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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4641-4651
By
From the Departments of Medicine, Biochemistry, and Orthopædic
Surgery, VA Medical Center and University of Minnesota Medical School,
Minneapolis, MN.
Stem cell localization, conservation, and differentiation is
believed to occur in niches in the marrow stromal microenvironment. Our
recent observation that long-term in vitro human hematopoiesis requires
a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize
that such HSPG may orchestrate the formation of the stem cell niche. We
compared the structure and function of HS from M2-10B4, a
hematopoiesis-supportive cell line, with HS from a nonsupportive cell
line, FHS-173-We. Long-term culture-initiating cell (LTC-IC)
maintenance was enhanced by PG from supportive cells but not by PG from
nonsupportive cells (P < .005). The supportive HS were
significantly larger and more highly sulfated than the nonsupportive
HS. Specifically, supportive HS contained higher 6-O-sulfation on the
glucosamine residues. In agreement with these observations, purified
6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly
maintained LTC-IC. In contrast, completely desulfated heparin,
N-sulfated heparin, and unmodified heparin did not support LTC-IC
maintenance. Moreover, the supportive HS promoted LTC-IC maintenance
but not differentiation of CD34+/HLA-DR
THE REGULATED proliferation, commitment,
and terminal differentiation of undifferentiated hematopoietic stem
cells (HSCs) occurs in intimate contact with the bone marrow (BM)
microenvironment. This results in preservation of the stem cell pool
while permitting controlled cell proliferation and
differentiation.1-5 Stem cells are thought to be localized
in stem cell niches or local area networks in the microenvironment,
where they interact with the components of their niche-including
stromal cells, extracellular matrix (ECM) proteins, and cytokines that
are present on stromal cells or bound to ECM macromolecules. Regional
variation in these components within the hematopoietic microenvironment
may create niches that are specific for cells at a given stage of
differentiation.6,7 Specific niches might exist that induce
conservation and maintenance of primitive progenitors and other niches
that promote proliferation and differentiation, depending on the
specific cytokines and matrix components present within it. At present,
the identity and structural characteristics of the macromolecules that
mediate the formation of such niches remain unknown.
Stromal cell lines that differ in their hematopoietic supportive
capability (reviewed in Deryugina and
Müller-Sieburg6) may represent niches containing
specific components critical for either survival, proliferation, or
differentiation of stem cells.8 To define components of
niches that support maintenance of primitive human hematopoietic
progenitors, we initiated studies to identify and compare the factors
produced by hematopoiesis-supportive and nonsupportive cells. We have
previously demonstrated that 50% of human long-term culture-initiating
cells (LTC-ICs) can be maintained for 5 to 8 weeks in vitro when
cultured in medium conditioned by human mixed cell type marrow stromal
feeders or by the murine BM stroma-derived fibroblast cell line M2-10B4
(the supportive cells).9-11 Furthermore, 100% of LTC-ICs
are maintained for 8 weeks in stroma-conditioned medium supplemented
with the heparin-binding cytokines interleukin-3 (IL-3) + macrophage
inflammatory protein-1 We have shown that one such microenvironmental factor essential for
maintenance of human LTC-ICs is a heparan sulfate proteoglycan (HSPG)
secreted into the SN of the supportive cells.17 HSPGs are a
ubiquitous component of all tissue microenvironments, including the BM
microenvironment,18-21 and are present both on cell
surfaces and in the ECM. They can mediate cell adhesion, bind and
modulate the activity of cytokines, and also bind diverse ECM
proteins.22 HSPGs at least partly mediate adhesion of
hematopoietic progenitors to stromal cells.7,23-25
