We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1α (MIP-1α), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1α binding ability. A specific MIP-1α-binding HS oligosaccharide preparation of Mr 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1α binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.
Introduction
We have demonstrated that specific heparan sulfates (HSs) are required for cytokine-mediated, long-term in vitro maintenance of human long-term culture-initiating cells (LTC-ICs).1-3 The same HSs are required for in vitro maintenance of nonobese diabetic-severe combined immunodeficient (NOD-SCID) repopulating stem cells present in human umbilical cord blood.4 These studies showed that an essential property of progenitor-supportive HS was its ability to bind several growth stimulatory cytokines and cell cycle inhibitory chemokines with optimal affinity.2 3 However, HS preparations are a mixture of polysaccharide chains that vary considerably in size and sulfation pattern. It therefore remained uncertain whether the stem cell supportive capability of HS was attributable to an oligosaccharide of defined length contained within the HS chains and whether it correlated with binding to one specific cytokine or chemokine.
Our group has shown that macrophage inflammatory protein-1α (MIP)-1α, a cell cycle inhibitory chemokine,5 is required for optimal long-term in vitro LTC-IC and lymphoid progenitor maintenance.6-8 Other studies showed that cell surface HS may influence the biologic activity of MIP-1α by inducing its oligomerization and enhancing its microenvironmental concentration.9 We have recently defined the structural features of HS oligosaccharides required for binding MIP-1α.10
In the present work, we examined the LTC-IC supportive capability of HS oligosaccharides defined by their size and differential ability to bind MIP-1α. We demonstrate that LTC-IC supportive HS oligosaccharides must measure at least 8 kDa and have the ability to bind MIP-1α.
Study design
Preparation and characterization of HS oligosaccharides
Oligosaccharides of defined length were prepared by digestion of porcine intestinal mucosal (PIM) HS (Mr, 33 kDa; Celsus, Cincinnati, OH). Briefly, the smaller dp2-14 oligosaccharides (kindly provided by Sarah Goodger, Paterson Institute, United Kingdom), composed of the sulfated domains, were released from intact HS by heparinase III. The larger oligosaccharides (Mr, 5-20 kDa) were prepared (with assistance from Brindra Mohani) by the addition of 20 μL platelet extract (provided by Paul Brenchley, University of Manchester, United Kingdom) to 10 mg PIM HS spiked with 200 000 dpm 3H-radiolabeled HS treated as described.10 Each of the latter fragments is a pool of oligosaccharides named according to mean and modal size, with minor species deviation by a maximum of ± 20% of the mean. We recently reported on the ability of these sizes of oligosaccharides to bind MIP-1α.10
LTC-IC assays
CD34+/HLA-DR− cells were obtained from bone marrow aspirates taken from adult volunteers1 after informed signed consent, as approved by the institutional Human Subjects Subcommittee of the Minneapolis VA Medical Center. Cells were plated in transwell inserts placed in 24-well plates and were cultured in stroma-free conditions in long-term bone marrow culture (LTBMC) medium supplemented with low-dose cytokines and a single chemokine (MIP-1α), as described.1 Various HS oligosaccharides were added to the medium (final concentration, 5 μg/mL). Medium was changed 5 days each week. After 5 weeks, the cells were recovered, counted, and plated at limiting dilutions on an irradiated stromal feeder layer (AFT024 cells) in 96-well plates.11,12 Cultures were maintained, and LTC-IC frequency was calculated as described.1
Statistical analysis
Data were analyzed using Prism software (GraphPad Software, San Diego, CA). Results were expressed as mean ± SEM. Significance of differences was determined by the 2-tailed t test and by the nonparametric Mann-Whitney U test.
Results and discussion
Total numbers of cells at the end of the 5-week culture period were comparable in presence or absence of the various oligosaccharides (data not shown). This is in agreement with our previous observations that HS does not change the cytokine-induced production of mature blood cells from CD34+/HLA-DR−progenitors1-3 and also indicates that the oligosaccharides were not cytotoxic.
