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HEMATOPOIESIS
From the Departments of Pathology and Developmental
Biology, Stanford University School of Medicine, Stanford, California.
Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB
HSC) are widely used for transplantation in the treatment of
malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their
numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G0 or G1
phase of the cell cycle. This has raised the question of whether a
subset of noncycling BM HSC is selectively released, or whether cycling
BM HSC are mobilized after M phase, but before the next S phase of the
cell cycle. To distinguish between these possibilities, mice were
treated with one dose of CY followed by daily doses of G-CSF, and
dividing cells were marked by administration of bromodeoxyuridine
(BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB
(LT)-HSC stained positively for BrdU, and therefore derived from cells
that divided during the treatment interval. Next, LT-HSC from mice
previously treated with a single dose of CY, which kills cycling cells,
and 3 daily doses of G-CSF, were nearly all killed by a second dose of
CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle.
Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA
content of MPB HSC suggests that these cells are mostly in
G1 phase. After CY/G-CSF treatment, virtually all BM LT-HSC
enter the cell cycle; some of these HSC then migrate into the blood,
specifically after M phase, and are rapidly recruited to particular
hematopoietic organs.
(Blood. 2001;97:2278-2285) In adult mammals, hematopoietic progenitors and
hematopoietic stem cells (HSC) migrate from bone marrow (BM) to the
periphery in response to several stimuli, including cytoreductive
drugs, such as cyclophosphamide (CY),1,2 and cytokines,
such as granulocyte colony-stimulating factor (G-CSF).3-5
G-CSF is widely used clinically to mobilize progenitors and HSC for
collection and transplantation, often in combination with CY, which
augments its effect.1,2 Despite common use, the mechanisms
of cytokine mobilization of HSC are poorly understood.
Combined administration of CY and G-CSF is associated with expansion of
the HSC pool.6 Following a single dose of CY and subsequent daily doses of G-CSF to mice, a large fraction of HSC in the
BM entered the cell cycle, leading to a more than 12-fold expansion in
long-term (LT) repopulating HSC in the BM after CY and 2 days of G-CSF
treatment. On the day after the dramatic 3-day expansion, the number of
BM LT-HSC began to decline, concomitant with a precipitous increase in
the number of LT-HSC in the blood and spleen. The rapid expansion of
LT-HSC in BM following administration of CY and G-CSF suggested that
most of these cells had entered the cell cycle.6 The
question of whether all LT-HSC entered the cell cycle after
CY/G-CSF, however, or whether a subset of quiescent HSC escaped
stimulation, was not addressed.
Intriguingly, despite the fact that HSC in the BM were cycling rapidly
at the time of mobilization, HSC released into the blood after
treatment with CY/G-CSF were either in the G0 or
G1 phase of the cell cycle.6 In addition,
hematopoietic progenitor and stem cells that appear in the blood
following treatment of mice or humans with various mobilizing
cytoreductive drugs or cytokines are in either the G0 or
G1 phase.7-14 At least 3 hypotheses could
explain the strong bias in favor of G0/G1 HSC
in the blood7: (1) HSC may be released from the BM in all
phases of the cell cycle, but S/G2/M-phase HSC might be
preferentially cleared, leaving G0/G1 cells
overrepresented in the blood; (2) alternatively, there could be a
preferential release of noncycling (G0) HSC into the blood,
leaving cycling cells behind in the BM; and (3) it is also possible
that all HSC enter the cell cycle in response to CY/G-CSF, and that
actively cycling cells migrate selectively to blood after M phase and
prior to the next S phase, thus placing them in G0 (if they
were to exit the cell cycle on mobilization) or in G1 phase.
