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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 147-155
HEMATOPOIESIS
Human LTC-IC can be maintained for at least 5 weeks in vitro when
interleukin-3 and a single chemokine are combined with O-sulfated
heparan sulfates: requirement for optimal binding interactions of
heparan sulfate with early-acting cytokines and matrix proteins
Pankaj Gupta,
Theodore R. Oegema Jr,
Joseph J. Brazil,
Arkaduisz Z. Dudek,
Arne Slungaard, and
Catherine M. Verfaillie
From the Departments of Medicine and Orthopædic Surgery and
Biochemistry, Veterans Affairs Medical Center Minneapolis, MN, and
University of Minnesota, Minneapolis, MN.
 |
Abstract |
We have shown that stromal O-sulfated heparan sulfate
glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell
"niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks
in the absence of stroma when O-S-GAGs are added to IL-3 and either
MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with
GAGs, 95 ± 7%; P < .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although
O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were
maintained for 5 weeks. When O-S-heparin was replaced by completely
desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC
maintenance at week 5 was not better than with cytokines alone.
Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin,
bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin.
However, the affinity of heparin for thrombospondin and PF4, and the
association and dissociation rates of heparin for PF4, were higher than
those of O-S-heparin. We conclude that (i) although cytokines may
suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend
not only on the ability of GAGs to bind proteins, but also on optimal
affinity and kinetics of interactions that affect presentation of
proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)
© 2000 by The American Society of Hematology.
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Introduction |
Stem cells may exist in "stem cell niches" in the
marrow microenvironment in vivo, where stromal cells, specific
extracellular matrix (ECM) components, and cytokines bound to stromal
cell surfaces or to ECM macromolecules provide the appropriate balance
of signals that preserves the stem cell pool while permitting
controlled proliferation and differentiation.1-4 A number
of cytokines, chemokines, and matrix components considered to be
essential for the localization, conservation, and proliferation of
primitive hematopoietic progenitors cells (HPC) are heparin-binding
proteins, including platelet factor 4 (PF4), basic fibroblast growth
factor (bFGF), hepatocyte growth factor (HGF), interleukin-3 (IL-3), vascular endothelial growth factor (VEGF), macrophage inflammatory protein-1 (MIP-1), thrombospondin (TSP), and fibronectin
(FN).5-20 Specific sulfation patterns of heparan sulfate
(HS) are required for binding and modulation of the activity of
cytokines that have activity on hematopoietic
progenitors.6,8,21,22 Additionally, the ability of HS to
modulate cytokine activity on target cells may depend on the affinity
and kinetics of association ("on" rate) between cytokines and
HS.23 O-sulfation of glycosaminoglycans (GAGs) is required
for binding of several growth factors, including PF4 and
platelet-derived growth factor.24,25 We have recently shown
that highly O-sulfated stromal cell-derived HS GAGs help to mediate the
formation of such "stem cell niches" by colocalizing specific
heparin-binding proteins with HPC, thereby orchestrating the controlled
growth and differentiation of stem cells.26
Of the numerous cytokines that modulate human HPC survival and
proliferation in vitro, the heparin-binding cytokine IL-3 and chemokines MIP-1 or PF4 are particularly critical. We have recently demonstrated that the human long-term culture-initiating cell (LTC-IC)-maintaining capability of stromal supernatant27,28 is improved by the addition of nanogram concentrations of IL-3 + MIP-1.29 In these stromal supernatant cultures, MIP-1
can be replaced by PF4,30 a GAG-binding chemokine that has
effects similar to MIP-1 on hematopoietic progenitors.31
Moreover, IL-3 and MIP-1 are required for the ex vivo survival of
human HPC with myeloid and lymphoid differentiation
potential.29,32,33 HS proteoglycans may be essential for
the localization of both MIP-1 and IL-3 within the marrow stromal
microenvironment9,26,34-36 and may interact with IL-3 to
augment adhesion and colocalization of HPC within the
microenvironment.37
Thrombospondin is a matrix protein present in serum, which is
synthesized by both hematopoietic and stromal cells, which directly mediates the adhesion of HPC.38 TSP in soluble or
immobilized form also modulates cytokine activity, synergizing with
stem cell factor (SCF) to stimulate HPC adhesion and proliferation but
inhibiting IL-3-mediated HPC proliferation.5 TSP has a
high affinity for binding to heparin and interacts with stromal HS
proteoglycans to increase HPC adhesion.37 HS GAGs also
influence the growth-modulatory activity of TSP, because low molecular
weight heparin neutralizes TSP-mediated inhibition of multilineage
colony-forming units-granulocyte, erythroid, macrophage, megakaryocyte
(CFU-GEMM) and CFU-megakaryocyte (CFU-MK) growth that is induced by
cytokines including IL-3, SCF, granulocyte-macrophage
colony-stimulating factor (GM-CSF), bFGF, and
thrombopoietin.39
We hypothesized that in the absence of stroma, specifically sulfated HS
GAGs are required for IL-3 + chemokine (MIP-1 or PF4)-mediated
LTC-IC maintenance and that this biological activity is dependent on
optimal binding characteristics of the GAGs for early-acting chemokines
(eg, PF4) and matrix components (eg, TSP). In the present studies, we
have examined the ability of purified, differentially sulfated heparin
GAGs to support IL-3 + chemokine-mediated LTC-IC maintenance and the
binding characteristics of these GAGs for cytokines and matrix
components essential for HPC localization and survival.
