|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Departments of Medicine/Hematology, Laboratory
Medicine, and Pathology, University of Liège, Belgium; and
Laboratory of Experimental Hematology, University of Antwerp, Belgium.
Engraftment of hematopoietic progenitor cells has been shown to
decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood
CD34+ cells. Migration toward medium conditioned by the
stromal-derived factor-1-producing cell line MS-5 was studied in
bovine serum albumin- and fibronectin (Fn)-coated transwells.
Migration was reduced in cycling CD34+ cells and long-term
culture-initiating cells (LTC-ICs) compared with their noncycling
counterparts across Fn but not across bovine serum albumin. Conversely,
Fn binding was higher in cycling CD34+ cells and LTC-ICs
compared with noncycling progenitor cells, while adhesion of both
subsets to bovine serum albumin was undetectable. The contribution of
Recent studies have indicated that entry of
cultured hematopoietic stem cells (HSCs) into active phases of the cell
cycle reduces their homing ability and subsequent long-term
repopulating potential. First, several investigators reported an
engraftment defect of stem cells that had been induced to cycle either
in vivo or ex vivo.1-4 Habibian et al5 later
demonstrated that murine stem cells transiting through S and
G2 phases acquired an engraftment defect that was
reversible after the completion of mitosis. Szilvassy et
al6 also observed a loss of long-term engraftment
associated with cell cycle activation. As for human stem cells, we
showed that transition from G0 to G1 of a first cell cycle resulted in a decrease of engraftment capacity in
immunodeficient mice.7 Also, Glimm et al8
provided evidence that expanded human HSCs were ineffective in
repopulation assays during S and G2/M phases. To date, the
explanation at the cellular and/or molecular level of these findings
has not been provided.
Specific interactions of HSCs with adhesion molecules expressed by
endothelial and stromal cells have been studied by numerous groups. A
model of HSC homing has progressively emerged.9,10 After
initial rolling on endothelial E and P selectins, HSCs adhere to the
luminal face of bone marrow endothelial cells through very late antigen
(VLA)-4/vascular cell adhesion molecule-1 and lymphocyte function-associated antigen-1/intercellular cell adhesion molecule-1 interactions. Adherent HSCs transmigrate through the basal lamina via
interactions of surface VLA-4 and VLA-5 with fibronectin (Fn). HSCs
undergo chemotactic intramedullary migration in response to
stromal-derived factor (SDF)-1 to reach adequate "niches" formed by bone marrow stromal cells expressing, among other ligands, vascular
cell adhesion molecule-1 11,12 and
Fn.13
Fn is recognized by 2 different Relationships between cell cycle transit and integrin expression and/or
function were suggested by several investigators,25,26 including our group.27 In nonhematopoietic tissues, it has
been well established that when adhesion is maximal cells are
immobilized and unable to migrate, while lower adhesion force may allow
cell locomotion.28-30 In this paper, we investigated
whether adhesion and migration of committed progenitor cells and
primitive long-term culture-initiating cells (LTC-ICs) were modulated
by cell cycle transit. Our data show that migration across Fn is
defective while static adhesion to Fn is increased in cycling
progenitor cells as compared with their noncycling counterparts.
Cells
Short-term suspension cultures
MS-5 conditioned medium The stromal MS-5 cell line31 was plated in 25 cm2 flasks in 7 mL -minimum essential medium (-MEM;
Biowhittaker) supplemented with 10% fetal bovine serum (Life
Technologies, Paisley, United Kingdom) at 37°C. At confluence, the
medium was completely replaced with fresh medium. After 7 days, the
conditioned medium (MS-5 CM) was harvested, spun at 2000g
for 10 minutes, and the supernatant frozen in small aliquots.
Concentration of SDF-1 in MS-5 CM was assayed using the Quantikine
human SDF-1 immunoassay kit (R&D Systems). The kit cross-reacts with
mouse SDF-1 whose amino acid sequence is 99% homologous to that of
human SDF-1. SDF-1 concentration was on average 76.5 ± 7.5 ng/mL in
MS-5 CM batches used for these studies.
Migration assays Migration assays were performed in 6.5-mm diameter 5-µm pore transwells (Costar, Cambridge, MA). Transwell filters were coated with 10 µg/cm2 Fn (Sigma) in phosphate-buffered saline (PBS; Biowhittaker). Control filters were coated with 1% fraction V BSA (Life Technologies) in PBS. Coating was done overnight at 4°C. The coating solution was aspirated and replaced by a 1% BSA solution in PBS at 37°C for 30 minutes to block nonspecific binding sites. After 2 washes in RPMI 1640 with HEPES 25 mM, 2 × 105 cells were plated in 100 µL Myelocult (Stem Cell Technologies) in the upper chamber of the transwell. The bottom compartment was filled with 600 µL MS-5 CM or-MEM with 10% fetal bovine serum as control medium.
