|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3294-3301
Dynamic Changes in Mouse Hematopoietic Stem Cell Numbers During Aging
By
Gerald de Haan and
Gary Van Zant
From the Blood and Marrow Transplant Program, Division of
Hematology/Oncology, University of Kentucky, Markey Cancer Center,
Lexington, KY; and the Department of Physiological Chemistry,
University of Groningen, Groningen, the Netherlands.
 |
ABSTRACT |
To address the fundamental question of whether or not stem cell
populations age, we performed quantitative measurements of the cycling
status and frequency of hematopoietic stem cells in long-lived C57BL/6
(B6) and short-lived DBA/2 (DBA) mice at different developmental and
aging stages. The frequency of cobblestone area-forming cells (CAFC)
day-35 in DBA fetal liver was twofold to threefold higher than in B6
mice, and by late gestation, the total stem cell number was nearly as
large as that of young DBA adults. Following a further  50% increase
in stem cells between 6 weeks and 1 year of age, numbers in old DBA
mice dropped precipitously between 12 and 20 months of age. In marked
contrast, this stem cell population in B6 mice increased at a constant
rate from late gestation to 20 months of age with no signs of
abatement. Throughout development an inverse correlation was observed
between stem cell numbers and the percentage of cells in S-phase.
Because a strong genetic component contributed to the changes in stem
cell numbers during aging, we quantified stem cells of 20-month old BXD
recombinant inbred (RI) mice, derived from B6 and DBA progenitor
strains, thus permitting detailed interstrain genetic analysis. For
each BXD strain we calculated the stem cell increase or decrease as mice aged from 2 to 20 months. Net changes in CAFC-day 35 numbers among
BXD strains ranged from an 10-fold decrease to an 10-fold increase. A genome-wide search for loci associated with this
quantitative trait was performed. Several loci contribute to the
trait putative loci map to chromosomes X, 2, and 14. We conclude that
stem cell numbers fluctuate widely during aging and that this has a
strong genetic basis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HEMATOPOIETIC STEM CELLS are generally
believed to possess self-replicating potential that not only sustains
lifelong blood cell production, but embodies a reserve potential
capable of meeting needs not normally encountered. For example, serial transplantation studies have shown that not only is a small fraction of
bone marrow stem cells from a single animal capable of restoring hematopoiesis in multiple lethally irradiated recipients, but that
single clones are capable of doing this in secondary, tertiary, and
quaternary hosts over a cumulative period that exceeds the lifespan of
the donor.1-3 Thus it may seem that stem cells escape deleterious changes culminating in the senescence that befalls somatic
cells of almost all other tissues during aging.4 But is
this really so? The question has been addressed repeatedly over the
years usually by transplantation assays, which are convenient, but bear
only partial similarity to natural changes accompanying aging.2,5-8 Normal hematopoiesis and engraftment in a
transplantation setting share a common dependence on the numbers of
stem cells present and their proliferative potential, either of which
may be independently affected by aging. As persuasive evidence of this,
during ontogeny, while stem cell numbers exponentially increased, the
proliferative potential of sorted human stem cells, as measured by the
number of progeny generated in vitro, was shown to decrease rapidly.9 In a similar vein, mouse C57BL/6 (B6) and
CBA fetal liver stem cells have a higher repopulating
ability than their adult marrow counterparts.10-13
Moreover, we have recently extended and confirmed data showing that
during a good part of the entire mouse lifespan stem/progenitor cell
cycling decreased from near a theoretical maximum to almost
undetectable levels,14,15 a finding consistent with
observations made in many other aging tissues.4
Surprisingly, stem cell frequencies, measured either by flow cytometry
in long-term bone marrow culture or by in vivo retransplantation
assays, have been shown to significantly increase during aging, at
least in the C57BL/6 mouse.14,16,17 This apparently
counterintuitive finding is not easy to explain, but we have recently
argued that the aging stem cell pool may have lower residual
proliferative potential or `quality', as it presumably has
collectively completed more cell cycles.18 A gradual loss of proliferative capacity in dividing cells has been attributed to an
erosion of telomeres, which may thus serve as a cell's mitotic clock.19,20 Ectopic expression of telomerase in normally
telomerase-negative fibroblasts has recently been shown to promote
telomere lengthening and, most importantly, a not yet defined, but
substantial, extension of proliferative potential.21 Other
findings support the notion that a telomere-driven mitotic clock may be
operative in the hematopoietic system, as it has been shown that
telomeres of hematopoietic cells shorten significantly during aging and
after bone marrow transplantation,20,22 despite the fact
that stem cells express low levels of telomerase.23-25
It has been established, using a variety of experimental approaches,
that a strong genetic component underlies the proliferation and
population size of stem cells in different mouse strains, including B6
and DBA/2 (DBA).26-30 We have recently mapped to chromosome 18 a locus affecting stem cell frequency in young adult
mice.30 To better understand the genetic regulation
underlying stem cell dynamics during aging of B6 and DBA mice, we
compared stem cell numbers obtained in young (2 months) and old (20 months) BXD recombinant inbred strains. Our data show that multiple
quantitative trait loci (QTLs) contribute to variation in changes in
stem cell numbers during aging.
| |
MATERIALS AND METHODS |
Mice used.
Young 6-week old female B6, DBA, and BXD recombinant inbred (RI) mice
were purchased from The Jackson Laboratory (Bar Harbor, ME). Twelve-
and 20-month old B6 and DBA mice were obtained from the National
Institute of Aging (Bethesda, MD). For measurements using 20-month old
BXD strains, 6-week old BXD mice were purchased from the Jackson
Laboratory and were aged in the animal facility of the University of
Kentucky. Mice were kept in microisolator cages and fed sterilized food
and acidified water. Mice with obvious tumors, or otherwise not
thriving, were excluded from the study. Twenty of the 26 existing BXD
strains were available for this analysis. BXD mice of strains 2, 5, 8, 13, 21, and 22 all died before reaching 20 months of age.
