Journal of Gerontology: BIOLOGICAL SCIENCES
2005, Vol. 60A, No. 4, 448–456
Copyright 2005 by The Gerontological Society of America
c-Kit Expression and Stem Cell Factor-Induced
Hematopoietic Cell Proliferation Are Up-Regulated
in Aged B6D2F1 Mice
Aleah L. Smith,1 Felicia M. Ellison,1 J. Philip McCoy, Jr.,2 and Jichun Chen1
1
Hematology Branch and 2Flow Cytometry Core Facility, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland.
Expression of c-Kit (CD117) and stem cell factor/c-Kit-mediated cell proliferation were tested in
vitro in young and old B6D2F1 mice to study the role of c-Kit signaling in hematopoietic stem
cell (HSC) senescence. Increasing age is associated with a significant increase in bone marrow
(BM) cells without affecting mature blood cells. The number of c-Kit-expressing BM cells
increased significantly in old mice when compared to young controls, to 201% in total BM cells,
261% in Lin cells, 517% in LinCD34þSca1þ progenitor cells, and 1272% in LinCD34Sca1þ
HSCs. Sorted LinSca1þCD117þ BM cells from an old mouse expanded 5-fold when cultured in
vitro for 72 hours with stem cell factor at 25 ng/ml, which was significantly higher than a 2.5-fold
expansion of the same cells from a young donor. HSCs and progenitor cells from B6D2F1 mice
maintain extremely high proliferative potentials and do not reach proliferative arrest at old age
during a normal life span.
W
HETHER hematopoietic stem cells (HSCs) undergo
senescence has been a debated issue for many years
(1,2). HSCs are a group of unique cells: Not only do they
have the ability to self-renew like other somatic cells, but
they also possess the ability to differentiate into progenitor
cells, and eventually into mature blood cells of various cell
lineages (3–7). In previous studies (3–7), HSCs from
C57BL/6 (B6)-based mouse strains showed no signs of
senescence, whereas HSCs from other strains showed
decreased functionality with increasing age. These strain
differences were the groundwork for genetic analyses that
defined several quantitative trait loci that regulate HSC
senescence (4,8,9). HSC functional ability is usually studied
in cell mixtures because of low HSC frequency and
unpredictable associations between HSC phenotype and
function (10–13). Thus, a significant decrease in HSC
functionality with increasing age is a practical definition for
HSC senescence.
An important pair of cytokine-receptor signaling molecules in the regulation of HSC function and senescence are
stem cell factor (SCF) and its receptor, c-Kit (CD117),
a transmembrane receptor tyrosine kinase expressed on
primitive HSCs and progenitor cells (14–18). Expression of
CD117 has been used as a marker to characterize HSCs in
mice and in humans (15,16,19–21). SCF and c-Kit interaction triggers a chain of signaling events which activate
downstream pathways leading to cell proliferation and/or
differentiation (14,22). Spontaneous mutations in mice of
the c-Kit gene result in the characteristic white spotting with
serious consequences, including functional failure in
hematopoiesis (17,18,23–25). Thus, c-Kit signaling plays
a significant role in maintaining normal hematopoiesis.
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In the current study, we specifically tested the effects of
aging on c-Kit expression and c-Kit signaling in bone
marrow (BM) hematopoietic cells. First, we found that the
proportion and total number of c-Kitþ cells are significantly
increased in aged B6D2F1 mice in various BM cell fractions.
We then sorted BM cells into LinSca1þCD117þ and
LinSca1þCD117 fractions and cultured them in vitro to
test cell expansion with or without SCF stimulation. Our data
support the view that HSCs from B6D2F1 mice have very
high proliferative potentials and do not reach proliferative
arrest even at an old age during a normal life span. Increasing
age is associated with an enlarged HSC pool and an enhanced
HSC response to SCF stimulation in B6D2F1 mice.
METHODS
Mice and Cell Preparation
Hybrid B6D2F1 mice were purchased from the Jackson
Laboratory (Bar Harbor, ME) and were housed in the
National Institutes of Health animal facility under standard
care and nutrition conditions. We used young (2–6 months)
and old (20–30 months) females in the current study.