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Methods |
Cell separation
Bone marrow was aspirated in preservative-free heparin from the
posterior iliac crest of healthy young volunteers after informed consent. Bone marrow mononuclear cells were separated by Ficoll-Hypaque centrifugation (s.g., 1.077), and CD34+ enriched cells were
obtained using Ceprate® LC CD34 selection columns
(CellPro Inc, Bothell, WA). CD34+/HLA-DR
cells were obtained by flow cytometry, using a FACStar
Plus® flow cytometry system equipped with a Consort 32 computer as
previously described.26,34
Stromal feeders and conditioned media
Human primary bone marrow stromal feeders were established from
human bone marrow mononuclear cells as previously described, irradiated
at 1250 rad when confluent, and maintained in long-term bone marrow
culture (LTBMC) medium.26,34 Supernatant from irradiated stromal cultures was harvested 2 to 3 days after a half-medium change,
centrifuged to remove cell debris, and frozen at  70°C until use.
Cytokines
Recombinant human cytokines used in long-term cultures included 500 pg/mL granulocyte colony-stimulating factor (G-CSF)
(Neupogen®; Amgen, Thousand Oaks, CA), 50 pg/mL GM-CSF
(Immunex Corp, Seattle, WA), 200 pg/mL SCF (a kind gift from Amgen), 50 pg/mL leukemia inhibitory factor (LIF; R & D Systems Inc, Minneapolis,
MN), 200 pg/mL MIP-1 (R & D Systems) and 2 ng/mL IL-6 (a kind gift
from Dr G. Wong, Genetics Institute).
As indicated, medium in some long-term cultures was supplemented with
G-CSF, GM-CSF, SCF, LIF, and IL-6 in the same concentrations as
described above, along with 5 ng/mL IL-3 (a kind gift from Dr G. Wong) and either 10 ng/mL MIP-1 or 200 ng/mL PF4 (purified from
human platelets).
Glycosaminoglycans
HS, chondroitin sulfate, and heparin were obtained from Sigma (St.
Louis, MO). The differentially sulfated heparins used in this study, derived from the same parent unmodified heparin molecule by
N-desulfation and N-reacetylation (O-sulfated heparin), N- and
O-desulfation and N-reacetylation (completely desulfated heparin), or
N- and O-desulfation and N-resulfation, were obtained from Seikagaku
America (Falmouth, MA). Disaccharide compositional analysis of the
unmodified heparin and differentially sulfated heparins (Seikagaku
America, personal communication) is shown in the
Table. The completely desulfated heparin
was largely composed of desulfated disaccharides. The O-sulfated
heparin was largely composed of O-disulfated disaccharides
{-UA(2-OSO3)-GlcNAc(6-OSO3)-} and
6-O-sulfated disaccharides {-UA-GlcNAc(6-OSO3)-} (where
the UA was mainly iduronic acid) and contained only trace amounts
(<1%) of the trisulfated disaccharides {-IdoA
(2-OSO3)-GlcNS (6-OSO3)-}, whereas unmodified heparin was mainly composed of trisulfated disaccharides, with a
smaller proportion of O-disulfated units. The N-sulfated heparin was
depleted of O-sulfated units (not shown). For several growth factors,
the regions of transition between highly sulfated GlcNS-containing blocks and unsulfated GlcNAc-containing blocks are believed to be
important for binding and biological activity of HS. Because of the
specificity of the sulfotransferase enzymes, the terminal GlcNAc
(adjacent to GlcNS) in such blocks may be 6-O-sulfated in such
regions.40,41 Disaccharides at such transition points thus are 6-sulfated, with or without 2-O sulfation of the hexuronic acid: Most disaccharides in O-sulfated heparin are identical to such
units and more closely resemble sulfation patterns of HS rather than
heparin.
The GAGs were added to long-term cultures at a final concentration of 5 µg/mL; we have previously shown this concentration to be in the
optimal range for LTC-IC maintenance.26
Preparation of synthetic proteoglycan-like molecules
GAGs and either ovalbumin or human albumin were dissolved (2 mg each) in 1 mL distilled water, and 20 mg of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) in 50 µL water
was added (all chemicals from Sigma). The mixture was shaken at 4°C
for 12 hours to couple GAGs to ovalbumin or albumin via the carboxyl
groups by amide bonds.42 Low molecular weight by-products
were removed by dialysis in 12 000-14 000 molecular weight cut-off
(MWCO) dialysis tubing against phosphate-buffered saline
(PBS). For use in cultures, the protein-coupled GAGs
(final concentration 5 µg/mL) were dissolved in fresh LTBMC medium
and sterile-filtered.
Long-term cultures
LTBMC medium.
The medium consisted of Iscove's modified Dulbecco's medium with
12.5% fetal calf serum, 12.5% horse serum, 2 mM L-glutamine, 1000 U/mL penicillin, 100 U/mL streptomycin, and 106
mol/L hydrocortisone.43
Conditioned medium cultures.