RI strains.
RI mice are a powerful genetic tool for gene mapping. We chose to study
the BXD strains because we have previously documented numerous
differences in both hematopoietic stem and progenitor cell parameters
and longevity parameters between the two progenitor strains from which
they were derived.14,26-28,30 Twenty-six BXD strains have
been generated by crossing C57BL/6 and DBA/2 inbred strains, and
through continuous inbreeding beginning at the F2 generation, each strain has evolved a unique combination of homozygous `B' or `D' alleles. The unique patterns of recombination in each RI
strain are maintained through continuous inbreeding at The Jackson
Laboratory and they are commercially available from this source. Over
the ensuing years since their generation, numerous laboratories have
used them to map either genetic markers or genes of interest. Thus, in
the context of the BXD RI strains, roughly 1,700 genotypes have been
mapped. As the mouse genome comprises about 1,500 to 1,600 centimorgans
(cM) in total, there is an average of about one marker per cM,
resulting in a high-density genetic map. It is therefore not surprising
that genes contributing relatively large quantitative effects can be
mapped in this set of RI strains without further genotyping of marker loci.
Genome-wide linkage statistics.
The frequencies of cobblestone area-forming cells (CAFC) day-35 (per
105 bone marrow cells) in young and old BXD mice was
measured as described below. The percent change, either positive or
negative, in CAFC day-35 frequency was calculated by comparing the data obtained in this study at 20 months of age with frequencies we obtained
in young (2 month old) BXD mice.30 It was this parameter, percent change, that became the phenotype of interest in our study. The
array of phenotypic results obtained from the set of BXD strains is
often referred to as a strain distribution profile (SDP). In the case
of a simple Mendelian trait, determined by a single locus, the
phenotypes may be expected to fall into two groups: those characteristic of each of the two progenitor strains (C57BL/6 and DBA/2
in this case), provided they differ for the trait in question. In this
case, the genotype (either `B' or `D') of a given BXD strain at the
locus of interest can be deduced by simply comparing the phenotype with
that of the two progenitor strains and determining which it matches. In
the case of complex traits such as the subject of the present study,
multiple loci make small individual contributions to the final
phenotype and thus, as we found here, the SDP may well include values
more extreme than either progenitor strain, as well as the full range
of intermediate values (see Fig 4). Phenotypes more extreme than either
of the progenitor strains are possible because of combinations of both positive and negative influences of individual quantitative trait loci
(QTLs) on the phenotype, and the fact that neither progenitor strain
may necessarily represent a pure combination of loci responsible for
either all of the positive or all of the negative effects on phenotype.
As discussed below, powerful computational software is now available
for mapping QTLs such that the user is not required to discriminate
between `B' and `D' phenotypes, rather the quantitative phenotypic
data for each RI strain serves as the starting point for analysis.
Irrespective of the complexity, the strategy for mapping is to select
the most similar, if not identical, SDP from the large list of
previously mapped genotypes mapped in BXD strains. A concordance between a phenotypic SDP and an existing genotypic SDP (map location) indicates the presence of a QTL at or near that location contributing to the phenotype. However, due to the limited number of BXD strains available, the number of SDPs to be compared, and consequently the
complexity of testing required to establish concordance, a close or
identical match in SDPs may occur by chance.31-33 As with all statistical comparisons, it is necessary to make a calculation of
the probability that the observed result was a false-positive. We
therefore calculated the genome-wide probability of obtaining the
observed linkages by random chance corresponding to an error threshold
of P = .05; that is, one chance in 20 of such a false-positive error. We did this using the robust nonparametric permutation method
developed by Churchill and Doerge,34 which is
implemented in the Map Manager QT (b15) software developed by
Manley.35
Interval mapping.
A subroutine of the Map Manager QT software, using computationally
efficient regression equations, was used for mapping the QTLs.35 The probability of linkage between our trait
(differences in CAFC frequency between young and old mice) and
previously mapped genotypes was estimated at 1-cM intervals along the
entire genome, except for the Y chromosome. The statistical power of
linkage of the phenotype to individual genotypes (point-wise linkage
statistics), such as those reported (see Table 2) for the three
principal loci uncovered in our interval mapping, should attain values
of between .000133 and .0000231 to reach a
level of genome-wide statistical significance. Linkages approximating
that level are deemed `suggestive' and are worthy of reporting,
although confirmation of linkage is required.32
The BXD genotype database.
The current mapping data files for BXD RI strains, compiled by R.W.
Elliott and B. Taylor, were downloaded from The Roswell Park Cancer Institute (Buffalo, NY;
ftp://mcbio.med.buffalo.edu/pub/MapMgr/data/).
Preparation of hematopoietic tissues and cells.
Fetal livers were harvested from day 13.5, 14.5, and 16.5 postcoitum
(pc) fetuses. To obtain fetuses, three to four females were introduced
into a cage with one male at 5:00 PM, and pregnant females,
identified by the presence of a vaginal plug, were isolated the next
morning at 9:00 AM. This timepoint was considered day 0.5 pc.
At the designated time, embryos were dissected and fetal livers
isolated. Three to five fetal livers were pooled and
disrupted by flushing through increasingly smaller gauge needles.
For experiments with bone marrow, cells were flushed from femora
obtained from three to six B6 or DBA mice, or from one to three BXD
mice, pooled, and used in the CAFC assay.
CAFC assay.
