Download Increased hematopoietic stem cell mobilization in aged

HEMATOPOIESIS
Increased hematopoietic stem cell mobilization in aged mice
Zhenlan Xing, Marnie A. Ryan, Deidre Daria, Kalpana J. Nattamai, Gary Van Zant, Lei Wang, Yi Zheng, and Hartmut Geiger
Hematopoietic stem and progenitor cells
(HSPCs) are located in the bone marrow
in close association with a highly organized 3-dimensional structure formed by
stroma cells, referred to as the niche.
Mobilization of HSPCs from bone marrow
to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF)
requires de-adhesion of HSPCs from the
niche. The influence of aging of HSPCs
on cell-stroma interactions has not been
determined in detail. Using a mouse model
of G-CSF–induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately
5-fold greater in aged mice. Competitive
mobilization experiments confirmed that
enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic
cells from aged mice correlated with reduced adhesion of hematopoietic progeni-
tor cells to stroma and with elevated
levels of GTP-bound Cdc42. These results might indicate that stroma–stem
cell interactions are dynamic over a lifetime and result in physiologically relevant
changes in the biology of primitive hematopoietic cells with age. (Blood. 2006;108:
2190-2197)
© 2006 by The American Society of Hematology
Introduction
Hematopoietic stem and progenitor cells (HSPCs) are located in
the bone marrow (BM) in close association with a highly organized
3-dimensional structure formed by stroma cells, referred to as the
niche.1,2 It has been demonstrated that these cell–cell interactions
are vital for the biology of HSPCs. Systemic administration of
cytokines and chemokines or cytotoxic agents mobilize HSPCs
from the BM into peripheral blood (PB), where they are collected
in clinically useful quantities for stem cell therapies.3,4 Mobilization of HSPCs in response to these factors requires the de-adhesion
of HSPCs from the niche.5,6
Evidence accumulating over the past decade has proven that
there is a measurable and successive functional decline in hematopoietic, intestinal, and muscle stem cell function.7,8 Given that stem
cell activity is necessary to replenish lost differentiated cells, it has
been hypothesized that aging of hematopoietic stem cells (HSCs)
leads to reduced stem cell renewal and thus reduced tissue
homeostasis in aged animals,7-11 which is emphasized by ageassociated anemia and a decline in function of immune cells in
elderly persons.12-19 HSC aging is intrinsic to the aged cell and
cannot be reverted by exposing HSCs from aged animals to a
young microenvironment.18-20 The ability of healthy, older patients
to undergo stem cell mobilization in response to granulocyte
colony-stimulating factor (G-CSF), the standard regimen used for
clinical HSPC mobilization, has thus far not been investigated in
detail because autologous hematopoietic stem cell transplantation
is most often administered to adults younger than 50 years and
rarely to patients older than 60 years.21,22 The limited available
information is not conclusive in terms of efficiency of mobilization
because it is based primarily on the analysis of mobilization of
elderly patients after severe chemotherapy23,24 and possibly also
because 2 different methods of determining mobilization efficiency
are used (colony-forming cell [CFC] frequency vs CD34⫹ cell
frequency in PB). Combining general clinical wisdom and these
published reports, it is anticipated that the ability to mobilize
HSPCs in response to G-CSF may be reduced in elderly patients,23-26 hampering the efficient collection of HSPCs for subsequent hematopoietic stem cell therapies. Whether a negative
correlation exists between age and mobilization is still under
debate.24,25
The mouse has been used in a wide variety of studies to model
human G-CSF–induced mobilization of HSCs, with an overall
good correlation between results obtained from murine studies and
subsequent results obtained in humans. By investigating mobilization proficiency in aged mice, we report here that aged mice show
not only decreased but increased efficiency in mobilizing hematopoietic progenitor cells (HPCs) and HSCs to PB.
From the Division of Experimental Hematology, Department of Pediatrics,
Cincinnati Children’s Hospital Medical Center and University of Cincinnati
College of Medicine, Cincinnati, OH; and the Departments of Internal Medicine
and Physiology, Markey Cancer Center, University of Kentucky, Lexington, KY.
and RO1 AG022859 (G.V.Z.), and by the Ellison Medical Foundation.
Submitted December 4, 2005; accepted May 20, 2006. Prepublished online as
Blood First Edition Paper, June 1, 2006; DOI 10.1182/blood-2005-12-010272.
Supported by the Mouse Core and the Flow Cytometry Core of the Division of
Experimental Hematology at Cincinnati Children’s Hospital Medical Center.
Supported in part by National Institutes of Health grants RO1 HL076604 (H.G.)
