Author contributions: J.S.C., S.H.K., and A.R.R.: collection and/or assembly of data, data analysis and interpretation; L.J.N. and B.L: data analysis and interpretation, conception and design, manuscript writing, and final approval of manuscript provision. J.S.C. and S.H.K. contributed equally to this work.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS October 23, 2012.
Daily, cells incur tens of thousands of DNA lesions caused by endogenous processes. Due to their long-lived nature, adult stem cells may be particularly susceptible to the negative impact of this constant genotoxic stress. Indeed, in murine models of DNA repair deficiencies, there is accumulation of DNA damage in hematopoietic stem cells and premature loss of function. Herein, we demonstrate that mice expressing reduced levels of ERCC1-XPF DNA repair endonuclease (Ercc1−/Δ mice) spontaneously display a progressive decline in the number and function of hematopoietic stem/progenitor cells (HSPCs). This was accompanied by increased cell death, expression of senescence markers, reactive oxygen species, and DNA damage in HSPC populations, illustrating cell autonomous mechanisms that contribute to loss of function. In addition, the bone marrow microenvironment of Ercc1−/Δ mice was not permissive for the engraftment of transplanted normal stem cells. Bones from Ercc1−/Δ mice displayed excessive osteoclastic activity, which alters the microenvironment in a way that is unfavorable to HSPC maintenance. This was accompanied by increased proinflammatory cytokines in the bone marrow of Ercc1−/Δ mice. These data provide novel evidence that spontaneous, endogenous DNA damage, if not repaired, promotes progressive attrition of adult stem cells via both cell autonomous and nonautonomous mechanisms. STEM CELLS2013;31:511–525
With aging, there is a decline in functional hematopoietic stem cells (HSCs) . As a consequence, HSCs from old donors have impaired differentiation capacity and impaired ability to regenerate the hematopoietic system of lethally irradiated hosts . This loss of regenerative capacity in the hematopoietic compartment has been attributed to intrinsic (cell autonomous) changes , suggesting that time-dependent accumulation of damaged organelles or macromolecules drives age-related decline in HSC function.
Multiple human diseases caused by inherited defects in DNA repair display accelerated aging of the hematopoietic system , strongly suggesting that DNA damage is one type of cellular damage that contributes to aging-related loss of HSC function. All cells in the body are continuously challenged by DNA damage as a consequence of the instability of DNA in an aqueous, oxidative environment and vulnerability to reactive electrophiles . DNA damage is repaired via numerous complex pathways. Given that tens of thousands of lesions that occur per nuclear genome per day, DNA repair may be inadequate to remove all of the damage over the lifetime of mammals. Long-lived cells such as adult stem cells may be particularly prone to accumulation of unrepaired DNA damage .
In mouse models of many genome instability disorders, there is spontaneous and progressive loss of HSC function [6, 7]. This includes mice with defects in transcription-coupled nucleotide excision repair, increased mutations in the mitochondrial genome, telomere maintenance, and nonhomologous end-joining of double-strand breaks. In other models, there is a loss of progenitor and HSC number. This includes mice with mutations in the translesion polymerase pol μ or regulatory factors in the Fanconi anemia pathway of DNA interstrand crosslink repair [8, 9]. However, the mechanism by which endogenous DNA damage affects HSC function is still not sufficiently understood, in particular in the absence of confounding factors such as impaired transcription or immunodeficiency. To address this gap in knowledge, we carefully examined the hematopoietic system of a DNA-repair deficient model, Ercc1−/Δ mice without the introduction of exogenous genotoxic stress.
Ercc1−/Δ mice express approximately 5% of the normal level of ERCC1-XPF DNA repair endonuclease, which is involved in nucleotide excision repair, DNA interstrand crosslink repair [10, 11], and the repair of double-strand breaks with 3′ overhangs . Ercc1−/Δ mice are healthy into adulthood (8 weeks) then begin to show numerous progressive symptoms associated with old age . By 21 weeks of age, Ercc1−/Δ mice exhibit symptoms, as well as functional, histopathological, ultrastructural, and gene expression changes that significantly correlate with those observed in 2–3-year-old mice, relative to young mice [13–16]. Therefore, Ercc1−/Δ mice offer a unique model to investigate the impact of endogenous DNA damage on hematopoietic stem/progenitor cell (HSPC) function that is pertinent to a human progeroid syndrome with strong correlations to normal aging. Herein, we establish that there is spontaneous and rapid loss of HSPC number and function in Ercc1−/Δ mice and provide evidence that this is due not only to changes in damaged HSPCs but also their niche.