Furthermore, a number of hematopoietic cytokines, such as IL-3,
granulocyte-macrophage colony-stimulating factor (GM-CSF), basic
fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF),
bind to HS.4,5,26,27 They may therefore form the backbone
of macromolecular complexes in the microenvironment that regulate cell
growth and differentiation.18,22,28-31
Structurally, HSPGs are highly heterogeneous macromolecules composed of
a core protein and covalently linked, sulfated HS side chains. These HS
glycosaminoglycans (GAGs) consist of repeating disaccharide units of
D-glucosamine (GlcN) and a hexuronic acid (UA) that is either
D-glucuronic acid (GlcA) or L-iduronic acid (IdoA). HSPGs from
different tissues of origin have unique structural characteristics due
to differences in their core proteins as well as in the nature, number,
and pattern of sulfation of the disaccharides, including variability in
N-, 6-O-, and, rarely, 3-O-sulfation of GlcN, 2-O-sulfation of IdoA,
and sometimes 2-O-sulfation of GlcA.32 It is believed that
these differences are responsible for highly specific interactions of
GAGs with diverse macromolecules.32,33
Specificity in the pattern of O-sulfation of HS is one of the important
determinants of its functional specificity and its ability to bind
cytokines and to modulate cell proliferation and differentiation.26,27,34-38 Bovine kidney HS has a high
degree of 6-O-sulfation of the first N-acetylated glucosamine adjacent to the terminal N-sulfated glucosamine at the interfaces between sulfated and nonsulfated blocks.39 We have recently shown
that this HS supports LTC-IC maintenance.17
We hypothesized that (1) differences in HS secreted by the
hematopoiesis-supportive and nonsupportive cells may be responsible for
the observed differences in LTC-IC maintenance by these two cell lines
and (2) the hematopoiesis-supportive activity of these HS may depend on
specific patterns of sulfation that allow colocalization of HSC with
cytokines and ECM components that support the conservation and
proliferation of HSC. Such HSPGs would thus form the backbone of a
functional stem cell niche. In this report, we describe the structural
and functional characteristics of heparan sulfate required to form a
functional microenvironment that supports long-term in vitro human
hematopoiesis.
Stromal Feeders and Cell Line Cultures
Long-Term Cultures
Cell separation.
BM was aspirated in preservative-free heparin from the posterior iliac
crest of healthy young volunteers after obtaining informed consent. BM
mononuclear cells were separated by Ficoll-Hypaque centrifugation
(specific gravity, 1.077) and enriched for
CD34+ cells using Ceprate LC CD34-avidin immunoadsorption
columns (CellPro Inc, Bothell, WA).41 The CD34+
enriched cell population was labeled with anti-CD34-phycoerythrin (PE)
and anti-HLA-DR-fluorescein isothiocyanate (FITC)
antibodies (Becton Dickinson, San Jose, CA) and
CD34+/HLA-DR Cytokines and glycosaminoglycans.
Recombinant human cytokines used in long-term cultures included
granulocyte colony-stimulating factor (G-CSF; Neupogen; Amgen, Thousand
Oaks, CA), GM-CSF (Immunex Corp, Seattle, WA), stem cell factor (SCF; a
kind gift from Amgen), leukemia inhibitory factor (LIF; R & D Systems
Inc, Minneapolis, MN), MIP-1 Culture system.
DR Structural Analysis of Proteoglycans
Purification of PGs from SN of supportive and nonsupportive cells.
For use in long-term cultures, PGs in the SN of irradiated supportive
and nonsupportive cells were radiolabeled (1 of 10 flasks were labeled)
on the sulfate groups by addition of 50 µCi/mL
Na235SO4 (ICN Biomedicals Inc,
Irvine, CA) in sulfate-replete (sulfate content of IMDM, 0.8 mmol/L)
LTBMC medium for 24 hours. PGs were purified by diethyl aminoethyl
(DEAE)-Sephacel anion exchange high-performance liquid
chromatography (HPLC; Beckman, Fullerton, CA), as previously described.17 For
structural analysis, PGs were labeled with 20 µCi/mL
3H-glucosamine (to label the GAG backbone; DuPont NEN,
Boston, MA) and 50 µCi/mL
Na235SO4 in LTBMC medium for 18 to
24 hours, before purification by anion exchange HPLC.