A total of 10 × 103 to 14 × 103
DR cells were suspended in a transwell insert with a
microporous 0.4-µm membrane placed in stroma-free wells of 6- or
24-well plates. Media in the lower well were replaced 5 times weekly
with 3 mL (for 6-well plates) or 0.8 mL (for 24-well plates) fresh
unsupplemented LTBMC medium, LTBMC medium supplemented with cytokines
alone or in combination with cell line-derived or artificially
synthesized PGs or GAG chains, or conditioned medium from M2-10B4 or
human stromal feeders. After 2 or 5 weeks of culture, progeny of
DR cells were recovered from the transwells by
vigorous washing and counted on a hemocytometer. Cells were plated in
methylcellulose cultures to enumerate colony forming cells (CFC) and in
limiting dilution analysis to determine the absolute number of LTC-IC
present, as described.28,44 All cultures were maintained at
37°C in humidified 5% CO2 atmosphere.
Short-term methylcellulose cultures.
Day 0 DR cells and the progeny of
DR cells recovered at 2 or 5 weeks from long-term
cultures were plated in methylcellulose-containing medium supplemented
with 30% fetal calf serum (FCS), 3 IU/mL erythropoietin, and 5 ng/mL IL-3 and CFC enumerated at day 14. All cultures were maintained at 37°C in humidified 5% CO2 atmosphere.
Enumeration of the absolute number of LTC-IC by limited dilution
analysis.
Day 0 DR cells (22 replicates per concentration) or
progeny of DR cells recovered from long-term
cultures were plated at limiting dilutions onto irradiated M2-10B4
feeders in 96-well plates, as described.26,28,34 Cultures
were fed by weekly replacement of 75 µL fresh LTBMC medium. After 5 weeks, all the medium was removed and the wells overlaid with
methylcellulose-containing medium supplemented with 30% FCS, 3 IU/mL
erythropoietin, and 5 ng/mL IL-3. All cultures were maintained at
37°C in humidified 5% CO2 atmosphere. Wells were
scored for the presence or absence of secondary CFC at day 14. The
absolute number of LTC-IC was calculated as the reciprocal of the
concentration of test cells that yields 37% negative wells, using
Poisson statistics and the weighted mean method.28,44
Iodination of differentially sulfated heparins
Heparins were coupled with tyramine14,15 and were
iodinated using carrier-free Na2125I (Amersham,
Arlington Heights, IL) and the Iodo-gen iodination reagent (Pierce,
Rockford, IL).14 Free 125I was removed from the
labeled HS by separation on a Sephadex G-25 column, followed by
exhaustive dialysis. The specific activity was 5 to 70 µCi/µg for
the iodinated GAGs.
Affinity coelectrophoresis (ACE)
Binding of iodinated heparins to cytokines including IL-3 (R & D
Systems), MIP-1, bFGF (R & D Systems), and VEGF (isoforms VEGF165 and VEGF121; kind gifts from Dr S. Ramakrishnan, University of Minnesota) and to ECM molecules including
TSP (a kind gift from Dr Robert Hebbel, University of Minnesota) and FN
(Sigma) was examined using ACE. ACE was performed as
described.26,45 Briefly, a 1% agarose gel (low melting
point Seaplaque; FMC, Rockland, MD) in 50 mM sodium MOPSO (Sigma), pH
7.0; 125 mM NaCl; 0.5% CHAPS buffer was cast with a strip well (for
HS) and a perpendicular 8-lane comb (for proteins). Protein samples
(TSP, bFGF or IL-3) prepared at twice the desired concentration in
MOPSO/NaCl/CHAPS electrophoresis buffer were mixed with an equal volume
of 2% agarose and allowed to gel in appropriate wells created by the
8-lane comb. The heparin preparation in MOPSO/NaCl/CHAPS
electrophoresis buffer containing bromophenol blue and sucrose was
added to the strip well. Electrophoresis was performed in a Hoefer
electrophoresis apparatus in MOPSO/NaCl running buffer prepared without
CHAPS at 330 mA, 45 to 60 V for 2.5 to 2.75 hours at 20°C to
25°C. Gels were air-dried and autoradiographed at 80°C.
For VEGF, FN, and MIP-1, the gels were cast in 50 mM MOPSO, pH 7.0;
50 mM sodium acetate (NaAc). Protein samples and the heparins were also
prepared in the same buffer. Electrophoresis was performed as above,
for 2 to 2.5 hours.
Determination of half-maximal binding (EC50).
125I-labeled heparin or O-sulfated heparin was
electrophoresed through varying concentrations of TSP (1-100 nM) in the
affinity coelectrophoresis system. The retardation
coefficient45 was calculated as
R = (M0 M)/M0, where M0 was
the mobility of free GAGs (taken as mobility in presence of ovalbumin),
and M was the mobility of GAGs through the TSP-containing lanes.
Mobility was measured as the distance from the loading well at the top
of the gel to the upper end (most retarded) of the GAG smear. Because retardation is a function of the binding affinity of the GAG to TSP, a
plot of retardation (binding) versus concentration of TSP was used to
calculate half-maximal binding concentration, as a measure of the
affinity of the 2 GAGs for TSP.