2190
Materials and methods
Animals
Young C57BL/6J mice were obtained from The Jackson Laboratory (Bar
Harbor, ME) and were subsequently housed in the animal barrier facility at
Cincinnati Children’s Hospital Medical Center (CCHMC). Aged C57BL/6J
animals were retired breeders derived from our own colony or were
obtained from The Jackson Laboratory and subsequently aged in the animal
barrier facility at CCHMC or obtained from the National Institute on Aging
(NIA) aged rodent colony (Harlan). B6.SJL(BoyJ) mice were obtained
either from the divisional stock (derived from animals obtained from The
Reprints: Hartmut Geiger, Division of Experimental Hematology, Department
of Pediatrics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave,
Cincinnati, OH 45229; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
Jackson Laboratory) or from the National Cancer Institute (NCI; C57BL/6
Ly5.2Cr). All animals were house in a pathogen-free environment in the
CCHMC barrier facility.
Mobilization
rhuG-CSF (Amgen Biologicals, Thousand Oaks, CA) was administered
intraperitoneally at 12.5 ␮g/mL in PBS/0.1% BSA at 100 ␮g/kg once a day
for 5 days. Animals were given injections of a volume in the range of
136 ␮L up to 240 ␮L per injection. HPC frequency in PB, BM, and spleen
(SPL) was determined by a CFC assay on day 6.
CFC assay
For the CFC assay, 10 or 20 ␮L PB, 2 ⫻ 104 whole BM cells, 1 ⫻ 105 SPL
cells, or 4 ⫻ 103 low-density BM cells in 20 ␮L Hanks balanced salt
solution (HBSS) were admixed with 80 or 90 ␮L HBSS and 1 mL colony
assay media (MethoCult GF M 3534; Stem Cell Technologies, Vancouver,
BC, Canada) containing 50 ng/mL rm SCF, 10 ng/mL rm IL-3, and
10 ng/mL rh IL-6 (all from PeproTech, Rocky Hill, NJ) and incubated at
37°C. Each tissue sample was analyzed in triplicate. Colonies of more than
50 cells were scored in PB assays after 7 to 8 days and in BM and SPL
assays after 9 to 10 days.
Flow cytometry
Immunostaining and flow cytometry analyses were performed according to
standard procedures, and cells were subsequently analyzed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA). To distinguish aged
from young cells in the competitive transplantation model, anti–Ly5.2
(clone 104, FITC conjugated; BD Biosciences) and anti–Ly5.1 (clone A20,
PE conjugated; BD Biosciences) monoclonal antibodies were used. For
lineage (Lin) analysis in tissues, anti-CD3⑀ (clone 145-2C11, PE-Cy7
conjugated), anti-B220 (clone RA3-6B2, APC conjugated), anti-CD11b
(clone M1/70, APC-Cy7 conjugated), and anti–Gr-1 (clone RB6-8C5,
APC-Cy7 conjugated, all from BD Biosciences) antibodies were used. For
the determination of chimerism in the HSC and HPC compartments,
low-density BM cells obtained from layering whole BM cells on a density
gradient (Histopaque 1083; Sigma, St Louis, MO) were incubated with a
cocktail of biotinylated antibodies specific for molecules on differentiated
hematopoietic cells (Lin cocktail) containing anti-CD11b (clone M1/70),
anti-B220 (clone RA3-6B2), anti-CD5 (clone 53-7.3), anti–Gr-1 (clone
RB6-8C5), anti-Ter119, and anti-CD3⑀ (clone 145-2C11; all from BD
Biosciences) antibodies and subsequently were incubated with anti–Sca-1,
clone E13-161.7, PE-Cy7–conjugated (eBiosciences, San Diego, CA),
anti–c-Kit, APC-conjugated, clone 2B8, and streptavidin APC-Cy7 (all
from BD Biosciences). For the determination of the expression of adhesion
receptors, low-density BM cells were individually incubated with biotinylated antibodies specific against CD184 (CXCR4), CD49d (clone R1-2), or
CD49e (clone 5H 10-27) and subsequently with PE-labeled Lin cocktail
antibodies, anti–Sca-1 PE-Cy7, anti–c-Kit APC, and streptavidin FITC. For
the determination of the genotype of CFC colonies, individual colonies
were picked out of the semisolid medium at day 9 of culture, resuspended in
1 mL PBS, and stained for Ly5.1/Ly5.2 contribution to determine the
contribution of aged and young cells. At least 100 live cells were
acquired per colony, and only colonies showing greater than 80%
contribution to either the Ly5.1 or the Ly5.2 population were scored. For
live/dead discrimination in all immunophenotyping experiments, cells
were resuspended in medium containing 5 ␮g/mL 7AAD (Molecular
Probes, Eugene, OR).