We have previously shown that nearly all of the reconstituting activity
of mobilized HSC isolated from CY/G-CSF-treated C57BL/Ka-Thy-1.1 mice
is contained in 2 phenotypically defined populations:
Thy-1.1loSca-1+Lineage Mice
HSC mobilization
Bromodeoxyuridine treatment
Tissue preparation and HSC isolation Tissue processing. Single-cell suspensions of BM and spleen cells were prepared as previously described.6 In a typical BrdU experiment, BM was pooled from 5 mice, and blood was pooled from the same 5 mice and from 5 to 15 additional mice. Antibodies. Monoclonal antibodies (mAbs) used in immunofluorescence staining were prepared from hybridomas and included 19XE5 (anti-Thy-1.1), 2B8 (anti-c-Kit), E13 (anti-Sca-1, Ly6A/E). Lineage marker Abs included KT31.1 (anti-CD3), GK1.5 (anti-CD4), 53-7.3 (anti-CD5), 53-6.7 (anti-CD8), Ter119 (antierythrocyte-specific antigen), 6B2 (anti-B220), 8C5 (anti-Gr-1), and M1/70 (anti-Mac-1). The mAbs BU-1 (anti-BrdU, Caltag, South San Francisco, CA) and fluorescein isothiocyanate (FITC)-conjugated antimouse polyclonal Ab (Caltag) were used in BrdU analyses. Biotinylated 3C11 (anti-c-Kit), was sometimes used for positive selection of HSC. 2B8 and 3C11 recognize nonoverlapping epitopes on c-Kit. HSC isolation. The HSC were isolated as described previously.18 Sca-1+ or c-Kit+ cells were sometimes enriched by positive selection using MACS (Miltenyi Biotec, Sunnyvale, CA) streptavidin-conjugated magnetic beads according to the manufacturer's instructions. Figure 1C shows staining profiles and approximate sorting gates used for isolation of CY/G-CSF day +4 BM (i, i') and blood (ii, ii') LT-HSC. Cell cycle analyses Cell cycle analyses were performed as described.17 Briefly, double-sorted HSC were stained with Hoechst 33342 (Molecular Probes, Eugene, OR) at 10 µg/mL for 45 minutes at 37°C in Hoechst medium.19 Pyronine Y (PY) was then added to 1.0 µg/mL, and the cells were incubated for an additional 15 minutes, prior to washing and analysis by flow cytometry.Quantitation of BrdU incorporation by HSC Cells were stained with BrdU and visualized by fluorescence microscopy as previously described.17 To obtain percentages of BrdU+ HSC, cell nuclei (stained blue with Hoechst 33342) were identified using the Hoechst filter, then BrdU+ (green) nuclei in the same field were counted using the FITC/Texas Red filter (Figure 1D). In each experiment, whole thymocytes sorted from mice not exposed to BrdU were used as negative controls and were always 100% negative (Figure 1D, i, i', and data not shown). Thymocytes from mice exposed to BrdU were used to control for possible nonspecific staining. Consistent with expectations,20 a significant fraction of thymocytes sorted from mice treated with BrdU alone for 5 days were BrdU+ (ii, ii', and data not shown). LT-HSC isolated from BM (iii, iii') or blood (iv, iv') of BrdU/CY/G-CSF-treated animals showed the characteristic BrdU punctate green nuclear staining.21 To rule out the possibility that residual staining from the FITC-conjugated anti-Thy-1.1 could yield false-positive staining, Thy-1.1lo BM cells were sorted directly onto a slide and observed to have no staining (data not shown).Reverse transcriptase-polymerase chain reaction analysis of cyclin D2 expression Nested reverse transcriptase-polymerase chain reaction (RT-PCR) on double-sorted LT-HSC (5 cells/sample) was performed as described.17 Primer sequences are as follows: sense primers, 5'AGA GAC CAT CCC GCT GAC TGC3'; 5'GAA AAG CTG TGC ATT TAC ACC3'. Antisense primers, 5'GCA GGC TGT TCA GCA GCA GAG3'; 5'GCT CAG TCA GGG CAT CAC ACG3'. RT primers, 5'TCC CGC ACG TCT GTA GGG GTG3'.Isolation and PKH-26-labeling of murine erythrocytes Red blood cells (RBC) from untreated C57BL/Ka-Thy1.1 mice were isolated by density centrifugation over a Ficoll cushion (Sigma, St Louis, MO). Purified RBC (108) were then labeled with the fluorochrome PKH-26, according to the manufacturer's recommendations (Sigma).Hematopoietic progenitor short-term homing assays Hematopoietic progenitor cells enriched for HSC (Lin /loSca-1+c-kit+) were
isolated from day +4 MPB of  -actin/eGFP transgenic mice. The
eGFP+ progenitor cells (45 000) and 150 000
PKH-26-labeled erythrocytes were sorted and injected together into the
retrorbital sinus of anethetized day +4 CY/G-CSF C57BL/Ka-Thy1.1
recipients. Samples of peripheral blood were collected from the tail
vein at the indicated time points (from 30 seconds to 3 hours). After 3 hours, mice were killed and tissues and organs were collected, as
indicated, for analysis. Quantitation of PKH-26+ RBC or
eGFP+ progenitors in blood and tissue samples was performed
by flow cytometry. Preliminary experiments demonstrated that samples
from uninjected control mice did not generate signals in the
PKH-26+ or eGFP+ gates.