Surface plasmon resonance.
The binding of GAGs to PF4 was examined on a BIAcore®
biosensor equipment (Pharmacia, Uppsala, Sweden) using surface plasmon resonance, which is highly sensitive and monitors real-time binding of
an analyte (GAG) to a ligand (PF4) immobilized on a sensor chip.46,47 This is measured in resonance units (RU), which correlates with the amount of analyte bound (1 RU = 1
pg/mm2). Binding of GAGs in solution to immobilized PF4 was
examined as described.26 The KD was calculated
using BIAevaluation 2.1 software (Pharmacia Biosensor, Uppsala, Sweden)
from the average of ka (association rate, "run-on"
phase) and kd (dissociation rate, "wash-out" phase)
kinetics: KD = av. kd/ka (ks).
Statistics.
Results of data are reported as the mean ± SEM. Levels of
significance were determined by the 2-sided Student's t test.
| |
Results |
HS in combination with IL-3 and MIP-1 supports long-term
maintenance of LTC-IC
In stroma-free cultures supplemented with IL-3 + MIP-1, only
22 ± 1% of LTC-IC were recovered after 5 weeks
(P < .001 vs day 0 LTC-IC frequency; Figure
1), even though 89 ± 3% of day-0 LTC-IC were maintained for 2 weeks. In cultures where 5 µg/mL HS was
added together with IL-3 + MIP-1, 121 ± 10% of input numbers of LTC-IC were maintained for up to 5 weeks (P = .002 vs
cultures with IL-3 + MIP-1 alone). A lower concentration of HS (1 µg/mL) was less effective (LTC-IC maintenance 73 ± 5% at 5 weeks; P = .001 vs 5 µg/mL HS). High concentrations of GAGs
(20 µg/mL HS) inhibited cell proliferation and LTC-IC maintenance
(data not shown). Further, maintenance of LTC-IC at week 5 in HS + IL-3 + MIP-1 cultures was similar to that seen in cultures supplemented with stroma-conditioned medium + IL-3 + MIP-1 (134 ± 12%).
LTC-IC expanded 4 ± 0.8-fold at 2 weeks in stroma-conditioned
medium-supplemented cultures, whereas LTC-IC expansion in HS + IL-3 + MIP-1 cultures was only 1.3 ± 0.1-fold at week 2. These
results suggest that, in the presence of HS, long-term in vitro
maintenance of LTC-IC can be obtained by addition of a single
growth-promoting cytokine (IL-3) and a single growth-inhibitory
chemokine (MIP-1). LTC-IC expansion, in contrast, may require the
presence of additional cytokines and/or factors present in
stroma-conditioned medium.
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| Fig 1.
LTC-IC maintenance in the presence of HS with IL-3 and
MIP-1.
A total of 10 000 to 20 000 DR cells were plated
in 0.4-µm transwell inserts in 6-well tissue culture clusters. Medium
in the lower chambers of the wells was replaced daily by either
stroma-conditioned medium (Stroma CM) or long-term bone marrow culture
(LTBMC) medium supplemented with or without 5 µg/mL HS. A total of 5 ng/mL IL-3 and 10 ng/mL MIP-1 were added to all cultures, including
Stroma CM. Cultures were harvested after 2 weeks (A) or 5 weeks (B) and
cells replated at limiting dilutions for estimation of LTC-IC
frequency, as described in Methods. Numbers within the bars indicate
the number of experiments. Comparison between LTBMC medium only and
other conditions: *P < .005.
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Cytokines alone induce short-term expansion of LTC-IC, but
O-sulfated GAGs are required for long-term LTC-IC maintenance
We have demonstrated that stroma-conditioned medium can be replaced
by a combination of low-dose cytokines (CK) + HS.34 We
therefore cultured DR cells for 2 to 5 weeks in
stroma-free cultures supplemented with IL-3 (5 ng/mL) and MIP-1 (10 ng/mL) with or without a combination of CK in the concentrations
detected in stroma-conditioned medium and with or without bovine kidney
HS. The combination of IL-3 + MIP-1 + CK but no HS induced a
2 ± 0.5-fold expansion of LTC-IC at 2 weeks (Figure
2). However, the majority of LTC-IC was
lost at 5 weeks (LTC-IC recovery 32 ± 2%). The addition of HS to
the combination of IL-3 + MIP-1 + CK resulted in a
2.8 ± 0.6-fold expansion of LTC-IC at 2 weeks. In contrast to
IL-3 + MIP-1 + CK, cultures supplemented with IL-3 + MIP-1 + CK + HS resulted in long-term maintenance of a significantly greater
proportion of LTC-IC (76 ± 7%) at 5 weeks (P < .001
vs IL-3 + MIP-1 + CK cultures).
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| Fig 2.
Modulation of LTC-IC maintenance by differentially
sulfated GAGs in the presence of multiple growth-promoting cytokines
with IL-3 and MIP-1.
