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Human Molecular Genetics Advance Access originally published online on December 21, 2006
Human Molecular Genetics 2007 16(3):286-294; doi:10.1093/hmg/ddl457
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Published by Oxford University Press 2006

Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background

Yong-Gang Yao1,*, Felicia M. Ellison1, J. Philip McCoy2, Jichun Chen1 and Neal S. Young1

1 Hematology Branch and 2 Flow Cytometry Core Facility, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1202, USA

* To whom the correspondence should be addressed at: National Heart, Lung, and Blood Institute, NIH, Bldg 10 CRC, Rm 3E-5140, 10 Center Drive, Bethesda, MD 20892-1202, USA. Fax: +1 3014968396; Email: yaoy3{at}nhlbi.nih.gov/ ygyaozh{at}yahoo.com

Received October 8, 2006; Accepted December 4, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Alterations in mitochondrial DNA (mtDNA) and consequent loss of mitochondrial function underlie the mitochondrial theory of aging. In this study, we systematically analyzed the mtDNA control region somatic mutation pattern in 2864 single hematopoietic stem cells (HSCs) and progenitors, isolated by flow cytometry sorting on Lin–Kit+CD34– parameters from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, to test the hypothesis that the accumulated mtDNA mutations in HSCs were strain-correlated and associated with HSC functional senescence during aging. An increased level of mtDNA mutations in single HSCs was observed in old B6 when compared with young B6 mice (P=0.003); in contrast, no significant age-dependent accumulation of mutations was observed in BALB mice (old versus young, P=0.202) and the level of mutations in both young and old BALB mice was close to that of old B6 mice (P>0.280). Cellular reactive oxygen species (ROS) in mouse HSCs could not be correlated with the level of mtDNA mutations in these cells, although B6 mice had a higher proportion of ROS– cells when compared with the BALB mice. Propagation assays of single HSCs showed B6 cells form larger colonies compared with cells from BALB mice, irrespective of age and mtDNA mutation load. We infer from our data that age-related mtDNA somatic mutation accumulation in mouse HSCs is influenced by the nuclear genetic background and that these mutations may not obviously correlate to either cellular ROS content or HSC senescence.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mitochondria, which provide the cell with most of its energy, are consequently also the major site of reactive oxygen species (ROS) generation. Mitochondria contain their own genome, and mitochondrial DNA (mtDNA) appears to accumulate somatic mutations over time (1–3). In the currently popular mitochondrial theory of aging, ROS are hypothesized to induce mtDNA alterations, leading ultimately to mitochondrial dysfunction and cellular and organ dysfunction (4–7). Indeed, numerous studies have provided evidence that mtDNA somatic mutations in aging cells and tissues are associated with increased level of ROS (1,8,9), despite controversies concerning this theory (10,11).

Recently, the mitochondrial theory of aging received more direct support from animal studies, in which aging was accelerated in mice that expressed a proofreading-deficient version of the mtDNA polymerase gamma (POLG) (12,13). Although POLG mutator mice accumulated mtDNA mutations in an approximately linear manner during postnatal life, massively increased oxidative stress has not been accompanied by the exponential accumulation of mtDNA mutations in these mutator mice (12,14). Induction of apoptosis triggered by accumulated mtDNA mutations, particularly in tissues with rapid cellular turnover, may be the mechanism for premature aging in POLG mutator mice (12). Transgenic mice that expressed a proofreading-deficient version of POLG targeted to heart developed dilated cardiomyopathy due to the increased level of mtDNA mutations in the heart (15); this mouse also showed no increase in oxidative stress in cardiac tissue (16) and had elevated numbers of apoptotic cells (17). However, the mtDNA mutation level in the POLG mutator mice was more than an order of magnitude greater than that observed with natural aging; furthermore, the phenotypes observed in the mtDNA mutator mice are shared with other premature aging mouse models (18,19), in which mtDNA mutations do not appear to be involved (20). As a result, some investigators have questioned whether the POLG mutator experiments are applicable to the mechanism of normal aging (20–22).