Statistical analysis The Student t test was used to compute the P values shown in Figure 2B.
MPB HSC are derived from dividing precursors To test whether G0/G1 HSC appearing in the blood following mobilization derive from noncycling (G0) BM LT-HSC that are selectively released into the blood, or are instead LT-HSC that after actively cycling in the BM are released into the blood after M phase of the cell cycle, mice were treated simultaneously with CY/G-CSF and with the thymidine analog BrdU, which is incorporated during DNA synthesis (Figure 1B,D). Virtually all (99.1 ± 0.5% SD) Thy-1.1loSca-1+Linc-Kit+Mac-1
cells isolated from the BM of day +4 CY/BrdU/G-CSF-treated mice stained positively for BrdU (Table 1).
Thy-1.1loSca-1+Linc-Kit+Mac-1
cells from MPB of CY/BrdU/G-CSF-treated mice were 98.5%
BrdU+ (Table 1, experiment 2), and
Thy-1.1loSca-1+Lin/loc-Kit+
cells (this population includes the
Thy-1.1loSca-1+c-Kit+Mac-1lo
transiently self-renewing HSC) were 97.8% BrdU+ (Table 1,
experiment 3). Thus, nearly all HSC isolated from MPB had divided at
least once during BrdU exposure, even though only a small percentage of
such cells are typically in the S, G2, or M phases of the
cell cycle at the time of their isolation6 (data not
shown). Given that the vast preponderance of HSC are in the BM until
day +3, and these are dividing,6 the most likely explanation for the presence of BrdU+,
G0/G1 phase HSC in the blood is that HSC
divided in the BM and migrated to the blood prior to their next
S phase.
If the MPB and splenic HSC are derived from dividing LT-HSC following
CY/G-CSF, then giving these mice a second dose of CY on day +2 of the
protocol should eliminate them, because CY preferentially kills
dividing cells.22 As reported previously, the initial dose
of CY on day LT-HSC isolated from the blood at CY/G-CSF day +4 express cyclin D2 messenger RNA MPB LT-HSC are found primarily in the G0/G1 fraction of the cell cycle,7-14 and appear to migrate into the bloodstream following completion of M phase. To determine whether MPB HSC remain in cycle in the bloodstream, or if entry into the bloodstream is accompanied by exit from the cell cycle (ie, entry into G0 phase), the expression of messenger RNA (mRNA) for cyclin D2, a marker for cycling cells,23 was assayed. In 2 experiments, nested RT-PCR performed on 5 cells per sample revealed that most CY/G-CSF day +4 BM and blood LT-HSC express cyclin D2 mRNA. Data from one of the experiments is shown in Figure 3. In this experiment, 7 of 10 BM and 9 of 10 blood LT-HSC samples were cyclin D2+. Little cyclin D2 mRNA from control splenic T cells was detected. In the second experiment (not shown), 12 of 12 BM and 12 of 12 blood samples were positive. Because cyclin D2 mRNA is rapidly degraded following exit from the cell cycle,24,25 these results are consistent with the notion that HSC isolated from blood are in G1 phase.
