): Compared with non-transduced () and vector-transduced () fibroblasts entering replicative senescence at ~60 population doublings, fibroblasts transduced with pBABE-hTERT-IRES-GFP () and with pOS-hTERT-IRES-GFP () bypassed senescence. The arrow indicates the time point of gene transfer.
Next, CD34+ CB cells were transduced with the retroviral vector pBABE-hTERT-IRES-GFP. GFP+ cells were fluorescence-activated cell (FAC)–sorted and cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, IL-6, and G-CSF. Transgene expression was stable, as indicated by GFP expression above 90% at the end of the culture (data not shown).
The endogenous telomerase activity of enriched CD34+ cells was moderate, being 51 ± 9% (n = 5) of the activity found in a representative telomerase-positive immortal cell line (NX). During the subsequent 6 days of cell expansion, enzyme activity was strikingly increased by more than 15.3 ± 6.5-fold (n = 5) compared with day 0, which was followed by a continuous decline thereafter. In CD34+ cells transduced with the hTERT gene, telomerase activity was significantly elevated relative to control cells throughout the culture (Figs. 2A and 2C). Considering the decline of endogenous telomerase activity, a peak fold increase of telomerase activity in ectopically hTERT-expressing cells was achieved by day 22, reaching 10.4 ± 3.1-fold (p < .05, n = 7) relative to vector-only transduced cells (Fig. 2B). Thus, the remaining ectopic telomerase activity was comparable with the levels that are found in immortalized cells, such as the phoenix cell line. Similar results were obtained using the pOS-hTERT-IRES-GFP vector (n = 3; data not shown).
Figure 2. Elevated levels of telomerase activity in CD34+ cord blood cells ectopically expressing hTERT. (A): Telomerase activity of CD34+ cord blood cells was analyzed using the telomerase repeat amplification protocol ELISAplus assay at indicated time points during in vitro expansion in the presence of stem cell factor, Flt-3, interleukin-3, interleukin-6, and G-CSF. TRAP-related ELISA absorbances were translated in relative telomerase activity values, which are expressed in relation to the immortal control cell line (NX). Black bars represent the hTERT-transduced cells, gray bars represent vector-only transduced controls, and white bars represent nontransduced cells. Values are means ± standard error from seven independent experiments, except where otherwise indicated. * Indicates significant differences in telomerase activity of hTERT-transduced cells compared with nontransduced and vector-transduced cells, respectively (p < .05). (B): The net effect of ectopic telomerase expression in relation to endogenous telomerase activity is demonstrated by the ratio of telomerase activities of hTERT- and vector-only transduced CD34+ cord blood cells from the seven experiments (A). * Indicates significant differences between indicated time points. (C): Representative Southern blot for one cord blood tissue indicating the typical, telomerase-mediated 6-nucleotide ladder in telomerase-positive samples. Negative control using RNase-treated extracts (+RNase) is shown for the hTERT-transduced specimen after 22 days in culture.
Telomere Length Dynamics in hTERT-Transduced CD34+ CB Cells
The average length of telomeric repeats was measured in transduced and nontransduced CD34+ cells at different time points during in vitro expansion using the flow-FISH method. Figure 3A shows typical telomere fluorescence histograms of hTERT overexpressing and control cells in the course of the expansion culture. The mean telomere length of enriched CD34+ CB cells was found to be 9.2 ± 0.2 kb (n = 6; Fig. 3B). Upon cell expansion, the mean telomere length of control cells was decreased by 1.1 ± 0.4 kb between days 10 and 22 of the culture (p < .05, n = 6). In CB cells ectopically expressing hTERT, the mean telomere length was found to be slightly increased in the range of 200 to 600 bp during different time points of cell expansion, which was significant by days 10 and 14 (p < .05) but not by day 22 (p = .3). However, despite increased telomerase levels, telomere attrition was not prevented in hTERT-transduced CB cells, resulting in a loss of 1.3 ± 0.4 kb over the culture period (p < .05, n = 6; Fig. 3B). Thus, overexpression of telomerase does not seem to stabilize the mean telomere length in dividing hematopoietic progenitor cells.