A total of 10 000 to 20 000 DR cells were plated
in 0.4-µm transwell inserts in 6-well tissue culture clusters. Medium
in the lower chambers of the wells was replaced daily by either
stroma-conditioned medium (Stroma CM) or by LTBMC medium supplemented
with or without a combination of cytokines (500 pg/mL G-CSF, 50 pg/mL
GM-CSF, 200 pg/mL SCF, 50 pg/mL LIF, and 2 ng/nl IL-6) and with or
without 5 µg/mL each of GAGs. A total of 5 ng/mL IL-3 and 10 ng/mL
MIP-1 were added to all cultures, including Stroma CM. Cultures were
harvested after 2 weeks (A) or 5 weeks (B). The equivalent of 500 to
1000 DR cells plated at day 0 were replated after
harvesting in methylcellulose cultures for estimation of CFC
generation, and the remaining cells were replated at limiting dilutions
for estimation of LTC-IC frequency, as described in Methods. Numbers
within the bars indicate the number of experiments. Comparison between
cytokines only and other conditions: *P < .002; comparison
between desulfated heparin and other conditions: § P < .001.
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We have recently shown that chemically differentially sulfated heparins
have different LTC-IC supportive capabilities, such that LTC-IC
maintenance is supported by O-sulfated heparin in combination with low
concentrations of CK but not by desulfated-, N-sulfated- or
unmodified-heparin.26 We therefore examined the effect of
completely desulfated and O-sulfated heparins on IL-3 + MIP-1-mediated LTC-IC maintenance (Figure 2). Addition of
O-sulfated heparin to IL-3 + MIP-1 + CK resulted in
1.7 ± 0.2-fold LTC-IC expansion at week 2, which was lower than
with HS. However, 95 ± 7% of LTC-IC were maintained at week 5. When desulfated heparin was combined with IL-3 + MIP-1 + CK,
2.5 ± 0.4-fold LTC-IC expansion was seen at week 2, but LTC-IC
recovery at week 5 (23 ± 6%) was no better than with cytokines alone.
When PF4 (200 ng/mL) was substituted for MIP-1 (10 ng/mL) in
stroma-free cultures, LTC-IC maintenance was similar to that seen in
the presence of MIP-1 (Figure 3). Most
of the LTC-IC were maintained in cultures supplemented with IL-3 + PF4 + CK in the presence of either HS or O-sulfated heparin (LTC-IC
supportive GAGs) but not in the presence of desulfated heparin (LTC-IC
nonsupportive GAG). Thus, both the heparin-binding chemokines MIP-1
and PF4 require the sequences present in O-sulfated HS to support
LTC-IC maintenance.
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| Fig 3.
Efficacy of differentially sulfated GAGs to modulate
LTC-IC maintenance in the presence of PF4.
A total of 10 000 to 20 000 DR cells were plated
in 0.4-µm transwell inserts in 6-well tissue culture clusters. Medium
in the lower chambers of the wells was replaced daily by LTBMC medium
supplemented with or without a combination of cytokines (500 pg/mL
G-CSF, 50 pg/mL GM-CSF, 200 pg/mL SCF, 50 pg/mL LIF, 2 ng/mL IL-6, and
200 pg/mL MIP-1) with 5 ng/mL IL-3 and 200 ng/mL PF4 and with or
without 5 µg/mL each of GAGs. Cultures were harvested after 5 weeks
and replated at limiting dilutions for estimation of LTC-IC frequency,
as described in Methods. Numbers within the bars indicate the number of
experiments. Comparison between day 0 and other conditions:
*P < .002.
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The generation of CFC from primitive progenitors is induced by
cytokines and does not require GAGs
A 2-fold expansion in the numbers of CFC generated was sustained for
2 to 5 weeks in cultures supplemented with IL-3 + MIP-1 (Figure
4). The addition of HS to IL-3 + MIP-1
did not affect CFC numbers. However, CFC generation was significantly
higher in the presence of stroma-conditioned medium + IL-3 + MIP-1
at both 2 and 5 weeks (P < .001 and P = .02,
respectively, vs HS + IL-3 + MIP-1). This suggests that the
increased CFC generation in the latter condition is due to the presence
of low concentrations of cytokines present in stroma-conditioned
medium. Indeed, when low-dose cytokines (known to be present in
stroma-conditioned medium) were added to IL-3 + MIP-1 in stroma-free
cultures, the generation of CFC at 2 to 5 weeks became comparable to
that seen in the presence of stroma-conditioned medium + IL-3 + MIP-1, although the size of the colonies generated in the presence
of stroma-conditioned medium was significantly larger. This may be due
to the presence in stroma-conditioned medium of additional cytokines,
such as flt-3 ligand, IL-11, and thrombopoietin, which were not added
to the stroma-free cultures. Overall, the presence of CK + IL-3 + MIP-1 induced a 10- to12-fold increase in CFC at 2 weeks and 6-fold
increase in CFC at 5 weeks, compared to CFC present in the input
population at day 0. CFC numbers were not affected by the further
addition of HS, O-sulfated heparin, or desulfated heparin to CK + IL-3 + MIP-1.
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| Fig 4.
Generation of CFC.