LTC-IC maintenance in the presence of the parent HS (Figure1; Table 1) was comparable to that seen with chemically modifiedO-sulfated (N-desulfated) heparin (from Seikagaku America, Falmouth, MA) that we have previously shown has optimal supportive activity.2 3 However, LTC-IC maintenance in presence of the small sulfated oligosaccharides (dp2-14) or the 5-kDa oligosaccharide was no better than in the absence of GAGs. Thus, these molecules had no activity, and they did not improve LTC-IC maintenance above that seen with cytokines alone. In contrast, LTC-IC maintenance was significantly improved by oligosaccharides of 7.5 kDa or more. The activity of the 10-kDa and 20-kDa oligosaccharides was comparable to that of parent HS. The activity of the 7.5-kDa oligosaccharides was significantly less than that of intact HS, indicating that the smallest size of oligosaccharides that optimally support long-term LTC-IC maintenance in vitro is contained within the 10-kDa oligosaccharide pool, which ranged from approximately 8-12 kDa.
![Fig. 1. Effect of HS oligosaccharides on LTC-IC maintenance in the presence of low-dose cytokines and MIP-1α. / CD34+/HLA-DR− cells (9000-10 000 cells/well) were plated in 0.4-μm transwell inserts in 24-well tissue culture clusters. Medium in the lower chambers of the wells was replaced 5 days each week by LTBMC medium supplemented with a combination of low-dose recombinant cytokines (500 pg/mL granulocyte-colony-stimulating factor [G-CSF], 50 pg/mL granulocyte macrophage-colony-stimulating factor [GM-CSF], 200 pg/mL stem cell factor [SCF], 50 pg/mL leukemia inhibitory factor (LIF), and 2 ng/mL IL-6) and 200 pg/mL MIP-1α, with or without 5 μg/mL indicated HS oligosaccharides or intact (parent) HS. Cultures were harvested after 5 weeks, and cells were replated at limiting dilutions for estimations of LTC-IC frequency, as described in “Study design.” The absolute number of LTC-ICs in the starting cell population at day 0 was 2.96 ± 0.3 per 100 DR− cells plated. The maintenance of LTC-IC after 5 weeks of culture in the presence of intact parent HS was 65% ± 15% of day 0 LTC-IC. n = 3 independent experiments. LTC-IC maintenance observed in the presence of the different oligosaccharides is expressed as a percentage of that seen with the intact parent HS molecule. Comparisons between No GAGs (cytokines alone) and other conditions and between intact (parent) HS and the other conditions are as indicated in Table 1. SEM bars are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/6/10.1182_blood-2002-08-2588/4/m_h80633985001.jpeg?Expires=1737566426&Signature=RzMOsrF~QzqlMuVjcXx2joj9kV4wzC-ui34DFdcUb2bTQWTOypqcOhm5d~9wlK9LbF5ON1tbchI2fzRi1OD5dpjZSOa4N4lHjAJPZLv2BPrHzQu0gHf4kLSV801XqWiopRPuc8I30rw4sq0g~qSIJY56T6cXkdezWGghwkWnme2CmNIEUgSDMwDpdG6PH0GuvUrTDJv~RyeQXWhRGGEWWxhkUJ6EcRZW8Ni-uYid5sLIGSzL4ZSjPTAEHPn0CXEdCfHEZyIJ2ovqGbo2K0uzKq3lvtE2D2zcWP7QAqZGye8VpZwsFlnYFWSSzYyPpXtF8cSwYWUGmB7bfqwplQyaSg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of HS oligosaccharides on LTC-IC maintenance in the presence of low-dose cytokines and MIP-1α.