MATERIALS AND METHODS
Ercc1−/Δ mice were generated by mating of heterozygous mice in two different inbred backgrounds to create isogenic F1 hybrids: Ercc1+/Δ FVB/n × Ercc1+/− C57Bl/6 (B6). Genomic DNA was isolated from a 1-mm ear plug of 10-14-day-old mice using a NucleoSpin 96 Tissue DNA extraction system (Macherey-Nagel, Inc.). Genotyping of the Ercc1 null allele was done by polymerase chain reaction (PCR) coamplification of the 3′ end of exon 7 from the WT allele and the neomycin resistance marker cloned into exon 7 of the targeted allele using primers specific for exon 7, neor, and intron 7 (5′-AGCCGACCTCCTTATGGAAA, 5′-TCGCCTTCTTGACGAGTTCT, and 5′-ACAGATGCTGAGGGCAGACT, respectively). WT (0.25 kb) and mutant (0.4 kb) fragments were separated by electrophoresis on a 2% agarose gel. The hypomorphic allele of Ercc1 (Δ) was amplified by adding a fourth primer (5′-CTAGGTGGCAGCAGGTCATC) to amplify the neomycin cassette in the mutant allele (0.5 kb). WT or heterozygous littermates were retained as aging-matched controls. All animal experiments were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Colony Forming Cell Assays
For the CFU-GM assay, bone marrow cells (1 × 104 per dish) were seeded into 35 mm dishes in MethoCult GF (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). For the pre-B assay, bone marrow cells (1 × 105 per dish) were plated in MethoCult M3630 (StemCell Technologies). All colony forming cell assays were performed in triplicate. The colonies were counted on day 7 for pre-B and day 12 for CFU-GM colonies.
Quantitative Real-Time PCR
Total RNA from LSK cells was isolated using RNeasy Micro kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Reverse transcription of total RNA was performed using QuantiTech reverse transcription kit (Qiagen). Primers for p16Ink4a were purchased from Applied Biosystems (Foster City, CA, http://www.appliedbiosystems.com).
Flow Cytometry Analysis
LSK cells were defined by staining with a lineage cocktail, Sca-1 and c-kit antibodies. LSK cells were further defined by staining with anti-CD34, CD48, and CD150 antibodies as previously described . Cell sorting was performed by Moflow (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com). The SA-β-Gal activity was assessed using C12FDG (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). The level of mitochondrial superoxide anion within LSK cells was assessed using MitoSOX (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Cell death was determined by Annexin V and PI staining. Intracellular cytokine levels were measured by flow cytometry (BD Bioscience, San Diego, CA, http://www.bdbiosciences.com). Briefly, bone marrow cells were stained with antibodies to lineage surface markers. Cells were then fixed, permeabilized, and stained with anti-TNF-α and IL-1α antibodies according to the manufacturer's instructions (e-BioScience, San Diego, CA, http://www.ebioscience.com). Cell populations were gated (e.g., CD4+, CD8+, and double positive cells) and the fraction of cells expressing TNF-α and IL-1α were measured. γ-H2AX was assessed by anti-phospho-histone H2AX (Ser139) antibody (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) using the Cytofix/Cytoperm plus Solution Kit (BD Bioscience, San Diego, CA, http://www.bdbiosciences.com).
Cell Cycle Analysis
Cell cycle analysis was performed by staining the cells with fluorescein isothiocyanate (FITC)-conjugated Ki-67 antibody (BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com/index_us.shtml). Briefly, LSK cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) followed by staining with Ki-67-FITC (BD Biosciences) and DAPI (Invitrogen).