Peripheral blood samples were obtained through retroorbital sinus bleeding. Complete blood counts were
performed using a Hemavet 1500 analyzer (Drew Scientific,
Oxford, CT). BM cells were flushed from two femurs and
two tibias of each mouse into 2 mL of flow buffer (2.68 mM
KCl, 1.62 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM
NaCl, 7.69 mM NaN3, and 1% bovine serum albumin) using
a 25-gauge needle, and filtered through a 90-lm nylon mesh
(Small Parts, Miami Lakes, FL) to prepare for single cell
suspensions. BM cells were counted using a model Z2
HEMATOPOIETIC CELL SENESCENCE
Coulter Counter (Coulter, Hialeah, FL) after erythrocytes
were lysed with ZAP-OGLOBIN II lytic reagent (Coulter).
Total BM cells were calculated based on the assumption that
two tibiae and two femurs contain 25% of all BM cells in
the body.
Flow Cytometry
BM cell composition was analyzed by flow cytometry.
Cells were incubated in Gey’s solution (130.68 mM NH4Cl,
4.96 mM KCl, 0.82 mM Na2HPO4, 0.16 mM KH2PO4, 5.55
mM dextrose, 1.03 mM MgCl2, 0.28 mM MgSO4, 1.53 mM
CaCl2, and 13.39 mM NaHCO3) for 10 minutes on ice to
lyse red blood cells, stained first with a premixed antibody
cocktail and then with diluted streptavidin-conjugated
Quantum Red (SAQR), and analyzed using a BD LSR II
flow cytometer (BD Biosciences, San Jose, CA). Monoclonal antibodies for murine CD3 (clone 145-2C11), CD4
(clone GK 1.5), CD8 (clone 53-6.72), CD11b (clone M1/
70), CD19 (clone ID3), CD34 (clone RAM34), CD45R
(B220, clone RA3-6B2), CD117 (c-Kit, clone 2B8),
erythroid cells (clone Ter119), granulocytes (Gr1/Ly6-G,
clone RB6-8C5), and stem cell antigen 1 (Sca1, clone E13161) were all purchased from BD Biosciences (San Diego,
CA). Each antibody was conjugated with fluorescein
isothiocyanate (FITC), phycoerythrin (PE), biotin, or
allophycocyanin (APC). SAQR was purchased from Sigma
(St. Louis, MO). Each acquisition was stopped when 20,000
or 1,000,000 cells were collected, depending on the type of
analysis and the availability of cells.
Cell Sorting
BM cells from two tibias and two femurs of each young
or old B6D2F1 mouse were flushed into 2 mL of Iscove’s
modified Dulbecco’s medium (IMDM; American Type
Culture Collection, Manassas, VA). Residual erythrocytes
were lysed using Gey’s solution, and cells were stained for
30 minutes on ice with a premixed antibody cocktail
containing Sca1- FITC, CD117-PE, and Lineage-Biotin
(CD3, CD4, CD8, CD11b, CD19, Gr1, and Ter119). After
a second staining with SAQR for 30 minutes, cells were
sorted into LinSca1þCD117þ and LinSca1þCD117
fractions using a MoFlo cell sorter (DAKO Cytomation,
Ft. Collins, CO). For each sample, 30–40 3 106 original
cells were stained for sorting.
SCF-Stimulated Cell Proliferation
Unfractionated BM cells, sorted LinSca1þCD117þ cells,
and sorted LinSca1þCD117 cells were cultured in 12-well
polystyrene tissue culture plates (Corning, Corning, NY) at
an initial concentration of 5000 cells per well in 2 mL of
Dulbecco’s modified essential medium (Cellgro Mediatech,
Herndon, VA) supplemented with 15% heat-inactivated
fetal bovine serum, 1% penicillin, 1% streptomycin, 1% Lglutamine, interleukin-3 at 5 lg/mL, and interleukin-6 at 5
lg/mL. Cells were cultured with or without the presence of
SCF at 25 ng/mL (R & D Systems, Minneapolis, MN) in
duplicate or triplicate (depending on the number of cells
available) at 378C with 5% CO2. After 2 days in culture,
cells were examined under a phase-contrast microscope
and were photographed. After 3 days in culture, cells were
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collected and counted under a light microscope to calculate
cell expansion (defined as the ratio between the number of
cells recovered and the number of cells used to initiate the
culture). Cultured cells were also stained with antibodies
and were analyzed by flow cytometry using similar antibody
combinations as described earlier to define the phenotype
of the expanded cells.