After incubation at 37°C during 3 hours, nonmigrating and migrating
cells were harvested from the top and bottom compartments,
respectively. Nonmigrating cells were recovered by 2 washes each
consisting of a 5-minute treatment with an enzyme-free cell
dissociation buffer (Life Technologies) at 37°C followed by vigorous
pipetting. In some experiments, nonmigrating cells remaining loosely
adherent to the interface filter or floating in the upper chamber (ie,
nonadherent cells) were separately harvested from cells that were
firmly adherent to the filter (ie, adherent cells). Nonadherent cells
were recovered by 2 gentle washes with PBS 1% BSA, and adherent cells
were harvested after a 5-minute treatment with cell dissociation
buffer. Harvested cells in all fractions were counted in
Fuchs-Rosenthal hemacytometers (total volume 3.2 µL). The percentage
of cells in each fraction was calculated relative to the total number
of cells plated. In selected experiments, stimulated CD34+
cells were preincubated with neutralizing antibodies against VLA-4
(clone P4C2, Life Technologies), VLA-5 (clone P1D6, Life Technologies),
CXCR4 (clone 12G5, Pharmingen, San Diego, CA), or isotype-matched mouse
immunoglobulin G (IgG) (Pharmingen) during 30 minutes at 4°C prior to
the migration assay.
Adhesion assays Fn (Sigma) was adsorbed at 4°C to wells of nontissue culture-treated 24-well plates (Becton Dickinson, Erembodegem, Belgium) at 10 µg/cm2, except where indicated, in PBS. Coating was done in the same conditions as described above for transwell filters, after which 1 × 105 to 2 × 105 cultured CD34+ cells were plated in RPMI 1640 containing 0.1% BSA at 37°C. After 1-hour incubation, nonadherent cells were harvested by 2 standardized washes using warm PBS 1% BSA. Adherent cells were then recovered after a 5-minute incubation in cell dissociation buffer at 37°C followed by vigorous pipetting. Adherent and nonadherent cells were counted in Fuchs-Rosenthal hemacytometers. Percent adhesion was calculated relative to the total number of cells plated. Adhesion on BSA-coated control plates was less than 2%. In some experiments, CD34+ cells were preincubated with blocking antibodies anti-VLA-4, anti-VLA-5, or control mouse IgG during 30 minutes at 4°C prior to the adhesion assay.Determination of adhesion and migration of CD34+ cells in different phases of the cell cycle Cells recovered in various fractions after adhesion or migration assays were incubated in PBS 0.6% IGEPAL CA-630 (Sigma) containing 50 µg/mL propidium idodide (PI) and 1 mg/mL ribonuclease (Boehringer, Mannheim, Germany). After a 30-minute incubation on ice in the dark, cells were run on a FACSort flow cytometer collecting FL-2 channel values. The percentage of cells in the different phases of the cell cycle was determined with Modfit software (Becton Dickinson). Absolute numbers of adherent cells in G0/G1, S, or G2/M were obtained by multiplying the percentage of adherent cells by the percentage of cells in G0/G1, S, or G2/M in the adherent fraction. The same procedure was used to obtain numbers of G0/G1, S, and G2/M cells in the nonadherent, migrating, and nonmigrating fractions. The migratory activity of cells in G0/G1, S, and G2/M was calculated from the ratio of the absolute number of migrating cells in G0/G1, S, or G2/M and the total number of cells (migrating plus nonmigrating) in G0/G1, S, or G2/M. Similarly, binding capacity of cells in G0/G1, S, and G2+M was obtained by dividing the number of adherent cells in G0/G1, S, or G2+M by the total number of cells (adherent plus nonadherent) in the corresponding phase.Colony-forming cell and LTC-IC assays Colony-forming cell assays were performed in Methocult H4435 (Stem Cell Technologies), which is supplemented with 50 ng/mL SCF, 20 ng/mL granulocyte-macrophage colony-stimulating factor, 20 ng/mL interleukin-3, 20 ng/mL interleukin-6, 20 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin. Absolute frequencies of LTC-ICs in cell suspensions recovered after migration assays were determined by limiting dilution analysis over M2-10B4 fibroblast feeder cells.32 Briefly, the fibroblast feeder cell line M2-10B4 (American Type Culture Collection, Rockville, MD) was cultured in RPMI 1640 with 10% FCS. Cells were irradiated at 50 Gy and plated in 96-well plates at 15 000 cells per well in 100 µL Myelocult (Stem Cell Technologies). Within a week, test cells were plated in limiting dilution at 24 wells per cell dose in another 100 µL Myelocult and maintained at 33°C in a 100% humidified atmosphere containing 5% CO2, with weekly half-medium change. After 5 to 6 weeks, medium was carefully aspirated from each well, followed by the addition of 200 µL Methocult H4435. After an additional 2 weeks, wells were scored for the presence or absence of hematopoietic colonies, and the frequency of LTC-ICs was calculated using the maximum likelihood estimator.33To determine the percent migration of cycling and noncycling LTC-ICs, hydroxyurea (HU) killing tests were performed as previously described,27 prior to migration assays. Briefly, 1 × 105 to 2 × 105 CD34+ cells were treated with 2 mg/mL HU (Sigma) in Iscoves medium with 1% BSA during 1 hour at 37°C. Control cells were incubated in medium only. After 2 washes in Iscoves medium with 1% BSA, cells were transferred in transwells and allowed to migrate during 3 hours. Exposition of the cells to HU did not interfere with migration (37.9% ± 4.5% migration in HU-treated cells vs 36.6% ± 