The CAFC assay, described by Ploemacher et al,36 was used
as published earlier, with minor modifications.14,30 In
brief, cells of the stromal cell line, FBMD-1, originally established by Neben et al,37 were seeded in 96-well plates (Costar,
Cambridge, MA) in Dulbecco's modified Eagle's medium
(DMEM) containing L-glutamine (GIBCO-BRL, Life
Technologies, Grand Island, NY), 5% horse serum, 15% fetal bovine
serum (sera from GIBCO-BRL), 10 4 mol/L
 -mercaptoethanol, 105 mol/L hydrocortisone
(Sigma, St Louis, MO), 80 U/mL penicillin, 80 µg/mL streptomycin
(both from GIBCO-BRL), and 25 mmol/L NaHCO3. Plates were
incubated at 33°C in 5% CO2, and used 10 to 14 days later. Bone marrow cells were seeded onto these preestablished stromal
layers in six dilutions, serially in threefold increments from 333 to
81,000 cells/well (40 wells per dilution). At this time, the medium was
switched from 5% horse serum and 15% fetal bovine serum to 20% horse
serum. After 1 week, all wells were evaluated for the presence or
absence of cobblestone areas, defined as colonies of at least five
small nonrefractile cells growing beneath the stromal layer. CAFC day-7
correspond to relatively committed progenitor cells, whereas CAFC
day-21 and 35 reflect increasingly more primitive cell
subsets.36,38,39 CAFC frequencies were calculated using
Poisson statistics.40 For calculations of total body stem
cell numbers in the fetus, the frequency of CAFC day-35 was multiplied
by fetal liver cellularity. For total body stem cell numbers in adult
mice, the CAFC day-35 frequency was first multiplied by the femoral
cellularity, and subsequently by a factor of 17, under the assumption
that one femur represents 6% of the total marrow.41
Measurement of progenitor cell cycling.
The percentage of CAFC day-7 in S-phase of the cell cycle was
determined by using a hydroxyurea suicide technique, as described previously.14,30 Briefly, 2 aliquots of marrow or fetal
liver cells were diluted to a concentration of 1 × 107 cells/mL. Hydroxyurea (Sigma) was added on 1 aliquot at
a concentration of 200 µg/mL, and both samples were incubated at
33°C for 1 hour. After incubation, the two-cell suspensions were
washed and a nucleated cell count was performed. A CAFC day-7 assay was
performed with the 2 aliquots, and the fraction of cells killed by
hydroxyurea was calculated by dividing the CAFC frequency in the
hydroxyurea-treated cell suspension by the frequency obtained with the
control cell aliquot.
| |
RESULTS |
Progenitor cell cycling in DBA and B6 mice during aging.
CAFC day-7 represent relatively committed progenitor cells that are
actively cycling, whereas CAFC day-35 reflect primitive stem cells that
are predominantly quiescent.14,36,38 To assess the
steady-state proliferation status of the stem cell compartment, we
measured the percentage of CAFC day-7 in S-phase
(Fig 1A). At all timepoints, except at 1 year, DBA/2 cells had a much higher cycling activity than B6 cells. The
percentage of cells in S-phase declined daily from gestational days
13.5 to 16.5 of fetal development.
View larger version (29K):
[in this window]
[in a new window]
| Fig 1.
Changes in the proliferative status and total number of
hematopoietic stem cells in B6 and DBA mice during aging. (A) The
percentage of CAFC day-7 in S-phase was measured at different
timepoints during development in the fetal liver (FL) and adult bone
marrow (BM) in B6 and DBA mice. (B) The total number of CAFC day-35 per
B6 ( ) and DBA ( ) mouse was calculated in the fetal liver (first
three datapoints) or adult bone marrow (other datapoints). Differences
between B6 and DBA values are significant (nonoverlapping 95%
confidence intervals) at all timepoints, except at 1 year.
|
|
Stem cell numbers in DBA and B6 mice during aging.
We have previously shown that CAFC day-35 numbers in the bone marrow
increase twofold to threefold as both B6 and DBA mice age from 2 to 12 months.14 In this study, we made more detailed measurements
of the dynamics of stem cell numbers by extending our analysis of CAFC
day-35 frequencies to three times during fetal development and to
20-month old mice. Because we were interested in assessing the total
hematopoietic stem cell pool size, we calculated the absolute number of
CAFC day-35 per mouse (Fig 1B). For fetuses, this was done by
multiplying the frequency of CAFC day-35 per 105 liver
cells, as shown in Table 1, with fetal
liver cellularity. Total body CAFC day-35 numbers for adult mice were
calculated assuming that the femur represents 6% of total
marrow.41 We did not quantify stem cell numbers in the
spleen, but we have previously established that during adult
steady-state hematopoiesis, the spleen contains less then 1% of the
total stem cell pool.42 Similarly, the resulting number in
fetuses is a modest underestimation of the total stem cell pool, as
small numbers of hematopoietic stem cells may also be found in other
fetal organs.43
Interestingly, the vast majority of stem cells found in young adult
bone marrow is already present in the fetal liver at day 16.5, a
finding supported by a recent study by Harrison and
Astle.44 It is apparent from Fig 1B that the stem cell pool
size is far from static during aging; rather, it is highly dynamic.
Importantly, the continuously changing size of this compartment appears
to be strongly influenced by genetic determinants. A seemingly
ever-expanding stem cell population was observed in B6 mice during the
20 months of observation, whereas in DBA mice, a clear maximum was
observed at 12 months, after which the stem cell compartment contracted.
Table 1 shows the relative frequency of CAFC day-35 numbers per
105 fetal liver or adult bone marrow cells that was used to
calculate the total stem cell pool per mouse as presented in Fig 1B.
Perhaps not surprisingly, CAFC day-35 frequency is high in the fetal
liver from mid to late fetal development, and subsequently is
approximately fivefold less in marrow, presumably due to the dilution
accompanying the dramatic increase in overall size of the newborn,
including hematopoietic tissue, and the demand for rapid blood cell
production. Quantitatively similar differences between fetal liver and
adult marrow stem cell frequencies have been reported using an in vivo competitive repopulating unit assay12 or by flow cytometry
analysis of stem cell markers.45 Interestingly, the
relative frequency of CAFC day-35 in both strains of mice drops by
50% from day 13.5 to day 14.5 in utero. This relative decline is
probably caused by an expansion of nonhematopoietic cells in the fetal
liver at this time, and it should be emphasized that the absolute
number of stem cells in the fetal liver increases substantially from day 13.5 to 14.5 (Fig 1B). Once in the bone marrow, stem cell frequencies in B6 mice increased continuously during aging. Initially, a qualitatively similar observation was made in DBA mice, but at 20 months, stem cell frequencies in this strain were severely reduced to
25% of values observed at 12 months. Although the stem cell
frequency within a strain changes significantly with age, there was no
evidence for increased intrastrain variation during aging, ie, the 95%
confidence limits at all timepoints remained between 75% and
140% of the estimated frequency from Poisson statistics (Table 1).