Preparation of HSPGs.
Purified, 3H- and 35S-labeled PGs were digested
by chondroitinase ABC (cABC; Seikagaku) and chromatographed on a
Sephadex G-50 (Sigma) column, and undigested HSPGs eluting at
V0 were collected. Free HS chains were released from the
core protein by treatment of the HSPG with sodium hydroxide in the
presence of sodium borohydride (NaBH4). Between 96% and
99% of the purified HS from the various peaks was digestible by
nitrous acid,43,44 indicating that this material was highly
purified for HS. The size of the HSPG and HS was estimated by gel
filtration chromatography on a 0.75 × 95 cm Sepharose CL-6B
column equilibrated in 4 mol/L guanidine hydrochloride and 0.05 mol/L
sodium acetate, pH 5.8. The approximate sizes of PG and GAG were
estimated by the methods of Heinegard and Hascall45 and
Wasteson,46 respectively.
Distribution of N-sulfation and O-sulfation in HS.
35S-and 3H-labeled HS from both
cells were subjected to low pH nitrous acid (pH 1.5) deaminative
cleavage of N-sulfated regions as described.44 The digested
oligosaccharides were resolved by gel filtration chromatography on a
0.75 × 110 cm Sephadex G-25 column equilibrated and eluted at a
rate of 6 mL/h with 0.2 mol/L ammonium acetate, pH 7.0; 0.3-mL
fractions were collected. The resulting oligosaccharides were analyzed
as described previously.47
CarboPac PA1 chromatography.
3H- and 35S-labeled CS/DS PGs in the SN of the
two cells were recovered from DEAE-Sephacel anion exchange HPLC,
dialyzed against water, and lyophilized. Analysis of sulfated
disaccharides was performed as described.48 Briefly, CS/DS
were digested using cABC, the resulting disaccharides were reduced by a
modified borohydride reduction reaction to stabilize unsaturated
disaccharides to alkali, and the reduced alditols were desalted using
Dowex 50 hydrogen. The disaccharides were resolved by HPLC using a
CarboPac PA1 column eluted at 1 mL/min with a sodium trifluoroacetic
acid gradient in 0.1 mol/L NaOH. Fractions of 1 mL were collected.
Disaccharides were detected by integrated pulse amperometry on a pulsed
electrochemical detector module and identified by comparison to known
disaccharide standards. The 3H and 35S
radioactivity incorporated in the disaccharides was measured, and the
relative metabolism of 3H-glucosamine by the 2 cells was
estimated by the ratio of 3H:35S in the
sulfated disaccharide peaks.48
2-O-Desulfation of GAGs
Iodination of HS Purified HS from the two cells was labeled with tyramine50,51 and iodinated using carrier-free Na2125I (Amersham, Arlington Heights, IL) and the Iodo-gen iodination reagent (Pierce, Rockford, IL).51 Free 125I was removed from the labeled HS by separation on a Sephadex G-25 column, followed by exhaustive dialysis. The specific activity was 3.4 µCi/µg for the iodinated supportive HS and 7.2 µCi/µg for the nonsupportive HS.Affinity Coelectrophoresis (ACE) Binding of 35S-labeled HS from the supportive and nonsupportive cells to cytokines including IL-3 (R & D Systems), PF4, MIP-1 , and bFGF (R & D Systems) and to ECM molecules including
thrombospondin (TSP; a kind gift from Dr Robert Hebbel, University of
Minnesota, Minneapolis, MN) and fibronectin (FN; Sigma) was examined
using ACE. To digest residual core proteins of the HSPGs and any other remaining proteins before ACE, 3H- and
35S-labeled HS preparations obtained from the two cells as
described above were further incubated with proteinase K (Sigma)
followed by heating at 100°C for 1 minute to inactivate the enzyme.