Blood cell production or hematopoiesis occurs in a hierarchical structure of cells based on a limited number of primitive stem cells, which continuously replenish mature erythrocytes, leucocytes and platelets by a process of commitment through progressively less primitive and more differentiated intermediate progenitor cells (23). The effect of aging on hematopoiesis long has been an active area of basic and clinical research interest (24–27). In an organism's lifetime, hematopoietic stem cells (HSCs) are mitotically quiescent, but they undergo a carefully regulated process of balanced self-renewal, required in order to maintain the stem cell pool and commitment to differentiate to mature progeny cells (23). Although injection of a single HSC into a lethally irradiated mouse can restore the entire lymphohematopoietic system (28,29), evaluation of the functionality of individual HSCs during aging is not trivial. A significant decrease in HSC functionality with age has been observed in previous studies by the measurement of homing, self-renewal, and lymphoid commitment (27,30–33).

We hypothesized that accumulated mtDNA somatic mutations in HSCs might underlie loss of stem cell function with age and perhaps be strain-correlated, suggesting modulation by the genetic background of the animal. Here, we analyzed mtDNA control region somatic mutation and hematopoietic colony formation in individual bone marrow (BM) HSCs from young and old C57BL/6 (B6) and BALB/cBy (BALB) mice, and we also measured the proportion of ROS-negative cells in different BM cell fractions. Our results indicate a remarkable difference in mtDNA somatic mutation levels in HSCs from B6 and BALB mice during normal aging.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
mtDNA somatic mutation in single HSCs in B6 and BALB mice
Using our recently established method for flow-cytometry sorting to single cells followed by DNA amplification and sequence determination for the mtDNA control region (34–36), we systematically analyzed mtDNA somatic mutation patterns in single HSCs from young and old B6 and BALB mice (Fig. 1; Supplementary Material, Table S1). We estimated the mtDNA mutation level in a population of single HSCs from each animal by two parameters: (i) the number of haplotypes identified and (ii) the proportion of cells harboring mutations when compared with the aggregate sequence. The first parameter corresponds to the total number of mutations (including nucleotide substitutions, insertions and deletions, or both) that have occurred within a population of cells, and the second may reflect effects from factors such as the time that a mutation occurred, genetic drift, and the magnitude of selection or clonal expansion of the subclones bearing a specific non-aggregate haplotype. These two parameters are not independent in regard to their biological significance. Drift and segregation may also influence the probability of detecting a cell with a particular mutation, as well as the proportion of cells with that mutation. Since nearly all the non-aggregate haplotypes occurred only once in an HSC cell population from each mouse in our current observation, both parameters yielded nearly identical results (Table 1; Supplementary Material, Table S1).


Figure 4571
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  		Figure 1. Screening for mtDNA sequence variation in single Lin–Kit+CD34– cells. (A) Representative staining and gating for single-cell sorting. (B) Two-step PCR amplification of single Lin–Kit+CD34– cells. Lanes 1–11 refer to different single cells and lane 12 is a negative control. (C) Mutation scoring based on the sequencing chromatographs. (D) Number of mtDNA haplotypes observed in a population of single Lin–Kit+CD34– cells from young and old B6 and BALB mice. The horizontal lines represent mean values.

 


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  		Table 1. Frequency of mtDNA mutations in single mouse HSCs and progenitors

 
Overall, there was a marginal strain effect (P<0.051) on mtDNA mutation level, as represented by the number of haplotypes per 100 cells, in which BALB mice (21.1±1.7) had a higher level of mutations than B6 mice (16.6±1.4). There was a significant age effect (P<0.002) on mtDNA mutations, as old mice (22.6±1.7) had more mtDNA mutations than young mice (15.2±1.4). The strain and age effects were mainly contributed by the lower level mtDNA mutation in young B6 mice, whereas the mutation frequency was similar among old B6, young BALB and old BALB mice. The unpaired t-test showed a statistically significant (P=0.003) increase of mtDNA mutation in single-cell populations from old B6 (21.92±1.63) compared with young B6 mice (11.34±1.87). This age-dependent mtDNA mutation accumulation pattern in B6 mice, however, was not evident in the BALB mice group: young BALB mice (19.03±1.99) had modestly fewer mtDNA haplotypes per 100 cells compared with old BALB mice (23.25±2.36), but the difference was not significant (P=0.202) (Fig. 1D). Furthermore, mtDNA sequence mutation level in young BALB mice was comparable to that of the old B6 mice (P=0.286>0.05), indicating an elevated level of somatic mutation in HSCs from young BALB mice. There was no significance difference between the old B6 and old BALB groups (P=0.656).

