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Blood, 1 June 2002, Vol. 99, No. 11, pp. 3947-3954
HEMATOPOIESIS
Genetically determined variation in the number of phenotypically
defined hematopoietic progenitor and stem cells and in their
response to early-acting cytokines
Els Henckaerts,
Hartmut Geiger,
Jessica C. Langer,
Patricia Rebollo,
Gary Van Zant, and
Hans-Willem Snoeck
From the Carl C. Icahn Institute for Gene Therapy and
Molecular Medicine, Mount Sinai School of Medicine, New York, NY; and
the Markey Cancer Center, University of Kentucky, Lexington.
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Abstract |
Quantitative trait analysis may shed light on mechanisms regulating
hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6
(B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of
primitive progenitor cells to the early-acting cytokines kit ligand,
flt3 ligand, and thrombopoietin. A significant quantitative trait locus
was found on chromosome 2 that could not be confirmed in congenic mice,
however, probably because of epistasis. Because it has been shown that
alleles of unknown X-linked genes confer a selective advantage to
hematopoietic stem cells in vivo in humans and in cats, we also
analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked
locus regulating the responsiveness of progenitor and stem cells to
early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice,
correlating genetic variation was found in the absolute number and
frequency of Lin Sca1++kit+ cells,
which are highly enriched in hematopoietic progenitor and stem cells,
and in the number of
LinSca1++kit cells, a
population whose biologic significance is unknown, suggesting that both
populations are functionally related. Suggestive quantitative trait
loci (QTLs) for the number of
LinSca1++ cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome
2 was confirmed in congenic mice. We thus demonstrated genetic
variation in the response to cytokines critical for hematopoiesis in
vivo and in the pool size of cells belonging to a phenotype used to
isolate essentially pure primitive progenitor and stem cells, and we
identified loci that may be relevant to the regulation of hematopoiesis
in steady state.
(Blood. 2002;99:3947-3954)
© 2002 by The American Society of Hematology.
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Introduction |
Hematopoiesis consists of an ordered series of
events in which primitive hematopoietic stem cells renew or
differentiate into mature blood cells of at least 8 lineages. The exact
mechanisms responsible for the regulation of self-renewal versus
differentiation of hematopoietic stem cells in vivo are unknown but may
involve stochastic mechanisms and a balance between stimulatory and
inhibitory signals regulating renewal, differentiation, and
apoptosis.1
One strategy to gain insight into the regulation of hematopoiesis is to
study naturally occurring genetic variation in the hematopoietic
system. This may lead to the identification of regulatory mechanisms
that are relevant in vivo. Pool size and cycling activity of the stem
cell compartment are under complex intrinsic genetic control in inbred
mouse strains.2-8 Putative stem cell pool size, as
determined by the day 35 cobblestone area-forming cell assay (CAFCd35),
varies widely among inbred mouse strains.2 This was not
the case for earlier appearing, and therefore more mature, CAFCd7,
indicating that the gene(s) involved act on the more primitive progenitor compartment.8 Using BXD recombinant inbred (RI) mouse strains, a locus involved in the regulation of CAFCd35 frequency was mapped to mouse chromosome 18, in a region syntenic with and corresponding to a critical segment of human chromosome 5q, which is
frequently deleted in myelodysplasia and acute myeloblastic leukemia.2 Muller-Sieburg and Riblet, using a different
assay in BXD mice, mapped 2 candidate loci regulating the number of long-term culture-initiating cells (LTC-IC) to mouse chromosome 1.3 Further evidence for the existence of intrinsic
genetic control of hematopoietic stem cell kinetics comes from
embryo-aggregated chimeric DBA/2 B6 mice. In these mice,
DBA/2-derived stem cells contribute significantly and stably to
hematopoiesis in young adults, but with aging, B6-derived hematopoiesis
becomes predominant, if not exclusive.5 Hematopoietic
recovery after the administration of the myelotoxic drug 5-fluorouracil
to young B6DBA/2 chimeras was significantly faster in the
DBA/2-derived stem cell compartment than in the B6-derived
compartment.6 Similarly, when chimeric bone marrow was
transplanted, early engraftment of irradiated hosts was predominantly
DBA/2 derived, significantly out of proportion to the DBA/2
representation in the marrow graft, further suggesting a proliferative
advantage for DBA/2-derived stem cells. Because the microenvironment in
these chimeric mice was identical for both DBA/2- and B6-derived stem
cells, stem cell-intrinsic mechanisms must be at the basis of these
differences.6 Genetic variation has also been demonstrated
in the repopulation capacity of hematopoietic stem cells. One of the
CXB RI strains had a significantly higher capacity for long-term
competitive repopulation in CByB6F1 recipient mice than any of the
other CXB strains.7 Loci affecting the efficiency of