ACE was performed as described.52 Briefly, a 1% agarose
gel (low melting point Seaplaque; FMC, Rockland, MD) in 50 mmol/L
sodium MOPSO (Sigma), pH 7.0, 125 mmol/L NaCl, 0.5% CHAPS buffer was cast with a strip well (for HS) and a perpendicular 8-lane comb (for
proteins). Protein samples 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 HS 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, submerged in Amplify solution (Amersham,
Arlington Heights, IL), redried, and autoradiographed at
 80°C on Kodak Biomax MS film using a Kodak Biomax TranScreen
LE intensifying screen (Eastman Kodak, Rochester, NY). In
other experiments, the binding of 125I-labeled cell line HS
to TSP and MIP-1 was examined by ACE.
Surface Plasmon Resonance (SPR) The binding of GAGs to PF4 was examined on 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.53,54 A change in mass at the surface of the chip upon binding of the analyte causes a change in refractive index, changing the angle at which plasmon resonance occurs. This is measured in resonance units (RU), which correlate with the amount of analyte bound (1 RU = 1 pg/mm2). For assessment of binding of GAG in solution to immobilized PF4, 1 µg/mL biotinylated PF4 in HEPES buffer containing 150 mmol/L NaCl, 1 mmol/L CaCl2, and 1 mmol/L MgCl2 was perfused over an SA5 (with immobilized streptavidin) sensor chip. GAGs in equilibration buffer (HEPES buffer as above with 0.005% surfactant p20) in a range of concentrations between 10 4 and 107 mol/L were
perfused over the chip at 20 µL/min. The KD was
calculated using BIAevaluation 2.1 software (Pharmacia Biosensor AB,
Uppsala, Sweden) from the average of ka (association or
run-on phase) and kd (dissociation or wash-out phase)
kinetics: KD = kd/ka (where ka is derived from the ks plot).
Adhesion of CD34+ Cells Unmodified or differentially sulfated heparins were conjugated to ovalbumin by amide linkage, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC).55 Briefly, GAG and ovalbumin were dissolved (2 mg each) in 1 mL distilled water and 20 mg of 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. EDC was removed by dialysis against phosphate-buffered saline (PBS). For adhesion experiments, 48-well plates were coated by overnight incubation at 37°C with 150 µL/well of 5% fatty acid free bovine serum albumin (BSA; Sigma) or 100 µg/mL ovalbumin-conjugated GAGs in Voller's bicarbonate buffer. For some experiments, plates were incubated for an additional 24 hours at 4°C. Wells were washed and blocked with 1% BSA in PBS before the adhesion assay. Fluorescence-activated cell sorting (FACS)-sorted CD34+ cells were labeled with 51Cr and resuspended in IMDM + 0.3% BSA, and 20,000 cells in 250 µL volume/well were allowed to adhere to the coated wells for 4 hours at 37°C. The total radioactivity present in an equivalent number of cells (20,000 cells in 250 µL volume) was measured separately. Nonadherent cells were removed by four to six gentle washings with PBS, and their removal was confirmed by visual inspection. Adherent cells were lysed by addition of 500 µL/well of 1% Triton X-100 in PBS and 51Cr radioactivity was measured. The percentage of adhesion was calculated as: % adhesion = cpm in adherent cell lysate × 100/total cpm in 20,000 cells plated.Binding of 125I-HS to CD34+ Cells CD34+ cells were suspended in 0.5 mL cold IMDM + 0.3% BSA, and 125I-HS from the supportive or nonsupportive cells was added. After incubation on ice with gentle mixing for 90 minutes (maximal binding was seen at 60 to 90 minutes in preliminary experiments; data not shown), the cells were washed with cold IMDM + 0.3% BSA and the bound radioactivity in the cell pellet counted using a gamma counter.Statistics Results of data are reported as the mean ± SEM. Levels of significance were determined by the two-sided Student's t-test.