To evaluate short- and long-term reconstituting activity of donor cells, recipient mice were lethally irradiated (Shepherd Mark I 68 irradiator, 137Cs γ source) 8–24 hours prior to transplantation. Bone marrow cells from 7- and 21 week-old Ercc−/Δ mice or their control littermates were transplanted by tail vein injection. For the competitive repopulation assay, test cells (CD45.1/2) from Ercc1−/Δ mice and control littermates were transplanted along with an equal number of competitor cells (CD45.1) into lethally irradiated recipient mice (CD45.2). Competitor cells, like the test cells were obtained from F1 mice (FVB/B6). To examine the fate of transplanted cells in nonablated hosts, 5 × 106 normal WT bone marrow cells (B6, CD45.1) were transplanted into nonirradiated recipient mice (CD45.1/2). Peripheral blood and bone marrow cells were collected from recipient mice at various times post-transplant, and the ratio of CD45.1/CD45.1.2 was determined by flow cytometry.
TRAP Staining and Immunohistochemistry
The femurs dissected from 7- and 21-week-old Ercc1−/Δ mice and WT littermates were fixed in 4% paraformaldehyde solution in phosphate buffered saline (PBS, pH 7.2) for 2 days and then decalcified in 10% EDTA (pH 7.5) for 10 days. After decalcification, they were embedded in paraffin and cut longitudinally to 5 μm thickness. For identification of osteoclasts, the sections were deparaffinized, dehydrated, and stained using TRAP staining kit (B-Bridge International) according to the manufacturer's instructions. For immunohistochemical staining of RANKL, bone marrow sections were immunolabeled overnight with goat polyclonal antibody against mouse RANKL (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com, 1:50) at 4°C. Subsequently, the bone sections were incubated with biotinylated anti-goat antibody (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and DAB substrate (Vector Laboratories) according to the manufacturer's instructions.
Enzyme-Linked Immunosorbent Assay
The levels of cytokines were measured using enzyme-linked immunosorbent assay. Serum samples from 17-week-old Ercc1−/Δ mice and control littermates (n = 3 per group) were diluted to 1:10 in PBS. Bone marrow fluid was collected as previously described with minor modifications . Absorbance was measured using a microplate reader set at 450 nm. Plotted is the average ± SEM.
Bone marrow cells from 5- to 6-week-old mice (B6) were seeded in a 96-well plate at a density of 2 × 105 cells per well and incubated with 50 ng/ml M-CSF (eBioscience) for 3 days. Serum from 17 week-old Ercc1−/Δ mice and control littermates were diluted in PBS samples and added to each well in the presence or absence of neutralizing antibodies (anti-IL-1α or anti-TNF-α; 1 mg/ml). The cells were incubated further for 3 days. Osteoclasts were identified by TRAP staining as described above and counted. The average of TRAP positive cells ± SEM is plotted.
All p values (statistical significance) were determined by Student's t test, Mann–Whitney U test, or Wilcoxon test; *, < .05; **, < .01; *** < .005.
A Hypomorphic Mutation in Ercc1 Leads to Progressive Attrition of HSPC Function
DNA repair-deficient Ercc1−/Δ mice have normal peripheral blood counts in young adulthood (7–9 weeks). By 21–24 weeks of age, the mutant animals had lymphopenia, indicative of hematopoietic dysfunction (Supporting Information Fig. S1). Accordingly, Ercc1−/Δ mice exhibit a slight but significant reduction (1.5-fold, p = .0007) in bone marrow cellularity at 7 weeks of age compared to littermate controls (Fig. 1A). The reduction was more pronounced in 21-week-old Ercc1−/Δ mice (4.4-fold, p < .0001). These data demonstrate that Ercc1−/Δ mice spontaneously develop progressive loss of hematopoiesis in the first 4–5 months of life.
Previous studies indicate that with aging, alterations in hematopoiesis are caused by a loss of HSPC function . We first assessed hematopoietic progenitor cell (HPC) function by performing the colony-forming assay. Bone marrow cells from 7-week-old Ercc1−/Δ mice had a significantly reduced ability to form colony forming unit-granulocyte-macrophage (CFU-GM) and pre-B colonies compared to control littermates (Fig. 1B, left panels). A more pronounced reduction in clonogenic capacity was seen in bone marrow cells from 21-week-old Ercc1−/Δ mice (Fig. 1B, right panels). In particular, formation of pre-B colonies was almost completely absent in 21-week-old Ercc1−/Δ mice (Fig. 1B, lower right panel). These data demonstrate a progressive decline in proliferative capacity of HPCs isolated from progeroid Ercc1−/Δ mice, similar to what was previously observed in the more severe Ercc1−/− knockout mice .