Proportion of ROS– cells in BM cell fractions of B6 and BALB mice
We quantified the proportion of ROS– cells in whole BM, Lin–, Lin–Kit+CD34– and Lin–Kit+CD34+ BM cells from young and old inbred B6 and BALB mice by the uptake of the fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCF-DA) to discern age and strain effects on cellular ROS content in different BM cell fractions. There was a general tendency of age-related decline in the proportion of ROS– cells in whole BM in both mouse strains (P>0.08; Fig. 2A). When ROS were measured in BM subfractions, we found that the proportion of ROS– cells in Lin– cell fraction was significantly lower in BALB (72.4±1.3%) than in B6 (78.2±1.6%) mice (P<0.03). Moreover, the two strains showed a significant difference with aging (P<0.01), as the percentage of ROS– cells in Lin– cell fraction increased with age in B6 mice [young (74.4±2.2%) versus old (82.1±2.2%)] but decreased in BALB mice (young, 76.3±1.8% versus old, 68.5±1.8%) (Fig. 2B). When ROS was measured in the further fractioned Lin–Kit+CD34– and Lin–Kit+CD34+ cell populations, we found that ROS– cell percentage was lower in Lin–Kit+CD34– cells (44.7±2.5% for B6; 35.9±2.0% for BALB) than in Lin–Kit+CD34+ cells (63.4±1.9% for B6; 60.6±1.5% for BALB) (Fig. 2B). There was no overall age difference in the proportion of ROS– cells among Lin–Kit+CD34– cells (P>0.23), which contain long-term functional HSCs, in either B6 or BALB mice. However, the proportion of ROS– Lin–Kit+CD34– cells was significantly (P<0.05) lower in BALB (35.9±2.0%) than in B6 mice (44.7±2.5%), suggesting that ROS are generally higher in HSCs from BALB mice.


Figure 4572
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  		Figure 2. Cellular ROS content in BM cells from young and old B6 and BALB mice. BM cells extracted from femurs and tibias of young and old B6 and BALB mice were stained with Lin-PE+CD34-PE-Cy5+CD117-APC+DCF-DA and analyzed by an LSR II flow cytometer. (A) A representative staining for whole BM cells showing the proportion of ROS– cells. (B) Summary of proportions of ROS– cells in whole BM, Lin–, Lin–Kit+CD34– and Lin–Kit+CD34+ cells. Results were shown as mean values with standard errors.

 
The proportion of ROS– cells in total BM cells showed very strong negative correlation to mtDNA mutation level observed in single HSCs: a coefficient of –0.82 between the number of haplotypes/100 cells and the proportion of ROS– cells in total BM cells (P<0.003). However, when we restricted our analysis to Lin–Kit+CD34– cells, we failed to observe such a correlation between ROS level and mtDNA mutation level (correlation coefficient=0.07, P=0.841). This result suggested that there might be no direct relationship between the cellular ROS level and the mtDNA mutation in mouse single HSCs.

Colony formation of single HSC cells in B6 and BALB mice
To functionally characterize HSCs and progenitor cells, we sorted single Lin–Kit+CD34– cells from young and old B6 and BALB mice and performed an in vitro culture assay in Dulbecco's modified essential medium (DMEM) supplemented with IL-3, IL-6 and SCF. The plating efficiency of individual cells from both B6 and BALB mice was similar (around 20%) and was not affected by age. As a measure of proliferative capacity, we visually grouped colonies into four grades (I–IV) according to the number of cells per colony (Fig. 3A). There was no obvious difference between the frequency of colonies in each grade for young and old mice from the same strain. However, B6 mice differed from BALB mice by having more large colonies belonging to grades III and IV and fewer small colonies to grades I and II (Fig. 3B).