Number of CFC in the starting DR population at day 0 and after 2 weeks (A) or 5 weeks (B) in culture. All cultures were
supplemented with 5 ng/mL IL-3 and 10 ng/mL MIP-1. The indicated
cultures were additionally supplemented with the low-dose cytokine
combination. Numbers within the bars indicate the number
of experiments. Comparison between HS and Stroma CM:
*P < .001 (at 2 weeks); §P = .02 (at 5 weeks).
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As for CFC generation, the addition of HS or O-sulfated heparin to IL-3 + MIP-1 + CK did not affect cell expansion (the total numbers of
terminally differentiated mature cells present in culture) at 2 to 5 weeks (data not shown). Cell expansion in cultures supplemented with
IL-3 + MIP-1 + CK was comparable to that seen in cultures supplemented with stroma-conditioned medium + IL-3 + MIP-1 (500- to
800-fold expansion at 5 weeks).
Cytokine-binding profiles of hematopoietically supportive GAGs
Because O-sulfated heparin but not the other modified heparins or
unmodified heparin supported LTC-IC, we next examined the cytokine- and
matrix component-binding capabilities of these heparins to examine if
differences in this capability correlated with their ability to support
LTC-IC maintenance. Desulfated heparin and N-sulfated heparin, which
did not support LTC-IC maintenance, showed no binding to MIP-1,
IL-3, VEGF, FN, or TSP by ACE (Figure 5).
N-sulfated heparin, but not desulfated heparin, bound bFGF to a small
extent. O-sulfated heparin, which supports LTC-IC maintenance, bound to
IL-3, bFGF, FN, TSP and, to a lesser extent, to VEGF and MIP-1.
Unmodified heparin, which possesses a high degree of both N- and
O-sulfation, also bound to all these proteins. We have already
shown,26 using surface plasmon resonance, that O-sulfated
heparin and unmodified heparin, but not desulfated heparin or
N-sulfated heparin, bind PF4.
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| Fig 5.
Binding of modified heparins to proteins by affinity
coelectrophoresis (ACE).
125I-labeled GAGs (HEP, unmodified heparin; De-S HEP,
completely desulfated heparin; N-S HEP, N-sulfated heparin; O-S HEP,
O-sulfated heparin) were electrophoresed through the proteins
(cytokines and matrix components) cast in agarose gels. Results are
shown for binding of the GAGs to 100 nM TSP, 25 nM bFGF, and 500 nM
each of VEGF, FN, IL-3, and MIP-1. Ovalbumin (500 nM) was used as a
negative control protein. The NaCl electrophoresis buffer was used for
TSP, bFGF, and IL-3, and the sodium acetate buffer was used for VEGF,
FN, and MIP-1. Migration of unbound GAGs that are not bound to
proteins is dependent on both size and charge. The average size of all
4 GAGs was comparable (8 to 12 kd). Because the negative charge on De-S
HEP is the least, its migration was slowest. N-S HEP has an
intermediate charge and migrated faster than De-S HEP. O-S HEP and HEP,
which are the most highly charged, migrated at comparable rates.
Binding to proteins was seen as retardation of the relative rate of
migration of the GAGs through the protein lanes, compared to its
migration through ovalbumin.
|
|
However, the binding profile of O-sulfated heparin was different than
that of heparin. At the concentrations examined, unmodified heparin
bound homogeneously to TSP, bFGF, VEGF, and FN, whereas O-sulfated
heparin bound heterogeneously to these proteins, resulting in a smear
in the protein lanes. Binding of both heparin and O-sulfated heparin to
IL-3 was seen as a smear. When labeled heparin was eluted from the
upper and lower portions of the smear and reelectrophoresed into a
separate IL-3-containing gel (see ref. 14 for method), labeled heparin
from the upper portion bound to IL-3, whereas that from the lowest
portion did not bind to IL-3 (data not shown). Thus, the uppermost
portion of the smear (most retarded) is indicative of the most avidly
bound subset of labeled molecules in the O-sulfated heparin, whereas
the lowest portion of the smear (least retarded) is indicative of
unbound O-sulfated heparin molecules, because it migrated at the same
rate as O-sulfated heparin in the ovalbumin lane. Thus, O-sulfated
heparin may contain GAG chains that bind to proteins over a range
of affinities, unlike the highly sulfated unmodified heparin, and
suggests that at least part of the binding requirements are provided by
N-sulfate groups.
The VEGF165 isoform contains a heparin-binding domain in
exon 7. VEGF121 isoform lacks this
exon.18,48,49 Both O-sulfated heparin and unmodified
heparin bound to VEGF165 but not to VEGF121. This indicates that the binding of both GAGs to VEGF165, as
seen on ACE, is dependent on the presence of the heparin-binding domain in this protein.
Recent studies demonstrate that the modulation of the biological
activity of cytokines by HS depends on the affinity and kinetics of association ("on" rate) between cytokines and
HS.50 We observed that both O-sulfated heparin and heparin
bound all the proteins examined above, but only O-sulfated heparin
supported LTC-IC maintenance. We therefore examined the relative
affinity and kinetics of binding of these GAGs for proteins required
for HPC localization and maintenance. The affinity of heparin for
TSP, a matrix protein, was 4-fold higher than the affinity of
O-sulfated heparin (half maximal binding {EC50} 4 nM vs
17 nM, respectively; Figure 6).