Data Analysis
Blood, spleen, and BM cellular composition data as well
as cell expansion data were analyzed using one-way or twoway analysis of variance platforms provided by the JMP
Statistical Discovery Software (SAS Institute, Cary, NC)
(26). Results are shown as mean and standard error,
and statistical significance was declared at p , .05 and
p , .01 levels.
RESULTS
Higher Proportion of CD117þ Cells in the
BM of Old B6D2F1 Mice
The primary focus of this study was to test the effects of
increasing age on c-Kit (CD117) expression and hematopoietic cell response to SCF stimulation, as the SCF/c-Kit
ligand/receptor pair is vital to the normal function of HSCs
and progenitor cells. First, we extracted BM from young and
old B6D2F1 mice and stained the cells with various
antibodies to test CD117 expression. Of the 1,000,000 cells
collected for each sample, the proportion of CD117þ cells
was slightly higher (not significant, p . .05) in old than in
young B6D2F1 mice within total BM cells (4.4 6 0.5% vs
3.4 6 0.4%; Figure 1, A and B), and was significantly
higher ( p , .05) in old than in young mice in Lin BM
cells (0.72 6 0.09% vs 0.43 6 0.08%; Figure 1, C and D).
When BM cells were further divided into LinCD34
and LinCD34þ cell fractions (Figure 2, A and B),
the proportions of CD117þ cells were significantly higher
( p , .05) in old versus young B6D2F1 mice in both the
LinCD34þ (0.58 6 0.08% vs 0.36 6 0.06%; Figure 2, C
and D) and the LinCD34 cell fractions (0.14 6 0.02% vs
0.08 6 0.02%; Figure 2, E and F). Furthermore, the
percentage of LinCD34þSca1þCD117þ progenitor cells
was three times higher (0.0363 6 0.0070% vs 0.0113 6
0.0031%, p , .05; Figure 2, C and D), whereas the
percentage of LinCD34Sca1þCD117þ HSCs was more
than eight times higher (0.0375 6 0.0110% vs 0.0045 6
0.0013%, p , .05; Figure 2, E and F), in old than in
young B6D2F1 mice. Thus, in old B6D2F1 mice,
CD117 expression was up-regulated in all fractions of
BM cells, especially in the fraction that contains the most
primitive HSCs.
More HSCs but Normal Mature Blood Cells in
Aged B6D2F1 Mice
In comparison to young mice, old B6D2F1 mice had
significant increases in total BM cells (159%, p , .01; Figure
3A), CD117þ cells (201%, p , .01; Figure 3B), LinCD117þ
cells (261%, p , .01; Figure 3C), LinCD34CD117þ cells
(286%, p , .01; Figure 3D), LinCD34þSca1þCD117þ cells
(517%, p , .01; Figure 3E), and LinCD34Sca1þCD117þ
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SMITH ET AL.
Figure 1. Bone marrow (BM) cells extracted from two tibiae and two femurs of each young and old B6D2F1 mice were first counted, and then stained with an
antibody cocktail containing CD34-fluorescein isothiocyanate (FITC) þ Sca1- phycoerythrin (PE) þ Lineage-Biotin/streptavidin-conjugated Quantum Red (CD3, CD4,
CD8, CD11b, CD19, Gr1, and Ter119) þ CD117- allophycocyanin (APC). Expression of CD117 on total BM cells (A and B) and on Lin BM cells (C and D) were
shown as flow cytometry dot plots. Data presented were representatives of five pairs of young (A and C) and old (B and D) mice used for the analysis.
HEMATOPOIETIC CELL SENESCENCE
451
Figure 2. After flow cytometry analysis, bone marrow cells were first gated into LinCD34 and LinCD34þ cell fractions (A and B). Expression of Sca1 and
CD117 on LinCD34þ (C and D) and LinCD34 (E and F) cells were shown as representatives of five pairs of young (A, C, and E) and old (B, D, and F) B6D2F1
mice used for the analysis.
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SMITH ET AL.
Figure 3. Calculated total bone marrow cells (A), CD117þ cells (B), LinCD117þ cells (C), LinSca1þCD117þ cells (D), LinSca1þCD117þCD34þ cells (E), and
LinSca1þCD117þCD34 cells (F) were shown as means with standard error bars from five young and five old B6D2F1 female mice. Young and old differences were
all at a p , .01 significance level except for LinSca1þCD117þCD34þ cells (E) which is at a p , .05 significance level.
cells (1272%, p , .05; Figure 3F). Notably, the increase in
LinCD34Sca1þCD117þ HSCs (Figure 3F) far exceeded the
increase in total BM cells (Figure 3A) in old B6D2F1 mice.
