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(The FASEB Journal. 1999;13:707-713.)
© 1999 FASEB

Research Communications

Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span

GERALD DE HAANa,b and GARY VAN ZANTa,*


a Blood and Marrow Transplant Program, Division of Hematology/Oncology, University of Kentucky Medical Center, Lexington, Kentucky 40536-0093, USA; and

b Department of Physiological Chemistry, University of Groningen, Groningen, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal somatic cells undergo replicative senescence in vitro but the significance of this process in organismic aging remains controversial. We have shown previously that hemopoietic stem cells of common inbred strains of mice vary widely in cycling activity and that this parameter is inversely correlated with strain-dependent mean life span. To assess whether cell cycling and life span are causally related, we searched for quantitative trait loci (QTLs) that contributed to variation of these traits in BXH and BXD recombinant inbred mice. Two QTLs, mapping to exactly the same intervals on chromosomes 7 and 11, were identified that were associated with variation of both cell cycling and life span. The locus on chromosome 11 mapped to the cytokine cluster, a segment that shows synteny with human chromosome 5q, in which deletions are strongly associated with myelodysplastic syndrome. These data indicate that steady-state cell turn-over, here measured in hemopoietic progenitor cells, may have a significant effect on the mean life span of mammals. de Haan, G., Van Zant, G. Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span.


Key Words: stem cells • longevity • cell proliferation • aging • quantitative trait locus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MATURE BLOOD CELLS in the circulation are derived from primitive hemopoietic stem cells residing in the bone marrow. A hierarchy of distinct stem cell subsets can be recognized in which the most primitive, long-term repopulating cells have a very low fraction of cells in S-phase, whereas more committed, but still pluripotent, progenitors have a much higher proliferative activity 1-3) . However, we have shown that the number of progenitor cells in S-phase varies widely between different inbred strains of mice 4-6) ; it is inversely correlated with mean mouse life span and decreases during aging in a strain-dependent fashion 5, 6) . In addition, we demonstrated in chimeric mice, constructed by aggregating embryos of strains with intrinsic differences in hemopoietic stem cell cycling, that the more proliferative stem cell population (DBA/2) underwent replicative senescence after ~1.5–2 years (about the normal life span of this strain), leaving all subsequent blood cell formation in the chimera to the coexistent, largely quiescent stem cells of the long-lived genotype (C57BL/6) (7) . Others have shown that the proliferative potential of individual human hemopoietic stem cells decreases greatly during development from fetal liver to cord blood to adult bone marrow, and this is inversely correlated with the mean telomere length of such cell populations (8) . Recent clinical findings have shown that accelerated telomere shortening occurs after stem cell transplantation where the relatively few stem cells present in the graft are subjected to intense replicative stress during engraftment 9, 10) . These observations on the kinetics of blood cell formation were reminiscent of the data originally presented by Hayflick et al. (11) on the kinetics of fibroblast growth. It was proposed that normal somatic cells have an upper limit to replication, and once this is reached, proliferation decreases and cells ultimately senesce. Recently, it has been shown that telomeres play a role in this process, since expression of telomerase in cells that normally do not express this enzyme delays or prevents senescence (12) . It has been suggested that this cellular mitotic clock also sets the limits of organismic life span, since there is an inverse correlation between the proliferation potential of fibroblasts and the age of the donor (13) . Collectively, these data indicate a potential relationship between cell proliferation and in vivo aging.

To demonstrate that cell proliferation is indeed associated with organismic life span, genes that play a role in both processes have to be identified. As a first step toward this goal, we searched for quantitative trait loci (QTLs)1 that contribute to hemopoietic progenitor cell cycling and mouse life span in BXD and BXH recombinant inbred mice. These genetically mosaic mice are derived from C57BL/6 (low cycling stem cells, long life span) and DBA/2 or C3H/He mice (high cycling stem cells, short life span). Typing these strains for a phenotype of interest may result in a map position harboring the gene that causes the phenotypic variation between the two strains. We demonstrate a strong negative correlation between mouse life span and stem cell cycling in BXD mice, and report the chromosomal map position of two loci, mapping to the same intervals on chromosomes 7 and 11, that were associated with both stem cell cycling and mouse life span. Independent confirmation of the involvement of the locus on chromosome 11 was obtained from the evaluation of BXH mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female C57BL/6J, DBA/2J, C3H/HeJ and recombinant inbred BXD/Ty and BXH/Ty mice were purchased from The Jackson Laboratory (Bar Harbor, Maine) and were used at an age of 6-8 wk. Mice were kept in microisolators and were fed sterilized food and water.