4.5% migration in control cells, n = 7). HU did not modify VLA-4, VLA-5, and CXCR4 expression or alter adhesion to Fn (data not shown). Migrating and nonmigrating cells were then replated in limiting dilution in LTC-IC assays. In each fraction, the percentage of cycling LTC-ICs was estimated from the proportion of LTC-ICs killed by HU treatment relative to the percentage of LTC-ICs in control cells. Migration of cycling LTC-ICs was calculated from the ratio of the number of cycling LTC-ICs in the migrating fraction and the total number (migrating plus nonmigrating) of cycling LTC-ICs. Similarly, to calculate the migratory activity of noncycling LTC-ICs, the number of noncycling LTC-ICs in the migrating fraction was divided by the total number (migrating plus nonmigrating) of noncycling LTC-ICs. Flow cytometric analysis of integrin and CXCR4 expression Fluorescein isothiocyanate (FITC)-conjugated anti-VLA-4 (CD49d, Coulter Immunotech, Marseille, France) and FITC-conjugated anti-VLA-5 (CD49e, Pharmingen) were used in combination with phycoerythrin (PE)-conjugated anti-CXCR4 (Pharmingen). Cells were incubated with antibodies or isotype-matched control IgG for 20 minutes on ice in the dark. Cells were washed in PBS 1% calf serum (Biowhittaker) and fixed in PBS 1% paraformaldehyde (Sigma). Data were acquired on a FACSort flow cytometer (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson). CXCR4+ cells were identified as those displaying a fluorescence above 99% of the isotypic control. Integrin density was expressed as the mean channel fluorescence ratio (MCFR) defined as MCF of integrin expression divided by MCF of fluorescence-matched isotypic control.Flow cytometric cell sorting Cultured CD34+ cells were sorted on the basis of cell cycle status. Briefly, cells were incubated in serum-free medium supplemented with 5 µg/mL Hoechst 33342 (Molecular Probes, Eugene, OR) during 30 minutes at 37°C. Cells in G0/G1 or in S/G2/M were sorted by a FACS Vantage (Becton Dickinson) equipped with a multiline UV laser (Innova Enterprise Ion Laser, Coherent, Santa Clara, CA) (351-364 nm) providing excitation for Hoechst. Hoechst signal was detected through a 424 ± 22 nm bandpass filter. The purity of sorted cells was confirmed by DNA staining of sorted cells with PI and always exceeded 90%.For cell sorting on the basis of VLA-5 expression, cultured CD34+ cells were labeled with biotinylated anti-VLA-5 (clone VC5; Pharmingen) and with streptavidin-FITC (Pharmingen), each step for 20 minutes at 4°C. Gating was set to include the lower and upper 20% of the VLA-5 histogram. Contamination between VLA-5low and VLA-5high fractions was 5% to 10%. In selected experiments where CD34+ cells were sorted simultaneously according to the level of VLA-5 expression and cell cycle status, Hoechst 33342 was added to anti-VLA-5-stained cells at the final concentration of 5 µg/mL during 30 minutes at 37°C, prior to cell sorting. Statistical analysis Results are reported as mean ± standard error of the mean (SEM). Comparisons were made using Student t tests. All P values are 2-sided.
Effect of fibronectin on the motility of cell cycle-activated progenitor cells It has been well established that CB progenitor cells reside in the G0/G1 phase of the cell cycle when freshly isolated.34 A 48-hour ex vivo culture in the presence of SCF, thrombopoietin, and FL was used to induce a large proportion of the cells into active phases of the cell cycle (> 40% of cells in S/G2/M in all experiments). The cloning efficiency of stimulated CD34+ cells was estimated in methylcellulose assays and was 90.5% ± 1.8% (n = 21). This must be compared with the cloning efficiency of freshly isolated CB CD34+ cells, which was 40.5% ± 3.5% in historical controls. An increase in cloning efficiency after a short-term cytokine stimulation has been observed repeatedly in our laboratory and is due to the loss of nonclonogenic cells quickly after culture initiation. The use of a highly pure population of clonogenic progenitors was essential to ascertain that the cells being studied were at the same level of differentiation.Migratory activity of cultured CD34+ cells was assayed in
transwells (Figure 1). Spontaneous
migration toward control medium was observed in 5.5% ± 1.4% and
18.0% ± 2.0% of the cells through BSA- and Fn-coated filters,
respectively (P < .05). Migration toward MS-5 CM was
increased up to 26.0% ± 3.4% through BSA (P < .05
compared with control medium) and up to 41.0% ± 3.3% through Fn
(P < .05, compared with control medium). Therefore,
migration of stimulated CD34+ cells was dependent both on a
chemoattractant gradient and on the presence of an adhesive substratum.
We confirmed that migration toward MS-5 CM was dependent on the
presence of SDF-1. The expression of SDF-1 receptor, CXCR4, was
measured by FACS analysis in cells recovered in the migrating and
nonmigrating fractions (Figure 2).
Migration toward MS-5 CM was, in part, dependent on the presence of
CXCR4 because 46.1% ± 5.6% of the cells migrating through an Fn
interface were CXCR4+ as compared with 23.6% ± 4.4%
for nonmigrating cells (P < .05, n = 7). As negative
control, spontaneous migration toward control medium was not related to
differences in CXCR4 expression: 26.6% ± 6.9% of migrating and
29.6% ± 5.6% of nonmigrating cells were CXCR4+,
respectively (P > .05). Migration toward MS-5 CM was
inhibited by a neutralizing antibody against CXCR4, across either Fn
(48.5% inhibition) or BSA (52.6% inhibition).