Despite the significant increases in BM hematopoietic
progenitor and stem cells, peripheral blood mature cell
concentrations remained normal in old B6D2F1 mice.
Complete blood count analyses revealed that concentrations
of red blood cells (9.7 6 0.8 vs 9.1 6 0.8), white blood
cells (8.5 6 0.8 vs 8.3 6 1.1), neutrophils (2.30 6 1.37 vs
1.78 6 0.39), lymphocytes (7.35 6 0.64 vs 5.90 6 0.8),
HEMATOPOIETIC CELL SENESCENCE
453
and platelets (891 6 44 vs 997 6 36) were relatively
comparable between young and old mice with no significant
difference. Blood hemoglobin concentration was slightly
higher in young (14.7 6 1.2) than in old mice (13.0 6 1.2),
but this difference was not statistically significant either.
Figure 4. Total bone marrow cells (A), sorted LinSca1þCD117þ cells (B),
and sorted LinSca1þCD117 cells (C) from young and old B6D2F1 mice were
each cultured in Dulbecco’s modified essential medium at 378C with 5% CO2
with or without the presence of stem cell factor at 25 ng/mL. Three days later,
cells were harvested and counted under a regular light microscope. Fold of cell
expansion was calculated based on the number of cells harvested and the number
of cells used to initiate the culture. Data are presented as means with standard
error bars from triplicate measurements.
SCF-Stimulated LinSca1þCD117þ Cell Proliferation
Is Elevated in Old B6D2F1 Mice
The fact that total numbers of LinCD34Sca1þCD117þ
and LinCD34þSca1þCD117þ cells were both significantly
increased in aged B6D2F1 mice provided evidence indicating that immature hematopoietic cell proliferation was
up-regulated in the BM of old B6D2F1 mice. One
possibility is that cells from old mice are more sensitive to
a normal cytokine environment. The other possibility is
that the old environment provides higher concentrations
of cytokines that stimulate cell proliferation. We cultured
total BM cells and sorted LinSca1þCD117þ cells and
LinSca1þCD117 cells in vitro for 3 days, with or without
the presence of SCF, to test the hypothesis that BM cell
response to SCF stimulation was up-regulated in old
B6D2F1 mice.
When unfractionated total BM cells were cultured, we
observed no age or SCF effect on cell proliferation (Figure
4A). Young and old cells expanded 1.57 6 0.09-fold and
1.90 6 0.15-fold, respectively, showing no significant age
effect. Cells expanded 1.77 6 0.12-fold and 1.70 6 0.12fold, respectively, with or without SCF (Figure 4A),
indicating that total BM cell proliferation was not stimulated
by the concentration of SCF used in the study. Similarly, for
sorted LinSca1þCD117 cells, there was no SCF effect on
cell proliferation, as cells expanded 1.74 6 0.13-fold and
1.68 6 0.13-fold, respectively, with or without SCF
simulation (Figure 4B). However, LinSca1þCD117 cells
from old B6D2F1 mice proliferated slightly better ( p , .01)
than did those from young B6D2F1 mice. On average, old
cells expanded 2.07 6 0.14-fold, whereas young cells
expanded 1.36 6 0.12-fold (Figure 4B).
For sorted LinSca1þCD117þ cells, we observed significant age ( p , .05) and SCF ( p , .05) effects on cell
proliferation (Figure 4C). On average, LinSca1þCD117þ
cells from old B6D2F1 mice expanded 4.87 6 0.79-fold
when cultured with SCF, which is significantly more ( p ,
.01) than the 2.49 6 0.56-fold expansion of young
LinSca1þCD117þ cells cultured with SCF (Figure 4C).
Without SCF, old LinSca1þCD117þ cells expanded
2.33 6 0.79-fold, slightly more (not significant) than the
1.52 6 0.61-fold expansion of young cells (Figure 4C).
We further analyzed cultured cells by flow cytometry for
the expression of CD117 and Sca1 on Lin cells (Figure 5).