HSC recovered from the BM at CY/G-CSF day +3, or from the blood or spleen at CY/G-CSF day +4, have elevated levels of total RNA compared to HSC isolated from BM of untreated animals To characterize further the cell cycle status of HSC appearing in the periphery following treatment with CY/G-CSF, double-sorted HSC were stained with Hoechst 33342 and PY (see "Materials and methods"), allowing simultaneous assessment of the fraction of cells with more than 2n DNA content (Hoechst 33342), as well as relative levels of total double-stranded RNA (PY26). As cells progress from G0 to G1, and from G1 to S, G2, and M phases, they accumulate RNA, mostly in the form of double-stranded ribosomal RNA,26 and so it is reasonable to propose that PY high cells have elevated protein synthesis. As shown in Figure 4 and Table 2, total RNA levels of G0/G1 and S/G2/M HSC isolated from CY/G-CSF-treated animals (day +3 to day +5) were always higher than RNA levels of corresponding populations from untreated mice in all tissues examined. In 3 experiments, LT-HSC from blood of CY/G-CSF day +4 and +5 animals had 50%, 13%, and 24% (mean = 29% ± 19% SD) higher PY staining than HSC from BM of untreated mice (Table 2), suggesting that MPB LT-HSC are in a more activated state than LT-HSC from BM of untreated mice.
Mobilized stem and progenitor cells exhibit extremely short transit times in the blood and migrate to specific hematopoietic organs If HSC are selectively mobilized following M phase, but are found in the blood primarily in G1 phase, then transit of MPB HSC in the blood must be relatively brief (ie, shorter than progression from G1 to S phase). To test directly the half-life of MPB HSC and progenitor cells in the bloodstream, we purified Lin/lockit+Sca-1+ hematopoietic
progenitor cells from MPB of mice transgenically expressing eGFP driven
by the -actin promoter. These mice express high levels of eGFP in
all cells,16 including HSC (Figure
5A), a fact that can be exploited to
track the in vivo migration of transplanted eGFP+ cells in
a wild-type host. FACS-sorted MPB eGFP+ hematopoietic
progenitors from day +4 CY/G-CSF-treated animals were injected
intravenously into wild-type recipients similarly treated with
CY/G-CSF. To control for the injection, normal erythrocytes, fluorescently tagged with the red fluorochrome PKH-26 were coinjected with the eGFP+ cells. Whereas PKH-26+
erythrocytes were easily detectable in the blood of the recipient at
the early time points (< 1 minute), eGFP+ progenitor cells
were not detected (Figure 5B). In fact, although PKH-26+
RBC were detectable in the bloodstream throughout the experiment, eGFP+ progenitors cells were never observed in the blood.
Importantly, eGFP+ progenitor cells were easily identified
in the spleen, liver, and BM when recipient mice were killed 3 hours
after intravenous injection (Figure 5C). In addition, the migration
pattern of transplanted MPB HSC was consistent with previous studies
showing that mobilization initiates migration of BM HSC to the spleen
through the blood.6 The eGFP+ stem and
progenitor cells were found primarily in the liver and spleen, and only
a small fraction of cells migrated to BM. The eGFP+ cells
were not detected in lymph nodes, Peyer patches, or thymus. Taken
together, these data demonstrate that the transit time of mobilized
stem and progenitor cells in the blood is very brief and suggest that
migration of HSC into spleen and liver may contribute to the rapid
clearance of HSC from the circulation.