mobilization of progenitor cells to the peripheral blood have been
identified on chromosomes 2 and 11.9
In humans and in cats, evidence for genetically determined regulation
of hematopoiesis comes from studies addressing X inactivation in the
hematopoietic system in vivo.10-13 In as many as 50% of aging women, progressive skewing of X inactivation occurs in the hematopoietic system.10,11 Similar data were obtained in
cats.12 Furthermore, skewed X chromosome inactivation in
all hematopoietic lineages also occurs after bone marrow
transplantation12 and after repeated chemotherapy with
busulfan in cats.13,14 Human female monozygotic twins that
show skewed X inactivation in the hematopoietic system with aging tend
to inactivate the X chromosome from the same parent.11,15
Thus, alleles on the X chromosome confer a growth, survival, or
reconstitution advantage to stem cells. Progressive skewing of X
inactivation in the hematopoietic system is therefore likely to be
caused to a large extent by hemizygous selection,12 ie, a
selective advantage of one X chromosome over another, whereas
stochastic mechanisms are less dominant.15
The aim of this study was to test the hypothesis that strain-dependent
variation would exist in the response of primitive hematopoietic
progenitor and stem cells, as defined by the
LinSca1++kit+
phenotype,16-18 to the early-acting factors kit ligand
(KL), flt3 ligand (flt3L), and thrombopoietin (TPO). These factors have
been shown to be critical to the function of the hematopoietic stem cell compartment in vivo.19-21 We show here that there is
indeed strain-dependent variation in the response to these early-acting factors and that at least one locus for this trait maps to the X
chromosome. In addition, we found strain-dependent variation in the
absolute number and frequency of LinSca1++
cells (c-kit+ and c-kit subpopulations). A
quantitative trait locus (QTL) for this trait on chromosome 2 was
confirmed in congenic mice.
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Materials and methods |
Mice
Female mice of strains C57BL/6J (B6), DBA/2J, and BXD RI, aged 6 to 8 weeks, were all purchased from Jackson Laboratories (Bar Harbor,
ME). The mice were maintained in a germ-free environment and fed ad
libitum. Experiments and animal care were performed in accordance with
the Mount Sinai Institutional Animal Care and Use Committee.
Antibodies and cytokines
Unconjugated Ter119 (erythroid), CD2 (T and NK cells), CD3, CD4,
CD8 (T cells), B220 (B cells), Ly6G/Gr1 (granulocytes), Mac1 (macrophages), phycoerythrin-conjugated Sca1, biotin-conjugated anti-c-kit, Cychrome-conjugated streptavidin, and fluorescein isothiocyanate-conjugated goat antirat antibodies were purchased from
PharMingen (San Diego, CA). Recombinant mouse flt3L and thrombopoietin (TPO) and anti-transforming growth factor- (anti-TGF-)
antibodies were purchased from R&D Systems (Minneapolis, MN).
Supernatants from BHK/HM-5, BHK/MKL (both a kind gift of Dr J. Matous,
University of Washington), and WEHI 3B (a kind gift of Dr S Tsai, Mount
Sinai School of Medicine, New York, NY) cells were used as a source of
granulocyte macrophage-colony-stimulating factor (GM-CSF), KL, and
interleukin-3 (IL-3), respectively.
Isolation of LinSca1+/+ cells
Femurs and tibias were flushed with Iscoves modified Dulbecco
medium (IMDM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal
calf serum (FCS). Low-density bone marrow cells, obtained after density
centrifugation, were stained with Ter119, CD2, CD3, CD8, CD4, B220,
Mac1, and Gr1 for 20 minutes at 4°C, washed, and stained with goat
antirat antibodies for 20 minutes at 4°C. After washing, the cells
were stained for 20 minutes at 4°C with phycoerythrin-conjugated Sca1
and biotin-conjugated CD117 (c-kit), washed with phosphate-buffered saline, and stained with streptavidin-Cychrome. Cells were sorted on a
MoFlo flow cytometer (Cytomation, Fort Collins, CO) at 30 psi sheath
pressure and at a rate of 12 000 to 15 000 events per second. Cells
with a low side scatter, a low-to-medium forward scatter (Figure
1, R1), a green (lineage) fluorescence
lower than the median fluorescence of cells stained with
isotype-matched control antibodies, and an orange (Sca1) fluorescence
twice the intensity (in terms of channel numbers) of the brightest
cells in control samples (Figure 1, R2) were sorted as
LinSca1++ cells. Cells falling in R1 and R2
with positive red (c-kit) fluorescence (Figure 1, R3) were sorted as
LinSca1++kit+ cells, whereas
cells falling in R1 and R2 with negative red fluorescence (Figure 1,
R4) were sorted as LinSca1++kit
cells. The absolute number of cells in each population was estimated by
recording the number of sorted cells, as indicated by the number of
sortable events on the counter of the cell sorter. The abort frequency
was less than 5% in the sorting conditions we used. Bone marrow pooled
from at least 2 mice was used in each experiment. Because the secondary
goat antirat antibodies were not blocked with rat immune globulins,
most Lin+ cells appear Sca1+ in Figure 1. This
blocking step was omitted because only lineage-negative cells were
isolated, and the blocking step did not affect the fluorescence or the
number of the cells in the sort windows in preliminary experiments.