Essentially, there was no CD117 or Sca1 expression on Lin
cells from cultured young (Figure 5A) and old (Figure 5B)
total BM cells. This was also the case in Lin cells from
cultured young (Figure 5C) and old (Figure 5D)
LinSca1þCD117 cells. Only cultured LinSca1þCD117þ
cells from young (Figure 5E) and old (Figure 5F) mice
contained larger cell fractions expressing both CD117 and
Sca1. However, the proportions of CD117þ and
CD117þSca1þ cells were much larger in cultured cells from
old B6D2F1 mice (Figure 5F).
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SMITH ET AL.
Figure 5. Cultured cells were harvested and stained with the antibody cocktail Sca1-fluorescein isothiocyanate (FITC)þ CD117-phycoerythrin (PE) þ LineageBiotin/streptavidin-conjugated Quantum Red (CD3, CD4, CD8, CD11b, CD19, Gr1, and Ter119). Expression of Sca1 and CD117 on Lin cells from cultured total
bone marrow cells (A and B), sorted LinSca1þCD117 cells (C and D), and sorted LinSca1þCD117þ cells (E and F) from young (A, C, and E) and old (B, D, and F)
B6D2F1 mice were shown as dot plots after 3 days culture in Dulbecco’s modified essential medium at 378C with 5% CO2 and stem cell factor at 25 ng/mL. Cells from
triplicate culture wells of each sample were pooled for the staining.
HEMATOPOIETIC CELL SENESCENCE
DISCUSSION
HSC senescence is an important issue with clinical
relevance. In human BM failure syndromes such as aplastic
anemia, myelodysplastic syndromes, and paroxysmal nocturnal hemoglobinuria, HSC number and function are both
reduced, leading to the development of anemia, neutropenia,
and thrombocytopenia. A potential cause for HSC functional failure is senescence under which HSCs experience
an irreversible loss in their capacity to proliferate.
Defining HSCs and progenitor cells by marker phenotypes has been a widely accepted practice in recent years.
Spangrude and colleagues (11,12) were the first to use cell
surface markers to define HSCs. More recently, Osawa and
colleagues (21) sorted out single cells, tested them in
a competitive engraftment assay, and found that cells with
long-term engraftment ability were found in the CD34
fraction of LinSca1þCD117þ cells, whereas the CD34þ
fraction of LinSca1þCD117þ cells contained less primitive
progenitor cells. In a separate experiment, Zhao and
colleagues (20) confirmed that long-term functional HSCs
are in the CD38þCD34 fraction of LinSca1þkitþ cells. In
addition, Orlic and colleagues (27) indicated that the
LinCD117þ cell fraction contains primitive HSCs, whereas
the LinCD117 cell fraction does not. In the case of HSC
senescence, Morrison and colleagues (28) found that old B6
mice had a 5- to 7-fold increase in cells with the HSC
marker phenotype and a 2-fold increase in HSC engraftment
in vivo. Results from our current study are consistent with
these findings from earlier studies that the HSC-containing
LinSca1þCD117þ cells are highly responsive to SCF
stimulation (Figure 4B).
As a key receptor, c-Kit plays a vital role in the regulation
of HSC proliferation and differentiation. This protooncogene lies on mouse chromosome 5 at the Whitespotting locus. Mutations at the White-spotting locus affect
proliferation and/or migration of cells during early embryogenesis and cause intrinsic defects in the HSC hierarchy
(17,29). Mutations at different portions of the c-Kit gene
result in various levels of functional loss, affecting embryonic survival and hematopoiesis (14,18,23,24). In our current study, increasing age caused significant increases in the
proportion and total number of CD117þ cells in various cell
fractions, especially the LinCD34Sca1þCD117þ primitive
HSCs (Figures 1 and 3). Our result is consistent with observations in B6 mice, where increasing age is associated
with an increased number and function of HSCs (4,9,28,30).
Two elements are to be considered in studying HSC
senescence: HSC number and functional ability per HSC. In
BALB mice, HSC frequency was unchanged and total HSCs
per mouse was slightly higher in old mice as a result of the
increased total BM cellularity. However, functional ability
per HSC was significantly reduced in old BALB mice and
accounted for the overall decline in HSC functionality (3,4).
In the current study, sorted LinSca1þc-Kitþ cells from old
B6D2F1 mice had a 5-fold expansion when cultured in vitro
for 72 hours in the presence of SCF, twice that of the 2.5fold expansion seen in sorted LinSca1þc-Kitþ cells from
young mice cultured under the same conditions (Figures 4
and 5). There are two possible explanations for this result: 1)
each old LinSca1þc-Kitþ cell proliferated twice as fast as
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each young LinSca1þc-Kitþ cell; 2) the old LinSca1þcKitþ cell population contains twice as many cells in the
proliferative state as young LinSca1þc-Kitþ cells.