We examined the relationship between LT-HSC expansion in the BM
and mobilization from BM to blood. By administering BrdU to mice
undergoing a mobilizing regimen of CY/G-CSF and then isolating BM and
MPB LT-HSC, the proliferation history of LT-HSC in both compartments
was determined. As previously reported, in otherwise untreated mice
placed on continuous oral BrdU for 5 days, about 40% of
Thy-1.1lo Sca-1+ Lin It remained formally possible that HSC may be released from the BM in all phases of the cell cycle, but S/G2/M-phase HSC are preferentially cleared, leaving G0/G1 cells overrepresented in the blood. If this were true, the earliest HSC appearing in the spleens of mice following mobilization with CY/G-CSF would be expected to have an S/G2/M fraction as high or higher than that of the BM, because the spleen is a primary destination for circulating HSC in mice.6,11,31-33 However, on the day that significant numbers of HSC abruptly appear in the blood and spleen following CY/G-CSF, only about one third as many splenic HSC as BM HSC are in S/G2/M.6 Further, if this hypothesis were true, one would expect that MPB HSC from splenectomized mice would have a higher percentage of S/G2/M cells than MPB HSC from intact mice, which has been shown not to be the case.7 Finally, the demonstration that MPB progenitors, the majority of which are in G1 phase of the cell cycle, are rapidly and uniformly cleared from circulation argues against selective clearance of S/G2/M-phase cells from the blood. The association of LT-HSC expansion with mobilization begs the question of whether mobilization follows cell division in all instances in which HSC migration is observed. In normal prenatal development, sequential migrations of pluripotent HSC are thought to be associated with expansion of the HSC pool.34,35 Following treatment of adult animals with some cytokines, however, mobilization can occur very rapidly. Within 30 minutes of administration, interleukin 8 (IL-8) causes mobilization of hematopoietic progenitors and LT-HSC in mice,36 and of hematopoietic progenitors in primates,37 albeit in numbers much lower than at the peak of CY/G-CSF mobilization.6 Thus, it might appear that IL-8 causes mobilization by a different pathway than CY/G-CSF or that it acts by bypassing the early steps of the CY/G-CSF pathway. However, it may be that only BM HSC that have recently undergone cell division are mobilized by IL-8. On the order of 8% of the approximately 80 000 LT-HSC in adult mouse BM6 enter the cell cycle each day.17,27 If 8% of these LT-HSC also complete M phase each day, and if half of these cells are rapidly mobilized by IL-8 treatment, MPB would contain a maximum of about 1.5 LT-HSC/µL blood after IL-8 (assuming a blood volume of 2 mL). This estimate appears consistent with published data on mobilization induced by IL-8.36 Therefore, even though there are clearly differences between mobilization induced by IL-8 and CY/G-CSF, postmitotic, G1-phase cells may migrate in both cases. About 60 000 LT-HSC appear in the spleen between days +2 and +3 of the CY/G-CSF treatment protocol (an ~100-fold increase), even though the number of LT-HSC in the blood at any moment during this interval averages fewer than 10 000.6 At a minimum, it would appear that the equivalent of the entire complement of LT-HSC in the blood would have to be delivered to the spleen 6 times in 24 hours. The percentage of cycling cells in the BM or spleen is insufficient to account for more than a small fraction of the ~100-fold increase in the number of splenic cells.6 Thus, the most likely explanation is that LT-HSC arrive in the spleen from the BM through the blood at a high rate of flux. This hypothesis was confirmed by directly measuring the clearance of mobilized stem and progenitor cells from MPB. Transferred eGFP+ progenitor cells were removed from the circulation of day +4 CY/G-CSF-treated wild-type recipients in less than 1 minute, whereas control PKH-26-labeled RBC were observed in the blood for at least 3 hours after injection, the observation interval. Importantly, the rapid clearance of mobilized eGFP+ progenitor cells from the circulation cannot be explained entirely by nonspecific clearance mechanisms, because significant migration of eGFP+ cells into the spleen and liver of the mobilized recipients was observed within 3 hours of transfer. Taken together, the above data suggest that MPB HSC are actively cycling G1-phase cells that migrate rapidly from the blood into specific hematopoietic organs. These data suggest that MPB HSC are in the circulation for only one or 2 passes prior to migration to other sites, whereas RBC are fated to circulate in the bloodstream without homing. Alternatively, LT-HSC may cycle in the BM, pass through M phase, and
enter G0 phase before or on arriving in the blood. A recent
study of human MPB CD34+ cells,12 only a
fraction of which are HSC,38 supports this view, whereas
another group concluded that human MPB CD34+ cells were in
G1 phase.13 It has been shown that human
CD34+Thy-1+Lin One might imagine that an early step in physiologic or drug-induced HSC
migration would involve weakening or detachment of specific adhesive
interactions between HSC and surrounding stromal cells and ECM
elements. There is reason to assume that BM LT-HSC, which express high
levels of c-Kit,18
We thank Libuse Jerabek for superb laboratory management, Veronica Braunstein for antibody production, Koichi Akashi and Dennise Dalma-Weiszhausz for careful review of the manuscript, David Parks for advice on flow cytometry, Stanley Tamaki and Nobuko Uchida for advice on DNA/RNA staining for flow cytometry, Allen Smith for advice on DNA repair, Jos Domen for helpful discussions, and Diana J. Laird for statistical advice.