Figure 4573
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  		Figure 3. Culture assay for single BM Lin–Kit+CD34– cells. (A) Four grades of colonies from single Lin–Kit+CD34– cells after 6-day culture. Each colony was grouped on the basis of the number of cells it harbored. (B) Calculated frequency of each colony grade with standard deviation in young and old B6 and BALB mice. Each of BALB mouse group contained three animals. For B6 mice, two old and eight young animals were used. Each animal was counted for 96 single Lin–Kit+CD34– cells, with the exception of two B6 young mice, whose BM cells were pooled together for staining and sorting, and we counted 480 single Lin–Kit+CD34– cells for these two mice.

 
mtDNA somatic mutation spectrum in single HSCs
To define whether there were specific mutational ‘hotspots’ in single HSCs during aging, as has been reported for the mtDNA control region in human fibroblasts, skeletal muscles and leukocytes (37–39), we enumerated somatic mutation ‘hits’ in cells from individual animals. As shown in Figure 4A, the site was given a score of 1 if a mutation at this site were present in a single mouse, irrespective of its frequency in single HSCs from the same mouse (which might be due to preferential clonal expansion). Mutations were almost evenly distributed over the entire mtDNA control region sequence. The majority of somatic mutations were transitions; indels (insertions and deletions) were sporadic and located at a region with the tandem repeat of the deleted or inserted nucleotide(s). Several sites, such as 15 572 and 15 836 [according to the numbering of a mouse complete mtDNA sequence AY172335 [GenBank] (40)], were affected four times in different young and old mice and might represent potential hot spots; however, scoring of the mutational spectrum might be inaccurate because of the small sample size.


Figure 4574
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  		Figure 4. mtDNA mutation spectrum in single Lin–Kit+CD34– cells from young and old B6 and BALB mice. (A) Mutation hits observed in all 31 animals studied in this study. Each site was scored according to the occurrence of the mutation in the animals, and the frequency of certain mutation in different cells from the same animal (which reflects clonal expansion) was ignored, for example, a site marked with 2 means this mutation was detected in two different animals. Sites were further denoted by different colors to highlight the mutation status other than nucleotide transition: red refers to the site with insertion and deletions (indels); blue refers to the site with transitions and indels; pink refers to the site with only transversions; yellow refers to the sites with transitions and transversions. (B) Frequency of transitions, transversions and indels according to the age and strain grouping. B6 old mice have higher frequency of transversions and indels compared with young B6 mice. There is a lower preferential occurrence of transition G>R (R means heteroplasmy for A and G) compared with the other transitions, for example, T>Y (Y means heteroplasmy for C and T).

 
We also counted the number of nucleotide substitutions and indels within each of the old and young mouse group according to strain (Supplementary Material, Table S1). Overall, transition G>A was less frequently observed than the other three transitions (C>T, T>C and A>G) in single cells. Nucleotide transversions and indels occurred more frequently in old B6 compared with young B6 mice, and the difference reached statistical significance (for indels, P=0.024; for transversions, P=0.045; Fisher's exact test). In BALB mice, however, there was no age-dependent difference in the occurrence of transversions and indels in single HSCs (Fig. 4B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Somatic mtDNA mutations, which have been observed in a variety of cells and tissues from mature animals, have been proposed as a biomarker of aging (9,41). In the current study, we analyzed mtDNA somatic mutations in single HSCs from young and old B6 and BALB inbred mice as an efficient method to detect low-frequency individual somatic mutations that are not well represented in tissue homogenates. We found that the age-dependent mtDNA mutation pattern was different in B6 and BALB mice: B6 mice acquired a significant level of mtDNA mutations with age, whereas BALB mice only showed a modest increase in mtDNA mutations over time, and furthermore, they demonstrated an elevated level of mtDNA mutations in young animals (Fig. 1 and Table 1). To our knowledge, this is the first report that provided direct evidence for the influence of genetic background on mtDNA somatic mutations during aging in mice. We only determined mtDNA somatic mutations in the control region sequence, and it is possible that mutations in coding region sequences may be differently affected by ROS or contribute to aging, especially if such mutations were deleterious and subject to strong selection pressure.