Figure 3. Telomere length dynamics in hTERT-transduced CD34+ cord blood cells analyzed by flow-FISH. (A): Representative flow-FISH histograms of nontransduced (neg), vector-only, and hTERT-transduced CD34+ cells corresponding to one cord blood sample at indicated time points. The difference between the mean fluorescence provided by the telomere-specific probe (gray) and the mean background fluorescence (white) is translated into indicated telomere fluorescence unit (TFUTRF) (kb) values based on calibration experiments using Southern blotting. (B): Mean TFUTRF (kb) values ± standard error from six experiments are shown during in vitro culture for hTERT-transduced (black bars) CD34+ cord blood cells and their vector-only (gray) and nontransduced (white bars) counterparts. * Indicates significant differences in telomere length between hTERT-transduced cells and nontransduced and vector-only transduced cells, respectively, and between indicated time points (p < .05). Abbreviation: FISH, fluorescence in situ hybridization.
Modulation of Telomerase Activity Does Not Influence Telomere Length Dynamics inAC133+ CB Cells
To exclude that the heterogeneity within the selected CD34+ cell population affects the outcome of observed telomere length dynamics, an additional set of experiments (n = 4) was performed in which CD34+ CB cells were sorted for GFP and AC133-PE fluorescence after transduction with the pOS-hTERT-IRES-GFP vector. In addition, transduction was performed with the same construct containing a dominant-negative mutant of hTERT (DN-hTERT).
As expected,AC133+GFP+ cells, which were transduced with the vector containing the wild-type hTERT gene, showed elevated levels of telomerase activity, whereas those transduced with the dominant-negative mutant had strikingly reduced or absent telomerase activity (Fig. 4A). Interestingly, the mean telomere length was not significantly affected (Fig. 4B) despite these highly different levels of telomerase activity, which confirms our observation that modulation of telomerase activity itself has only limited impact on telomere length dynamics in hematopoietic progenitor cells during in vitro culture.(Stefan Zimmermanna,b, Ste)
b Albert-Ludwigs-University, Department of Biology, Freiburg, Germany;
c Max-Planck Institute for Immunobiology, Hans-Spemann Laboratories, Freiburg, Germany
Key Words. Telomere length ? Telomerase ? Flow-FISH ? Cord blood ? Aging ? Stem cells
Correspondence: Dr. Uwe Martens, Freiburg University Medical Center, Department of Hematology/Oncology, Hugstetterstr. 55, D-79106 Freiburg, Germany. Telephone: 49-761-270-3401; Fax: 49-761-270-3418; e-mail: martens@ukl.uni-freiburg.de
ABSTRACT
Hematopoietic stem cells (HSCs) have a remarkable regenerative potential to replace most mature blood cells continuously throughout life. However, serial bone marrow transplantation experiments in mice associated with shortening of telomere repeats provide strong evidence that HSCs are not immortal cells but have a limited replication potential . Telomeres are the molecular caps at the ends of chromosomes that are composed of repetitive TTAGGG sequences and associated proteins . Telomeres stabilize and protect chromosome ends from end-to-end fusions, recombination, and degradation. The maintenance of telomeres throughout many cycles of cell division requires the enzyme telomerase, which consists of two core components, the RNA-subunit, hTR, containing the template, and a catalytic protein subunit, hTERT . In general, telomerase activity is absent in most somatic human cells, and programmed shortening of telomeres has been observed in dividing cells and with aging . In contrast, most tumor and embryonic stem cells circumvent replicative senescence by expression of hTERT, which leads to stabilization of telomeres and acquisition of an immortal phenotype .
Unlike normal somatic cells, telomerase activity has been detected at low levels in hematopoietic progenitor cells, which is upregulated in response to cytokine stimulation . However, despite telomerase activity, telomere shortening is not prevented but delayed on proliferation of hematopoietic cells . Replicative stress such as bone marrow reconstitution after autografting or allografting has been shown to result in accelerated telomere shortening . In addition, progressive telomere shortening has been reported in patients with bone marrow failure syndromes, such as aplastic anemia .