CD34+/HLA-DR− cells (9000-10 000 cells/well) were plated in 0.4-μm transwell inserts in 24-well tissue culture clusters. Medium in the lower chambers of the wells was replaced 5 days each week by LTBMC medium supplemented with a combination of low-dose recombinant cytokines (500 pg/mL granulocyte-colony-stimulating factor [G-CSF], 50 pg/mL granulocyte macrophage-colony-stimulating factor [GM-CSF], 200 pg/mL stem cell factor [SCF], 50 pg/mL leukemia inhibitory factor (LIF), and 2 ng/mL IL-6) and 200 pg/mL MIP-1α, with or without 5 μg/mL indicated HS oligosaccharides or intact (parent) HS. Cultures were harvested after 5 weeks, and cells were replated at limiting dilutions for estimations of LTC-IC frequency, as described in “Study design.” The absolute number of LTC-ICs in the starting cell population at day 0 was 2.96 ± 0.3 per 100 DR− cells plated. The maintenance of LTC-IC after 5 weeks of culture in the presence of intact parent HS was 65% ± 15% of day 0 LTC-IC. n = 3 independent experiments. LTC-IC maintenance observed in the presence of the different oligosaccharides is expressed as a percentage of that seen with the intact parent HS molecule. Comparisons between No GAGs (cytokines alone) and other conditions and between intact (parent) HS and the other conditions are as indicated in Table 1. SEM bars are shown.
Effect of HS oligosaccharides on LTC-IC maintenance in the presence of low-dose cytokines and MIP-1α.
CD34+/HLA-DR− cells (9000-10 000 cells/well) were plated in 0.4-μm transwell inserts in 24-well tissue culture clusters. Medium in the lower chambers of the wells was replaced 5 days each week by LTBMC medium supplemented with a combination of low-dose recombinant cytokines (500 pg/mL granulocyte-colony-stimulating factor [G-CSF], 50 pg/mL granulocyte macrophage-colony-stimulating factor [GM-CSF], 200 pg/mL stem cell factor [SCF], 50 pg/mL leukemia inhibitory factor (LIF), and 2 ng/mL IL-6) and 200 pg/mL MIP-1α, with or without 5 μg/mL indicated HS oligosaccharides or intact (parent) HS. Cultures were harvested after 5 weeks, and cells were replated at limiting dilutions for estimations of LTC-IC frequency, as described in “Study design.” The absolute number of LTC-ICs in the starting cell population at day 0 was 2.96 ± 0.3 per 100 DR− cells plated. The maintenance of LTC-IC after 5 weeks of culture in the presence of intact parent HS was 65% ± 15% of day 0 LTC-IC. n = 3 independent experiments. LTC-IC maintenance observed in the presence of the different oligosaccharides is expressed as a percentage of that seen with the intact parent HS molecule. Comparisons between No GAGs (cytokines alone) and other conditions and between intact (parent) HS and the other conditions are as indicated in Table 1. SEM bars are shown.
Significance of differences (P)
| dp2 | dp4 | dp6 | dp8 | dp10 | dp14 | 5 kDa | 7.5 kDa | 10 kDa | 15 kDa | 20 kDa | HS | O-S hep | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Versus no GAG* | NS | NS | NS | NS | NS | NS | NS | .005 | .018 | .007 | .0003 | .0002 | .0005 |
| Versus no GAG* | NS | NS | NS | NS | NS | NS | NS | .01 | .01 | .01 | .01 | .008 | .005 |
| Versus HS* | .006 | .017 | .0001 | .004 | .0001 | .0006 | .006 | .035 | NS | .0057 | NS | — | NS |
| Versus HS1-160 | < .05 | < .05 | .008 | .008 | .008 | .008 | < .05 | .02 | NS | .008 | NS | — | NS |
| dp2 | dp4 | dp6 | dp8 | dp10 | dp14 | 5 kDa | 7.5 kDa | 10 kDa | 15 kDa | 20 kDa | HS | O-S hep | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Versus no GAG* | NS | NS | NS | NS | NS | NS | NS | .005 | .018 | .007 | .0003 | .0002 | .0005 |
| Versus no GAG* | NS | NS | NS | NS | NS | NS | NS | .01 | .01 | .01 | .01 | .008 | .005 |
| Versus HS* | .006 | .017 | .0001 | .004 | .0001 | .0006 | .006 | .035 | NS | .0057 | NS | — | NS |
| Versus HS1-160 | < .05 | < .05 | .008 | .008 | .008 | .008 | < .05 | .02 | NS | .008 | NS | — | NS |
NS indicates not significant; —, not calculated.