Stem cell numbers in old BXD recombinant inbred mice.
The genomes of BXD mice consist of a mosaic of homozygous B6 and DBA
segments. Phenotyping BXD mice for a specific trait, followed by a
linkage analysis of this trait with polymorphic markers previously
mapped in these strains, may identify loci genetically linked with the
observed phenotypic variation, and thus provide a map
location.46 We have recently used 2-month old BXD RI mice
to map loci that contribute to the observed variation in CAFC day-35
numbers between young B6 and DBA mice. The principal locus mapped to
chromosome 18, approximately 19 cM from the centromere.30 In the present study, we followed a similar strategy to map loci associated with changes of stem cell numbers during aging in old B6 and
DBA mice. We verified the reproducibility of quantifying CAFC day-35
numbers over an extended period of time by retesting selected BXD
strains and both progenitor strains. Figure
2 shows that the CAFC assay as used in these experiments is reliable, as data were highly reproducible over a 2-year period. All data-points fall within the 95% confidence limits obtained by linear regression analysis.
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
Testing the reproducibility of the CAFC assay. CAFC
day-35 frequencies were measured in two independent experiments over a
2-year interval using six different mouse strains as marrow donors. In
each experiment cells from three mice were pooled. Results are given as
mean values, and 95% confidence intervals are indicated.
|
|
Figure 3B shows the CAFC day-35 frequency
per 105 bone marrow cells in the 20-month old BXD strains
that were available for analysis. Mice of the six missing strains
(BXD-2, 5, 8, 13, 21, and 22) had died before 20 months. Figure 3A is
shown for comparison and presents data obtained with 2-month old BXD
strains, as published previously.30 It is immediately
apparent that the variation in stem cell frequency among the strains is
much higher in old than in young BXD mice, very much like the data
obtained with the parental strains (compare with Fig 1B). The range in
CAFC day-35 frequency varies approximately 16-fold in young BXD strains (BXD-29 having the lowest, and BXD-11 having highest stem cell numbers), but this increases to approximately 340-fold in old BXD mice.
BXD-29 remained the lowest with .063 CAFC day-35/105 cells,
and BXD-15 was the highest with 21.1 CAFC day-35/105 cells.
For comparison, these values translate into a total CAFC day-35 pool
size per mouse of only 100 for BXD-29 and of 82,000 for BXD-15.
View larger version (27K):
[in this window]
[in a new window]
| Fig 3.
CAFC day-35 frequency in bone marrow of young and old BXD
mice. CAFC day-35 frequencies were determined in the bone marrow of
2-month (A) and 20-month (B) old BXD mice. The results presented in (A)
are shown for comparison and have been published.30
|
|
Change in BXD stem cell numbers during aging.
An aim of this study was to map loci that contribute to the
age-dependent stem cell dynamics in B6 and DBA mice. To this end, we
calculated the percentage change of CAFC day-35 frequency in old BXD
mice (Fig 3B) compared with the value observed in their young
counterparts (Fig 3A) to obtain the relative changes. In addition, we
performed this calculation for less primitive CAFC day-7 and day-21
subsets (Fig 4). Figure 4A depicts the
change in CAFC day-7 frequencies during aging. Old BXD mice have
essentially similar progenitor cell frequencies as young mice, but when
more primitive CAFC day-21 frequencies are compared, an increased
variation is apparent (Fig 4B). Frequencies of this cell stage in old
B6 mice increased to 300% compared with young, whereas this population is reduced to 50% in old DBA mice. Old BXD mice show a similar pattern; in some strains CAFC day-21 frequencies increase twofold to
threefold (B6 phenotype), whereas in others, a DBA-like reduction is
observed. Several strains show an intermediate phenotype, and old BXD-6
and BXD-15 mice show a greater expansion than parental B6. When
primitive CAFC day-35 frequencies were measured, the variation among
strains was even more extreme (Fig 4C). Old BXD-6 and BXD-15 mice
exhibited an exceptional expansion of stem cell numbers, whereas the
stem cell pool in BXD-29 declined more dramatically (approximately
10-fold). This variation cannot be due to experimental error in the
assay, as CAFC day-7 frequencies, scored in the same plates using the
same limiting-dilutions, are constant during aging and thus serve as an
internal control. In addition, we find no evidence that intrastrain
variation, which potentially could cause extreme outliers, increases
during aging because the 95% confidence intervals of CAFC day-35
frequencies in B6 and DBA mice remained stable throughout their
lifespan (Table 1).
View larger version (19K):
[in this window]
[in a new window]
| Fig 4.
Changes in progenitor and stem cell numbers in BXD mice
during aging. CAFC day-7, 21, and 35 were measured in young (2 months)
and old (20 months) BXD mice, and the percentage increase or decrease
in cell numbers during aging was calculated. (A, B, and C) Show
relative changes in the numbers of CAFC day-7, day-21, and day-35,
respectively. Note that the Y-axis for all three panels is the same.
Three young BXD mice were used for all datapoints. The number of old
BXD mice per datapoint was 1 (seven strains), 2 (five strains), or 3 (eight strains). The most extreme deviations from young CAFC day-21 and
35 numbers were seen in strains from which three animals were used
(BXD-6, -15, and 29).
|
|
Mapping loci that control population dynamics of stem cells during
aging.
A genome-wide search for linked loci was performed using the data
presented in Fig 4C as the phenotype of interest.