Bone marrow cells were flushed from femora obtained from three mice, pooled, and used in the cobblestone area-forming cell (CAFC) assay.

CAFC assay
The CAFC assay, as described by Ploemacher et al. (14) , was slightly modified, as published earlier (15) . This in vitro assay allows quantification of primitive hemopoietic cell subsets of various developmental stages. Cells of the stromal cell line FBMD-1 (16) were seeded in 96-wells plates (Costar, Cambridge, Mass.) in Dulbecco' modified Eagle's medium containing L-glutamine (Life Technologies, Grand Island, N.Y.), supplemented with 5% horse serum and 15% fetal bovine serum (both from Life Technologies), 10-4 mol/l ß-mercaptoethanol, 10-5 mol/l hydrocortisone (Sigma, St Louis, Mo.), 80 U/ml penicillin and 80 µg/ml streptomycin (both from Life Technologies), and 25 mmol/l NaHCO3. Plates were incubated at 33°C in 5% CO2 and used 10-14 days after seeding. Bone marrow cells were harvested from the mice and a nucleated cell count was performed. To measure the percentage of cells in the S-phase of the cell cycle, we used a hydroxyurea suicide technique, as described previously (5) . To this end, two identical samples were diluted to a concentration of 1 x 107 cells/ml. Hydroxyurea (Sigma) was added to one sample at a concentration of 200 µg/ml and both samples were incubated at 33°C for 1 h After incubation, both cell suspensions were washed and a nucleated cell count was performed again. Based on this cell count, the CAFC assay was initiated. Bone marrow cells were seeded on the stromal layer in six dilutions, each cell concentration threefold apart (81,000 cells/well being the highest cell concentration, 333 cells/well the lowest). At this time the medium was switched from 5% horse serum and 15% fetal bovine serum to 20% horse serum. For each cell dilution, 40 replicate wells were used. After 1 wk, all wells were evaluated for the presence or absence of cobblestone areas, defined as colonies of at least five small nonrefractile cells that grow underneath the stromal cells. Colonies scored after 7 days (i.e., CAFC day 7) have been shown to be derived from pluripotent progenitor cells 3, 17) . The fraction of cells killed by hydroxyurea was calculated by dividing the estimated CAFC frequency in the hydroxyurea-treated cell suspension by the control value.

Linkage analysis using BXD RI strains
The percentage of CAFC day 7 in S-phase was calculated as described above, and the strain distribution pattern was used to search genome-wide for QTLs that affect the cycling of this cell stage. This was done by entering the values for all 26 BXD strains into the software program MapManager QT (b15) (18) , which computes regression statistics between variation in phenotype and variation in alleles at loci that have already been mapped. At present, some 1700 loci have been mapped in the BXD strains. Similarly, mean life span data for the BXD strains obtained from a study published by Gelman et al. (19) were used to search for QTLs associated with mean life span.

Confirmation of linkage using BXH RI strains
We tested all 12 BXH RI strains for progenitor cell cycling in a manner similar to that described above. After an initial genome-wide screen, Iapls2-37 on chromosome 10 was found to be linked to the trait. Next, a composite interval mapping strategy was used to search for additional loci with modifying effects on cell cycling. This was done as recently described (20) , by performing a second genome-wide search, but now controlling for Iapls2-37. Thus, an analysis is conducted to search for loci to explain remaining variation not accounted for by the primary QTL (i.e., Iapls2-37). To assess the effects of such secondary loci on the primary QTL, the interval mapping procedure on chromosome 10 was repeated by correcting for these modifiers.

Linkage statistics
To establish criteria for suggestive and significant linkage (21) , a permutation test was performed using MapManager QT (1000 permutations at 1 cM intervals (22) . This test compares the peak LOD score obtained for a given data set with the peak LOD scores obtained for 1000 random permutations of the same data set (20) . To assess genome-wide significance, point-wise probabilities (P) for both RI sets for the same interval were pooled using Fisher's equation: {chi}2 = -2(lnpBXD + lnpBXH), with 4 degrees of freedom (20) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of variation in hemopoietic progenitor cell cycling in BXD recombinant inbred strains
The percentage of CAFC day 7 in S-phase among the BXD strains ranged from undetectable to ~60% of the population (Fig. 1 ). Some strains (BXD 9, 19, 20, 25, 30) had fewer cells in S-phase than parental C57BL/6, and conversely, several strains (BXD 1, 5, 6, 11, 16, 21, 31, 32) had more cells in S-phase than did DBA/2. Since no mendelian distribution was observed, it was clear that the variation in progenitor cell cycling between C57BL/6 and DBA/2 is caused by multiple, quantitative loci. The extreme differences between `outlier' BXD strains probably results from the random shuffling of different combinations of B6 and DBA alleles that affect cell cycling, so that some strains inherit all the low cycling alleles and other strains inherit all the high cycling alleles. It is also apparent that B6 mice must contain some alleles associated with high cycling that are masked by more dominant low cycling alleles (otherwise strains that have a lower cycling than B6 would not have been found); conversely, DBA mice must contain some low cycling alleles.