That HSC function does not decline with increasing age is
not an uncommon phenomenon. In the mouse model, inbred
B6 and D2 mice, the parental strains of B6D2F1 mice, have
distinctive phenotypes of HSC senescence (30). D2 mice are
short-lived but have higher cobblestone area forming cell
frequencies in fetal liver cells in comparison to B6 mice
(30,31). B6 mice have twice as many LinSca1þc-Kitþ and
LinSca1þc-Kit cells in their BM than do D2 mice (32,33),
but D2 LinSca1þc-Kitþ cells contain twice as many cells
with cobblestone area forming cell activity (9,30). Mice
transplanted with 1000 D2 LinSca1þc-Kitþ cells recover
much faster than do animals transplanted with the same
number of B6 cells. Data from early studies using B6-based
strains showed that marrow or spleen grafts from aged mice
produced antibody-forming cells as effectively as did grafts
from younger donors in recipients. In addition, grafts from
aged and younger donors gave similar responses when
stimulated with various doses of antigens (6,7). Marrow cell
lines from old donors also functioned as well as those from
young donors after transplantation into either W/Wv anemic
or lethally-irradiated normal recipients (7). In the B6-D2
allophenic mice, D2 HSCs showed high functionality early
in life, but were then eclipsed by HSCs of the B6 genotype
(34). When B6-D2 allophenic marrow cells were engrafted
into lethally irradiated B6D2F1 recipients, D2 hematopoiesis was dominant early after transplantation but B6
hematopoiesis ascended over the subsequent months to
become dominant later in life (35,36). Our data, which show
a higher response of old LinSca1þc-Kitþ cells to SCFstimulated cell proliferation (Figure 4), is consistent with
previously published results and may represent the dominant
fashion by which the B6 phenotype is inherited.
In a recent report, Chen and colleagues (37) found that
freshly isolated CD4þ and CD8þ T cells from aged B6 mice
expressed increased levels of chemokines such as interferonc-inducible protein 10 and macrophage inflammatory protein
(MIP)-1a and -1b, and that the age-related difference in T-cell
chemokine expression has an important functional consequence. In studying in vitro proliferative potential of human
skin biopsies from members of the Baltimore Longitudinal
Study of Aging, Smith and colleagues (38) found a significant
decline in proliferative potential with donor age in females,
but not in males. In addition, the proliferative potential was
significantly greater for donors under the age of 30 years
when compared with all donors over the age of 30 years (38).
We speculate that HSCs from B6D2F1 and other B6-based
strains may have higher proliferative potentials or lower
proliferative rates. This might also be the case for the skin
biopsies from some of the older men in the study by Smith and
colleagues (38). The larger HSC pool in old B6D2F1 mice is
associated with insignificantly lower mature cell counts in the
peripheral blood. It is possible that the enlarged HSC pool in
old B6D2F1 mice is a response to the aged environment, in
which up-regulation in HSC proliferation is necessary to
maintain hematopoiesis at a normal level.
We studied the effect of aging on HSC number and functional response to SCF stimulation in vitro in B6D2F1 mice.
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SMITH ET AL.
Increasing age is associated with a significant increase in the
total number of HSCs and progenitor cells. Sorted HSCs from
old B6D2F1 mice also showed significantly higher levels of
response to SCF stimulation in vitro. Thus, not only the HSC
pool size, but also the functional ability per HSC, are
increased in aged B6D2F1 mice during a normal life span.
ACKNOWLEDGMENTS
We thank Dr. Neal S. Young for his critical review and constructive
comments. We also thank Ms. Leigh Samsel (Flow Cytometry Core
Facility) and Mr. David Caden (Laboratory Animal Medicine & Surgery) of
the National Heart, Lung, and Blood Institute for technical assistance in cell
sorting and complete blood counts.
Address correspondence to Dr. Jichun Chen, Hematology Branch,
National Heart, Lung, and Blood Institute, National Institutes of Health,
Building 10, Clinical Research Center Room 3-5132, 10 Center Drive,
Bethesda, MD 20892-1202. E-mail: [email protected]
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Received October 1, 2004
Accepted December 8, 2004
Decision Editor: James R. Smith, PhD