P values determined by t test.
P values determined by Mann-Whitney Utest.
We have recently reported that the characteristic structure of HS oligosaccharides that bind MIP-1α consists of 2 sulfated end domains separated by a central N-acetylated domain and that the minimum size of the HS oligosaccharide required for this binding is approximately 8.3 kDa.10 This is in contrast to fibroblast growth factor-2 (FGF-2), in which a single sulfated domain of HS is capable of activating the biologic activity of the cytokine.13 In the current study, we correlated the LTC-IC-maintaining capability of these oligosaccharides with their MIP-1α-binding ability. The lack of LTC-IC supportive activity of the smaller oligosaccharides (dp2-5 kDa) was consistent with the absence of structural features required for MIP-1α binding in these molecules. In contrast, oligosaccharides (10 kDa and 20 kDa) and the intact HS that bound MIP-1α did support LTC-IC maintenance optimally. These oligosaccharides have the structural features necessary for MIP-1α binding, and their affinity for MIP-1α is comparable to that of intact HS.10 That the 7.5-kDa oligosaccharides had some LTC-IC–maintaining activity, although this activity was lower than that of intact HS, may be attributable to the minority of larger fragments within this pool and is in keeping with the observation that the average size required for binding MIP-1α is slightly larger than 7.5 kDa.10
Interestingly, the LTC-IC–maintaining activity of the 15-kDa oligosaccharide was lower than that of intact HS and the 10-kDa oligosaccharide. A smaller proportion of the 15-kDa oligosaccharide bound avidly to MIP-1α compared with intact HS or the 10-kDa oligosaccharide (Figure 2). This indicates that the LTC-IC-maintaining capability of HS oligosaccharides is not only related to their size, it likely requires optimal MIP-1α binding achieved by a specific HS structure. One possible explanation for the lower binding and activity of the 15-kDa oligosaccharide is that the method used to generate the oligosaccharides might have resulted in structural differences between the sized pools. For example, the specificity of the heparanase enzyme may be changed at the higher pH used to obtain the larger oligosaccharides, resulting in a different structure at cleavage sites. Determination of sequence-specific differences between the 10-kDa and 15-kDa oligosaccharides will be of interest.
![Fig. 2. Binding of HS oligosaccharides to MIP-1α. / An MIP-1α (BB10010) affinity gel column was prepared by mixing 100 μg MIP-1α with 100 μg heparin in 500 μL coupling buffer (0.1 M HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 80 mM NaCl, pH 7.0) and was incubated for 20 minutes at room temperature. MIP-1α was then bound to Affi-Gel 10, and the column was prepared as previously described.14 A control column was prepared with the MIP-1α omitted. 3H-labeled HS chains were digested with platelet heparanase under different pH conditions, and the resultant HS fragments were size-fractionated on a Cl-6B Sepharose gel filtration column. Intact HS and fragments of 20 kDa,14 kDa, 10 kDa, and 5 kDa were applied to the MIP-1α (BB10010) and to a control Affi-Gel column in 0.15 M NaCl and were eluted with a stepwise NaCl gradient. The percentage of material eluting at more than 0.15 M NaCl has been averaged from 2 experiments, after subtraction of the control results. Binding of the different oligosaccharides to MIP-1α is expressed as a percentage of the binding of the intact parent HS molecule. Standard error bars are shown. Data are from the experiments described in Stringer et al.10](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/101/6/10.1182_blood-2002-08-2588/4/m_h80633985002.jpeg?Expires=1737566426&Signature=CMUC4tQ8LjK0vfqRmxjn4EOXbnMYRn8uDBzVzAEFvxDrMjfnZjFg2-0B2jp6JO3ctbUY1vSrZQ8yVUTO7oUG2DzYchN-8hcoS7e8opp-geYzYsThZlAZOIdAP89a6fZdSnT881wkDwyfRlgUWwQVZldbKvu9oLonSqdsgZAT7uNRctWL2jcsDNPFZr4zTXEauNZG4YSz7DGRlsVbmjqZHfdOOls66N1Hs9~RpP4LG9vPbuXTEjPAQPKAW7YxQn23-ciCs7Oixr7-ibSJCnbZTcNz-KvZKPs687uoua6qZgyXR1kXUIQaot-16cmaBARN0-GCQI9ItvldIYfgAgMY5A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Binding of HS oligosaccharides to MIP-1α.