Similar to the strain effect on mtDNA mutation accumulation in single Lin–Kit+CD34– cells, we observed a significant strain difference in the level of cellular ROS content in the same cell type, in which the proportion of ROS– cells was lower in BALB than in B6 mice (Fig. 2B). In BALB mice, there was a slightly increased level of mtDNA mutation in single Lin–Kit+CD34– cells with age (Fig. 1D), and we observed fewer ROS– cells within this cell fraction in old animals compared with young animals, but the difference was not statistically significant. Despite that mtDNA mutation frequency in Lin–Kit+CD34– cells was significantly increased with age in B6 mice (Fig. 1D), there was no clear age effect on the frequency of ROS– cells in the same cell population (Fig. 2B). This result was not expected according to the hypothesis of a vicious cycle between mtDNA mutation and ROS (4,5,41) but was consistent with recent observations that the ROS level in the PLOG mutator mice was normal, notwithstanding the exponential accumulation of mtDNA somatic mutations (12,14,16). Although the somatic mutations in the mtDNA control region might not have a direct effect on ROS production, the high level of mtDNA mutations in B6 old mice would be expected to be associated with an elevated level of ROS in old animals according to the mitochondrial theory of aging, but this result was not observed. Others have shown that intracellular ROS concentration has an important regulatory role in mitochondrial oxidative phosphorylation and cell function and is itself under stringent control (42,43). Intriguingly, the single-cell culture assay demonstrated a strong effect of strain genetic background on the proliferative potential of HSC, as reflected in standard colony formation under the same culture conditions, with single Lin–Kit+CD34– cells from B6 mice forming much larger colonies compared with BALB mice, unrelated to age or the numbers of mtDNA mutations (Fig. 3). A parsimonious interpretation of above results would suggest that acquired mtDNA mutations are epiphenomenal rather than causal for an important functional difference in HSCs from these mice. On this point, we would argue that the senescence of mouse HSCs during aging may be unrelated to mtDNA mutations.

To date, besides a well-studied large deletion in mtDNA (9,44), reported results are in conflict regarding the pattern of age-associated mtDNA mutations in humans and mice. Attardi and colleagues (37–39) identified several age-dependent specific point mutations in the mtDNA control region in some human cells, but specific point mutations in skeletal muscle during normal aging were not observed by others (45). In liver from B6 mice (46), there was an increasing level of mtDNA mutation with age, and one mutation [16012 G>A according to the numbering of sequence AY172335 [GenBank] (40)] occurred in two of the three old mice analyzed. In contrast, another group (47) failed to identify any specific point mutations in mtDNA control region in a variety of tissues from aged B6 mice. The explanation for these discrepancies is unclear, but different techniques used for mutation screening could be the major factor. The very large number of single cells analyzed in our study should allow a tentative summary of the mtDNA mutation spectrum in mouse HSCs. In general, mutations appeared to accumulate randomly and evenly across mtDNA control region sequence; most of nucleotide sites were singlets, and we only observed a few sites mutated in single cells from two or more different animals irrespective of age (Fig. 4). The seeming age-dependent specific mutation 16012 G>A described by Khaidakov et al. (46) was found only once in our study. We would conclude from our data that there is no age-dependent specific mtDNA control region mutation in mouse single HSCs.