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| Fig 6.
Affinity of unmodified and O-sulfated heparins for
thrombospondin.
The affinity of unmodified or O-sulfated heparin for TSP was examined
using affinity coelectrophoresis. 125I-labeled GAGs were
electrophoresed through varying concentrations of TSP (0 to 100 nM).
Half-maximal binding (EC50; a measure of affinity) was
calculated as the concentration of TSP (in nM) at which retardation of
migration of the GAGs (R; a function of binding) was 50%.
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Because the chemokine PF4 is effective in supporting maintenance of
LTC-IC in vitro, its binding was also investigated. By ACE, the
affinity of heparin for PF4 was higher than the affinity of O-sulfated
heparin (not shown). We therefore further examined the real-time
binding kinetics between PF4 and unmodified or O-sulfated heparin using
surface plasmon resonance (Figure 7). These
studies demonstrated that the affinity of heparin for PF4 was 5-fold
higher than the affinity of O-sulfated heparin (KD, 17 nM
vs 90 nM). Moreover, the rates of association ("on" rate;
ka) and dissociation ("off" rate; kd) of
heparin were 2- to 2.5-fold higher than those of O-sulfated heparin,
for PF4.
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| Fig 7.
Binding of unmodified and O-sulfated heparins to PF4.
After a stable baseline tracing was established by perfusion of
unsupplemented buffer over biotinylated PF4 immobilized on a
streptavidin chip in a BIAcore® biosensor equipment, a
range of concentrations of unmodified heparin (HEP) or O-sulfated
heparin (O-S HEP) were perfused over the chip. Binding of each GAG was
measured in resonance units (1 RU = 1 pg/mm2) in
triplicate at each concentration at 5 different concentrations.
Representative binding curves are shown for the following
concentrations of GAGs: 20.8 µmol/L HEP and 31.3 µmol/L O-S HEP.
The perfusion of GAGs resulted in a shift in the tracing due to binding
of the GAGs to PF4 (association phase; shaded area labeled A). When GAG
perfusion was stopped and perfusion with unsupplemented buffer resumed,
the tracing returned downward as the bound GAGs dissociated from PF4
(dissociation phase; shaded area labeled D). Perfusion of heparin
resulted in a sharp up-slope, indicating rapid association, and a sharp
down-slope following cessation of heparin perfusion, indicating rapid
dissociation. In contrast, perfusion of O-sulfated heparin resulted in
more gradual up-slope and down-slope, indicating slower association and
dissociation from PF4. The table shows the median values for
KD, ka (association rate) and kd
(dissociation rate).
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| |
Discussion |
The present studies demonstrate that, in the absence of stroma, a
combination of specific HS GAGs with IL-3 and MIP-1 or PF4 is
sufficient and necessary for the maintenance of the majority of input
numbers of human LTC-IC for up to 5 weeks. LTC-IC expansion requires
the presence of additional growth-promoting cytokines that are
detectable in stroma-conditioned medium.
Results presented here are consistent with our recent observation that
stroma-derived, O-sulfated HS GAGs improve LTC-IC maintenance in
stroma-free cultures.26,34 In support of the importance of
such O-sulfated GAGs for hematopoiesis, HS from the AFT024 stromal cell
line, which supports multipotential and transplantable progenitors,2-4,51-53 is also enriched in such O-sulfated
HS.54 Use of either HS or HS coupled to ovalbumin or human
serum albumin in combination with IL-3 + MIP-1 ± CK resulted in
equivalent LTC-IC maintenance, CFC generation, and cell expansion (data
not shown). This indicates that (i) the LTC-IC maintaining activity is
attributable to the HS side chains of HS proteoglycans and (ii) that
the multimeric structure formed by coupling of HS chains to a
"core" protein may not be more active than single HS chains in
solution. Consistent with previous studies from our group, GAGs were
not required for the cytokine-induced generation of mature blood cells
and committed progenitors, or for short-term expansion of more
primitive LTC-IC.
Both 2-week expansion and 5-week maintenance of LTC-IC were slightly
superior in stroma-conditioned medium + IL-3 + MIP-1 cultures,
compared to stroma-free cultures supplemented with cytokines and GAGs.
This suggests that additional cytokines, such as flt-3 ligand or
thrombopoietin, or additional unidentified stromal factors may further
enhance support of primitive hematopoietic
progenitors.3,51,52
Of note, we demonstrate that IL-3, MIP-1, and PF4 require O-sulfated
GAGs for their LTC-IC-maintaining capability. Other investigators have
shown that the reconstituting ability of stem cells is lost when
cultured in vitro with high concentrations of IL-3 in the absence of
stroma.55,56 In contrast, microenvironmentally presented
IL-3 produced by genetically engineered stromal cells does not exhaust
primitive progenitors.57 Thus, whereas IL-3 alone may be
detrimental to the in vitro maintenance of primitive stem cells, in the
presence of O-sulfated GAGs and/or other matrix molecules (added to the
cultures or produced by stromal feeders), IL-3 supports HPC in vitro.