Competitive transplantation
Two- to 4-month-old B6.SJL(BoyJ) (recipient) mice were irradiated with a
total dosage of 11.75 Gy (7 Gy ⫹ 4.75 Gy, 4 hours apart), and cells were
subsequently transplanted into the retro-orbital sinus in a volume of 200 ␮L
in HBSS/2% FCS. For BM transplants, whole BM cells were harvested and
pooled from the tibia and the femur of 2- to 3-month-old B6.SJL(BoyJ) and
25-month-old B6 mice (3 mice each age group) in HBSS supplemented
with 2% FCS. Equal numbers of BM cells (2 ⫻ 106 cells of each, aged and
INCREASED MOBILIZATION WITH AGE
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young) were transplanted into irradiated recipients. For PB transplants, PB
was harvested and pooled from 4 mobilized mice each and subjected to red
blood cell lysis. Irradiated recipients underwent transplantation with equal
volumes of PB cells from young and aged mice with a total of 800 ␮L PB
per recipient to ensure engraftment. After transplantation, mice were
housed in a pathogen-free environment in the CCHMC barrier facility.
Transendothelial migration
The murine cervical high-endothelial venule cell line (mHEVc) was
cultivated as previously described.27 Transwell inserts (8 ␮m, polycarbonate membrane; Corning Glassworks, Corning, NY) were preincubated in
RPMI 1640 supplemented with 20% FCS for at least 1 hour. Four thousand
endothelial cells were seeded on the transwell. Confluence of the endothelial monolayer after 4 days was confirmed microscopically and by the
ability of the monolayer to retain red blood cells (greater than 95%
retention). Low-density BM cells obtained from layering whole BM cells
from mobilized mice on a density gradient (Ficoll-Paque plus 1077;
Amersham Biosiences, Freiburg, Germany) were seeded at a density of
1 ⫻ 106 cells/well on top of the transwell in RPMI 1640 supplemented with
20% FCS. CFC frequency was determined in parallel. Murine SDF-1
(PeproTech) was added to the lower chamber (100 ng/mL) where indicated.
Transwell chambers were subsequently incubated at 37°C, migrated cells
were harvested 18 hours later, and the cell count was determined and
analyzed for CFC frequency. Lymphocyte migration across endothelial
cells was linear from 0 to 24 hours; thus, the 18-hour time point chosen for
our experiments was in the linear range and resulted in a reasonable
percentage of cells capable of migrating.28
CAFC progenitor adhesion assay
For the cobblestone area–forming cell (CAFC) progenitor adhesion assay,
FBMD-1 cells were seeded in IMDM supplemented with 15% FCS and 5%
horse serum (both from Gibco, Grand Island, NY) at a density of 1000 cells
per well in a 96-well plate, as previously described.29 After 1 week in
culture, confluence of the FBMD-1 monolayer was verified microscopically. Whole BM cells were diluted in CAFC medium (IMDM, supplemented with 20% horse serum [Gibco] and 10⫺5 M hydrocortisone ␴) at
3000, 1500, 750, and 375 cells per well (15 wells per cell concentration)
onto the FBMD-1 stroma cell line. To determine progenitor cell adhesion,
nonadherent cells were washed from the FBMD-1 stroma cells after 2 or
4 hours, and fresh CAFC medium was added to each well. The frequency of
total HPCs and adherent HPCs was determined by counting the frequency
of cobblestone areas at day 7 on the FBMD-1 stroma cell line. Initial
experiments confirmed that the percentage of adherent plus nonadherent
HPCs totaled 100% of the initially plated HPC frequency (data not shown).
Rho-GTPase effector domain pull-down assay
Relative levels of GTP-bound Rac1, Rac2, and Cdc42 were determined by
an effector pull-down assay, as previously described.30,31 Briefly, whole BM
cells or lineage-negative (Lin⫺) cells were lysed in a buffer containing 1%
Triton X-100 and incubated with glutathione bead–bound GST-PAK1.
Bound (activated) and unbound (nonactivated) RhoGTPases were probed
by immunoblotting with antibodies specific for Rac1 (clone 102), Cdc42
(clone 44), ␤-actin (clone C4; all BD Biosciences), and Rac2 (polyclonal;
Upstate Biotechnology, Lake Placid, NY), and the relative amount of each
protein was quantified by densitometry. To obtain lineage-depleted BM
cells, low-density BM cells (Ficoll Histopaque 1083; Sigma) pooled from 4
to 6 mice were incubated with biotinylated rat antimouse antibodies (Lin
Abs) against surface receptors on differentiated hematopoietic cells (Lin
cocktail: anti-CD11b, clone M1/70, anti-B220, clone RA3-6B2, anti-CD5,
clone 53-7.3, anti–Gr-1, clone RB6-8C5, anti-Ter119, anti-CD3⑀, clone
145-2C11; all from BD Biosciences) and subsequently with sheep anti–rat
IgG antibodies coupled to magnetic beads (Dynabeads; Invitrogen, Carlsbad, CA). Cells were exposed to a magnetic field, and Lin⫺ cells were
harvested and lysed.