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| Figure 1.
Sort windows used to isolate LinSca1++,
LinSca1++kit+, and
LinSca1++kit cells.
Cells with low to medium forward scatter (FSC) and low side scatter
(SSC) (R1), negative green (lineage) fluorescence, orange (Sca1)
fluorescence higher than twice the fluorescence level of cells stained
with control antibodies (not shown) (R2), and positive red (c-kit)
fluorescence (LSK+, R3) or negative red fluorescence
(LSK, R4) were isolated. For the isolation of
LinSca1++ cells, cells falling into R1 and R2
were isolated.
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Pre-colony-forming cell assay
Lin-Sca1++ or
LinSca1++kit+ cells were cultured
in triplicate at approximately 50 cells per well in flat-bottom,
96-well plates in serum-free media (StemPro34; Gibco BRL), 50 ng/mL
flt3L, 50 ng/mL TPO, and 10% BHK/KL supernatant (containing kit
ligand). Because the BHK/KL supernatant contained 10% FCS, the actual
serum concentration in these cultures was still 1%. Three hours after plating, the exact number of cells per well was determined by visually
counting the cells at × 40 magnification. After 5 days of liquid
culture at 37°C and 5% CO2, the cells were counted, and
500 cells were plated in methylcellulose cultures containing IMDM, IL-3
(10% of WEHI 3B supernatant), GM-CSF (10% of BHK/HM-5 supernatant),
KL (10% of BHK/MKL supernatant), erythropoietin (2 U/mL), FCS (20%),
anti-TGF- (10 µg/mL), and 2-mercaptoethanol (106
M). After 8 days of incubation at 37°C in a humidified incubator with
5% CO2, the cultures were scored for colony formation. The same lot of fetal bovine serum was used in all the experiments.
Statistical analysis
Student t test for unpaired samples was used, unless
indicated otherwise.
Congenic mice
Congenic mice were generated by marker-assisted
back-crossing22,23 using B6 and BXD-31Ty as founders.
These B6.DBA/2-(D2Mit133-D2Mit200) congenic animals have a segment of
chromosome 2 between 66.7 and 102 cm, derived from DBA/2 on a B6
background, and were obtained after 5 generations of male back-crossing
and genotype-based selection with genetic markers spaced approximately
20 cM apart. Analysis of polymorphic simple sequence repeat elements
was made by polymerase chain reaction using primers obtained from
Research Genetics (Huntsville, AL).
Quantitative trait analysis using BXD RI strains
Recombinant inbred strains were generated by repeated inbreeding
of F2 mice derived from 2 parental inbred strains, C57BL/6 (B6) and
DBA/2 in the case of BXD RI strains. The genome of RI strains is
composed of a patchwork of homozygous chromosome segments derived from
either progenitor strain, with each of the RI lines having a unique
combination of patches from the progenitor. Genetic maps are available
for each RI strain that allow the mapping of traits specified by loci
at which the parental strains are polymorphic.24,25 Linkage analysis is performed by determining the relevant trait in each
of the strains of a set of RI strains, which results in a strain
distribution pattern (SDP). The data are then analyzed using Map
Manager QTb29ppc software developed by Manly.26 This software statistically analyzes the linkage of a given trait with previously typed polymorphic loci in the RI strains, which are inherited from either parental strain. This allows the assignment of a
trait to a corresponding map position and the calculation of
statistical significance.27
Statistical analysis.
Because of the limited number of RI 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.27,28 As with all statistical comparisons, it is necessary to make a calculation of the probability that the observed result was a false-positive. Therefore, the genome-wide probability of obtaining the observed linkages by random
chance, corresponding to an error threshold of P = .05, is
calculated using the robust nonparametric permutation method developed
by Churchill and Doerge,27 which is implemented in the Map
Manager QTb29ppc software developed by Manly et
al.26 The peak logarithm of the likelihood of odds ratio
(the LOD score) of the correctly ordered data obtained in our study is
compared with the peak LOD scores computed for 5000 random permutations of the same data. As an example of this kind of analysis, if the actual
data gave an LOD score of 6 and only 1 in 1000 random permutations exceeded this value, the genome-wide probability of a false-positive would be approximately 0.001. The P = .5 error threshold,
a level considered suggestive of a QTL, corresponds to a LOD score that is exceeded by the highest LOD scores of half the
permutations.27
Interval mapping.
A subroutine of the Map Manager QTb29ppc software, using
computationally efficient regression equations, is used for mapping the
QTLs. The probability of linkage between our trait under study 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) should attain values of at least 0.0001 to reach a level of
genome-wide statistical significance.27,28
Mapping databases.