PGs from Hematopoiesis-Supportive Cells But Not From Nonsupportive Cells Support LTC-IC Maintenance Proteoglycans purified from the SN of the two cells were evaluated for their ability to support LTC-IC maintenance. A combination of supportive cell PGs and the picogram concentrations of cytokines found in stromal SN supported maintenance of the same number of LTC-IC as cultures fed with unfractionated stromal SN. However, supportive cell PG alone or cytokines alone were unable to maintain LTC-IC (Fig 1). In contrast, when purified PGs from the SN of the nonsupportive cells were combined with the same cytokines, no increase in LTC-IC maintenance was seen over that with cytokines alone. Maintenance of LTC-IC in cultures supplemented with nonsupportive cell PGs + cytokines or with supportive cell PGs + cytokines in the present study was comparable to the LTC-IC maintenance observed in the presence of unfractionated nonsupportive cell SN and supportive cell SN previously described by our group.13 This indicates that a combination of cytokines at concentrations found in stromal SN + purified PGs recreates the hematopoiesis-supportive capabilities of unfractionated SN from the two cell lines.
Supportive HS Are Larger and More 6-O-Sulfated Than Nonsupportive HS Because the LTC-IC maintaining activity of the supportive PGs is due to their HS,17 we examined the differences in HS from the supportive and the nonsupportive cells. Upon anion exchange HPLC, 35SO4- and 3H-glucosamine-labeled PG from SN of the supportive cells eluted as a single major peak, whereas PGs from the nonsupportive cells eluted in three peaks (labeled A, B, and C; data not shown). The three peaks in PGs from the nonsupportive cells and comparable regions from the supportive cells' PGs were analyzed separately.Size of HPSGs and GAGs. The size of HSPGs from the two cells was similar (average molecular weight [Mr], 160 to 210 kD). However, the average size of supportive HS (45 kD) was larger than nonsupportive HS (both peak A [22 kD], which contains the majority of its HS, and peak B [33 kD], which constitutes a smaller proportion of its HS). Degree of sulfation of HS.
Sulfation, determined as the ratio of dpm of 35S-sulfate to
3H-glucosamine, was higher in PGs from the supportive cells
than from the nonsupportive cells in three independent experiments. Because different cell types can have different glucosamine pool sizes
and fluxes, the specific activity of the sulfated disaccharides generated by cABC was used to correct the measured incorporation of
3H-glucosamine in GAGs by the supportive cells and the
nonsupportive cells, as outlined by Midura et al.48 The
average ratio of 3H:35S in the sulfated
disaccharides ( Sulfation pattern of HS. To determine if supportive and nonsupportive HS differ in sulfation pattern, we digested 3H-glucosamine- and 35SO4-labeled HS with low pH nitrous acid. The resulting oligosaccharides were resolved by Sephadex G-25 column chromatography. At low pH, nitrous acid cleaves HS at GlcNSO3 residues, leaving regions with GlcNAc as intact oligosaccharides.44 When the resulting oligosaccharides and disaccharides are resolved by G-25 gel filtration chromatography,47 large undigested oligosaccharides (>8 monosaccharide residues) containing contiguous GlcNAc residues elute in the excluded volume (pool I in Fig 2), disaccharides and free sulfate derived from regions containing contiguous GlcNSO3 residues elute in the total volume (pool III), and oligosaccharides of 3-8 monosaccharides derived from cleavage of alternating [-(UA-GlcNSO3-UA-GlcNAc)n-] or variably spaced regions containing both GlcNAc and GlcNSO3 residues elute in the included volume (pool II; fractions between vertical bars). The 3H-glucosamine in pool II is indicative of the proportion of the total carbohydrate backbone contained in such oligosaccharides. The 35S in pool II oligosaccharides is largely as 6-O-sulfated GlcN, rather than as 2-O-sulfated UA,39,47 and is therefore representative of the extent of 6-O-sulfation on GlcNAc residues adjacent to the terminal GlcNSO3 residue.39,56