Table 2 summarizes the results of this
linkage analysis and lists the loci most strongly associated with the
trait. These putative loci on chromosomes 2, 14, and X reached LOD
scores of 3.1, 2.4, and 2.4, respectively, values associated with
suggestive, but not significant, linkage.32,33 This is
likely to result from the involvement of multiple loci at which B6 and
DBA mice differ. The change in stem cell pool size during aging in
these mice is probably the result of interaction of the QTLs listed in
Table 2, as well as others not detected in our analysis. To resolve these loci, more meiotic events will have to be generated (and progeny
tested) than can be evaluated in the limited context of the 20 BXD
strains.
View this table:
[in this window]
[in a new window]
|
Table 2.
Quantitative Trait Loci Most Strongly Associated
With the Variation in the Change in CAFC Day-35 Frequency Between
B6 and DBA Mice During Aging
|
|
| |
DISCUSSION |
In this study, we have quantified stem cell populations during fetal
development and adult aging in short-lived DBA and long-lived B6 mice.
Because of their normally quiescent nature and their capability to
extensively self-renew, large fluctuations in the size of the stem cell
pool were not expected. Surprisingly however, we observed dynamic
changes in the number of stem cells during the lifespan of these
strains of mice, as stem cell numbers accumulated continuously in aging
B6 mice, but dropped to embryonic values in old DBA mice. We have
previously reported that progenitor cells from DBA mice show a marked
reduction in cycling activity from 2 to 12 months of age.14
Together with the present observation of declining CAFC day-35 numbers,
this suggests that hematopoietic cell production in DBA mice generally
enters a period of decline after 12 months, possibly leading to
senescence. Although, as yet, we formally cannot prove the significance
of these findings for in vivo stem cell function, the concept of
sencescence is strengthened by observations made in B6 DBA
embryo-aggregated chimeras, where initially stable blood cell chimerism
is established, but DBA stem cells slow and then cease blood cell
production after 1 year.27 Thus our findings here
reiterate both temporally and quantitatively what we have previously
reported using chimeras and firmly establish that primitive
hematopoietic stem cell characteristics are significantly affected by
aging, presumably due to their replicative history. The effect of aging
on stem cells is highly strain-specific however, and our present study
was aimed at providing insight into the genetic predispostion of stem
cell aging. Not surprisingly, the complex pattern of changes in stem
cell numbers was found to be caused by multiple loci. Putative loci
were shown to map to chromosomes X, 2, and 14, but the restricted
genetic power of BXD analysis to map variation in traits that are
influenced by many loci, precluded attaining significant linkage with
any of these genomic intervals. We found no linkage to any marker on
chromosome 18, to which we have previously mapped a locus affecting CAFC day-35 frequency in young mice,30 suggesting the two
traits are independent. Our finding of linkage to an interval on the X-chromosome deserves further comment. Studies by Abkowitz et al47 in female heterozygous Safari cats (Geoffrey x
domestic) have recently shown an X-linked locus that appears to
regulate stem cell kinetics by conferring a competitive advantage to
stem cells bearing a euchromatic Geoffrey X chromosome (and lyonization of the domestic-derived counterpart). This competitive growth advantage
was observed indirectly as a predominance of mature blood cells with
this X-inactivation pattern and became apparent only after 3 to 6 years.47 Excessive skewing of X-inactivation patterns,
attributed to a declining stem cell population, has also been observed
in blood cells of 40% to 60% of normal women over 60 years old who
are heterozygous for X-linked G6PD alleles.48,49 While
intriguing, it remains to be established whether the X-linked locus,
which we report here, is involved in the dynamic regulation of stem
cell numbers during aging in cats or humans, an association that
requires independent confirmation of linkage.
Primitive hematopoietic stem cells, with the possible exception of
those in the fetus, have a very low turnover rate and are generally
believed to have a self-renewal probability (P) of .5, resulting in adequate hematopoietic blood cell replacement and a static
pool size.50 It has been suggested that P, a value not possible to measure directly, can temporarily fluctuate to meet
needs imposed by perturbation, such as stem cell transplantation or
administration of cytotoxic drugs in cancer therapy.1,51 Our data here suggest that values for P can change dramatically during normal aging, as the stem cell pool size is dynamic. In the
fetal liver, it must initially be considerably higher than .5 (and may
be close to 1.0), given the exponential daily expansion of stem cell
numbers. Expansion of stem cells in fetal liver of DBA mice is
particularly noteworthy. In adult marrow, however, a more complex
pattern is observed. In B6 mice, in which the stem cell compartment
expands at a constant rate (Fig 1), P appears to be slightly
higher than .5 during at least 20 months of life. In DBA mice, the stem
cell compartment increases less dramatically between the initial
expansion in fetal liver and 1 year of age. Thus, P in DBA
adult mice must be lower than P in adult B6 during the first 12 months, but nonetheless greater than .5. Second, and more importantly,
it must be less than .5 thereafter, when stem cell numbers decline. It
remains to be determined whether the contracting stem cell pool is
responsible for the shorter lifespan of DBA mice or whether it is
merely a correlative symptom of it. If a mitotic clock, potentially
provided by telomere length,19,20 sets the maximal number
of divisions stem cells may undergo, a prediction would be that a
population of fast cycling stem cells is exhausted earlier than a
population of slowly cycling stem cells. In this respect, it is
interesting to note that DBA stem cells have a threefold to fourfold
higher cycling activity than B6 cells, and this line of reasoning was
used to explain extinction of DBA-derived hematopoiesis in B6DBA
chimeras.14,26,30 Preliminary measurements in aged B6D2F1
mice, which are longer-lived than B6, show an even more pronounced stem
cell increase during normal aging (data not shown). It should be
emphasized however, that the relationship between lifespan and changes
in stem cell numbers as suggested by these data is likely more
complicated. For example, BXD-29 and BXD-15, the two most extreme
strains analyzed here, have comparable cell cycle
kinetics52 and lifespans.53 The complex
relationships between these parameters suggest the existence of
intricate regulatory mechanisms controlling alterations of in vivo stem
cell numbers during aging and the findings reported here are but a
first step in unravelling them. For example, the complexity obviously
does not preclude identification of genetic mechanisms that play a role
in this process, and the present study offers genetic approaches for
subsequent analyses that are currently being pursued.
| |
ACKNOWLEDGMENT |
The authors thank Drs Stephen J. Szilvassy and Craig T. Jordan for
critically reading the manuscript and Dr Rob Williams for invaluable
assistance with MapManager software.
| |
FOOTNOTES |
Submitted April 14, 1998; accepted January 7, 1999.