Mapping databases were downloaded from
www.nervenet.org/papers/bxn.html.29
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Results |
Responsiveness to early-acting factors and number of phenotypically
defined progenitor cells in B6 and DBA/2 mice
A pre-colony-forming cell (pre-CFC) assay was used to measure
proliferation and differentiation capacity of
LinSca1++kit+
cells,16-18 isolated by flow cytometric cell sorting
according to the sort windows shown in Figure 1, from B6 and DBA/2
mice. In this assay,
LinSca1++kit+ cells were cultured
in liquid cultures supported by the early-acting factors KL, flt3L, and
TPO.19-21 After 5 days of culture, the cells were counted
and plated in methylcellulose assays supported by KL, IL-3, GM-CSF,
erythropoietin, and neutralizing anti-TGF- antibodies for the
determination of CFC content. B6 and DBA/2 mice were chosen for these
studies because of the known strain-dependent variation in the
hematopoietic system between these 2 mouse strains2-6,8,9 and because of the availability of a relatively large set of BXD RI
strains together with a dense map of polymorphic
markers.24-26,29
LinSca1++kit+ cells from B6 mice
proliferated better in response to flt3L, KL, and TPO than
LinSca1++kit+ cells from DBA/2
mice (P = .01, n = 12, paired t test; Figure 2A). The difference in CFC generation was
not statistically significant (n = 12, Figure 2A). Most colonies
generated were myeloid, with 20% to 40% macroscopic colonies derived
from high proliferative potential cells. During these experiments, we
also recorded the number of sorted
LinSca1++kit+ events, as
determined on the counter on the cell sorter, as well as the frequency
of LinSca1++kit+ cells. The
frequency (not shown) and the absolute number (Figure 2B) of
LinSca1++kit+ cells were 2-fold
higher in B6 than in DBA/2 mice (P = .04, n = 8).
Long-term repopulating stem cells are enriched in the
LinSca1++kit+ fraction of bone
marrow cells.16-18 In addition, a
c-kit subpopulation of
LinSca1++ cells has been identified by
Randall and Weissman that does not proliferate in vitro and that has no
detectable long-term repopulating capacity.30 In addition,
in our hands, these cells did not respond to flt3L, KL, or TPO at all
(n = 16, not shown). The biologic significance of this population is
unknown. We noticed, however, that the number of
LinSca1++kit cells was also
2-fold higher in B6 than in DBA/2 mice (P = .04, n = 8,
Figure 2B). When, in a separate set of experiments, the total number of
LinSca1++ cells was quantified, the number in
B6 mice was again 2-fold higher than in DBA/2 mice
(P = .02, n = 6, Figure 2B). These data suggest that the
size of the LinSca1++ population as a whole
is genetically determined. An alternative explanation might be that B6
and DBA/2 mice simply differ in the level of expression of Sca1, so
that it would appear that DBA/2 mice have a lower number of
Sca1++ cells. However, the range of expression levels of
Sca1 on Lin cells in B6 and DBA/2 mice (Figure 2C) was
identical. It is therefore unlikely that the observed 2-fold variation
in the numbers of LinSca1++ cells was caused
by variation in the relative level of expression of the Sca1 antigen.
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| Figure 2.
Proliferative capacity and number of
LinSca1++kit+ and
LinSca1++kit cells in B6 and
DBA/2 mice.
(A) Number of cells and CFCs obtained after 5 days of liquid culture of
50 LinSca1++kit+ cells from B6
and DBA/2 mice supported by KL, flt3L, and TPO. Results are given as
mean ± SEM (n = 12 independent triplicate experiments;
*significantly different from B6, paired t test). (B)
Absolute number of LinSca1++,
LinSca1++kit+, and
LinSca1++kit cells in B6 and
DBA/2 mice as determined by flow cytometric cell sorting (mean ± SEM, n = 8 independent experiments in which the bone marrow pooled
from at least 2 mice was used; *significantly different from B6,
paired t test). (C) Range of expression levels of Sca1 on
Lin bone marrow cells from B6 and DBA/2 mice.
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Quantitative trait analysis in BXD RI strains
To further investigate potential genetically determined variation
in the cytokine responsiveness and number of
LinSca1++ cells, we performed quantitative
trait analysis using BXD RI strains. Twenty-eight BXD RI strains were
analyzed for responsiveness to the early-acting factors flt3L, KL, and
TPO, for the absolute number and frequency of
LinSca1++ cells, and for the fraction of
c-kit+ cells among LinSca1++
cells. In all RI strains, at least 3 independent experiments were
performed in triplicate using bone marrow pooled from at least 2 individual mice in each experiment. The strain distribution patterns
are shown in Figure 3. Because
LinSca1++kit cells do not
respond to early-acting cytokines at all (n = 16), only
LinSca1++kit+ cells are
responsible for cell proliferation and CFC generation from
LinSca1++ cells. Table
1 summarizes the map positions
obtained.