To better characterize the effect of Fn, cells remaining in the top
chamber after 3 hours of transmigration were fractionated into
nonadherent and adherent fractions. Less than 2% adherent cells could
be recovered from a BSA-coated filter (n = 10). In contrast, in the
presence of 10 µg/cm2 Fn, 10.8% ± 1.6% (n = 14) of
input cells were strongly adherent to the transwell filter. With
increasing Fn concentrations (Figure 3),
the proportion of migrating cells rose from 27.9% to 44.9%, together
with the appearance of an adherent fraction (up to 12.9% of total
input cells at a 20 µg/cm2 Fn concentration). Thus, at
high Fn concentrations, although the proportion of migrating cells
increased, a significant percentage of nonmigrating cells was fully
adherent to the transwell filter.
The expression of VLA-4 and VLA-5 integrins was analyzed in adherent,
nonadherent, and migrating CD34+ cells coexpressing CXCR4.
VLA-4 was brightly expressed by most of the cells (> 99%) in all 3 fractions and with similar MCFR (data not shown). Small differences
were observed in VLA-5 antigen density among the 3 fractions: MCFR of
VLA-5 was 3.6 ± 0.7 in the nonadherent fraction, 4.7 ± 0.8 in
adherent cells, and 2.8 ± 0.2 in the migrating fraction
(P < .05 compared with adherent cells, n = 5) (Figure
4). Therefore, the inability of a subset of CD34+ CXCR4+ cells to migrate across Fn was
not caused by down-regulation of either VLA-4 or VLA-5. On the
contrary, VLA-5 was slightly overexpressed in the adherent fraction.
Adhesion and migration of cycling progenitor cells are inversely correlated We next studied Fn binding and migratory activity of activated CB CD34+ cells in relation with cell cycle status. Following adhesion assay, the cell cycle status of adherent and nonadherent cells was established by DNA staining with PI (n = 18). The adhesion capacity to Fn of CB CD34+ cells increased with cell cycle progression: 14.7% ± 2.6% in G0/G1, 18.7% ± 2.7% in S (P < .05 compared with G0/G1) and 31.5% ± 4.6% in G2/M (P < .05 compared with both G0/G1 and S) (Figure 5A). Adhesion to a BSA-coated plate was always less than 2%.
The same experimental design was used to assess the migratory activity of stimulated CB CD34+ cells in different phases of the cell cycle. Following migration assay toward MS-5 CM in an Fn-coated transwell, cells recovered from the top and bottom chambers were assayed for cell cycle status by FACS analysis. Migration decreased with cell cycle progression: 44.5% ± 2.5% in G0/G1, 39.1% ± 3.2% in S (P < .05 compared with G0/G1), and 31.0% ± 2.7% in G2/M (P < .05 compared with both G0/G1 and S) (Figure 5B). This was not due to an inhibitory effect of MS-5 CM on the cycling status of migrating CD34+ cells. Indeed, in control experiments, when stimulated CD34+ cells were incubated for 3 hours in MS-5 CM, the proportion of cells in S/G2/M (47.0% ± 0.3%) was not different from that of cells incubated in control medium (46.9% ± 1.0%, n = 2). Moreover, when the filter was coated with BSA, migration toward MS-5 CM was similar in all phases of the cell cycle (26.9% ± 3.7% in G0/G1, 30.5% ± 3.1% in S, and 26.4% ± 5.2% in G2/M, Figure 5C). These results demonstrate that Fn overall facilitated transmigration of activated CD34+ cells but at the same time reduced the migrating ability of S/G2/M cells, whose binding capacity was upregulated as compared with cells in G0/G1. Adhesive and migratory activities of ex vivo-cultured CB
CD34+ cells were compared with those of unstimulated
CD34+ cells (Table 1).
Adhesion to Fn was higher after 48 hours of ex vivo culture
(P < .05). Migration toward MS-5 CM across Fn declined
significantly (P < .05), while no changes in motility were observed in BSA-coated transwells. This confirms a specific change
in Fn interaction during cell cycle progression of progenitor cells
ex vivo.
It has been suggested that culture of hematopoietic cells with Fn
causes accelerated cell cycle progression.20,35,36 To exclude the possibility that contact with Fn by itself was responsible for the higher cycling status of adherent or nonmigrating cells (ie,
cells staying in contact with Fn throughout adhesion or migration assays), activated CD34+ cells were stained with Hoechst
33342 and sorted into G0/G1 and S/G2/M subpopulations before measuring the adhesive and
migratory capacities of each fraction separately. Cycling cells had a
3-fold higher adhesion capacity (50.2% ± 5.1%) compared with
noncycling cells (16.0% ± 2.2%, P < .05) (Figure
6).Transmigration toward MS-5 CM across
BSA was similar for cycling (19.0% ± 1.1% migration) and
noncycling cells (19.4% ± 0.9% migration, P > .05),
whereas in Fn-coated transwells cycling cells had a reduced migratory capacity as compared with the noncycling cells (13.8% ± 2.0% vs 33.3% ± 5.8%, P < .05) (Figure 6). Migration of
cycling cells across Fn was even significantly lower than across BSA
(13.8% ± 2.0% vs 19.0 ± 1.1%, P < .05). This
further suggests that full adhesion to Fn of cycling CD34+
cells inhibited their migration.