Submitted September 1, 1999; accepted December 13, 2000.
Supported by National Institutes of Health grant 5R01 HL-58770 to I.L.W. D.E.W. was supported by National Institute of Allergy and Infectious Diseases Training grant 5T32 AI-07290. S.H.C. is supported by the Medical Scientist Training Program grant 5T32 GM-07365 at Stanford University School of Medicine. A.J.W. is supported by the American Cancer Society, grant PF-00-017-01-LBC.
I.L.W. has declared a financial interest in Amgen and Systemix.
D.E.W. and S.H.C. contributed equally to this work.
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: Irving L. Weissman, Department of Pathology, Beckman B257, Stanford University School of Medicine, Stanford, CA 94305; e-mail: irv{at}stanford.edu.
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  I. L. Weissman and J. A. Shizuru The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases Blood, November 1, 2008; 112(9): 3543 - 3553. [Abstract] [Full Text] [PDF]
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S. Huygen, O. Giet, V. Artisien, I. Di Stefano, Y. Beguin, and A. Gothot Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture Blood, September 26, 2002; 100(8): 2744 - 2752. [Abstract] [Full Text] [PDF]
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E. Henckaerts, H. Geiger, J. C. Langer, P. Rebollo, G. Van Zant, and H.-W. Snoeck Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines Blood, May 13, 2002; 99(11): 3947 - 3954. [Abstract] [Full Text] [PDF]
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D. E. Wright, E. P. Bowman, A. J. Wagers, E. C. Butcher, and I. L. Weissman Hematopoietic Stem Cells Are Uniquely Selective in Their Migratory Response to Chemokines J. Exp. Med., May 6, 2002; 195(9): 1145 - 1154. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1, and placed on BrdU-containing
water for the duration of treatment. In one experiment (Table 
) and
PKH-26+ RBC (
). Day +4 CY/G-CSF-treated wild-type mice
were anesthetized and injected intravenously with sorted
eGFP+ progenitor cells and PKH-26-labeled erythrocytes, as
described in "Materials and methods." Samples of peripheral blood
were obtained from the recipient mouse at the indicated time points.
The frequency of eGFP+ or PKH-26+ at each time
point was determined by flow cytometry. Data are expressed as the
number of eGFP+ or PKH-26+ cells per
106 total nucleated blood cells. (C) In vivo homing of MPB
stem and progenitor cells. eGFP+ progenitor cells were
infused into anesthetized day +4 CY/G-CSF-treated wild-type
recipients, as in panel B. After 3 hours, animals were killed and the
number of eGFP+ cells in each organ was determined
by flow cytometry. Data are presented as the percent of total
eGFP+ cells recovered from each organ, as indicated. ILN
indicates inguinal lymph nodes; MLN, mesenteric lymph nodes; PP, Peyer
patches.
4-integrin, and CD44 (D.E.W., S.J.M., I.L.W., unpublished data, October 1996), have interactions of significant avidity with stromal membrane-bound Steel
factor, vascular cell adhesion molecule 1 (VCAM-1), and hyaluronate,
respectively, as each of these ligands is ubiquitous in the
BM.