It is still not completely clear how GAGs modulate LTC-IC maintenance
in vitro. We have previously hypothesized that GAGs may colocalize
cytokines and matrix proteins with HPC and/or modulate the activity of
cytokines.26,34 Although IL-3 is not detectable in soluble
form in stromal supernatants,58 stromal HS proteoglycans bind and present IL-3 in a biologically active form to hematopoietic cells9,35,59 and augment the localization of clonogenic
progenitors with IL-3.37 These studies, and our observation
that HPC-supportive O-sulfated heparin directly binds IL-3 and
MIP-1, support our colocalization hypothesis. GAGs may also modulate
the activity of localized cytokines on target
cells.8,21,22,60 Previous studies have shown that
heparin-induced dimerization of FGF molecules induces receptor
activation and cell proliferation61 and that dimers of IL-3
may be required for high-affinity receptor binding.62 The
interaction of IL-3 with HPC-supportive GAGs may thus promote or
modulate the biological activity of the cytokine via diverse mechanisms. Highly sulfated heparin also directly inhibits expression of cytokines and cell cycle proteins in target cells,63
suggesting yet another mechanism for GAG-mediated modulation of the
proliferation and differentiation-inducing effects of exogenously added
cytokines on HPC.
Structural features of O-sulfated heparin and unmodified heparin
(Table) may account for the observed differences in their affinity,
kinetics, and profiles of protein binding. These differences may be
responsible for our observation that O-sulfated heparin, but not
unmodified heparin, supports LTC-IC maintenance, even though both GAGs
bind the same cytokines and matrix molecules. Nearly all the individual
GAG chains in unmodified heparin possess an adequate number of highly
sulfated ("default" sulfated)64 disaccharide units
with sulfation at N-, 2-O- and 6-O- positions {-IdoA
(2OSO3)-GlcNS (6OSO3)-} (Table) for the various
proteins to bind to the GAG chains at the concentrations examined. In
contrast, O-sulfated heparin chains infrequently possess such
"default" sulfated disaccharides (Table). The binding of proteins
to O-sulfated heparin chains is likely to occur to more
"selective" protein-binding regions related to the O-sulfated
disaccharides, which are 6-sulfated, with or without 2-O sulfation: The
majority of disaccharides in O-sulfated heparin are identical to such
units and resemble sulfation pattern at the "transition points"
of HS. The variability in the ability of subsets of O-sulfated heparin
chains to bind proteins, as seen on ACE, might be indicative of the
presence or absence of such binding regions on individual O-sulfated
heparin chains. Consistent with this notion, the slow "on" time
(association rate) seen for binding of PF4 suggests that proteins may
take time to find such binding sites on O-sulfated heparin, and the
slow "off" time (dissociation rate) suggests that, once bound,
proteins may not come off rapidly.
Recent studies have demonstrated that HS possessing fast "on"
rate and high-affinity binding sites for bFGF do not activate the
biological activity of bFGF on target cells, whereas HS with a slow
"on" rate, low-affinity binding sites have this
capability.50 Separation of the 2 types of binding sites in
microheterogenous populations of HS, by enzymatic digestion with
heparinase that digests the high-affinity sites, restored the ability
of the HS to activate bFGF. We demonstrate that the O-sulfated heparin, which optimally supports HPC maintenance and expansion, binds relevant
growth factors and matrix molecules with a lower affinity and a slower
"on" rate than unmodified heparin. This suggests that the higher
degree of sulfation in heparin64 is detrimental to the
LTC-IC-maintaining ability of GAGs. O-sulfated heparin represents an
"average" structure and sulfation pattern, required for binding
of multiple cytokines and matrix proteins. The lower protein-binding
affinity of O-sulfated heparin than of the more highly sulfated
unmodified heparin may be essential for optimal delivery of proteins to
cell surface GAGs or receptors, and the slow dissociation rate may help
mediate accumulation of bound proteins in close proximity to the
progenitors.50,65 Taken together, differences between
unmodified heparin and O-sulfated heparin are consistent with the
concept that GAG-mediated support of human HPC in vitro (and likely
other target cells) depends not only on the ability of GAGs to bind
proteins, but also on optimal binding affinity and kinetics of
interactions with early-acting cytokines and matrix components.
| |
Acknowledgments |
The authors acknowledge the excellent technical assistance of Brad
Anderson and of the Biomedical Imaging and Processing Laboratory, University of Minnesota, and thank Dr James D. San Antonio (Jefferson Medical College, Philadelphia, PA) for helpful discussions.
| |
Footnotes |
Submitted June 23, 1999; accepted September 8, 1999.
Supported by the US Department of Veterans Affairs, Washington, DC;
grants R01-HL-48738, P01-CA-65493, and AR-32372 from the National
Institutes of Health, Bethesda, MD; the Minnesota Medical Foundation,
the University of Minnesota Bone Marrow Transplant Research Fund, and
the University of Minnesota Hospital and Clinic, Minneapolis, MN.
C.M.V. is a scholar of the Leukemia Society of America.
Reprints: Pankaj Gupta, Hematology/Oncology Section
111E, VA Medical Center, 1 Veterans Drive, Minneapolis, MN 55417; e-mail: gupta013{at}gold.tc.umn.edu.
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.
| |
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Abstract
Introduction