In summary, based on mtDNA mutation number and type, cellular ROS content and the colony-formation capacity of single Lin–Kit+CD34– cells from young and old inbred mice, our results are not supportive of a simple theory of mitochondrial aging in stem cells. The quantity of mutations observed was not related to or insufficient to suggest a ‘vicious cycle’ between ROS production and mtDNA damage in HSCs. On the contrary, we found that the mtDNA mutation process, ROS level and the colony-formation capacity in Lin– Kit+CD34– cells were more influenced by the strain genetic background. mtDNA control region sequence mutations may represent an end result of natural aging rather than be causative of age-related degeneration of the hematopoietic system in the mouse.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals
Inbred C57BL/6 (B6) and BALB/cBy (BALB) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and were housed and bred at the National Institutes of Health animal facility (Bethesda) under standard care and nutrition condition. Mice were used in the following age ranges: old B6 (24–28 months); young B6 (2–3 months); old BALB (23–25 months); young BALB (2–3 months). We also used National Institute on Aging animal colony maintained by Harlan, Inc. (Indianapolis, USA) as an alternative source of young and old B6 and BALB mice.

BM cell preparations
BM cells were flushed from two femurs and two tibias of each mouse into 2 ml of flow buffer (2.68 mM KCl, 1.62 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 7.69 mM NaN3 and 1% BSA) using a 25-gauge needle and filtered through a 90 µm nylon mesh (Small Parts Inc., Miami Lakes, FL, USA) to prepare for single-cell suspensions. Cells were then incubated in Gey's solution (130.68 mM NH4Cl, 4.96 mM KCl, 0.82 mM Na2HPO4, 0.16 mM KH2PO4, 5.55 mM dextrose, 1.03 mM MgCl2, 0.28 mM MgSO4, 1.53 mM CaCl2 and 13.39 mM NaHCO3) for 10 min on ice to lyse red blood cells. Cells were counted using a Vial-Cell analyzer (Beckman-Coulter Corp, Hialeah, FL, USA), whereas total BM cells per mouse was calculated on the basis of the assumption that two tibiae and two femurs contain 25% of all BM cells in the body.

ROS measurement
Monoclonal antibodies for murine CD3 (clone 145-2C11), CD4 (clone GK 1.5), CD8 (clone 53-6.72), CD11b (clone M1/70), CD19 (clone ID3), CD34 (clone RAM34), CD117 (Kit, clone 2B8) and erythroid cells (clone Ter119) were all purchased from BD Biosciences (San Jose, CA, USA). Each antibody was conjugated with fluorescein isothyocyanate, phycoerythrin (PE), CyChrome or allophycocyanin (APC). The level of cellular ROS was measured by using the fluorescent probe DCF-DA. In brief, BM cells were first incubated with a pre-mixed antibody cocktail [CD34-PE-Cy5+lineage (CD3, CD4, CD8, CD11b, CD19, Gr1, Ter119)-PE+CD117 (Kit)-APC] on ice for 30 min and were then incubated with 20µM DCF-DA at 37°C for 30 min. After washing with FACS buffer (PBS supplemented with 5% FBS and 0.05% NaN3), the cells were analyzed on a BD-LSR II flow cytometer (Becton Dickinson, San Jose, CA, USA).

Single-cell sorting
BM cells from individual donors were flushed into Iscove's modified Dulbecco's medium, ATCC, Manasses, VA, USA). After lysing erythrocytes with Gey's solution, cells were stained for 30 min on ice with the CD34-PE-Cy5+lineage (CD3, CD4, CD8, CD11b, CD19, Gr1, Ter119)-PE+ CD117-APC antibody cocktail and then sorted by a MoFlo cell sorter (Dako-Cytomation Inc., Ft Collins, CO, USA).