Defective migration of cycling LTC-ICs Transwell assays toward MS-5 CM were set up with cultured CD34+ cells. Cells recovered in the nonmigrating and migrating fractions were replated in secondary LTC-IC assays. Migration of LTC-ICs toward MS-5 CM was similar across BSA- (40.5% ± 6.4%) and Fn-coated transwells (40.8% ± 4.7%, n = 3). Thus, contrary to what was observed for committed progenitors, Fn appeared to have no enhancing effect on the transmigration of overall LTC-ICs.The cell cycle status of LTC-ICs recovered in the migrating and
nonmigrating fractions was determined by HU killing assays (Figure
7). In the presence of Fn, migration of
noncycling LTC-ICs was 48.0% ± 2.9% while only 20.5% ± 1.1%
of cycling LTC-ICs were able to migrate toward MS-5 CM
(P < .05, n = 4). On the opposite, in BSA-coated
transwells, migration was increased in cycling LTC-ICs (53.7% ± 5.2%), compared with noncycling LTC-ICs
(27.9% ± 1.4%) (P < .05, n = 4). Thus, Fn had a
positive effect on the migration of noncycling LTC-ICs and an
inhibitory effect on that of cycling LTC-ICs. We had previously
reported that Fn binding is 3-fold higher in cycling LTC-ICs compared
with their noncycling counterparts.27 Overall, our data
suggest that the inverse relationship between adhesion and migration of
cycling progenitor cells holds true for the more primitive LTC-IC
subset.
Adhesion and migration of cycling CD34+ cells are independently regulated by VLA-4 and VLA-5 integrins in dividing cells Activated CD34+ cells were preincubated with nonspecific IgG or with antibodies blocking either VLA-4 or VLA-5 and assayed for transmigration activity. In all assays, the cell cycle status of migrating and nonmigrating cells was assessed by PI staining to establish whether the effect of blocking antibodies was present in all phases of the cell cycle (Figure 8A). Interestingly, while the inhibition provided by anti-VLA-4 antibody was similar in G0/G1 (22.3% ± 4.0% inhibition), in S (26.1% ± 8.5%), and in G2/M (17.6% ± 6.0%) (n = 5, P > .05), anti-VLA-5 antibody was less effective in blocking migration of cells in G2/M (9.9% ± 4.6% inhibition) compared with cells in G0/G1 (36.2% ± 2.6%, P < .05) or in S phase (40.3% ± 6.6%, P < .05).
Similar experiments were performed to establish the contribution of VLA-4 and VLA-5 in mediating adhesion of activated CD34+ cells in various phases of the cell cycle. Anti-VLA-4 treatment resulted in 30.9% ± 11.1% inhibition of Fn binding in G0/G1 and 38.3% ± 1.1% in S phase, whereas no inhibition was observed for cells in G2/M (Figure 8B). Anti-VLA-5 was slightly more effective in blocking cells in S (82.2% ± 3.3% inhibition) and G2+M (84.9% ± 5.0%), compared with G0/G1 cells (71.0% ± 4.1% inhibition, P < .05). When comparing migration and adhesion data, the most striking difference was observed for CD34+ cells in G2/M: VLA-5 mediated strong adhesion but not migration of G2/M cells, while the reverse was shown for VLA-4, ie, VLA-4 was involved in migration but not in static adhesion of this cell subset. Altogether, these results suggest that the effect of VLA-4 and VLA-5 integrins in mediating adhesion and migration of stimulated progenitor cells onto Fn was modulated in relationship to cell cycle status and independently of each other. Chemotaxis of VLA-5dim and VLA-5high cells subfractionated on the basis of cell cycle status The observation of a subset of CD34+CXCR4+VLA-5high cells unable to migrate across Fn (Figure 4) led us to speculate that maximal VLA-5 expression induced static adhesion to Fn and defective motility. To test this hypothesis, CD34+ cells were sorted into VLA-5high and VLA-5low subsets using a nonblocking antibody (clone VC5; Pharmingen), and migration of both fractions toward MS-5 CM was assayed across Fn and BSA (Table 2). Migration across Fn was higher in the CD34+ VLA-5low compared with the CD34+VLA-5high fraction (P < .05), while migration of each subset was similar across BSA. However, compared with the VLA-5dim fraction, the VLA-5high fraction had a 2-fold enrichment in cycling cells, which might then underlie their defective migration capacity. Similar findings have already been reported in murine HSCs fractionated on the basis of VLA-5 and other adhesion molecules37 and are mostly due to larger size and higher fluorescence background of cycling cells compared with noncycling cells.
To analyze independently the effect of cycle status and VLA-5
expression on transmigration across Fn,
CD34+VLA-5dim and
CD34+VLA-5high cells were each subfractionated
into G0/G1 and S/G2+M fractions (Figure 9). The migratory activity of
each of the 4 fractions was assessed toward MS-5 CM across Fn-coated
transwells (Table 3). The data clearly
show that the expression level of VLA-5 had no influence on the
chemotaxis of activated CD34+ cells across Fn, while
transit in S/G2+M negatively affected cell motility
independently of VLA-5 expression density.