Supported by the Department of Internal Medicine, University of
Kentucky Hospital and the Lucille P. Markey Cancer Center. G.dH. is a
fellow of the Netherlands Organisation for Scientific Research (NWO).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Gary Van Zant, PhD, Blood
and Marrow Transplant Program, Division of Hematology/Oncology,
University of Kentucky, Markey Cancer Center, 800 Rose St, Lexington,
KY 40536-0093; email: gvzant1{at}pop.uky.edu.
| |
REFERENCES |
1.
Siminovitch L, Till JE, McCulloch EA:
Decline in colony forming ability of marrow cells subjected to serial transplantation into irradiated mice.
J Cell Comp Physiol
64:23, 1964
2.
Harrison DE:
Normal function of transplanted mouse erythrocyte precursors for 21 months beyond donor life spans.
Nature New Biol
237:220, 1972[Medline]
[Order article via Infotrieve]
3.
Osawa M, Hanada K, Hamada H, Nakauchi H:
Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Science
273:242, 1996[Abstract]
4.
Rubin H:
Cell aging in vivo and in vitro.
Mech Ageing Dev
98:1, 1997[Medline]
[Order article via Infotrieve]
5.
Harrison DE, Astle CM, DeLaittre J:
Effects of transplantation and age on immunohemopoietic cell growth in the splenic microenvironment.
Exp Hematol
16:213, 1988[Medline]
[Order article via Infotrieve]
6.
Harrison DE:
Long-term erythropoietic repopulating ability of old, young, and fetal stem cells.
J Exp Med
157:1496, 1983[Abstract/Free Full Text]
7.
Mauch P, Botnick LE, Hannon EC, Obbagy J, Hellman S:
Decline in bone marrow proliferative capacity as a function of age.
Blood
60:245, 1982[Free Full Text]
8.
Boggs DR, Saxe DF, Boggs SS:
Aging and hematopoiesis. II. The ability of bone marrow cells from young and aged mice to cure and maintain cure in W/Wv.
Transplantation
37:300, 1984[Medline]
[Order article via Infotrieve]
9.
Lansdorp PM, Dragowska W, Mayani H:
Ontogeny-related changes in proliferative potential of human hematopoietic cells.
J Exp Med
178:787, 1993[Abstract/Free Full Text]
10.
Micklem HS, Ford CE, Evans EP, Ogden DA, Papworth DS:
Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice.
J Cell Physiol
79:293, 1972[Medline]
[Order article via Infotrieve]
11.
Rebel VI, Miller CL, Thornbury GR, Dragowska WH, Eaves CJ, Lansdorp PM:
A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse.
Exp Hematol
24:638, 1996[Medline]
[Order article via Infotrieve]
12.
Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM:
Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term.
Exp Hematol
25:293, 1997[Medline]
[Order article via Infotrieve]
13.
Rebel VI, Miller CL, Eaves CJ, Lansdorp PM:
The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts.
Blood
87:3500, 1996[Abstract/Free Full Text]
14.
de Haan G, Nijhof W, Van Zant G:
Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: Correlation between lifespan and cycling activity.
Blood
89:1543, 1997[Abstract/Free Full Text]
15.
Tejero C, Testa NG, Hendry JH:
Decline in cycling of granulocyte-macrophage colony-forming cells with increasing age in mice.
Exp Hematol
17:66, 1989[Medline]
[Order article via Infotrieve]
16.
Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL:
The aging of hematopoietic stem cells.
Nat Med
2:1011, 1996[Medline]
[Order article via Infotrieve]
17.
Harrison DE, Astle CM, Stone M:
Effects of age on transplantable primitive immuno-hematopoietic stem cell (PSC) numbers and function.
J Immunol
142:3833, 1989[Abstract]
18.
Van Zant G, de Haan G, Rich IN:
Alternatives to stem cell renewal from a developmental viewpoint.
Exp Hematol
25:187, 1997[Medline]
[Order article via Infotrieve]
19.
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB:
Telomere length predicts replicative capacity of human fibroblasts.
Proc Natl Acad Sci USA
89:10114, 1992[Abstract/Free Full Text]
20.
Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM:
Evidence for a mitotic clock in human hematopoietic stem cells: Loss of telomeric DNA with age.
Proc Natl Acad Sci USA
91:9857, 1994[Abstract/Free Full Text]
21.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE:
Extension of life-span by introduction of telomerase into normal human cells [see comments].
Science
279:349, 1998[Abstract/Free Full Text]
22.
Notaro R, Cimmino A, Tabarini D, Rotoli B, Luzzatto L:
In vivo telomere dynamics of human hematopoietic stem cells.
Proc Natl Acad Sci USA
94:13782, 1997[Abstract/Free Full Text]
23.
Morrison SJ, Prowse KR, Ho P, Weissman IL:
Telomerase activity in hematopoietic cells is associated with self-renewal potential.
Immunity
5:207, 1996[Medline]
[Order article via Infotrieve]
24.
Engelhardt M, Kumar R, Albanell J, Pettengell R, Han W, Moore MA:
Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells.
Blood
90:182, 1997[Abstract/Free Full Text]
25.
Yui J, Chiu CP, Lansdorp PM:
Telomerase activity in candidate stem cells from fetal liver and adult bone marrow.
Blood
91:3255, 1998[Abstract/Free Full Text]
26.
Van Zant G, Eldridge PW, Behringer RR, Dewey MJ:
Genetic control of hematopoietic kinetics revealed by analyses of allophenic mice and stem cell suicide.