The effect of Ercc1 mutation on hematopoietic lineage determination was examined using CD11b and B220 as markers of myeloid and lymphoid lineages, respectively. The percent of CD11b+ and B220+ cells in the peripheral blood of Ercc1−/Δ and normal littermates were similar at 7 weeks of age (Fig. 1C, upper panel). However, the lineage differentiation of Ercc1−/Δ mice was significantly skewed away from lymphoid lineages in favor of myeloid lineages by 21 weeks of age (Fig. 1C, lower panel). The skewed myeloid differentiation and reduced pre-B colony formation in Ercc1−/Δ mice mimic changes that occur in rodents with aging [19–21].
To further assess HSPC function, bone marrow cells from Ercc1−/Δ mice and control littermates were transplanted into lethally irradiated recipient animals. Survival of the recipient animals was measured as an index of the capacity of the donor cells to repopulate the recipients' hematopoietic system. As expected, bone marrow cells from 7- and 21-week-old control wild-type (WT) littermates were able to fully rescue the lethally irradiated recipients (Fig. 2A, open circles). Bone marrow cells from 7-week-old Ercc1−/Δ mice were sufficient to provide radioprotection (or short-term repopulating cells) (Fig. 2A, upper panel, closed diamonds): recipient survival was 100% until 8 weeks post-transplantation. However, their ability to support long-term survival of lethally irradiated recipients was partially impaired (60% recipient survival at >12 weeks post-transplantation). Of note, as the Ercc1−/Δ mice reached an age of 21 weeks, the radioprotective capacity of their bone marrow cells was substantially decreased (only 20% of the recipients were alive 5 weeks post-transplant) (Fig. 2A, lower panel, closed diamonds). Furthermore, the bone marrow cells from 21-week-old Ercc1−/Δ mice showed complete loss of long-term repopulating capacity and failed to rescue any of lethally irradiated recipient animals beyond 7 weeks post-transplant. These results indicate that while Ercc1−/Δ mice have functional HSPCs at 7 weeks of age, they are lost or become exhausted by the time Ercc1−/Δ mice are 21-week-old.
Competitive repopulation assays were also conducted. Bone marrow cells from Ercc1−/Δ mice or control littermates (CD45, 1/2) were mixed in equal proportion with WT competitor cells (CD45.1) and then transplanted into lethally irradiated recipient animals (CD45.2). Competitor bone marrow cells (CD45.1) were obtained from the first-generation cross of C57BL/6 (CD45.1) and FVB (CD45.1) mice to minimize potential skewing due to variation in genetic background. Bone marrow cells from control littermates were able to compete equally with the competitor cells at 4, 8, (data not shown), and 12 weeks (competitor, 42.94%; WT littermate, 49.65%) and >1-year post-transplant (competitor, 44.94%; WT littermate, 47.95%) (Fig. 2B), indicating that there is no competitive advantage or disadvantage resulting from histocompatibility mismatches. In contrast, Ercc1-/Δ bone marrow cells were almost completely outcompeted by the competitor cells at all time points analyzed post-transplantation (Fig. 2B). Collectively, these data support that there is a spontaneous and progressive loss of functional HSPCs in Ercc1-/Δ mice.
A Hypomorphic Mutation in Ercc1 Leads to Progressive Attrition of HSPC Number
We next used multicolor flow cytometry to measure the number and frequency of stem and progenitor populations in the bone marrow compartment of Ercc1-Δ mice. The gating strategy used to define various stem/progenitor cell populations is shown in Figure 2C. Lineage−Sca-1+c-kit+ (LSK) cells are composed of HSCs and a heterogeneous mix of multipotent/committed progenitors. LSK cells can be further subdivided into more primitive stem cell populations based on the expression of CD150, CD48, and CD34 [22, 23] (Fig. 2C). Meanwhile, lineage− Sca-1− IL-7R− c-kit+ cells can be further divided on the basis of the expression of CD34 and Fc receptor (FcR), yielding common myeloid progenitors (CMP; CD34+, FcR(low/−)), granulocyte-monocyte progenitor cells (GMP; CD34+, FcR+), and megakaryocyte-erythroid progenitor cells (MEP; CD34−, FcR(low/−)) . The absolute number of each type of cell in the bone marrow compartment of Ercc1−/Δ mice and littermate controls at two ages were plotted in Figure 2D. A moderate decrease in the absolute number of LSK cells (∼ 2-fold, p = .013) was observed in 7-week-old Ercc1−/Δ mice compared to control littermates (Fig. 2D, upper panel). SLAM LSK cells (CD150+, CD48−, LSK), which are currently considered to be the most primitive HSCs , were also modestly reduced (∼ 3-fold, p = .014) in 7-week-old Ercc1−/Δ mice compared to control littermates (Fig. 2D, upper panel). Bone marrow cells from 7-week-old Ercc1−/Δ mice displayed a modest but significant reduction in the number of HPCs, such as common lymphoid progenitor cells (defined as Lin−, IL−7Rα+, c-Kit+, Sca-1+) (p = .009)  and CMP cells (p = .04) but not GMP or MEP cells (Fig. 2D, upper panel).