Because telomerase plays a critical role in overcoming growth limitations attributable to telomere erosion, several normal cell types, such as fibroblasts, endothelial cells, and epithelial cells, have been immortalized with telomerase without signs of malignant transformation . Such attempts may open a range of opportunities for the use of telomerase-immortalized cells in research, tissue engineering, and treatment of age-related diseases. Particularly, activation of telomere maintenance mechanisms might pose a greater potential for adult stem cells similar to advantages of embryonic cells, which are highly telomerase positive and immortal . In this study, we manipulated the endogenous telomerase activity in hematopoietic progenitor cells by ectopic transfer of hTERT as well as by a dominant-negative mutant. As a result, telomere length dynamics were only minimally influenced despite striking differences in telomerase activity. This suggests that telomerase function is more tightly regulated in HSCs compared with various other cell types. Furthermore, we provide evidence that telomerase activity is directly associated with proliferative capacity and lineage differentiation.
MATERIALS AND METHODS
Ectopic Transfer of hTERT in CD34+ CB Cells Increases the Levels of Telomerase Activity
To evaluate the function of two hTERTs containing retroviral vectors, we transduced primary human fibroblast cells that are telomerase-negative with the pBABE-hTERT-IRES-GFP and the pOS-hTERT-IRES-GFP constructs, respectively. Similar to previous reports , we found for both vectors that ectopic transfer of hTERT-induced telomerase activity, which was accompanied by substantial telomere elongation in the range of 2–2.5 kb as measured by flow-fluorescence in situ hybridization (flow-FISH; Figs. 1A and 1B). As a result, both constructs were able to immortalize human diploid fibroblasts, whereas control cells entered senescence after approximately 60 population doublings (Fig. 1C).
Figure 1. Immortalization of human diploid fibroblasts using ectopic transfer of hTERT. The HK-1 primary human fibroblast cell line was cultured as described previously . (A): Whereas untransduced (neg) and vector-transduced cells showed no detectable telomerase activity (*), fibroblasts transduced with pBABE-hTERT-IRES-GFP (dark gray bar) and with pOS-hTERT-IRES-GFP (black bar) exhibited similar levels of telomerase activity as measured by telomerase repeat amplification protocol ELISAplus assay 4 weeks after retroviral transduction. (B): Mean telomere length analyzed by flow-fluorescence in situ hybridization technique at this time point in nontransduced (white bar), vector-transduced (gray bar), pBABE-hTERT-IRES-GFP–transduced (dark gray bar), and pOS-hTERT-IRES-GFP–transduced (black bar) cells. (C): Compared with non-transduced () and vector-transduced () fibroblasts entering replicative senescence at ~60 population doublings, fibroblasts transduced with pBABE-hTERT-IRES-GFP () and with pOS-hTERT-IRES-GFP () bypassed senescence. The arrow indicates the time point of gene transfer.
Next, CD34+ CB cells were transduced with the retroviral vector pBABE-hTERT-IRES-GFP. GFP+ cells were fluorescence-activated cell (FAC)–sorted and cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, IL-6, and G-CSF. Transgene expression was stable, as indicated by GFP expression above 90% at the end of the culture (data not shown).
The endogenous telomerase activity of enriched CD34+ cells was moderate, being 51 ± 9% (n = 5) of the activity found in a representative telomerase-positive immortal cell line (NX). During the subsequent 6 days of cell expansion, enzyme activity was strikingly increased by more than 15.3 ± 6.5-fold (n = 5) compared with day 0, which was followed by a continuous decline thereafter. In CD34+ cells transduced with the hTERT gene, telomerase activity was significantly elevated relative to control cells throughout the culture (Figs. 2A and 2C). Considering the decline of endogenous telomerase activity, a peak fold increase of telomerase activity in ectopically hTERT-expressing cells was achieved by day 22, reaching 10.4 ± 3.1-fold (p < .05, n = 7) relative to vector-only transduced cells (Fig. 2B). Thus, the remaining ectopic telomerase activity was comparable with the levels that are found in immortalized cells, such as the phoenix cell line. Similar results were obtained using the pOS-hTERT-IRES-GFP vector (n = 3; data not shown).