Cell
35:639, 1983[Medline]
[Order article via Infotrieve]
27.
Van Zant G, Holland BP, Eldridge PW, Chen JJ:
Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice.
J Exp Med
171:1547, 1990[Abstract/Free Full Text]
28.
Phillips RL, Reinhart AJ, Van Zant G:
Genetic control of murine hematopoietic stem cell pool sizes and cycling kinetics.
Proc Natl Acad Sci USA
89:11607, 1992[Abstract/Free Full Text]
29.
Muller-Sieburg CE, Riblet R:
Genetic control of the frequency of hematopoietic stem cells in mice: Mapping of a candidate locus to chromosome 1.
J Exp Med
183:1141, 1996[Abstract/Free Full Text]
30.
de Haan G, Van Zant G:
Intrinsic and extrinsic control of hemopoietic stem cell numbers: Mapping of a stem cell gene.
J Exp Med
186:529, 1997[Abstract/Free Full Text]
31.
Lander ES, Schork NJ:
Genetic dissection of complex traits.
Science
265:2037, 1994[Abstract/Free Full Text]
32.
Lander ES, Kruglyak L:
Genetic dissection of complex traits: Guidelines for interpreting and reporting linkage results.
Nat Genet
11:241, 1995[Medline]
[Order article via Infotrieve]
33.
Belknap JK, Mitchell SR, O'Toole LA, Helms ML, Crabbe JC:
Type I and type II error rates for quantitative trait loci (QTL) mapping studies using recombinant inbred mouse strains.
Behav Genet
26:149, 1996[Medline]
[Order article via Infotrieve]
34.
Churchill GA, Doerge RW:
Emperical threshold values for quantitative trait mapping.
Genetics
138:963, 1994[Abstract]
35.
Manley KF:
A Macintosh program for storage and analysis of experimental mapping data.
Mamm Genome
4:303, 1993[Medline]
[Order article via Infotrieve]
36.
Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL:
Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse.
Blood
78:2527, 1991[Abstract/Free Full Text]
37.
Neben S, Donaldson D, Sieff C, Mauch P, Bodine D, Ferrara J, Yetz-Aldape J, Turner K:
Synergistic effects of interleukin-11 with other growth factors on the expansion of murine hematopoietic progenitors and maintenance of stem cells in liquid culture.
Exp Hematol
22:353, 1994[Medline]
[Order article via Infotrieve]
38.
Down JD, Ploemacher RE:
Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: Differential effects of host conditioning with gamma radiation and cytotoxic drugs.
Exp Hematol
21:913, 1993[Medline]
[Order article via Infotrieve]
39.
Ploemacher RE, van der Loo JC, van Beurden CA, Baert MR:
Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo.
Leukemia
7:120, 1993[Medline]
[Order article via Infotrieve]
40.
Fazekas de StGroth:
The evaluation of limiting dilution assays.
J Immunol Methods
49:R11, 1982[Medline]
[Order article via Infotrieve]
41.
Chervenick PA, Boggs DR, Marsh JC, Cartwright GE, Wintrobe MM:
Quantitative studies of blood and bone marrow neutrophils in normal mice.
Am J Physiol
215:353, 1968
42.
de Haan G, Dontje B, Engel C, Loeffler M, Nijhof W:
Prophylactic pretreatment of mice with hematopoietic growth factors induces expansion of primitive cell compartments and results in protection against 5-fluorouracil-induced toxicity.
Blood
87:4581, 1996[Abstract/Free Full Text]
43.
Marcos MA, Morales-Alcelay S, Godin IE, Dieterlen-Lievre F, Copin SG, Gaspar ML:
Antigenic phenotype and gene expression pattern of lymphohemopoietic progenitors during early mouse ontogeny.
J Immunol
158:2627, 1997[Abstract]
44.
Harrison DE, Astle CM:
Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: Models for human umbilical cord blood.
Blood
90:174, 1997[Abstract/Free Full Text]
45.
Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL:
The purification and characterization of fetal liver hematopoietic stem cells.
Proc Natl Acad Sci USA
92:10302, 1995[Abstract/Free Full Text]
46.
Silver L:
Mouse Genetics, Concepts and Applications. New York, NY, Oxford, 1995.
47.
Abkowitz JL, Taboada M, Shelton GH, Catlin SN, Guttorp P, Kiklevich JV:
An X-chromosome gene regulates hematopoietic stem cell kinetics.
Proc Natl Acad Sci USA
95:3862, 1998[Abstract/Free Full Text]
48.
Champion KM, Gilbert JG, Asimakopoulos FA, Hinshelwood S, Green AR:
Clonal haemopoiesis in normal elderly women: Implications for the myeloproliferative disorders and myelodysplastic syndromes.
Br J Haematol
97:920, 1997[Medline]
[Order article via Infotrieve]
49.
Gale RE, Fielding AK, Harrison CN, Linch DC:
Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.
Br J Haematol
98:512, 1997[Medline]
[Order article via Infotrieve]
50.
Potten CS, Loeffler M:
Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.
Development
110:1001, 1990[Abstract/Free Full Text]
51.
Iscove NN, Nawa K:
Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion.
Curr Biol
7:805, 1997[Medline]
[Order article via Infotrieve]
52.
de Haan G, Van Zant G:
Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal lifespan.
FASEB J
13:707, 1999[Abstract/Free Full Text]
53.
Gelman R, Watson A, Bronson R, Yunis E:
Murine chromosomal regions correlated with longevity.
Genetics
118:693, 1988[Abstract/Free Full Text]
54. Mouse Genome Database (MGD), Mouse Genome Informatics, Bar
Harbor, ME, The Jackson Laboratory. World Wide Web (URL:
http://www.informatics.jax.org), February 1998
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Bhattacharya, A. Czechowicz, A.G. L. Ooi, D. J. Rossi, D. Bryder, and I. L. Weissman
Niche recycling through division-independent egress of hematopoietic stem cells
J. Exp. Med.,
November 11, 2009;
(2009)
jem.20090778v3.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B.P. Levi and S.J. Morrison
Stem Cells Use Distinct Self-renewal Programs at Different Ages
Cold Spring Harb Symp Quant Biol,
January 15, 2009;
(2009)
sqb.2008.73.049v1.