While HPCs in 21-week-old Ercc1−/Δ mice were not markedly altered compared to 7-week-old Ercc1−/Δ mice, there was a substantial further reduction in CD34-LSK (∼ 28 fold, p = .021) and SLAM LSK cells (∼ 30 fold, p = .023) (Fig. 2C, 2D), both of which are highly enriched for long-term repopulating HSPCs. These results demonstrate a time-dependent attrition of Ercc1−/Δ HSPCs, in particular the more primitive stem/progenitors cells.
A Hypomorphic Mutation in Ercc1 Increases the Turnover of HSPCs
Most HSPCs reside in a noncycling quiescent state, but prolonged regenerative demands could draw a greater fraction of HSPCs into active cell cycling, leading to premature loss of this stem progenitor cell population . Given the more dramatic decline in the number of primitive HSPCs than intermediate progenitors in Ercc1−/Δ mice (Fig. 2C, 2D), we asked whether there is increased turnover of Ercc1−/Δ HSPCs between 7 and 21 weeks of age. We tested this by measuring the cell cycle profile of LSK cells isolated from 14 to 16-week-old mice. The proportion of LSK cells in the quiescent state (G0) was significantly reduced in Ercc1−/Δ animals compared to control littermates (from 81% to 58%; Fig. 3). This coincided with an increase in LSK cells in the G1 (from 10% to 23%) and S/G2/M phase (from 9% to 19%). Interestingly, the active cycling of Ercc1−/Δ LSK cells coincided with the period of time in which there was a significant decrease in HSPC pool size (Fig. 2), suggesting that Ercc1−/Δ HSPCs may undergo cell death and/or senescence rather than self-renewal.
A Hypomorphic Mutation in Ercc1 Leads to Cell Death and Premature Senescence of HSPCs
To determine if the attrition of HSPCs is due to cell death, LSK cells isolated from 16-week-old Ercc1−/Δ mice and control littermates were stained with Annexin V and propidium iodide (PI). Ercc1−/Δ mice showed only a modest increase in the percent of early apoptotic cells (Annexin V+, PI-cells; 2% vs. 0.8%). The percent of late apoptotic (Annexin V+, PI+) and dead cells (Annexin V−, PI+) was significantly increased in LSK cells from Ercc1−/Δ mice compared to normal littermates (Fig. 4A).
We next determined the extent of senescence in Ercc1−/Δ HSPCs by measuring senescence-associated β-galactosidase (SA-β-gal) activity. SA-β-gal was not significantly increased in LSK cells from Ercc1−/Δ mice at 7 weeks of age (Fig. 4B). In contrast, LSK cells from 21-week-old Ercc1−/Δ mice displayed a dramatic increase in SA-β-gal activity compared to age-matched WT counterparts (Fig. 4B, 2.5-fold, p = .001). This was accompanied by a significant upregulation of p16INK4a (Fig. 4C). At 7 weeks of age, the p16INK4a level in Ercc1−/Δ LSK cells was approximately sixfold higher than control littermates (p = .02). A more marked increase in the level of p16INK4a was detected in Ercc1−/Δ LSK cells from 21-week-old mice (20-fold increase, p = .03).