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| Figure 3.
Strain-distribution patterns in BXD mice.
Strain-distribution pattern of (A) the proliferation of
LinSca1++ cells in response to KL, flt3L, and
TPO (expressed as cell number after 5 days of culture per 50 input
LinSca1++ cells), (B) the absolute number of
LinSca1++ cells, (C) the relative fraction of
LinSca1++ cells, and (D) the fraction of
c-kit+ cells among LinSca1++
cells in 8- to 10-week-old BXD RI mice (mean ± SEM, n = 3
independent experiments in which the bone marrow pooled from at least 2 mice was used).
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There was wide phenotypic variation among BXD RI strains for
proliferative capacity in response to flt3L, KL, and TPO. Cell number
per 50 input LinSca1++ cells after 5 days of
culture ranged from 124 ± 72 (BXD6) to 4369 ± 2119 (BXD5) (Figure
3A). CFC output completely paralleled cellular proliferation, and no
strain-dependent variation was noted in the secondary cloning
efficiency (not shown). It was surprising that up to 35-fold variation
in the proliferative capacity was seen among BXD RI strains (Figure
3A), whereas there was only a small difference in this trait among the
2 progenitor strains (Figure 2A), indicating that this is a multigenic,
quantitative trait. For multigenic traits, phenotypic spread among BXD
strains actually represents the phenotypic spread in the F2 generation, not of the progenitor strains. Therefore, extensive phenotypic variation can be seen within the RI strains, even when the phenotype of
the 2 progenitor strains is not significantly different for that trait.
The reason is that RI strains may have most of the positive or most of
the negative alleles contributing to a given trait, whereas the 2 progenitor strains may have both positive and negative alleles for that
trait.24,25 A major QTL for proliferative capacity was
found on chromosome 2, with a likelihood ratio statistic (LRS) in the
significant range as determined by permutation analysis. Interval
mapping showed the highest LRS value (18.8) between D2Mit495 (73.2 cM)
and D2Mit411 (77.6 cM) (Table 1). The donor of the high allele was B6.
Among the 28 BXD RI strains studied, the number of
LinSca1++ cells varied from 721 ± 86
(BXD6) to 6472 ± 447 (BXD28) cells per femur (Figure 3B). The
percentage of LinSca1++ cells in the bone
marrow (Figure 3C) correlated well with the absolute number of
LinSca1++ cells (r = 0.870,
P < .0001, not shown). This correlation indicates that
the fraction of LinSca1++ cells is a good
measure of the absolute number of
LinSca1++ cells and that our data cannot be
explained by genetically determined variation in bone marrow
cellularity. In addition, the range of expression of Sca1 was the same
in all BXD strains, indicating again that differences in the expression
levels of Sca1 cannot explain the variation in the number of
LinSca1++ cells. Although there was some
variation in the ratio of c-kit+/c-kit cells
among LinSca1++ cells in BXD RI strains, no
statistically significant differences were found (Figure 3D; only
strains in which 3 experiments were performed are shown). Variation in
the fraction of c-kit-expressing LinSca1++
cells thus cannot explain the 35-fold variation in proliferative capacity of LinSca1++ cells. The 9-fold
variation in the number of LinSca1++ cells
among BXD RI strains compared with the only 2-fold difference between
the 2 progenitor strains, B6 and DBA/2, suggest that the number of
LinSca1++ cells is a complex multigenic trait.
Quantitative trait analysis was performed for the number of
LinSca1++ cells using the average values for
the 3 independent experiments performed in each BXD strain (Table 1).
This analysis revealed a highly suggestive QTL for the number of
LinSca1++ cells on chromosome 4 (Table 1).
The highest value of the LRS (15.9) by interval mapping was found
between D4Mit33 (77.5cM) and D4Mit343 (79 cM). This was just short of
significant, for which an LRS of 16.6 was required according to
permutation analysis.27 The frequency of
LinSca1++ cells mapped to the same region of
chromosome 4 with a suggestive level of significance (LRS 12.7). The
donor of the high allele was DBA/2. A second suggestive QTL (LRS 9.4 for absolute number and 11.4 for frequency of
LinSca1++ cells) was found on chromosome 2, between D2Mit495 (73.2 cM) and D2Mit411 (77.6 cM). The donor of the
high allele at this locus was B6. A third, weaker QTL was found on
chromosome 7 between D7Mit17 (51 cM) and D7Mit238 (53 cM); B6 was the
donor of the high allele (LRS 9.2). The level of significance of the
association with all 3 loci was in the suggestive range according to
published criteria. This means that the association is likely but needs confirmation.25,27,28 However, the same 3 loci were also
found, again with a level of statistical significance in the suggestive range, when each round of mapping was analyzed individually. Therefore, each of these QTLs has been confirmed in successive, independent rounds
of linkage analysis.25
Interestingly, the QTL on chromosome 2 for the number of
LinSca1++ cells overlaps with the QTL on
chromosome 2 for the proliferative capacity of
LinSca1++ cells. In addition, there was a
significant correlation between proliferation and the number and
frequency of LinSca1++ cells among BXD RI
strains (Figure 4A; data shown for
absolute number of LinSca1++ cells), and the
pattern of LRS values along chromosome 2 was virtually identical for
both traits (Figure 4B). Taken together, these data may suggest that
genetic variation in the responsiveness to early-acting cytokines
contributes to the regulation of the absolute number of
LinSca1++ cells among BXD RI strains.