In this report, we have shown that cycling committed progenitor cells as well as LTC-ICs harvested after a 2-day expansion culture presented defective migration across Fn toward MS-5 CM. We can rule out the hypothesis that G0/G1 progenitor cells passively migrate at a higher rate because of their smaller size. Indeed, the higher migration rate of noncycling progenitor cells was observed when the transwell filter was coated with Fn whereas, across BSA-coated filters, migration of noncycling progenitor cells was similar, or even lower in the case of LTC-ICs, compared with that of their cycling counterparts. The impaired migratory activity of cycling progenitor cells was not due to a reduced response to SDF-1 because, again, migration across BSA toward MS-5 CM was not defective in this cell population. Thus, the migration defect of cycling progenitor cells was observed as a result of an active interaction with Fn. Indeed, when adhesion to Fn was studied in relation to cell cycle status, it was observed that adhesion of cycling committed progenitor cells and LTC-ICs was higher compared with their noncycling counterparts. A number of studies have been devoted to delineating the different factors influencing migration rate.30,38 Migration requires coordinated attachment of the cell front and release of binding sites at the cell rear. Cell-substratum adhesion strength is critical to this process because strong binding will retain cells while lower adhesive force will allow contact with the matrix and easier detachment of adhesion complexes. The differences in adhesion and migration described here in cycling and noncycling cells may thus indicate that adhesion strength to Fn increases during cell cycle transit, leading to static binding and impairment of cell migration. It has been shown that bone marrow implantation is dependent on intramedullary migration followed by selective retention in specific endosteal niches.24 If we assume that migration and adhesion require balanced interactions of both VLA-4 and VLA-5 with Fn, our data may suggest that inactivation of VLA-5-mediated migration of progenitor cells in G2/M may lead to a selective homing defect, while the inability of VLA-4 to mediate static binding to Fn in G2/M CD34+ cells might result in defective retention in the bone marrow. Conversely, VLA-4 and VLA-5 were simultaneously active in other phases of the cell cycle, in both adhesion and migration assays. It should be noted, however, that because LTC-ICs represent a small fraction of total CD34+ cells, we cannot provide direct evidence that the observed changes of integrin function with cell cycle status in cultured CD34+ cells were also present at the LTC-IC level. When comparing the behavior of committed progenitors and of LTC-ICs in transmigration assays, it was observed that migration of LTC-ICs toward MS-5 CM across BSA was higher than that of CD34+ cells. This may suggest that LTC-ICs are more responsive to SDF-1 than more committed progenitors, which is in accordance with data reported by Peled et al39 showing that among the CD34+ population the CD38low subset had an increased migratory activity toward SDF-1. In addition, Fn coating of the transwell filter was not effective in stimulating LTC-IC migration toward MS-5 CM above levels observed across BSA-coated filters, whereas in more committed progenitors Fn induced a 2-fold increase in migratory activity. This difference may result from stronger adhesion of LTC-ICs than of colony-forming cells to Fn, as described in a previous paper by our group27 and in other reports.40 Fn may increase migration of mildly adherent LTC-ICs while retaining strongly adherent LTC-ICs onto the filter, with no net change in overall LTC-IC migration. Yong and coworkers41,42 have studied the migratory activity of unstimulated and stimulated progenitor cells isolated from various sources. A consistent finding of these studies was that proliferating progenitor cells migrated more efficiently than nonproliferating ones. This difference with our results may arise from the fact that migration was assessed across endothelial cells by Yong and colleagues whereas in the present work migration was studied in Fn-coated transwells. Thus, it is not possible to draw general rules on the migration ability of cycling and noncycling progenitor cells. Instead, it is likely that the transmigratory activity will depend on the cell type or the extracellular matrix protein across which migration occurs. In addition, transendothelial migration may represent initial stages of homing, while migration across Fn may be more important for the final localization of stem/progenitor cells in the bone marrow environment. Therefore, extrapolation of these data to in vivo homing mechanisms must be done cautiously. An apparent discrepancy in our results is that most of the changes in migration of total CD34+ cells occurred during the G2/M phase while, in the LTC-IC subset, a similar modulation was observed during S phase. We assume that these differences are due to different procedures used for cell cycle phase identification. In LTC-IC experiments, cells were treated with HU, which selectively eliminates cells in S phase, prior to the migration assay. We believe that results obtained with this procedure demonstrate unambigously that LTC-ICs in S phase have decreased migratory activity across Fn. In the case of total CD34+ cells, cells were allowed to migrate, after which the cell cycle status of migrating and nonmigrating cells was determined by PI staining. Our results show a gradual decrease in migration during transit from G1 to S and from S to G2/M. Thus, it may be possible that during a 3-hour migration assay S phase cells remained in the top chamber and gradually accumulated in G2/M, which then would lead to an overestimation of the migration defect of G2/M cells. This hypothesis is supported by another study in which we have shown that synchronized CD34+ cells execute a complete transition from mid S phase to G2/M in approximately 3 hours.43 The data presented in this paper have important implications for the engraftment of ex vivo-expanded progenitor cells. It has been suggested by different authors that VLA-5 plays a physiologic role in stem cell trafficking.10,15,37,44 Because other studies have observed an engraftment defect of cycling stem/progenitor cells, our data may suggest that this is partly caused by increased static adhesion and impaired motility across Fn. Whether such a phenomenon applies to the interaction of cycling progenitor cells to other adhesion molecules remains the subject of further studies and should provide more detailed knowledge of the homing mechanisms of expanded HSCs.