A Hypomorphic Mutation in Ercc1 Leads to Increased Reactive Oxygen Species and DNA Damage in HSPCs
Cells from aged organisms have increased oxidative damage, which arises from endogenously produced reactive oxygen species (ROS) . ROS can drive cell senescence and/or death . In addition, ROS can drive HSPCs into the cell cycle, compromising their quiescent state . We, therefore, examined whether ROS is increased in LSK cells from Ercc1−/Δ mice compared to normal controls. Indeed, we observed a trend of increasing mitochondrial superoxide anion in LSK cells from 7-week-old Ercc1−/Δ mice compared to littermate controls (Fig. 5A). LSK cells from 21-week-old Ercc1−/Δ mice exhibited a more pronounced increase in the level of mitochondrial ROS relative to littermate controls.
We next measured γH2AX, a marker of DNA strand breaks, in HSPCs from 16- and 21-week-old mice. A modestly higher proportion of LSK cells were positive for γH2AX in 16-week-old Ercc1−/Δ mice compared with their normal littermate counterparts (8.7% vs. 2.7%) (Fig. 5B, top histograms). However, by 21 weeks of age, there was a much more dramatic increase in the fraction of LSK cells staining positively for γH2AX in Ercc1−/Δ mice compared to control littermates (24.5% vs. 4.3%) (Fig. 5B, lower histograms). Importantly, the accumulation of γH2AX was observed predominantly in LSK cells but not in other subpopulations of HSPCs such as Lin−Sca-1−c-kit+ cells (myeloid progenitors). We were unable to analyze levels of γ-H2AX in SLAM LSK or CD34-LSK cells due to their very low frequency of this population of cells in 21-week-old Ercc1−/Δ mice (Fig. 2). Collectively, these data support the conclusion that Ercc1−/Δ mice display an age-dependent increase in oxidative stress and spontaneous accumulation of DNA damage preferentially in stem/progenitor cell population.
A Hypomorphic Mutation in Ercc1 Also Leads to Loss of HSPCs Via Noncell-Autonomous Mechanisms
While the results shown above indicate cell autonomous defects within the Ercc1−/Δ HSPCs, we asked whether the fate of Ercc1−/Δ HSPCs is also influenced by their microenvironment, that is, via a cell non-autonomous manner. For this purpose, transplantation experiments were performed with or without conditioning regimen, as illustrated in Figure 6A. When normal donor cells (CD45.1) were transplanted into unconditioned WT recipients (CD45.1/2), donor-derived cells (CD45.1) made a modest contribution to the recipient's peripheral blood cells (13.6%) 5 weeks post-transplant but were barely detectable (∼ 0.05%) in the peripheral blood 9 weeks post-transplant and thereafter (Fig. 6B, upper). In contrast, normal donor cells (CD45.1) showed a higher contribution in unconditioned Ercc1−/Δ recipients at 5 weeks post-transplant (∼ 46%) (Fig. 6B, upper). However, the contribution of donor cells to Ercc1−/Δ recipient's hematopoietic system was drastically reduced thereafter and donor cell chimerism was almost undetectable (<1%) at 9 weeks post-transplantation (Fig. 6B, upper). Because stable engraftment is dependent upon successful lodging and expansion of donor HSPCs in the recipient's bone marrow, we monitored the frequency of donor cells in the recipient's bone marrow. Donor cells were barely detectable in the bone marrow of WT recipient mice (<0.01%) (Fig. 6B, lower left). This is expected since the stem cell niches in WT mice have already been occupied by the endogenous HSPCs. A higher percentage of donor cells (0.32%) were detected in the bone marrow of Ercc1−/Δ recipients. However, among the donor-derived cells in the bone marrow of Ercc1−/Δ recipients, donor-derived LSK cells were virtually undetectable (<0.1%) (Fig. 6B, lower right), suggesting the possibility that the microenvironment of Ercc1−/Δ mice is not permissive for stable and productive engraftment of transplanted donor HSPCs.