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  		Figure 1. Strain distribution pattern for CAFC day 7 cycling in 26 BXD and parental C57BL/6 (B6) and DBA/2 strains. Bars indicate standard error of the mean. Note that many strains have a smaller or larger population of CAFC day 7 in S-phase than the parental strains.

Hemopoietic progenitor cell cycling is inversely correlated with mean mouse life span
Our data as presented in Fig. 1 were compared with data obtained by Gelman et al. (19) . In this study, life span measurements of 22 BXD strains (10–20 mice per strain) were performed under carefully controlled housing conditions. For example, sentinel animals were present at all times to monitor for microbial pathogens. Histological necropsies revealed a wide range of death causes without any clear patterns. Figure 2 shows that, in young BXD mice, progenitor cell cycling and mean strain life span were significantly inversely correlated (P=0.0093), as we previously found in a survey of eight commonly used inbred strains (6) . Approximately 30% of the variation in mean life span could be explained, and indeed predicted, by differences in cycling (r2 = 0.29).



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  		Figure 2. Progenitor cell cycling and mean life span are inversely correlated in BXD recombinant inbred mice. The data as shown in Fig. 1 were related to data obtained by Gelman et al. (19) on the mean life span of 22 BXD strains. A significant negative linear correlation was demonstrated (Y= 60.605 - 0.06 X, r2 = 0.29, P=0.0093). The numbers next to each circle refer to the BXD strain numbers. The black squares represent the parental C57BL/6 (B6) and DBA/2 (DBA) mice (49) . No life span data were available for BXD strains 13, 20, 21, and 25. The dotted lines represent the 95% confidence intervals.

Mapping of loci associated with progenitor cell cycling and mouse life span
The strain distribution patterns for progenitor cell cycling and life span as shown in Figs. 1 and 2 were used to search for QTLs most strongly associated with these traits using MapManager QT (b15) software (18) . A genome-wide search for linkage with progenitor cell cycling revealed four putative loci; one major QTL on chromosome 11, reaching a genome-wide threshold required for suggestive linkage 21, 22) , and three weaker ones on chromosomes 4, 7, and 9 (Table 1 ). Similarly, the data of Gelman et al. (19) were used to search for QTLs affecting life span. Four regions were identified; two major QTLs on chromosomes 2 and 7, also reaching the genome-wide levels required for suggestive linkage, and two weaker ones on chromosomes 4 and 11 (Table 1) . These data were gathered during 1982–1986 and published in 1988, when the mouse genetic map was not dense and mapped traits in the BXD set in particular were sparse. The QTL on chromosome 11 was not detected in the original study because insufficient markers on that chromosome were available at that time (19) . In addition, the authors had to omit data from BXD strains 31 and 32 from their analysis since these strains had not been genotyped well enough at that time.


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  		Table 1. Quantitative trait loci most strongly associated with hemopoietic progenitor cell cycling and mean mouse life span in BXD recombinant inbred micea

It is striking that of the four loci that we found in each individual search, two were shared by both traits: one on chromosome 11 (near D11Ncvs76) and one on chromosome 7 (near D7Ncvs38). Subsequent interval mapping of both progenitor cell cycling and mouse life span resulted in a remarkably similar linkage pattern, with coincident peaks at chromosome 11, 31.0 cM (Fig. 3 A) and at chromosome 7, 6.0 cM (not shown).



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  		Figure 3. A) QTLs affecting progenitor cell cycling and mouse life span map to the same interval on chromosome 11. LOD score plot showing results of the interval mapping of CAFC day 7 cycling (open squares) and mean life span (closed squares) on chromosome 11. The corresponding map position of orthologous genes in human are shown at the top of the figure. B) Genotypic segregation of BXD strains according to the distribution of C57BL/6 (B) and DBA/2 (D) alleles at D7Ncvs38 and D11Ncvs76. Black bars indicate hemopoietic progenitor cell cycling (left y axis, ± standard deviation). Hatched bars show strain life span (right y axis, ± standard deviation). Long-lived strains type B for both loci and have a low percentage of cycling progenitors. Shorter lived strains type D for both loci and have a high percentage of cycling progenitors.