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| Figure 4.
Responsiveness to early-acting factors and number of
LinSca1++ cells in BXD RI strains.
(A) Correlation between the responsiveness to flt3, KL, and TPO and
number of LinSca1++ cells per femur among BXD
RI strains. (B) LRS along chromosome 2 (chr 2) for the number of
LinSca1++ cells per femur (top) and
proliferation (bottom).
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Confirmation of a locus on chromosome 2 determining the number of
LinSca1++kit+ cells in
congenic mice
Congenic mice22,23,25 were constructed in the
laboratory of GvZ, where a segment from chromosome 2 between D2Mit133
(66.7 cM) and D2Mit200 (102 cM), encompassing the region containing a
QTL for the number of LinSca1++ cells and for
their proliferative capacity (Table 1), from DBA/2 mice was crossed
onto the B6 background (B6.DBA/2-[D2Mit133-D2Mit200] mice). We
determined whether introgression of this DBA/2-derived segment of
chromosome 2 onto the B6 background would change the number of
LinSca1++,
LinSca1++kit+, and
LinSca1++kit cells and their
responses to flt3L, KL, and TPO toward the values obtained for DBA/2
(Figure 2). Mice congenic for the distal region of chromosome 4 or
chromosome 7 are not yet available.
As shown in Figure 5, congenic mice had
significantly fewer LinSca1++kit+
cells (P = .0075) than B6 mice, whereas the difference in
the number of LinSca1++kit
cells (P = .1) and total
LinSca1++ cells (P = .07) was at
the limit of statistical significance by paired t test. Our
data thus confirm that the segment of chromosome 2 between 66.8 and 107 cM contains at least one gene that contributes to the regulation of the
pool size of LinSca1++kit+ cells
and possibly also of
LinSca1++kit cells, that this
gene shows allelic variation between B6 and DBA/2 mice, and that DBA/2
was the donor of the low allele for this trait.
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| Figure 5.
Number of LinSca1++,
LinSca1++kit+, and
LinSca1++kit cells in congenic
mice.
(A) Average number (mean ± SEM, n = 8) of
LinSca1++,
LinSca1++kit+, and
LinSca1++kit cells per femur in
B6 and B6.DBA/2-(D2Mit133-D2Mit200) congenic mice (the latter are
indicated in the figure as B6.D2-chr2). Cell numbers were determined by
cell sorting. P values (paired t test) are given
at the tops of the figures.
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We anticipated seeing lower proliferative capacity and CFC generation
in LinSca1++kit+ cells from
congenic mice than from B6 mice. However, proliferation and CFC
generation were identical in B6 and congenic mice (not shown, n = 8).
The QTL on chromosome 2 for proliferative capacity could thus not be
confirmed in congenic mice.
Evidence for X-linked genetic factors in the regulation of the
response of primitive hematopoietic stem and progenitor cells to
early-acting cytokines in mice
Alleles of unknown X-linked genes confer a selective advantage to
hematopoietic stem cells in vivo in humans and in
cats.10-15 No models or in vitro assays are available to
investigate X-linked regulation of hematopoiesis in the mouse.
Furthermore, quantitative trait analysis with the available number of
BXD RI strains does not detect all QTLs involved in complex
traits.24,25 We therefore tried to establish a model to
detect X-linked allelic variation in the hematopoietic stem cell
compartment by investigating whether reciprocal male B6D2F1 and D2B6F1
mice differed in the number of
LinSca1++kit+ and
LinSca1++kit cells and in the
responsiveness of LinSca1++kit+
to early-acting cytokines. Female D2B6F1 and B6D2F1 mice are heterozygous in all their loci, and X inactivation is
random.31 Therefore, the phenotype of reciprocal female F1
hybrids will be the same, unless there is allelic variation in
imprinted genes, so that a parent-of-origin effect is observed.
Reciprocal male F1 hybrids, however, differ in the origin of the unique
X chromosome (B6 in B6D2F1 mice, and DBA/2 in D2B6F1 mice). Phenotypic
variation between reciprocal male F1 hybrids is thus most likely caused by allelic variation at X-linked loci. Theoretically, it is also possible that allelic variation at Y-linked loci exists.