6-O-Sulfation Is Essential for the LTC-IC Maintaining Capability of GAGs We next examined if these differences in sulfation patterns have functional implications for the ability of GAGs to support LTC-IC. We evaluated the ability of chemically modified heparins, which are selectively desulfated at one or more positions, to maintain LTC-IC. A combination of unmodified heparin and cytokines did not significantly increase LTC-IC maintenance over that seen with cytokines alone (Fig 3A). This suggested that the common, highly N- and O-sulfated heparin motif [-IdoA(2-OSO3)-GlcNSO3(6-OSO3)-] may not be optimal for LTC-IC maintenance. A completely N- and O-desulfated, N-reacetylated heparin (desulfated heparin) of comparable chain length also did not have any LTC-IC maintaining activity. Completely N- and O-desulfated, N-resulfated heparin, which is largely depleted of 2-and 6-O-sulfate groups but retains N-sulfation (N-sulfated heparin), had only partial activity. In contrast, a combination of cytokines with N-desulfated, N-reacetylated heparin, which retains 2- and 6-O-sulfate but not N-sulfate groups (O-sulfated heparin), maintained LTC-IC to the same extent as unfractionated stromal SN or HS from the supportive cells. Highly 6-O-sulfated bovine kidney HS maintained 70% to 80% LTC-IC. The optimal concentration of bovine kidney HS for support of LTC-IC maintenance was in the range of 5 µg/mL, because higher (20 µg/mL) or lower (1 µg/mL) concentrations were less effective (data not shown). In contrast to their effects on LTC-IC maintenance, the addition of PGs from the two cells, chemically desulfated heparins, or bovine kidney HS to cytokines did not change the number of colony-forming cells (CFCs) or mature cells generated over 5 weeks compared with cultures using cytokines alone (data not shown).
O-Sulfated HS Selectively Bind Early-Acting Cytokines and ECM Components and Directly Mediate Adhesion of CD34+ Cells We then examined if these GAGs might contribute to the colocalization of primitive progenitors with early-acting cytokines and matrix components, thereby facilitating the formation of an appropriate microenvironmental niche. Affinity coelectrophoresis experiments demonstrated that supportive 35S-labeled HS bound to IL-3, MIP-1, PF4, TSP, and bFGF. In contrast, nonsupportive
35S-labeled HS bound only PF4 and bFGF but not IL-3,
MIP-1, or TSP (Fig 4A). Neither HS bound
to FN.
We demonstrate that the addition of the hematopoiesis-supportive
cell-derived PGs but not the nonsupportive cell derived PGs increases
LTC-IC maintenance significantly. Compared with nonsupportive HS, the
supportive HS that are responsible for this effect are larger and are
more highly sulfated, specifically on the 6-O-position of GlcN located
adjacent to modified regions having GlcNSO3. HS possessing
these characteristics bind both ECM components and cytokines important
for growth of primitive hematopoietic progenitors and mediate the
direct adhesion of CD34+ cells to HS. Therefore, HSPGs with
large, highly 6-O-sulfated HS side chains may be central components of
the human hematopoietic stem cell niche in which conservation,
proliferation, and differentiation of primitive progenitors is
regulated. This conclusion is supported by a number of observations.
The authors acknowledge the excellent technical assistance of Laurel
Deloria and Brad Anderson and thank Dr James D. San Antonio (Jefferson
Medical College, Philadelphia, PA) for helpful discussions.
Submitted December 31, 1997;
accepted August 12, 1998.
Address reprint requests to Pankaj Gupta, MD, Hematology/Oncology
Section, VA Medical Center, One Veterans Drive, Minneapolis, MN 55417, e-mail: gupta013{at}gold.tc.umn.edu; or Catherine M. Verfaillie, MD,
Department of Medicine, Box 806 UMHC, 420 Delaware St SE, Minneapolis,
MN 55455, e-mail: verfa001{at}maroon.tc.umn.edu.
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Top
Abstract
Introduction
Di-4S and 
Robbing Peter to pay Paul?
Blood
81:3169, 1993
.
Nature
361:79, 1993
-binding domain in heparan sulphate.
Biochem J
310:497, 1995