Figure 3B shows how the BXD strains segregate into the two extreme genotypes for these two loci on chromosomes 7 and 11. C57BL/6 alleles at both D7Ncvs38 and D11Ncvs76 were associated with a longer life span (mean of 765 days) and low progenitor cell cycling (9.2%), whereas the corresponding DBA/2 alleles predisposed animals to a 25% shorter life span (mean of 592 days) and high progenitor cycling (32.4%). Linkage to D2Rik63 and Ms6hm, the other two life span QTLs (Table 1) , predisposed to a long life span in strains that had the D alleles (i.e., opposite the parental phenotype), and these markers were not associated with cell cycling.

Analysis of variation in hemopoietic progenitor cell cycling in BXH strains
C3H/He mice show an even more pronounced progenitor cell proliferation than do DBA/2 mice 4-6) , and therefore we also evaluated variation in cell cycling in BXH recombinant inbred mice (derived from C57BL/6 and C3H/He). Figure 4 A depicts the strain distribution pattern for the 12 BXH strains. No life span data for BXH strains are available, so we could only perform a linkage analysis for the cycling trait. As described above, a genome-wide search was carried out using the data presented in Fig. 4A . Significant genome-wide linkage (LOD=3.7, point-wise P=7.3 x 10-5, genome-wide P=0.007) was found with a locus on chromosome 10, Iapls2-37, mapping ~60 cM from the centromere (Fig. 4B ). This is very close to the Steel locus encoding the hemopoietic cytokine c-kit ligand (also known as stem cell factor or mast cell growth factor). Since it was obvious from the BXH phenotypes that more than one locus was involved in the variation in cell cycling and because we had obtained sufficient statistical power that allowed us to suspect the involvement of a locus on chromosome 10, we used the composite interval mapping approach, as recently described by Williams et al. (20) . In such an analysis, a second search is initiated for loci associated with the phenotypic variation that is unaccounted for by the primary QTL. To do this we performed a second genome-wide search using all BXH data, but now controlling for Iapls2-37. Using this approach, linkage was observed with loci on chromosome 11 (Iapls1-74, Scya3, and Xmv42, peak LOD score 2.8, point-wise P value 0.00036), bracketing the peak shown in Fig. 3A obtained in the BXD set. Thus, we obtained independent evidence of the involvement of a locus on chromosome 11 in progenitor cell cycling (the combined LOD score was 7.34, point-wise P value = 8.37 x 10-7, genome-wide P<0.001). When we controlled for the loci on chromosome 11, the LOD score for the locus on chromosome 10 in the BXH set greatly improved (to LOD = 6.45, point-wise P=4.93 x 10-8, genome-wide P<0.001, Fig. 4B ), again confirming the veracity of the chromosome 11 QTL. We propose to name the QTL on chromosome 7 `stem cell proliferation 1' (Scp1), the locus on chromosome 11 Scp2, and the locus on chromosome 10, mapped in the BXH set, Scp3.



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  		Figure 4. A) Strain distribution pattern for CAFC day 7 cycling in 12 BXH strains. Values for parental C57BL/6 (B6) and C3H/He are indicated. B) LOD score plot showing the results of the primary interval mapping of CAFC day 7 cycling in BXH mice on chromosome 10 (open squares). Results of the composite interval mapping controlling for modifying loci on chromosome 11 are indicated by filled squares.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Hemopoietic stem cells of different inbred strains of mice demonstrate a genotype-specific proliferative activity that is inversely correlated with organismal life span 5, 6) . We have mapped loci associated with cell cycle variation between C57BL/6, DBA/2, and C3H/He mice, and demonstrate that two of these loci also contribute to life span variation in these strains. These results argue compellingly for a causal relationship between hemopoietic cell cycling and mouse life span, and provide evidence for the importance of replicative cycling in the aging process in vivo.

Our earlier studies have shown that if highly cycling hemopoietic stem cells of short-lived DBA/2 genotype are put in competition with slowly cycling stem cells of C57BL/6 genotype in the common environment of embryo aggregation chimeras, mature blood cells of the DBA/2 genotype disappear from the circulation at a time roughly equivalent to the maximal life span of that strain (7) . These and other results have led us to argue that true self-renewal of individual hemopoietic stem cells may not exist and that the number of stem cells is finite (23) . The aging of stem cells (i.e., decline of stem cell quality) would be dependent on the rate of cycling of these cells, which we have shown to be highly strain dependent and inversely correlated with organismal life span. Stem cells of long-lived C57BL/6, whether measured by long-term repopulating ability 24, 25) , cell surface phenotype determined by flow cytometry (26) , or using the CAFC assay 5, 27) , have not been shown to be detrimentally affected by aging, whereas stem cell activity of shorter lived CBA/J (25) and DBA/2 5, 27) strains clearly declines with age.