To rule out the possibility that the poor engraftment in unconditioned WT and Ercc1−/Δ recipients was due to differences in genetic backgrounds between donor and recipient mice, we tested the compatibility of the strains under conditions of lethal irradiation. The majority of peripheral blood cells in conditioned (10 Gy) control littermate recipients were of donor origin (Supporting Information Fig. S2, upper panel). Furthermore, there was robust engraftment in the bone marrow as evidenced by a donor-dominated (>93%) chimerism and the presence of LSK cells in the bone marrow of conditioned control littermate recipients (Supporting Information Fig. S2, lower panel). Transplantation into lethally conditioned Ercc1−/Δ recipients is not technically feasible, since Ercc1−/Δ mice are hypersensitive to radiation . Therefore, in subsequent experiments, the ERCC1 mice were conditioned with 3 Gy total body irradiation. At 3 Gy, Ercc1−/Δ recipient mice could survive up to 7–9 weeks after transplantation. At 9 weeks post-transplantation, Ercc1−/Δ mice had a significantly reduced number of LSK cells in their bone marrow as compared with similarly conditioned WT recipients (Fig. 6C). These results reinforce the view that the microenvironment of Ercc1−/Δ mice does not provide an optimal milieu for HSPCs.
Osteoblasts provide the niche necessary to retain HSCs in a quiescent state [29, 30]. An imbalance of osteoblast/osteoclast activity is detrimental to long-term HSC maintenance [31, 32]. To determine whether the observed nonautonomous defects are associated with a shift in the balance of osteoclast and osteoblast activity, bone/bone marrow sections were stained for tartrate resistant acid phosphatase (TRAP), a marker of osteoclast activity. TRAP positive cells were barely detectable in the bone marrow of normal mice at 7 and 21 weeks of age (Fig. 6D, left panel). In contrast, 21-week-old Ercc1−/Δ mice demonstrated a considerably higher level of TRAP staining (Fig. 6D, lower right panel).
Interestingly, the serum from Ercc1−/Δ mice was more potent in promoting osteoclast formation than serum from littermate controls (Fig. 6F, p = .001), suggesting that humoral factors in Ercc1−/Δ mice may play a role in shifting the balance toward osteoclast formation. Ercc1−/Δ mice displayed an increased level of several proinflammatory cytokines in their serum (Fig. 6E). TNF-α and IL-1α were the most upregulated (>2 fold, p < .05). Addition of antibodies against TNF-α and IL-1α to the serum of Ercc1−/Δ mice attenuated osteoclast formation in vitro (Fig. 6F) demonstrating a causative role for these cytokines in promoting osteoclastogenesis. Of note, the TNF-α neutralizing antibody had a more potent inhibitory effect on osteoclast formation than the IL-1α antibody. However, the combination was additive. Since the serum level of the cytokines may not accurately reflect the characteristic of bone marrow microenvironment, we further measured cytokine expression in the bone marrow itself. In bone marrow supernatant, TNF-α and IL-1α were higher in Ercc1−/Δ mice than in littermate controls (Fig. 6G). To determine the source of the cytokine production in the bone marrow of Ercc1−/Δ mice, TNF-α and IL-1α expression were analyzed in various lineages of bone marrow cells. Flow cytometric analysis revealed that TNF-α and IL-1α were predominantly expressed by CD4 and CD8 expressing cells in Ercc1−/Δ bone marrow (Fig. 6G). In assays done with other cell lineage markers (e.g., B220+, CD11b+, Gr-1+), we found no significant difference.
RANKL, the primary mediator of osteoclast formation, was also expressed at high levels in the bone marrow of Ercc1−/Δ mice (Fig. 6H). Interestingly, the increased expression of RANKL was observed in Ercc1−/Δ mice at 7 weeks of age, which was before the appearance of TRAP positive cells. These findings support the observation that the bone marrow of Ercc1−/Δ mice is not conducive to HSPC engraftment and maintenance.
As humans age, there is loss of bone marrow cellularity, increased cell turnover and cell death in the hematopoietic compartment, and myeloid skewing . These characteristics indicate a loss of regenerative capacity in the hematopoietic system with aging. Many genetic diseases caused by mutations in genome maintenance mechanisms show involvement of the hematopoietic system , leading to the hypothesis that DNA damage contributes to aging-related loss of HSC function. In support of this, XpdTTD mice defective in nucleotide excision repair and transcription, Ku80−/− mice missing nonhomologous end-joining of double-strand breaks, G3 mTR−/− mice missing telomerase, and polγExo mice missing the exonuclease of the mitochondrial polymerase γ show accelerated loss of HSC function but not number . This led to the view that HSCs accumulate DNA damage, which directly affects HSC function [1, 35, 36]. In contrast, there is attrition of HSCs in polμ−/− mice defective in DNA damage tolerance, as well as Atm−/− and p53+/m mice with altered DNA damage response [4, 8]. Lig4Y288C mice, defective in nonhomologous end-joining of double-strand breaks, spontaneously develop bone marrow failure and lose HSCs as a consequence of increased HSC turnover .