The numbers of LinSca1++kit+ and
LinSca1++kit cells in female
and male F1 hybrids were intermediate between those for the parental strains, indicating that it is unlikely that X-linked genes regulate the number of LinSca1++kit+ or
LinSca1++kit cells in vivo (not
shown, n = 8). Proliferation (Figure
6A) and CFC production (Figure 6B) were
similar in female B6D2F1 and D2B6F1 mice (n = 4). This is in
accordance with complete heterozygosity in autosomal loci and random X
inactivation in female F1 hybrids.31 In contrast to female
F1 hybrid mice, however, proliferation (Figure 6A) and CFC generation
(Figure 6B) in LinSca1++kit+
cells from male D2B6F1 mice were significantly higher than in LinSca1++kit+ cells from male
B6D2F1 mice (n = 8). Because male D2B6F1 and B6D2F1 mice are
heterozygous in all their autosomal loci but differ in the origin of
the X chromosome (B6 in B6D2F1 mice and DBA/2 in D2B6F1 mice), these
data suggest that at least one X-linked locus contributes to the
regulation of the response of
LinSca1++kit+ cells to
early-acting factors and that the donor of the high allele is DBA/2. As
mentioned before, genetic variation in Y-linked genes could
theoretically also explain our data. Although this cannot be formally
excluded, the involvement of Y-linked genes is less likely given the
paucity of somatically active genes on the Y chromosome.32
In addition, if allelic variation between DBA/2 and B6 mice on the Y
chromosome plays a role, then one would expect a differential effect of
sex on this trait in B6 and DBA/2 mice, which was not the case (not
shown).
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| Figure 6.
Difference in proliferative capacity in reciprocal male
and female F1 hybrid mice.
Cell proliferation (A) and CFC generation (B) per 50 input
LinSca1++kit+ cells isolated from
male and female D2B6F1 and B6D2F1 hybrid mice. Results are given as
mean ± SEM. P values indicated on the figure were
obtained by paired t test (n = 4 for female D2B6F1 and
B6D2F1 mice; n = 8 for male D2B6F1 and B6D2F1 mice).
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Discussion |
In this study, we have identified 2 significant traits involving
the hematopoietic stem and progenitor cell compartment that show
quantitative variation: (1) responsiveness to early-acting hematopoietic cytokines and (2) absolute number of
LinSca1++ cells (both c-kit+ and
c-kit subpopulations).
Variation in the fraction of LinSca1+ cells
among inbred mouse strains was already noted previously by Spangrude
and Brooks.33 LinSca1++ cells
are highly enriched in stem and progenitor cells16-18 but are still heterogeneous. Long-term repopulating cells are thought to be
LinSca1++kithigh,1,16-18
though some reports suggest that a kit precursor of
kithigh repopulating stem cells exists.34
LinSca1+kitlow cells have been
shown to be enriched in early-lymphoid precursors,35 though lymphoid-committed precursors have recently been identified in
the Linkithigh population as
well.36 A
LinSca1+kit population has been
described by Randall and Weissman30 in steady state bone
marrow. This mystery population is quiescent, increases in
size with aging, does not proliferate in vitro, and does not contain
long-term repopulating activity. We did indeed reproduce these findings
(J.C.L. and H.-W.S., unpublished observations, December 2001).
The functional significance of this
LinSca1++kit population is
unknown. We show here that at least 3 loci determine the total number
of LinSca1++ cells: one on chromosome 2, one
on chromosome 7, and one on chromosome 4. Because we found that there
was little variation in the ratio of kit+/kit
cells among LinSca1++ cells, despite a
10-fold variation in the total number of
LinSca1++ cells in BXD strains (Figure 3), we
conclude that the number of
LinSca1++kit+ and
LinSca1++kit cells are
regulated by at least partially overlapping genetic mechanisms. Indeed,
the number of LinSca1++kit+ and
LinSca1++kit cells is 2-fold
higher in B6 than in DBA/2 mice. These mechanisms are not necessarily
identical, nor do they necessarily affect each subpopulation to the
same extent. In congenic mice, we find a significant effect of the
DBA-derived segment of chromosome 2 on the number of
LinSca1++kit+ cells, but the
effect on the number of
LinSca1++kit and total
LinSca1++ cells numbers is at the limit of
statistical significance, possibly suggesting that this locus mainly
regulates the number of
LinSca1++kit+ cells. This will,
of course, be reflected in the size of the total
LinSca1++ population, for which we have
demonstrated linkage on chromosome 2 in BXD RI strains. Although the
biologic significance of a small change in the number of
LinSca1++ cells caused by a single locus may
be limited, the combined effect of many loci affecting pool size of
LinSca1++ cells, as in those BXD mice with
extreme values, is likely to be important. In addition, the importance
of our data lies in the fact that we show that multiple loci affecting
the number of LinSca1++ cells exist. This may
lead to identification of the mechanisms that regulate the number of
LinSca1++ cells and of the kit+
and kit subpopulations thereof.