Single-cell PCR amplification and sequencing
Single Lin–Kit+CD34– cells were sorted into each well of a 96-well plate containing 50 µl of lysis buffer [10 mmol/l Tris–HCl (pH 8.0), 50 mmol/l KCl, 100 µg/ml proteinase K, 1% Triton X-100]. After an incubation at 56°C for 20 min to digest the cell, the whole plate was then heated at 96°C for 8 min to inactivate proteinase K in the lysate. Two-step nested PCR was used to generate enough DNA for sequencing mtDNA control region in single cells. The first PCR was performed in 30 µl of reaction mixture, which contains 5 µl of cell lysate, 400 µM of each dNTP, 1x LA PCRTM Buffer II (Mg2+ plus), 1 unit of TaKaRa LA TaqTM with proofreading activity (Takara Bio. Inc.), and 0.5 µM of each forward and reverse outer primer (mF15335: 5'- GTCTTGTAAACCTG AAATGAA –3' and mR171: 5'-AATGTGCTTGATACCCTCTCC-3'). PCR amplification was performed on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) with the following cycles: one cycle of 94°C for 3 minute; then 35 cycles of 94°C for 30 sec, 50°C for 40 sec and 72°C for 1 min with a 5 sec increase per cycle; and ending with a full extension cycle of 72°C for 10 min. The second PCR was performed in 50 µl of reaction mixture containing 400 µM of each dNTP, 1x LA PCRTM Buffer II (Mg2+ plus), 2 units of TaKaRa LA TaqTM, 0.5 µM of each forward and reverse inner primers (mF15368: 5'-CAAGACATCAAGAAGAAGGA-3' and mR16: 5'-TTTCAGTGCTTTGCTTTGTTA-3') and 5 µl of first PCR product under the same amplification condition as the first PCR, but with an annealing temperature of 52°C and an extension time of 90 s at 72°C per cycle.

The second PCR products were purified using the QIA quick PCR purification kit (Qiagen, Valencia, CA, USA) and were directly sequenced by using BigDye Terminator v3.1 Cycle Sequencing Kit on a 3100 DNA sequencer (Applied Biosystems) according to the manufacturer's manual. Three inner primers (mF15387: 5'-AGCTACTCCCCACCACCAGC-3'; mF15713: 5'-ATCCTCCGTGAAACCAACAA-3'; mR15797: 5'-TTTGATGGCCCTGAAGTAAG-3') and primer mR16 were used to overlap the sequence of the whole mtDNA control region. Sequence variation was scored relative to the mouse complete mtDNA reference sequence (AY172335 [GenBank] ) (40) on the basis of the original sequencing chromatograph. Because the limitation of the resolution of mtDNA heteroplasmy in the chromatograph, we only scored these sites with a heteroplasmy over 10% (Fig. 1). Extreme care was taken during PCR to avoid potential contamination.

Hematopoietic stem cell culture
Individual Lin–Kit+CD34– cells from B6 and BALB mice were sorted into each well of a 96-well plate containing 100 µl of complete DMEM (Cellgro Media Tech, VA, USA) supplemented with 15% heat-inactivated fetal bovine serum, 1% penicillin, 1% streptomycin, 1% L-glutamine, 3 ng/ml IL-3, 5 ng/ml IL-6 and 25 ng/ml SCF (R&D Systems, Minneapolis, MN, USA). Cells were cultured at 37°C with 5% CO2 for 6 days. Colonies from single cells were captured under a phrase-contrast microscope and were scored into four grades according to the number of cells in each colony: grade 1, 20 or fewer cells/well; grade 2, 21–70 cells/well; grade 3, 71–150 cells/well; grade 4, 151 or more cells/well (Fig. 3A). Plating efficiency was defined as the number of wells with cell colonies divided by the total number of wells plated.

Statistics
Data collected from various assays were statistically analyzed using the JMP statistical discovery software using multi-factor least square models for main effects and interactions (SAS Institute Inc., Cary, NC, 00). For mtDNA mutations in single Lin–Kit+CD34– cells, we focussed on the comparison of the number of the mutations (or haplotypes) that identified in a given number of single HSCs from the young and old mice. Results were shown as mean values with standard errors. Statistical significance was declared at P<0.05 level.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The authors thank Ms Leigh Samsel from Flow Cytometry Core Facility and Mr Keyvan Keyvanfar from Hematology Branch, National Heart, Lung and Blood Institute for technical assistance in cell sorting.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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