To map loci that contributed to this strain-dependent variation in proliferative activity of stem cells, we used BXD and BXH recombinant inbred mice. The analytical power of using these recombinant inbred strains resides in the fact that the genotype is fixed, so that one can go back to study a different trait in the same genetic background. This is clearly emphasized by our current study, where we compared a strain distribution pattern for cell cycling with life span data obtained 15 years ago (19) . This advantage has been underscored by Plomin et al. (28) , who have established a BXD recombinant inbred-QTL Cooperative Data Registry that enables comparison of longevity data to other traits reported in the literature concerning the BXD series (28) . To date, they have not been able to correlate any of some 137 BXD-phenotypes with longevity (29) . In this report we present the first such significant correlation. Our analysis resulted in the identification of four putative intervals associated with cell cycling. It is worth noting that when these were compared with those obtained by searching for life span loci, two of four independently mapped QTLs associated with these traits in BXD mice mapped to the same intervals. The individual QTLs reached the statistical criteria required for suggestive linkage, and combining BXD and BXH data by using composite interval mapping strategies resulted in the identification of a significantly linked QTL on chromosome 11 20, 21) . The strain distribution pattern for stem cell cycling that we present in Fig. 1 is almost an exact mirror image of the pattern reported by Muller-Sieburg (30) , where the frequency of long-term culture-initiating cells in BXD strains was measured. At present we have no clear explanation for this finding.

Together with our finding that, in young animals from eight standard inbred strains of mice, progenitor cell cycling and life span are inversely correlated (6) , this in vivo study suggests strongly that genes affecting the intrinsic rate of cell cycling also affect the rate of aging of mammalian organisms. The loci we have mapped contain genes that regulate cell cycling in at least one, but potentially more, critical renewing tissues. The nature of these genes remains to be elucidated and is under study. The genomic segment encompassing D11Ncvs76 contains the mouse cytokine cluster (i.e., genes for interleukins 3, 4, 5, 13, and granulocyte-macrophage colony-stimulating factor map here) and is syntenic to human 5q31. Perhaps not coincidentally, we have recently mapped a QTL significantly associated with hemopoietic stem cell (CAFC day 35) frequency to a region of mouse chromosome 18 that is also syntenic to human 5q (6) . Deletions of this chromosomal region in humans are associated with the 5q- myelodysplastic syndrome 31-37) .

Aging is clearly a multifactorial process 19, 38-42) , but our study suggests that, like recent advances made in this field in Caenorhabditis elegans and Saccharomyces cerevisiae 43, 44) , it is open to genetic analysis in mammals. Our findings support a model in which the rate of aging of an organism is partly the result of proliferation kinetics of certain critical tissues/organs. Inherent differences in cell cycle kinetics may determine the speed at which Hayflick's mitotic clock ticks 11, 45) and telomeres shorten 12, 13, 46) , but may also alter the rate at which potentially fatal DNA damage accumulates 39, 47, 48) . Genetic and/or environmental variations affecting either of these parameters may alter potential life span.


   ACKNOWLEDGMENTS
 
This work was supported by funds provided by the Lucille P. Markey Cancer Center, the University of Kentucky Hospital, and the Department of Internal Medicine. G.d.H. is supported by a fellowship awarded by The Netherlands Organization for Scientific Research. The authors thank Dr. Rob Williams for invaluable assistance with MapManager, Dr. Stephen Szilvassy for critically reading the manuscript, and Melissa Flett for statistical analyses.


   FOOTNOTES
 
* Correspondence: Blood and Marrow Transplant Program, Division of Hematology/Oncology, 800 Rose Street, University of Kentucky Medical Center, Lexington, KY 40536-0093, USA. E-mail: gvzant1{at}pop.uky.edu

1 Abbreviations: CAFC, cobblestone area-forming cell; QTLs, quantitative trait loci; Scp1, stem cell proliferation 1 locus on chromosome 7; Scp2, locus on chromosome 11; Scp3, locus on chromosome 10 mapped in the BXH set.

Received for publication September 29, 1998. Revision received November 23, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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