The correlation between the response to flt3L, KL, and TPO and the
number of LinSca1++ cells among BXD RI
strains and the overlapping QTLs for both traits raise the possibility
that the responsiveness to early-acting cytokines plays a role in
determining the number of LinSca1++ cells.
However, a phenotype could be reproduced in mice congenic for the locus
on chromosome 2 for the number of
LinSca1++kit+ and
LinSca1++kit cells, but not for
proliferative capacity in response to KL, flt3L, and TPO. The most
likely explanation for the lack of a proliferative phenotype in the
congenic mice is epistasis with other loci that show allelic
variation.25,37 In addition, a locus was found on the X
chromosome that clearly determines the response of
LinSca1++kit+ cells to
early-acting factors, but it has no effect on the number of
LinSca1++kit+ or
LinSca1++kit cells. Our data
thus suggest that genetic variation in the response to early-acting
cytokines does not determine the number of
LinSca1++kit+ or
Lin-Sca1++kit cells. This is in accordance
with the data of Miller et al,38 who showed that the
number of stem cells, as defined by the
LinSca1+WGA+ phenotype, is
similar in W mutant and wild-type mice. The correlation between the
response to early-acting factors and the number of LinSca1++ cells is most likely explained by
the fact that linked, but distinct, QTLs on chromosome 2 are major
determinants for both traits.
Other QTLs related to hematopoiesis have been mapped to this segment of
chromosome 2. In several studies,8,39 a suggestive QTL on
chromosome 2 at approximately 50 cM, regulating the increase of the
number of CAFCd35 with aging in B6 and DBA/2 mice has been reported.
Hasegawa et al9 identified a significant QTL that controls
the efficiency of mobilization of hematopoietic progenitor cells in
response to GM-CSF on chromosome 2 between 46 and 86 cM.9
It was recently shown that the mobilization of hematopoietic stem cells
occurs after the M-phase of the cell cycle.40 Therefore, it is possible that an allele responsible for a higher responsiveness to early-acting factors also causes more efficient mobilization of
hematopoietic stem cells. Potential candidate genes in this region of
chromosome 2 known to play a role in hematopoiesis include jag1 (encoding the Notch ligand Jagged-1),41
bmp2 (encoding BMP-2),42 and the il1
complex (encoding IL-1 and IL-1, respectively).43
QTL analysis, as reported here and by others,2-6,8,9
demonstrates differential genetic regulation of the size of the stem
and progenitor cell compartment, as defined by immunophenotype on one
hand and by functional assays on the other hand. Our data show that
LinSca1++kit+ cells are more
numerous in B6 than in DBA/2 mice, confirming an earlier report
in which the WGA++Linrho123low
phenotype was used to isolate and identify the most primitive hematopoietic precursors.6 Yet,
LinSca1++kit+ cells from DBA/2
mice repopulate syngeneic recipients more efficiently than those of B6
mice and generate more CAFCd35.44 In addition, DBA/2 mice
show faster recovery after the administration of 5-fluorouracil than B6
mice44 and contain more CAFCd35 and LTC-IC than B6
mice.2,3,8 Conversely, data in allophenic mice suggest
that on aging, B6-derived stem cells are more efficient at sustaining
hematopoiesis.4,5 In CXB mice, long-term repopulation
potential does not correlate with LTC-IC and CAFC numbers
either.7 QTL analysis and subsequent identification of
genes responsible for quantitative variation in the stem cell
compartment, as defined by function and by phenotype, may provide novel
insight into the regulation of hematopoiesis by helping to resolve
these discrepancies. A possible explanation may be that the number of
LinSca1++kit+ cells is a
reflection of stem and progenitor cell pool size in steady state
conditions, whereas assays such as CAFC, LTC-IC, and long-term
repopulation assay45 reflect, among other things, the
capacity of the stem cell compartment to respond to hematopoietic stress.
The answer to whether the putative X-linked locus we identified here is
the same as the locus that causes skewed X inactivation on aging in
cats and in humans will have to await gene identification of the loci
involved. However, given the importance of early-acting cytokines such
as KL, flt3L, and TPO in vivo,19-21 allelic variation in
one or more X-linked genes affecting the responsiveness to these
cytokines is an attractive candidate mechanism for the large genetic
component in the skewed X inactivation in elderly females in
humans11,15 and in cats.12 Our study suggests
that an easy assay measurement of the responsiveness to early-acting
cytokines in vitromay allow mapping and identification of X-linked
loci involved in the regulation of hematopoiesis.
In summary, we demonstrated genetic variation in the response to
cytokines critical for hematopoiesis in vivo and in the pool size of
cells belonging to a phenotyp |
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Abstract
Introduction