Ercc1−/Δ mice are immunocompetent, have normal telomere lengths, transcription, and DNA damage response [37, 38], allowing us to focus on the impact of unrepaired endogenous DNA damage on the hematopoietic system. Ercc1−/Δ mice have early onset and progressive loss of HSPC function and number (Figs. 1, 2). This provides evidence that endogenous DNA damage, if not repaired, can drive premature exhaustion of HSPCs.
Several factors associated with HSPC dysfunction were discovered to be perturbed in LSK cells from Ercc1−/Δ mice (Figs. 4, 5). γH2AX is elevated in LSK cells from 21-week-old Ercc1−/Δ mice but not younger animals or progenitor cells, indicative of age-dependent DNA damage accumulation in the HSPC subsets. ROS is elevated in HSPCs of progeroid Ercc1−/Δ mice compared to littermate controls. Deletion of the DNA damage signaling proteins ATM or MDM2, or mutation of the telomerase protein DKC1 also leads to increased ROS in HSCs and stem cell depletion [39–41].
Expression of the p16INK4a is associated closely with cellular senescence . p16INK4a expression was significantly elevated in Ercc1−/Δ LSK cells, which has been shown to attenuates HSC self-renewal .
DNA damage is thought to drive aging-related loss of HSC function via a cell autonomous mechanism [1, 3, 44], which is consistent with all of the above. However, in other tissue-specific, adult stem cell populations, the niche is also critical for maintaining a reservoir of stem cells and tissue regenerative capacity . Thus we also examined cell non-autonomous mechanisms for hematopoietic failure in the Ercc1−/Δ mice. Normal HSPCs were not able to establish sustainable, productive engraftment in Ercc1−/Δ mice, indicating that defects in the microenvironment contribute to the loss of HSPC number and function. This was supported by the observations that osteoclastic activity was significantly increased in Ercc1−/Δ mice compared to WT littermates as were proinflammatory cytokines in the serum of Ercc1−/Δ mice that drive osteoclastogenesis. Deletion of the cell cycle regulator retinoblastoma (RB) gene leads to osteoclast-mediated destruction of the osteoblastic niche, displacement of the HSCs, and myeloid expansion , which are similar to changes observed in Ercc1−/Δ mice.
Defective homing of Ercc1−/Δ HSPC via downregulation of homing-associated molecules could be another potential explanation for the hematopoietic deficiencies observed in Ercc1−/Δ mice. Unexpectedly, we found that the HSPCs from 15- and 21-week-old Ercc1−/Δ mice express significantly high levels of CXCR4 (Supporting Information Fig. 3), which is one of the critical mediators of HSC homing . This suggests that the dysfunction of Ercc1−/Δ HSPCs is not likely mediated by downregulating the expression of CXCR4. Because the majority of intravenously injected cells are trapped in the lung and liver, with very limited number of Ercc1−/Δ HSPCs, it was technically difficult to quantify homing of transplanted Ercc1−/Δ HSPCs.
FoxO−/−, Bmi-1−/−, and Lig4Y288C mice, which exhibit premature aging-like phenotypes, also undergo significant attrition in HSC numbers [7, 48, 49]. In contrast, it was reported that the number of HSCs increases in normal aged B6 mice, although there is considerable strain to strain variation .
In summary, analysis of the hematopoietic system of Ercc1−/Δ mice provides evidence that endogenous DNA damage promotes the loss of HSPCs through both cell autonomous and nonautonomous mechanisms. The heretofore unappreciated contribution of cell nonautonomous mechanisms of HSPC attrition could have important implications for elderly patients and patients with genome instability disorders requiring bone marrow transplantation.
We thank Chelsea Feldman for technical assistance. This study was supported in part by research funding from Department of Defense (W81XWH-09-1-0364) to B.C.L and NIH (ES016114) to L.J.N. This project used the UPCI flow cytometry facility and was supported in part by award P30CA047904.