The authors thank Dr E. Ponthier and all the nurses and physicians of the obstetrical department of the Clinique St Joseph, Liège, for providing CB samples. The expert technical assistance of M. Lenjou, Antwerp University Hospital, in Hoechst sorting is also acknowledged.
Submitted July 19, 2001; accepted November 1, 2001.
Supported by grants from the National Fund for Scientific Research (Brussels, Belgium), the University of Liège Center against Cancer, and the Belgian Federation against Cancer. O.G. and S.H. were supported by Télévie fellowships. Y.B. and A.G. are Research Director and Associate Scientist, respectively, of the National Fund for Scientific Research.
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.
Reprints: André Gothot, University of Liège, Laboratory Hematology, CHU Sart Tilman B35, 13, avenue de l'Hôpital, B-4000 Liège, Belgium; e-mail: agothot{at}ulg.ac.be.
1. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol. 1995;23:461-469[Medline] [Order article via Infotrieve].
2.
Ramshaw HS, Rao SS, Crittenden RB, et al.
Engraftment of bone marrow cells into normal unprepared hosts: effects of 5-fluorouracil and cell cycle status.
Blood.
1995;86:924-929
3.
Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ.
Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts.
Blood.
1996;87:30-37
4.
Kittler EL, Peters SO, Crittenden RB, et al.
Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts.
Blood.
1997;90:865-872
5.
Habibian HK, Peters SO, Hsieh CC.
et al. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit.
J Exp Med.
1998;188:393-398
6.
Szilvassy SJ, Meyerrose TE, Grimes B.
Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells.
Blood.
2000;95:2829-2837
7.
Gothot A, van der Loo JCM, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in NOD/SCID mice.
Blood.
1998;92:2641-2649
8.
Glimm H, Oh IH, Eaves CJ.
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0).
Blood.
2000;96:4185-4193 9. Peled A, Grabovsky V, Habler L, et al. The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest. 1999;104:1199-1211[Medline] [Order article via Infotrieve].
10.
Peled A, Kollet O, Ponomaryov T, et al.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice.
Blood.
2000;95:3289-3296
11.
Simmons PJ, Masinovsky B, Longenecker BM, et al.
Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells.
Blood.
1992;80:388-395
12.
Papayannopoulou T, Nakamoto B.
Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin.
Proc Natl Acad Sci U S A.
1993;90:9374-9378
13.
Nilsson SK, Debatis ME, Dooner MS, et al.
Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ.
J Histochem Cytochem.
1998;46:371-377 14. Williams DA, Rios M, Stephens C, Patel VP. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature. 1991;352:438-441[CrossRef][Medline] [Order article via Infotrieve]. 15. van der Loo JC, Xiao X, McMillin D, et al. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest. 1998;102:1051-1061[Medline] [Order article via Infotrieve].
16.
Kovach NL, Lin N, Yednock T, Harlan JM, Broudy VC.
Stem cell factor modulates avidity of
17.
Levesque JP, Leavesley DI, Niutta S, Vadas M, Simmons PJ.
Cytokines increase human hematopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.
J Exp Med.
1995;181:1805-1815
18.
Schofield KP, Rushton G, Humphries MJ, Dexter TM, Gallagher JT.
Influence of interleukin-3 and other growth factors on 19. Cui L, Ramsfjell V, Borge OJ, et al. Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: role of activation of very late antigen (VLA)-4 and VLA-5. J Immunol. 1997;159:1961-1969[Abstract].
20.
Levesque JP, Haylock DN, Simmons PJ.
Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors.
Blood.
1996;88:1168-1176
21.
Prosper F, Stroncek D, McCarthy JB, Verfaillie CM.
Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of
22.
Strobel ES, Mobest D, von Kleist S, et al.
Adhesion and migration are differentially regulated in hematopoietic progenitor cells by cytokines and extracellular matrix.
Blood.
1997;90:3524-3532 23. Voermans C, Gerritsen WR, von dem Borne AE, van der Schoot CE. Increased migration of cord blood-derived CD34+ cells, as compared to bone marrow and mobilized peripheral blood CD34+ cells across uncoated or fibronectin-coated filters. Exp Hematol. 1999;27:1806-1814[CrossRef][Medline] [Order article via Infotrieve].
24.
Nilsson SK, Johnston HM, Coverdale JA.
Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches.
Blood.
2001;97:2293-2299
25.
Yamaguchi M, Ikebuchi K, Hirayama F, et al.
Different adhesive characteristics and VLA-4 expression of CD34+ progenitors in G0/G1 versus S+G2/M phases of the cell cycle.
Blood.
1998;92:842-848 26. Becker PS, Nilsson SK, Li Z, et al. Adhesion receptor expression by hematopoietic cell lines and murine progenitors: modulation by cytokines and cell cycle status. Exp Hematol. 1999;27:533-541[CrossRef][Medline] [Order article via Infotrieve]. 27. Giet O, Huygen S, Beguin Y, Gothot A. Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin. Exp Hematol. 2001;29:515-524[CrossRef][Medline] [Order article via Infotrieve].
28.
Giancotti FG, Ruoslahti E.
Elevated levels of the
29.
DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA.
Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength.
J Cell Biol.