An MIP-1α (BB10010) affinity gel column was prepared by mixing 100 μg MIP-1α with 100 μg heparin in 500 μL coupling buffer (0.1 M HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 80 mM NaCl, pH 7.0) and was incubated for 20 minutes at room temperature. MIP-1α was then bound to Affi-Gel 10, and the column was prepared as previously described.14 A control column was prepared with the MIP-1α omitted. 3H-labeled HS chains were digested with platelet heparanase under different pH conditions, and the resultant HS fragments were size-fractionated on a Cl-6B Sepharose gel filtration column. Intact HS and fragments of 20 kDa,14 kDa, 10 kDa, and 5 kDa were applied to the MIP-1α (BB10010) and to a control Affi-Gel column in 0.15 M NaCl and were eluted with a stepwise NaCl gradient. The percentage of material eluting at more than 0.15 M NaCl has been averaged from 2 experiments, after subtraction of the control results. Binding of the different oligosaccharides to MIP-1α is expressed as a percentage of the binding of the intact parent HS molecule. Standard error bars are shown. Data are from the experiments described in Stringer et al.10
Binding of HS oligosaccharides to MIP-1α.
An MIP-1α (BB10010) affinity gel column was prepared by mixing 100 μg MIP-1α with 100 μg heparin in 500 μL coupling buffer (0.1 M HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 80 mM NaCl, pH 7.0) and was incubated for 20 minutes at room temperature. MIP-1α was then bound to Affi-Gel 10, and the column was prepared as previously described.14 A control column was prepared with the MIP-1α omitted. 3H-labeled HS chains were digested with platelet heparanase under different pH conditions, and the resultant HS fragments were size-fractionated on a Cl-6B Sepharose gel filtration column. Intact HS and fragments of 20 kDa,14 kDa, 10 kDa, and 5 kDa were applied to the MIP-1α (BB10010) and to a control Affi-Gel column in 0.15 M NaCl and were eluted with a stepwise NaCl gradient. The percentage of material eluting at more than 0.15 M NaCl has been averaged from 2 experiments, after subtraction of the control results. Binding of the different oligosaccharides to MIP-1α is expressed as a percentage of the binding of the intact parent HS molecule. Standard error bars are shown. Data are from the experiments described in Stringer et al.10
The current findings are consistent with our previous observation thatO-sulfated (N-desulfated) heparin, which more closely resembles HS and measures approximately 10 kDa, supports LTC-IC and NOD-SCID reconstituting progenitor maintenance.1-4Additionally, this is the first study suggesting that the large HS-binding sites isolated for MIP-1α,10PF4,15 and interleukin-8 (IL-8)16 may be important for the biologic activity of chemokine(s) on primitive progenitors.
Finally, this study suggests that binding to chemokines that act on hematopoietic progenitors (such as MIP-1α, monocyte chemoattractant protein (MCP)–1,4,17 IL-8,4and platelet factor 4 (PF4)3) might be useful as a screening strategy to short-list oligosaccharides before a direct assessment of activity on primitive progenitors.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-08-2588.
Supported by the United States Department of Veterans Affairs and by Cancer Research (United Kingdom).
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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