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XING et al
Statistical analyses
Paired Student t tests were used to determine the significance of the
difference between means.
Results
Mobilization of progenitors is increased in aged mice
In the standard mobilization assay, which closely resembles that
used in humans, young (2- to 3-month-old) and aged (20- to
26-month-old) C57BL/6 (B6) mice were given G-CSF (100 ␮g/kg)
once a day by intraperitoneal injection for 5 days, and PB was
collected on day 6. Given that the mean lifespan of these mice is
approximately 800 days,32 26 months of mouse age corresponds to
a human age of approximately 70 years. The frequency of HPCs in
PB was determined by a standard CFC assay. The frequency of
HPCs in PB of mobilized aged mice was elevated 3-fold over the
frequency found in PB of young mice (Figure 1A). Interestingly,
the aged animals mobilized increased numbers of HPCs despite
slight, but significant, anemia (indicated by a reduced red blood cell
[RBC] count; Figure 1B). Age-associated anemia has been previously reported in mice and humans.33,34 Age-associated anemia was
still prevalent in mobilized aged mice (Figure 1B).
G-CSF–induced mobilization leads to a redistribution of primitive hematopoietic cells from BM to the spleen.35 Thus, the
frequency of CFCs in spleen and BM was determined for young
and aged mice before and after mobilization. Aged mice showed a
4-fold increase in CFC frequency in spleen over the frequency in
young animals (Figure 1C). After mobilization, CFC frequency in
the spleen in young animals was increased 4-fold over the
premobilization frequency, whereas the frequency in mobilized
Figure 1. Aged mice mobilize increased numbers of HPCs to PB in response to
G-CSF. (A) Frequency of CFCs in 20 ␮L PB in young and aged mobilized (5-day
G-CSF at 100 ␮g/kg per day intraperitoneally; PB analysis on day 6) and nonmobilized mice, n ⫽ 10 for young nonmobilized and mobilized mice, n ⫽ 3 for 22-monthold mobilized mice, and n ⫽ 7 (3 at 20 months, 1 at 23 months, 3 at 26 months) for
mobilized aged mice. (B) White blood cell (WBC) and RBC counts in young and aged
mobilized and nonmobilized mice, n ⫽ at least 4 per value. (C) CFC frequency in
spleen in young and aged mobilized and nonmobilized mice, n ⫽ 3. (D) CFC
frequency in BM in young and aged mobilized and nonmobilized mice, n ⫽ 3. Values
shown are mean ⫾ 1 SEM. *P ⬍ .05.
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
aged mice was increased 3-fold (Figure 1C). The increase in the
CFC content in the spleen of mobilized mice was accompanied by a
moderate 19% increase in the total cell splenic number after
mobilization in aged animals (2.1-2.5 ⫻ 108 cells per spleen) and a
50% increase in young mice (1.4-2.1 ⫻ 108 cells per spleen). The
relative increase in CFC frequency in SPL in mobilized aged mice
might have been reduced because of space restrictions given the
high total splenic CFC numbers in these mice. An explanation for
the increased splenic CFC content in nonmobilized aged mice
might be a consequence of the slight anemia in aged mice but also
warrants further investigation. Possibilities include an altered
splenic microenvironment in aged mice or an altered homing
ability of aged HPCs to spleen. As in spleen, a significant, but much
lower, age-related increase in CFC content was detected in the BM
of nonmobilized animals. A slight increase in the CFC frequency
was detected in the BM of young mice as a result of the
mobilization regimen (Figure 1D), in contrast to a slight decrease
in BM of aged mice after mobilization. We conclude from these
results that quantitative aspects of HPC distribution in response to
mobilization are altered in aged animals.
Mobilization of hematopoietic stem cells is increased
in aged mice
Stem cells are defined by their ability to self-renew and to support
multilineage differentiation; therefore, stringent testing of stem cell
activity and frequency is best accomplished by transplanting
putative HSCs into lethally irradiated recipients. To unequivocally
compare the frequency and differentiation capacity of HSCs
mobilized to PB in young and old mice, standard competitive
repopulation assays were performed. To that end, equal volumes of
PB from young (2- to 3-month-old) mobilized B6.SJL(BoyJ) and
aged (25- and 26-month-old) mobilized B6 mice were transplanted
into lethally irradiated B6.SJL(BoyJ) animals (Figure 2A). Because
hematopoietic cells from B6.SJL(BoyJ) mice expressed the Ly5.1
allele and hematopoietic cells from B6 animals expressed the Ly5.2
allele of CD45 cell surface marker, B6.SJL cells could be
distinguished from B6 cells by flow cytometry using antibodies
specific for the Ly5 alleles. Young B6.SJL(BoyJ) mice are identical
to young B6 mice in their mobilization efficiency and
characteristics.36
Analysis of the contribution of the transplanted mobilized HSCs
to chimerism in PB in the recipient animals was measured up to
9 months after transplantation, when hematopoiesis in the
periphery was sustained by transplanted long-term repopulating
stem cells.37 HSCs mobilized to PB in aged animals contributed
on average to 80% to hematopoiesis in the PB of recipients
5 months after transplantation (Figure 2B). Further analyses of
PB from the recipient animals revealed that mHSCs from aged
mice had multilineage differentiation ability because they
contributed to the T, B, and myeloid lineages (Figure 2C).
One cohort of mice that underwent competitive transplantation
was killed 9 months later, and chimerism in various hematopoietic
tissue and the hematopoietic stem and progenitor cell compartments was determined by flow cytometry (Table 1). HPCs
(Lin⫺S⫺K⫹ cells) were identified by immunophenotyping as cells
negative for lineage markers (Lin⫺) and for the Sca-1 receptor (S⫺)
and positive for the c-Kit receptor (K⫹), whereas HSCs were
negative for lineage markers and positive for the Sca-1 and the
c-Kit receptors (Lin⫺S⫹K⫹). The chimerism supported by HSCs
mobilized to PB in aged mice (Ly5.2-positive cells) in the
hematopoietic tissues analyzed ranged from 77% in spleen to 96%
in thymus and was on average 86% in PB and BM and in the
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
INCREASED MOBILIZATION WITH AGE
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Figure 3. Experimental setup of the competitive mobilization experiment. Data
are presented in Table 2.
Figure 2. Aged mice mobilize a higher number of HSCs to PB in response to
G-CSF. (A) Experimental setup of competitive transplantation of mobilized PB cells.
(B) Flow cytometric analysis of chimerism in PB of mice that underwent competitive
(50/50) transplantation with mPB from aged (Ly5.2⫹) and young (Ly5.1⫹) mice 5
months after transplantation. (C) Determination of the percentages of T, B, and
myeloid (MY) cells in PB cells derived from the transplanted mobilized HSCs from
aged mice in the recipient 5 months after transplantation by flow cytometry. Data are
a summary of 2 independent experiments; in each, n ⫽ 3 for the number of donors
per age group from which PB was collected and n ⫽ 4 for the number of recipients.
One recipient animal had graft failure and was excluded from the analysis. Values
shown are mean ⫾ 1 SEM. *P ⬍ .05.
hematopoietic stem (Lin⫺S⫹K⫹) and progenitor cell (Lin⫺S⫺K⫹)
compartment in BM (Table 1). This percentage of contribution is
significantly increased over the 50% contribution expected in case
of an equal frequency of HSCs mobilized to PB in young and aged
mice. Thus, long-term repopulating HSCs were at least 5-fold
enriched (85% vs 15%) in PB from aged mice after mobilization
over PB from young mobilized mice. These data are in good
agreement with the increase in CFC frequency in PB of aged mice
after mobilization (Figure 1A).
Enhanced mobilization proficiency with age is intrinsic
to the stem cell
To determine whether the age-associated increased ability to
mobilize was intrinsic to HSCs or was driven by extrinsic changes
in the BM microenvironment, a competitive mobilization assay
was performed (Figure 3).36 Lethally irradiated mice underwent
Table 1. Chimerism in hematopoietic tissue 9 months
after transplantation
HPCs from young and aged mice do not differ in their
migratory behaviors
To mobilize, HSPCs must leave their stromal niche and migrate
through the endothelial barrier into the circulation.3-5 It is believed
that in mobilized mice, a positive gradient of stroma-derived
factor-1 (SDF-1) supports the migration of primitive cells from BM
across the endothelial barrier into blood vessels.39 To determine the
underlying mechanism leading to increased mobilization proficiency of aged HPCs, transendothelial migration assays were
performed. Low-density BM cells derived from mobilized animals
were seeded on top of a membrane with an 8-␮m pore size with or
without a monolayer of endothelial cells.27,40 Cells seeded on an
endothelial cell monolayer were further exposed to a positive
SDF-1 gradient (Figure 4A). Cells from the bottom well were
harvested 18 hours after seeding. The frequency of HPCs in the
population seeded on top and in the migrated population was
Table 2. Competitive mobilization in mice transplanted with equal
number of young and old BM cells
Contributions from HSCs mobilized
to PB in aged mice, %
Tissue
86.4 ⫾ 3.0
Peripheral blood
87.3 ⫾ 4.0
Bone marrow
Bone marrow progenitor cells,
competitive transplantation with equal numbers of young and aged
BM cells, and premobilization chimerism in PB in the recipient
animals was determined 3 months after transplantation. As previously reported and confirmed in these experiments, HSCs from
aged animals contributed to only 30% chimerism in PB in the
recipient animals, resulting in a 1:2 ratio in favor of young cells
(Table 2).18,38 These competitively reconstituted animals were
subsequently mobilized by G-CSF, and the CFC frequency in PB
was determined. PB of the mobilized reconstituted animals contained on average 58 CFCs per 20 ␮L, which is an almost 2-fold
increase over the 30 CFCs per 20 ␮L PB after mobilization seen in
young animals (Figure 1A). The genetic origin (young/aged) of the
mobilized CFCs was determined by analyzing individual colonies
by flow cytometry (Table 2). On average, 85% of all CFCs
mobilized to PB were derived from cells of aged mice, strongly
arguing that increased mobilization proficiency is not a simple
consequence of the increased CFC content in aged BM and is to a
large extent intrinsic to the aged primitive hematopoietic cell.
Lin⫺S⫺K⫹
86.5 ⫾ 7.1
Bone marrow stem cells, Lin⫺S⫹K⫹
85.4 ⫾ 4.3
Spleen
77.1 ⫾ 6.4
Thymus
96.2 ⫾ 1.9
Values shown are mean ⫾ SEM. n ⫽ 3 mice.
Contribution to PB before
mobilization, %
Contribution to CFCs after
mobilization, %
Aged cells
31 ⫾ 2
85 ⫾ 1
Young cells
69 ⫾ 2
15 ⫾ 1
Aged was defined as 26 months; young, as 2 months. Contribution of aged and
young BM cells to PB before mobilization and of CFCs derived from aged and young
cells to PB after mobilization, analyzing at least 50 CFCs per recipient. Data are
derived from 3 recipient animals that underwent competitive transplantation with BM
cells pooled from 3 donors each. Values shown are mean⫾ 1 SEM.
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XING et al
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
Figure 4B). Taken together, these data do not support differences in
migratory activity between HPCs from young and aged mobilized
mice as the mechanism of increased mobilization in aged mice.
Aged HPCs are impaired in their adhesion to stroma cells
Figure 4. Young and aged HPCs do not differ with respect to their ability to
cross an endothelial cell layer. (A) Experimental setup for the transwell-migration
assay. (B) Percentage of CFCs from mobilized young (2-3 months) and aged (24
months) mice migrating in 18 hours into the lower well of the transwell. n ⫽ 3. Values
shown are mean ⫾ 1 SEM. LDBM indicates low-density BM cells.
determined by a CFC assay. HPCs in BM from mobilized aged
mice did not exhibit a significant difference in their ability to
migrate through the membrane in the absence (aged 19% vs young
22%) or in the presence of the endothelial cell monolayer (aged
13% vs young 13%) or in the presence of the endothelial cell
monolayer with an SDF-1 gradient (aged 26% vs young 38%;
We next compared the adhesive properties of aged and young
HPCs. To this end, we measured adhesion of HPCs to a bone
marrow–derived stroma cell line (FBMD-1) using the CAFC
adhesion assay, which is a modification of the CAFC assay.41 The
CAFC assay is a well-established, limiting-dilution–type in vitro
surrogate assay to measure progenitor cell activity.41 For the CAFC
adhesion assay, BM cells were plated on top of a monolayer of
confluent FBMD-1 cells, and the nonadhesive cells were washed
off after 2 or 4 hours (Figure 5A). HPC frequency was subsequently determined at day 7, and the frequency of adhesive HPCs
was compared with the initial HPC frequency.42 BM cells from
nonmobilized aged mice showed a significant 2-fold reduction in
the ability to adhere to FBMD-1 cells after 4 hours (Figure 5B).
To correlate changes in adhesion/mobilization efficiency with
changes in the expression of adhesion receptors, the level of
surface expression of the surface receptors CXCR4 (the receptor
for SDF-1), CD49d (␣4 integrin), and CD49e (␣5 integrin) on aged
and young HSCs (Lin⫺S⫹K⫹) and HPCs (Lin⫺S⫹K⫺) was compared using flow cytometry (Figure 5C). These tested surface
molecules are involved in regulating HSPC adhesion/mobilization.3 The ratio of the mean fluorescence intensity (MFI) was
determined for each surface receptor on aged and young cell
populations. CD49d expression was significantly reduced on aged
HSCs, but not on aged HPCs, whereas CXCR4 and CD49e did not
show significant changes in the expression between aged and
young HSPCs (Figure 5C). These results suggest that reduced
adhesion of aged HPCs is independent of the expression levels of
these surface receptors.
Increased level of activation of Cdc42 in aged primitive
hematopoietic cells
Adhesion and de-adhesion of HSPCs from stroma requires significant changes in the cytoskeleton. These intracellular changes are
regulated in part by outside-in signaling of receptors sensing the
Figure 5. HPCs from aged mice are reduced in their ability to adhere to stroma. (A) BM cells from mobilized and nonmobilized aged (21-27 months) and young (2-3
months) mice were subjected to CAFC adhesion assay. Experimental setup. (B) Percentage of adherent HPCs after 2 or 4 hours determined as CAFC day 7 cells using the
CAFC adhesion assay. n ⫽ at least 3 for each age group. Values shown are mean ⫾ 1 SEM. (C). Ratio of the MFI of CXCR4, CD49d, CD49e on HPCs and HSCs from aged
(21-22 months) and young (2-3 months) mice. No change in the MFI of expression between cells from young and aged animals resulted in ratio of 1, indicated by the dashed
line. n ⫽ 3 mice each aged and young for CXCR4, and n ⫽ 4 mice each aged and young for CD49d and CD49e. *P ⬍ .05.
BLOOD, 1 OCTOBER 2006 䡠 VOLUME 108, NUMBER 7
cellular environment (eg, adhesion receptors, cytokines, chemokines), which then result in inside-out reactions of the cell and
finally in de-adhesion. Integrins, growth factor receptors, and
chemokine receptors have been shown to signal this outside-in
information through members of the family of small Rho
GTPases.43,44 Small Rho GTPases, including Rac1, Rac2, and
Cdc42, are involved in the regulation of adhesion in various
mammalian cell types, including hematopoietic cells.40,45-47
They act as molecular switches by cycling between an active
GTP-bound form and an inactive GDP-bound form. Upon
activation by distinct signals originating from these “environment sensing” receptors, active GTP-bound GTPases ultimately
regulate actin polymerization, integrin clustering, and adhesion
receptor–mediated adhesion.48,49
To further investigate possible mechanisms leading to the
reduced adhesion of aged HPCs, the activity of the small
RhoGTPases Cdc42, Ra1, and Rac2 was analyzed in BM cells
and cell populations highly enriched for HPCs (Lin⫺ cells) from
2- to 3-month, 13-month, and 21- to 27-month-old mice using an
effector-domain GST fusion pull-down protocol as previously
described.31 A significant increase in the GTP-bound form of
Cdc42 was detected in BM cells and in Lin⫺ cells from aged
animals (Figure 6A-C), whereas the levels of activity of Rac1
and Rac2 did not change with age (data not shown). These
results indicate that elevated levels of the active form of Cdc42
are associated with reduced adhesion and increased mobilization efficiency.
Discussion
Hematopoietic stem cell transplantation is most often administered
to patients younger than 50 and rarely to patients older than 60
years of age21,22 because the toxicity of the chemotherapy/
conditioning regimen has caused unacceptable morbidity in older
patients. In contrast, the average age of patients at diagnosis of
neoplasms (eg, leukemia, lymphoma) who are candidates for stem
cell transplantation ranges from 65 to 70 years.50 Given that in
recent years the adverse effects of the conditioning and the
mobilization regimens for transplantation have been improved
because of better treatment regimens and new therapeutic concepts
such as the minigraft,26,51 older patients can now be expected to
better tolerate transplantation regimens and thus be more likely to
undergo stem cell therapy and G-CSF–induced stem cell mobilization. Because the mouse is regarded as a model for human
G-CSF–induced mobilization, we compared the ability of aged and
INCREASED MOBILIZATION WITH AGE
2195
young mice to mobilize hematopoietic stem and progenitor cells to
PB in response to G-CSF.
Aged mice showed increased G-CSF–induced mobilization
efficiency for HPCs and HSCs. The data presented suggest that PB
in aged, mobilized mice contains at least a 5-fold higher concentration of long-term repopulating hematopoietic stem cells than the
PB of mobilized young animals and that the enhanced mobilization
efficiency is intrinsic to the HSC. This intrinsic mechanism might
be a direct mechanism, such as reduced adhesion of aged cells, or in
an indirect one in which stem cells from aged animals are able to
alter their microenvironments in a way that leads to reduced
adhesion and enhanced mobilization. Further experiments will
have to distinguish between such possibilities.
We and others recently described a 2- to 3-fold reduced homing
ability of aged BM HSCs in comparison with young BM
HSCs,18,19,38,52,53 resulting in reduced contribution of aged cells to
hematopoiesis when equal numbers of BM cells from aged and
young mice were transplanted into young recipients. The 77% to
96% levels of contribution of aged mobilized HSCs in recipients
that underwent competitive transplantation with mobilized PB cells
further suggests that mHSCs from aged mice are spared from this
homing defect of BM stem cells of nonmobilized aged mice, that
their homing abilities are actually enhanced, or that HSC mobilization in aged mice is even greater than that predicted from our data.
What might be the underlying molecular mechanisms for
enhanced HPC mobilization in aged mice? Cell adhesion and cell
migration are fundamental to the process of mobilization.5,6,40,54
Our experiments revealed that though HPCs from aged animals did
not show an increased ability to migrate to or cross an endothelial
cell layer, HPCs from aged animals are significantly deficient in
their ability to adhere to stroma cells, thus implying that reduced
adhesion of primitive hematopoietic cells to stroma might be the
underlying cause of enhanced mobilization proficiency. Reduced
adhesion of HSPCs from aged animals to stroma could also explain
the fact that young BM cells transplanted into nonablated aged
recipients are able to engraft, whereas young BM cells transplanted
into young nonablated animals are not because young cells might
be able to push less adhesive HSPCs in aged animals out of their
niches.55 Given that aged mice do not show increased spontaneous
mobilization to PB (Figure 1A), we conclude that the reduced
adhesion of HPCs from aged mice is necessary, but not sufficient,
for increased spontaneous mobilization and that only the combination of the action of G-CSF and reduced adhesion will result in
enhanced mobilization.
Our results indicate that associated reduced adhesion and
enhanced mobilization correlate with elevated levels of Cdc42
Figure 6. Increased activity of Cdc42 in primitive hematopoietic cells in aged mice. (A) BM cells from young (2- to 4-month-old) and aged (20- to 27-month-old) mice were
subjected to an effector domain pull-down assay and subsequently probed by immunoblotting. (B) Quantification of the relative amount of the active Cdc42-GTP bound form by
densitometry. n ⫽ 3 for young mice, and n ⫽ 4 for aged mice. Values shown are mean ⫾ 1 SEM. *P ⬍ .05. (C) Lin⫺ BM cells (enriched for HPCs) isolated from pooled BM cells
from each experiment in which 4 young, middle-aged, and aged mice (2, 13, and 22 months, respectively) were subjected to an effector domain pull-down assay and
subsequently probed by immunoblotting. Data are representative of 2 independent experiments.
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XING et al
activity in primitive hematopoietic cells from aged mice. We
recently reported a knock-out animal model system resulting in
constitutively elevated levels of Cdc42 activity in HPCs (Cdc42
GAP⫺/⫺ mice). HPCs from these mice showed reduced adhesion to
fibronectin.30 Thus, we speculate that the reduced stromal adhesion
of HPCs from aged animals might be a consequence of elevated
activity of Cdc42 in HPCs in aged mice.
What might be the cause for the age-related increase in Cdc42
activity? Integrins, growth factor receptors, and adhesion molecules have been shown to signal outside-in information through
Cdc42; thus, signaling through Cdc42 is embedded in a network
and not in a linear cascade. In addition, it has been speculated that
Cdc42 affects progenitor cell–cell contacts in skin by stabilizing
␤-catenin.56 Recently published gene expression profiles of stem
cells from young and aged animals,36 together with gene expression
data obtained in our own laboratories (data not shown), suggest that
HSPC cell aging is accompanied by changes in the expression of
signal transducers. Further experiments will be necessary to
correlate age-related changes in any of these gene categories with
functional consequences on Cdc42 activity and adhesion.
In summary, the data presented here demonstrate that HSC
and HPC mobilization proficiency is increased in aged mice and
that it is intrinsic to the stem cell. Our data support a need for
further studies aimed at determining the mobilization proficiency in healthy middle-aged and aged persons. Our results
also indicate that reduced adhesion to the stroma/niche might be
the mechanism involved in increased HPC mobilization proficiency in aged mice and that reduced adhesion might be a
consequence of increased levels of activated Cdc42 in HPCs
from aged animals. The interaction of primitive hematopoietic
cells with their niche might be regarded as dynamic over the
lifespan of the individual and might perhaps confer other
phenotypes associated with primitive hematopoietic cells in
aged animals.8
Acknowledgments
We thank Michael Jansen for providing the mHEVc cell line and
for help with the transendothelial migration assay. We thank
Susanne Wells, David Williams, and Jose Cancelas for critical
comments on the manuscript and Matthew Hodgson for editorial
assistance. H.G. is a New Scholar in Aging of The Ellison
Medical Foundation.
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