1993;122:729-737 30. Palecek SP, Loftus JC, Ginsberg MH, Lauffenburger DA, Horwitz AF. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature. 1997;385:537-540[CrossRef][Medline] [Order article via Infotrieve]. 31. Itoh K, Tezuka H, Sakoda H, et al. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp Hematol. 1989;17:145-153[Medline] [Order article via Infotrieve].
32.
Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM.
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood.
1989;74:1563-1570 33. Taswell C. Limiting dilution assays for the determination of immunocompetent cell frequencies. J Immunol. 1981;126:1614-1619[Abstract]. 34. Traycoff CM, Abboud MR, Laver J, Clapp DW, Srour EF. Rapid exit from G0/G1 phases of cell cycle in response to stem cell factor confers on umbilical cord blood CD34+ cells an enhanced ex vivo expansion potential. Exp Hematol. 1994;22:1264-1272[Medline] [Order article via Infotrieve].
35.
Yokota T, Oritani K, Mitsui H, et al.
Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains.
Blood.
1998;91:3263-3272
36.
Schofield KP, Humphries MJ, de Wynter E, Testa N, Gallagher JT.
The effect of
37.
Orschell-Traycoff CM, Hiatt K, Dagher RN, et al.
Homing and engraftment potential of Sca-1(+)lin( 38. Huttenlocher A, Sandborg RR, Horwitz AF. Adhesion in cell migration. Curr Opin Cell Biol. 1995;7:697-706[CrossRef][Medline] [Order article via Infotrieve].
39.
Peled A, Petit I, Kollet O, et al.
Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
Science.
1999;283:845-848 40. Van der Sluijs JP, Baert MR, Ploemacher RE. Differential adherence of murine hematopoietic stem cell subsets to fibronectin. Exp Hematol. 1994;22:1236-1243[Medline] [Order article via Infotrieve].
41.
Yong KL, Watts M, Shaun TN, et al.
Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31).
Blood.
1998;91:1196-1205
42.
Yong K, Fahey A, Reeve L, et al.
Cord blood progenitor cells have greater transendothelial migratory activity and increased responses to SDF-1 and MIP-3 43. Huygen S, Beguin Y, Gothot A. Regulation of adhesion and migration of human long-term culture-initiating cells during cell cycle synchronization [abstract]. Exp Hematol. 2000;28:55[Medline] [Order article via Infotrieve].
44.
Papayannopoulou T, Priestley GV, Nakamoto B, et al.
Synergistic mobilization of hemopoietic progenitor cells using concurrent
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. Foguenne, I. Di Stefano, O. Giet, Y. Beguin, and A. Gothot Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of {alpha}4 integrin- and CXCR4-mediated engraftment in NOD/SCID {beta}2-microglobulin-null mice Haematologica, February 1, 2009; 94(2): 185 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shi, Y. Zhao, T. Ishii, W. Hu, S. Sozer, W. Zhang, E. Bruno, V. Lindgren, M. Xu, and R. Hoffman Effects of Chromatin-Modifying Agents on CD34+ Cells from Patients with Idiopathic Myelofibrosis Cancer Res., July 1, 2007; 67(13): 6417 - 6424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Forde, B. J. Tye, S. E. Newey, M. Roubelakis, J. Smythe, C. P. McGuckin, R. Pettengell, and S. M. Watt Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells Blood, March 1, 2007; 109(5): 1825 - 1833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Pirson, F. Baron, N. Meuris, O. Giet, E. Castermans, R. Greimers, I. Di Stefano, A. Gothot, and Y. Beguin Despite Inhibition of Hematopoietic Progenitor Cell Growth In Vitro, the Tyrosine Kinase Inhibitor Imatinib Does Not Impair Engraftment of Human CD133+ Cells into NOD/SCID{beta}2mNull Mice Stem Cells, July 1, 2006; 24(7): 1814 - 1821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fukuda, H. E. Broxmeyer, and L. M. Pelus Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1{alpha}(CXCL12)/CXCR4 axis Blood, April 15, 2005; 105(8): 3117 - 3126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Ahmed, S. J. Ings, A. R. Pizzey, M. P. Blundell, A. J. Thrasher, H. T. Ye, A. Fahey, D. C. Linch, and K. L. Yong Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model Blood, March 15, 2004; 103(6): 2079 - 2087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Weigel-Kelley, M. C. Yoder, and A. Srivastava {alpha}5{beta}1 integrin as a cellular coreceptor for human parvovirus B19: requirement of functional activation of {beta}1 integrin for viral entry Blood, December 1, 2003; 102(12): 3927 - 3933. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
1 integrins expressed by HSCs: VLA-4
and VLA-5. Previous studies have highlighted a dominant role either for
VLA-4 
2 mM 2-mercaptoethanol (all
from Biowhittaker, Petit-Rechain, Belgium). Cells were stimulated by a
combination of 100 ng/mL SCF (Amgen, Thousand Oaks, CA), 50 ng/mL
thrombopoietin (Amgen), and 100 ng/mL flt3-ligand (FL; R&D Systems,
Abingdon, United Kingdom) and maintained at 37°C in a 100%
humidified atmosphere with 5% CO2. The percentage of
CD34+ cells up to 48 hours in culture was always more
than 95%.
) cells fractionated on the basis of adhesion molecule expression and position in cell cycle.
Blood.
2000;96:1380-1387