Figure 2. Elevated levels of telomerase activity in CD34+ cord blood cells ectopically expressing hTERT. (A): Telomerase activity of CD34+ cord blood cells was analyzed using the telomerase repeat amplification protocol ELISAplus assay at indicated time points during in vitro expansion in the presence of stem cell factor, Flt-3, interleukin-3, interleukin-6, and G-CSF. TRAP-related ELISA absorbances were translated in relative telomerase activity values, which are expressed in relation to the immortal control cell line (NX). Black bars represent the hTERT-transduced cells, gray bars represent vector-only transduced controls, and white bars represent nontransduced cells. Values are means ± standard error from seven independent experiments, except where otherwise indicated. * Indicates significant differences in telomerase activity of hTERT-transduced cells compared with nontransduced and vector-transduced cells, respectively (p < .05). (B): The net effect of ectopic telomerase expression in relation to endogenous telomerase activity is demonstrated by the ratio of telomerase activities of hTERT- and vector-only transduced CD34+ cord blood cells from the seven experiments (A). * Indicates significant differences between indicated time points. (C): Representative Southern blot for one cord blood tissue indicating the typical, telomerase-mediated 6-nucleotide ladder in telomerase-positive samples. Negative control using RNase-treated extracts (+RNase) is shown for the hTERT-transduced specimen after 22 days in culture.
Telomere Length Dynamics in hTERT-Transduced CD34+ CB Cells
The average length of telomeric repeats was measured in transduced and nontransduced CD34+ cells at different time points during in vitro expansion using the flow-FISH method. Figure 3A shows typical telomere fluorescence histograms of hTERT overexpressing and control cells in the course of the expansion culture. The mean telomere length of enriched CD34+ CB cells was found to be 9.2 ± 0.2 kb (n = 6; Fig. 3B). Upon cell expansion, the mean telomere length of control cells was decreased by 1.1 ± 0.4 kb between days 10 and 22 of the culture (p < .05, n = 6). In CB cells ectopically expressing hTERT, the mean telomere length was found to be slightly increased in the range of 200 to 600 bp during different time points of cell expansion, which was significant by days 10 and 14 (p < .05) but not by day 22 (p = .3). However, despite increased telomerase levels, telomere attrition was not prevented in hTERT-transduced CB cells, resulting in a loss of 1.3 ± 0.4 kb over the culture period (p < .05, n = 6; Fig. 3B). Thus, overexpression of telomerase does not seem to stabilize the mean telomere length in dividing hematopoietic progenitor cells.
Figure 3. Telomere length dynamics in hTERT-transduced CD34+ cord blood cells analyzed by flow-FISH. (A): Representative flow-FISH histograms of nontransduced (neg), vector-only, and hTERT-transduced CD34+ cells corresponding to one cord blood sample at indicated time points. The difference between the mean fluorescence provided by the telomere-specific probe (gray) and the mean background fluorescence (white) is translated into indicated telomere fluorescence unit (TFUTRF) (kb) values based on calibration experiments using Southern blotting. (B): Mean TFUTRF (kb) values ± standard error from six experiments are shown during in vitro culture for hTERT-transduced (black bars) CD34+ cord blood cells and their vector-only (gray) and nontransduced (white bars) counterparts. * Indicates significant differences in telomere length between hTERT-transduced cells and nontransduced and vector-only transduced cells, respectively, and between indicated time points (p < .05). Abbreviation: FISH, fluorescence in situ hybridization.
Modulation of Telomerase Activity Does Not Influence Telomere Length Dynamics inAC133+ CB Cells
To exclude that the heterogeneity within the selected CD34+ cell population affects the outcome of observed telomere length dynamics, an additional set of experiments (n = 4) was performed in which CD34+ CB cells were sorted for GFP and AC133-PE fluorescence after transduction with the pOS-hTERT-IRES-GFP vector. In addition, transduction was performed with the same construct containing a dominant-negative mutant of hTERT (DN-hTERT).
As expected,AC133+GFP+ cells, which were transduced with the vector containing the wild-type hTERT gene, showed elevated levels of telomerase activity, whereas those transduced with the dominant-negative mutant had strikingly reduced or absent telomerase activity (Fig. 4A). Interestingly, the mean telomere length was not significantly affected (Fig. 4B) despite these highly different levels of telomerase activity, which confirms our observation that modulation of telomerase activity itself has only limited impact on telomere length dynamics in hematopoietic progenitor cells during in vitro culture.(Stefan Zimmermanna,b, Ste)