[Abstract]
[PDF]
|
|
|
|
|
|
|
I. Roeder, K. Horn, H.-B. Sieburg, R. Cho, C. Muller-Sieburg, and M. Loeffler
Characterization and quantification of clonal heterogeneity among hematopoietic stem cells: a model-based approach
Blood,
December 15, 2008;
112(13):
4874 - 4883.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Guerrettaz, S. A. Johnson, and J. C. Cambier
Acquired hematopoietic stem cell defects determine B-cell repertoire changes associated with aging
PNAS,
August 19, 2008;
105(33):
11898 - 11902.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M Jawad, G Giotopoulos, C Cole, and M Plumb
Target cell frequency is a genetically determined risk factor in radiation leukaemogenesis
Br. J. Radiol.,
September 1, 2007;
80(Special_Issue_1):
S56 - S62.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. P. Zediak, I. Maillard, and A. Bhandoola
Multiple prethymic defects underlie age-related loss of T progenitor competence
Blood,
August 15, 2007;
110(4):
1161 - 1167.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Dumble, L. Moore, S. M. Chambers, H. Geiger, G. Van Zant, M. A. Goodell, and L. A. Donehower
The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging
Blood,
February 15, 2007;
109(4):
1736 - 1742.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Liang, G. Van Zant, and S. J. Szilvassy
Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells
Blood,
August 15, 2005;
106(4):
1479 - 1487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Geiger, G. Rennebeck, and G. Van Zant
Regulation of hematopoietic stem cell aging in vivo by a distinct genetic element
PNAS,
April 5, 2005;
102(14):
5102 - 5107.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Smith, F. M. Ellison, J. P. McCoy Jr., and J. Chen
c-Kit Expression and Stem Cell Factor-Induced Hematopoietic Cell Proliferation Are Up-Regulated in Aged B6D2F1 Mice
J. Gerontol. A Biol. Sci. Med. Sci.,
April 1, 2005;
60(4):
448 - 456.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Roeder, L. M. Kamminga, K. Braesel, B. Dontje, G. de Haan, and M. Loeffler
Competitive clonal hematopoiesis in mouse chimeras explained by a stochastic model of stem cell organization
Blood,
January 15, 2005;
105(2):
609 - 616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Henckaerts, J. C. Langer, J. Orenstein, and H.-W. Snoeck
The Positive Regulatory Effect of TGF-{beta}2 on Primitive Murine Hemopoietic Stem and Progenitor Cells Is Dependent on Age, Genetic Background, and Serum Factors
J. Immunol.,
August 15, 2004;
173(4):
2486 - 2493.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Henckaerts, J. C. Langer, and H.-W. Snoeck
Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice
Blood,
July 15, 2004;
104(2):
374 - 379.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Boulton, C. Cole, A. Knight, H. Cleary, R. Snowden, and M. Plumb
Low-penetrance genetic susceptibility and resistance loci implicated in the relative risk for radiation-induced acute myeloid leukemia in mice
Blood,
March 15, 2003;
101(6):
2349 - 2354.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. I. D. Rossi, T. Yokota, K. L. Medina, K. P. Garrett, P. C. Comp, A. H. Schipul Jr, and P. W. Kincade
B lymphopoiesis is active throughout human life, but there are developmental age-related changes
Blood,
January 15, 2003;
101(2):
576 - 584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Henckaerts, H. Geiger, J. C. Langer, P. Rebollo, G. Van Zant, and H.-W. Snoeck
Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines
Blood,
May 13, 2002;
99(11):
3947 - 3954.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. J. Morrison, D. Qian, L. Jerabek, B. A. Thiel, I.-K. Park, P. S. Ford, M. J. Kiel, N. J. Schork, I. L. Weissman, and M. F. Clarke
A Genetic Determinant That Specifically Regulates the Frequency of Hematopoietic Stem Cells
J. Immunol.,
January 15, 2002;
168(2):
635 - 642.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Geiger, J. M. True, G. de Haan, and G. Van Zant
Age- and stage-specific regulation patterns in the hematopoietic stem cell hierarchy
Blood,
November 15, 2001;
98(10):
2966 - 2972.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. I. D. Rossi, K. L. Medina, K. Garrett, G. Kolar, P. C. Comp, L. D. Shultz, J. D. Capra, P. Wilson, A. Schipul, and P. W. Kincade
Relatively Normal Human Lymphopoiesis but Rapid Turnover of Newly Formed B Cells in Transplanted Nonobese Diabetic/SCID Mice
J. Immunol.,
September 15, 2001;
167(6):
3033 - 3042.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Levin, L. Cocault, C. Demerens, C. Challier, M. Pauchard, J. Caen, and M. Souyri
Thrombocytopenic c-mpl{-}/{-} mice can produce a normal level of platelets after administration of 5-fluorouracil: the effect of age on the response
Blood,
August 15, 2001;
98(4):
1019 - 1027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Zaucha, C. Yu, G. Mathioudakis, K. Seidel, G. Georges, G. Sale, M.-T. Little, B. Torok-Storb, and R. Storb
Hematopoietic responses to stress conditions in young dogs compared with elderly dogs
Blood,
July 15, 2001;
98(2):
322 - 327.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Vickers, E. McLeod, T. D. Spector, and I. J. Wilson
Assessment of mechanism of acquired skewed X inactivation by analysis of twins
Blood,
March 1, 2001;
97(5):
1274 - 1281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. de Haan, S. J. Szilvassy, T. E. Meyerrose, B. Dontje, B. Grimes, and G. Van Zant
Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers
Blood,
August 15, 2000;
96(4):
1374 - 1379.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION