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1997 90: 1858-1866

Influence of Interleukin-3 and Other Growth Factors on 41 Integrin-Mediated Adhesion and Migration of Human Hematopoietic Progenitor Cells
Karen P. Schofield, Graham Rushton, Martin J. Humphries, T. Michael Dexter and John T. Gallagher
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
Inuence of Interleukin-3 and Other Growth Factors on a 4 b 1 IntegrinMediated Adhesion and Migration of Human Hematopoietic Progenitor Cells
By Karen P. Schoeld, Graham Rushton, Martin J. Humphries, T. Michael Dexter, and John T. Gallagher
The mechanisms by which hematopoietic progenitor cells are normally anchored in stromal niches and yet can be mobilized by specic growth factors are poorly understood. It is likely, however, that integrins and their extracellular matrix (ECM) ligands play a key role in this process, and recent evidence suggests that integrin function is modulated by signals originating from activated growth factor receptors. We have now examined this further by studying the role of growth factors on a4b1 integrin-mediated adhesion of human CD34" hematopoietic progenitor cells to specic recombinant bronectin fragments coated onto tissue culture dishes. Cells were prepared from cord blood and peripheral blood harvests. During a 30-minute adhesion assay a mean of 74% of CD34 cells attached to the so-called H120 fragment of bronectin, which contains the strongest a4b1 integrinbinding sequence. The level of cell adhesion was signicantly reduced by low concentrations of interleukin-3 (IL3) (2.5 to 10 ng/mL), whereas stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) at these concentrations did not affect adherence of the cells. Migratory behavior of CD34 cells was examined using bronectin fragments adsorbed onto a Transwell lter. The H120 fragment supported much higher levels of cell migration than the H0 fragment of bronectin, which contains a weak a4b1 integrin binding sequence. Over a 16-hour assay, migration of peripheral blood progenitor cells was increased slightly by SCF and by G-CSF. However, a marked stimulation was observed with IL-3, which signicantly increased migration. Similar effects were noted with cord blood cells, although a small proportion of cells were able to migrate in the absence of growth factors. These results indicate that there is a highly selective and functional link between the a4b1 integrin and IL-3/IL-3receptor that could affect the position of stem and progenitor cells in the marrow stroma and inuence their growth and development. 1997 by The American Society of Hematology.
NTERACTIONS BETWEEN hematopoietic progenitor cells and the bone marrow stroma in which they reside are recognized to be of physiologic importance not only for their normal proliferation and differentiation, but also for maintenance of the hematopoietic stem cell.1,2 These interactions are dynamic, but the mechanisms of cell adhesion to stroma are poorly understood and, likewise, there is little information on the underlying processes that govern cell migration and egress into the peripheral circulation. It is likely that highly regulated interactions between adhesion receptors on the progenitor cells and their extracellular matrix ligands play key roles. Foremost among adhesion receptors are the integrins that mediate attachment to specic matrix proteins. Recent evidence linking signaling pathways from integrins and growth factor receptors suggests that cooperation between the pathways is essential for normal cell development and function.3 The ability of integrins to transfer information in both directions between the extracellular matrix and the intracellular compartment adds complexity, but allows for the possibility of growth factors providing an inside-out signal, which could relay to integrins resulting in changes in their ligand binding activity.4,5 This would enable

From the Departments of Medical Oncology and Experimental Haematology, Paterson Institute, Manchester; and Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK. Submitted January 14, 1997; accepted April 30, 1997. K.P.S. is supported by a grant from the Cancer Research Campaign. Address reprint requests to Karen P. Schoeld, MD, CRC Department of Medical Oncology and Experimental Haematology, Paterson Institute for Cancer Research, Wilmslow Rd, Manchester, M20 4BX, UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 1997 by The American Society of Hematology. 0006-4971/97/9005-0020$3.00/0
growth factors, which themselves may be sequestered in discrete stromal environments by extracellular matrix molecules such as heparan sulfate, to provide a level of control over the localization and movement of hematopoietic progenitor cells that would not be provided by adhesion receptors alone.6 This cross-talk between growth factors and integrins may play a role in the dynamics of stem cell mobilization and homing. Both a4b1 (very late activation antigen-4 [VLA-4]) and a5b1 (VLA-5) integrins are present on CD34 hematopoietic progenitor cells, but a4bb1 is expressed at a higher level and is retained for longer periods during subsequent myeloid maturation.7,8 The role of a4b1 and a5b1 integrins in adhesion of hematopoietic progenitor cells has been described in several studies. Both a4b1 and a5b1 can mediate adhesion of CD34 progenitors to bronectin and a4b1 can also bind to vascular cell adhesion molecule-1 (VCAM-1), present on the surface of activated endothelial cells.7,9 Long-term culture initiating cells and colony-forming unit (CFU)-mix progenitors adhere to the bronectin COOH-terminal heparin-binding domain10 and primitive murine CFU-spleen (CFU-S) day 12 colony-forming cells adhere to the a4b1 binding sequence in bronectin.11 The importance of a4b1 is also highlighted in a study that showed that the addition of anti-a4b1 antibodies to long-term bone marrow cultures abrogated lymphopoiesis and retarded myelopoiesis.12 Much less is known about how integrins are involved in promoting migration of hematopoietic progenitor cells, although a4b1 may have a role, as it is known to mediate lymphocyte migration on bronectin and VCAM-113 and it has also been shown that a4b1 antibodies selectively mobilize hematopoietic progenitors into the blood.14 The general importance of b1 integrins in hematopoiesis is indicated by the nding that chimeric mice generated with b1 integrin-decient embryonal stem cells lack b1 0/0 cells in the blood, spleen, thymus, and marrow, as the cells were unable to migrate to these sites.15 The role of a4 integrins in migration is shown by a study in which the cytoskeletal linkages with the a4 cytoplasmic domain, rather than the equivalent domains in a2 and a5 , mediated cell migration.16

Blood, Vol 90, No 5 (September 1), 1997: pp 1858-1866

Blood 0037

5h3c$$$721

07-31-97 21:46:01

WBS: Blood
EFFECT OF IL-3 ON a4b1 ADHESION 1859
The integrin a4b1 binds to two main sites in the alternatively spliced IIICS region of bronectin, which are represented by the CS1 sequence, the strongest a4b1 binding site, and the CS5 sequence, which is a weaker binding site.17-19 A third weak a4b1 binding site is in the adjacent Hep II domain and is represented by the peptide sequence HI.20 Minimal active peptide sequences have been dened for each of the three sites: LDV for CS1, REDV for CS5, and IDAPS for H1. Here we have used two different recombinant proteins from the IIICS region as ligands for a4b1 ; H120, which contains all three binding sites and HO, which contains H1 alone, thus enabling assessment of the effect of strong and weak a4b1 binding sequences on attachment and migration of CD34 cells. Although hematopoietic progenitor cells expressing CD34 are heterogeneous and contain cells already committed to specic lineages, they represent the earliest population that is readily isolated in sufcient numbers for adhesion and migration assays.21,22 We have isolated CD34 cells from human umbilical cord blood or peripheral blood stem cell harvests for use in a short-term adhesion assay in which the cells attach in the presence or absence of growth factors to culture surfaces coated with H120 and H0. We have also determined the effect of growth factors on the ability of CD34 cells to migrate across lters coated with these recombinant proteins. The growth factors chosen for this study, stem cell factor (SCF), interleukin-3 (IL-3), and granulocytecolony stimulating factor (G-CSF) have all been used clinically for their ability to mobilize hematopoietic progenitor cells.23-25 On the basis of our results, we propose that there is a functional link between the IL-3 receptor and the a4b1 integrin.

MATERIALS AND METHODS

Cloning and expression of recombinant HepII/IIICS variants. cDNA clones for the ve different variants of the HepII/IIICS region of human bronectin were synthesized using reverse transcriptasepolymerase chain reaction (RT-PCR) amplication of primary human skin broblast mRNA as described by Mould et al.26 The PCR products were ligated into pUC119 and transformed into E coli JM109. Individual clones containing each of the variants were identied by restriction analysis and sequenced. Inserts were subcloned into the EcoRI site of the pGEX-2T expression vector and used to transform JM109. Recombinant clones were expressed as glutathione-Stransferase fusion proteins. These were isolated by glutathione afnity chromatography, thrombin digestion, and elution of the recombinant HepII/IIICS proteins from a heparin-agarose afnity column. The H120 (containing H1, CS1, and CS5 sequences) and HO (containing the H1 sequence only) variants were used for adhesion and migration experiments. Isolation and analysis of CD34 cells. Umbilical cord blood collected into heparin was layered over Ficoll (Lymphoprep; Nycomed, Birmingham, UK) and the interface mononuclear cells were collected following density gradient centrifugation. CD34 cells were isolated using a magnetic separation column according to the manufacturers instructions (mini-Macs; Miltenyi Biotec, Bergisch Gladbach, Germany), which resulted in average purity of 82% CD34/ cells. Aliquots from peripheral blood progenitor cell harvests mobilized with cyclophosphamide and G-CSF were treated in the same way, but did not require density gradient centrifugation. For maximum purity in the migration experiments, CD34 cells isolated by

mini-Macs were sorted from a Facs Vantage cell sorter (Becton Dickinson, San Jose, CA) using a second uorochrome-conjugated CD34 antibody, anti-HPCA-2 (Becton Dickinson) and control IgG (Becton Dickinson). Antibodies were added to the cells in phosphatebuffered saline (PBS)/0.5% bovine serum albumin (BSA) and left for 10 minutes at room temperature. The cells were then washed twice with PBS/0.5% BSA before sorting. All experiments with cord blood were performed using fresh cells, but aliquots from a frozen batch of peripheral progenitor cells were thawed and used as necessary. The viability of these cells was normal when stained with trypan blue. For three-color uorescence-activated cell sorting (FACS) analysis, CD34 cells isolated from the mini-Macs column were resuspended in PBS/0.5% BSA and uorochrome-conjugated antibodies were added; anti-CD34 uorescein isothiocyanate (FITC) (Becton Dickinson), anti-VLA4 (CD49d) phycoerythrin (PE) (Becton Dickinson), and anti-CD38 tricolor (TC) (CALTAG, San Francisco, CA). Control cells were labeled with the corresponding uorochrome-conjugated IgG. The cells were washed twice with PBS/ 0.5% BSA and analyzed using the PC lysis program (Becton Dickinson). Cells from the murine hematopoietic progenitor cell line, FDCPmix, which had not been subjected to an antibody enrichment procedure, were also used in the adhesion assay after washing three times in serum-free medium. Cell adhesion assay. Assays were performed in 96-well microtiter plates (Costar, Cambridge, MA). Wells were coated for 60 minutes at room temperature with 100 mL aliquots of recombinant H120 or HO proteins at 15 mg/mL in Dulbeccos PBS, and nonspecic binding sites blocked for 30 minutes at room temperature with 100 mL of 10 mg/mL heat-denatured BSA.17 In one experiment, human bronectin (50 mg/mL) was used as the ligand (Sigma, Poole, Dorset, UK). CD34 cells recovered from the mini-Macs were resuspended to between 106 to 106/mL in serum-free medium (Bio-Whittaker, Wokingham, UK; X-vivo 10). The following growth factors were added to the cell suspension separately or in combination: rhIL-3 (Sandoz, Basel, Switzerland) at 10 ng/mL, rhIL-6 (Sandoz) at 200 U/mL, rhG-CSF (Amgen, Thousand Oaks, CA) at 500 ng/mL, and rh SCF (Amgen) at 100 ng/mL. Conditioned medium (2%) containing IL-3 was added to the FDCP-mix cells. Growth factors were added at room temperature 30 minutes before starting the attachment assay. One hundred microliter aliquots of cells were added to the wells in duplicate and incubated for 30 minutes at 37C in a 5% CO2 incubator. In one experiment, cells were incubated for 5 minutes. Nonadherent cells were removed by shaking the plate and washing twice with 100 mL PBS and bound cells were removed by aspiration with PBS. Both fractions were counted with a hemocytometer and adhesion expressed as a percentage of bound cells/(unbound / bound cells). For inhibition of cell adhesion to the recombinant proteins, 10 mg of blocking anti-VLA a4 (HP2/1) and anti-b1 (3S3) antibodies (Serotec, Oxford, UK) together with a control IgG were added to the CD34 cells at 4C and left for 15 minutes before the adhesion assay. Cell migration assay. Migration experiments were performed using lters of 5 mm diameter pore size in 6.5-mm Transwell chambers (Costar). The membranes were coated on both sides by incubation with the recombinant bronectin fragments (coating solution concentration 15 mg/mL) H120 or HO in Dulbeccos PBS for 60 minutes and nonspecic binding sites were blocked with BSA as described for the adhesion assay. FACS sorted CD34 cells were resuspended to 105 to 106/mL in serum-free medium and 100 mL aliquots were placed in the upper chamber of the Transwells. Growth factors used separately or in combination as described for the adhesion assay were placed in the upper and lower chambers. The cells were incubated at 37C in 5% CO2 for either 4 hours or

1860 SCHOFIELD ET AL

Fig 1. Subpopulations of CD34 cells expressing a4 (PE, 2) and CD38 (tricolor, 3).
16 hours. Cells on the upper surface of the membrane were xed with methanol and removed using a cotton-bud. The membranes were then washed and stained with 0.1% crystal violet. Cells on the underside of the membrane were counted visually using a low power eld and those that had dropped into the lower chamber were counted with a hemocytometer. Migration was expressed as the total numbers of these cells. Blocking anti-a4 and anti-b1 antibodies used to inhibit migration were added to the cells at 4C and left for 15 minutes before the migration assay. Additional antibodies were also added to the cells in the upper chamber after 2 hours.

RESULTS

FACS analysis of CD34 cells. CD34 cells isolated from cord blood with the mini-Macs column were triple-labeled with uorescent antibodies (see Materials and Methods) to CD34, VLA-4, and the CD38 antigen, which is a differentiation marker for more mature hematopoietic progenitor cells. Cells were gated by size on the lymphocyte population and by uorescence on the CD34 cell population and the two additional uorescence parameters (2 and 3 ) were used to analyze subpopulations of a4 and CD38/ cells, respectively (Fig 1). Virtually all (99.8%) cells expressing CD34 also expressed a4 integrin including the CD380, more immature phenotype. The proportion of CD34/CD380 cells shown in Fig 1 was 10.5%, a greater value than reported in some studies,21,22 but consistent with a mean of 9.7% { 1.4%, which we obtained for three cord bloods. It can clearly be seen that all CD34 cells including those more obviously lacking the CD38 antigen are positive for a4 integrin. In CD34/ cells isolated from three peripheral blood stem cell harvests, a mean of 1.04% { 0.35% of cells were negative for the CD38 antigen and the majority of this minor subpopulation (84%) also expressed a4 integrin. Adhesion of CD34 cells to a4b1 binding sequences of bronectin; effect of growth factors. Cord blood CD34 cells suspended in serum-free medium attached efciently to the
H120 fragment used at concentrations from 5 to 15 mg/mL, and at the end of a 30-minute incubation at 37C, a mean of 65% of cells were adherent at the highest concentration of H120 (Fig 2A). When whole bronectin (50 mg/mL) was used as a ligand, the cells attached less efciently (mean of 21% adherent cells) compared with the H120 fragment in the same experiment (mean of 70% adherent cells). Cells also attached less efciently to the H0 fragment. Coating concentrations of 5 and 10 mg/mL of H0 were not signicantly different from the BSA control (14% attached cells) and at 15 and 30 mg/mL, only 24% and 25% of cells were adherent (Fig 2A). The specicity of the interaction between a4b1 integrins and the H120 fragment was conrmed by almost complete inhibition of adhesion of CD34 cells that had been preincubated with blocking anti-a4 and anti-b1 antibodies, whereas adhesion in the presence of control antibodies was only marginally inhibited (Fig 2B). CD34 cells are, therefore, able to attach via the a4b1 to the H120 fragment in the absence of growth factors in serumfree medium in this short-term 30-minute assay. We then went on to determine the effects of growth factors on cell adhesion. In the initial experiments, CD34 cells from peripheral blood were used and the cells were incubated with the growth factor(s) for 30 minutes before starting the adhesion assay. Adhesion in the presence of the combination of IL-3 (10 ng/mL), SCF (100 ng/mL), G-CSF (500 ng/mL), and IL-6 (200 U/mL) was reduced by a mean of 26% on the H120 fragment and by 31% on the HO fragment. Cells showed no attachment to the BSA control (Fig 2C). In a second series of experiments CD34 cells from peripheral blood were used and their adhesion to bronectin fragments was measured in the presence of single growth factors or a growth factor combination. It was clear that IL-3 (10 ng/mL) was the only growth factor that signicantly reduced cell adhesion (reduction 48% P .01), and this effect was not masked when SCF (100 ng/mL) or G-CSF (500 ng/ mL) was added to the cells together with IL-3 (Fig 2D). At a high concentration (500 ng/mL), SCF increased the number of adherent cells by a mean of 16% (P .05). No signicant changes were observed with G-CSF (Fig 2D). IL-3 alone was also found to strongly inhibit attachment to H120 of CD34 cells from cord blood (63% to 73% reduction, Table 1) and concentrations of IL-3 as low as 2.5 ng/mL produced a mean reduction in adhesion of 25% (not shown). Overall, in six separate experiments with CD34 cells from cord blood or peripheral blood, cell adhesion was consistently and signicantly reduced by IL-3 (P .001; Table 1). In one experiment with cord blood cells when the period of adhesion was reduced to 5 minutes, a mean of 25% of CD34 cells attached to H120 and the diminished response with IL-3 was already discernible, although statistically insignicant. In the same experiment, adhesion increased to a mean of 60% at 30 minutes without growth factors and was reduced by IL-3 to a mean of 42% (not shown). Cells from the murine hematopoietic progenitor cell line FDCP-mix gave a similar value to CD34 cells for adhesion to H120 in serum-free medium after 30 minutes (mean of 67% attached cells) and showed a similar reduction in adhesion with IL-3 (mean of 17% attached cells).

EFFECT OF IL-3 ON a4b1 ADHESION 1861
Fig 2. (A) Adhesion of CD34 cells isolated from mini-Macs to BSA and H0 and H120 fragments coated at different concentrations. All adhesion experiments were for 30 minutes at 37C. All histograms in all gures for adhesion and migration represent the mean of duplicate wells SD. Students t-test was used to calculate P values. (B) Adhesion of CD34 cells to H120 fragment coated at 15 mg/mL showing inhibition by blocking anti-a4 and anti-b1 antibodies, but not by IgG control. (C) Adhesion of CD34 cells to BSA and H0 and H120 fragments coated at 15 mg/mL with and without a mixture of growth factors; IL-3 (10 ng/mL), SCF (100 ng/mL), G-CSF (500 ng/mL), and IL-6 (200 U/mL). Reduction in adhesion to H120 induced by growth factors was signicant (P .05). (D) Adhesion of CD34 cells to H120 fragment coated at 15 mg/mL with IL-ng/mL, SCF 100 ng/mL, and G-CSF 500 ng/mL added together and separately at different concentrations. Changes in adhesion were signicant for all growth factors (P .005), IL-ng/mL (P .01), IL-ng/mL (P .005), and SCF (P .05).
Migration of CD34 cells on a4b1 binding sequences; effect of growth factors. The different abilities of growth factors to modulate adhesion of CD34 cells to bronectin fragments prompted us to examine their effect on cell migration through 5-mm Transwell lters coated with either the H120 or H0 fragments. Cells were FACS-sorted for the CD34 antigen following their initial isolation with the mini-Macs column
so that all cells were CD34/ (Fig 3). In the rst experiments, cells from peripheral blood were used and in the absence of growth factors, little or no cell migration was observed (Fig 4A). However, addition of a combination of growth factors resulted in a marked stimulation of migration through lters coated with the H120 fragment, which was approximately vefold that occurring through lters coated with BSA

1862 SCHOFIELD ET AL

Table 1. Loss of Adhesion of CD34 Cells to H120 Due to IL-3
% Adherent Cells in Serum-Free Medium % Adherent Cells With IL-3 % Reduction in Adhesion With IL-3

CB CB CB PB PB PB

92.1 68.4 83.7 62.8 65.4 71.6
24.8 22.7 30.4 32.8 41.6 43.7
73 66.8 63.6 47.7 36.4 38.9
CD34 cells isolated from individual CB, and PB samples were preincubated in the presence or absence of IL-ng/mL for 30 minutes before attaching to the H120 fragment. IL-3 resulted in consistent loss of adhesion (P .001). Abbreviations: CB, cord blood; PB, peripheral blood.
(P .01) (Fig 4A). In a separate experiment when growth factor-stimulated migration on BSA, H120, and H0 fragments was compared, again it was clear that H120 facilitated a growth factor induced migration to a much greater extent than either BSA (P .001) and the H0 fragment, the latter supporting only 28% of the migration that occurred on H120 (P .001) (Fig 4B). This is consistent with a predominant role for the stronger a4b1 binding sequences of H120 in promoting the migratory phenotype. The effect of IL-3, G-CSF, and SCF used separately and in combination on the migration of peripheral blood CD34 cells on the H120 fragment is shown in Fig 5A. All growth factors stimulated cell migration, but IL-3 was clearly the most effective single growth factor, giving a threefold increase over SCF or G-CSF. Combinations of other growth factors with IL-3 did not provide an additional signicant stimulus over that seen with IL-3 alone (Fig 5A). When different concentrations of growth factors were used, IL-3 was found to be active at 1 ng/mL, and its effects at this low concentration were greater than SCF at 100 ng/mL and G-CSF at 1,000 ng/mL (Fig 5B). Qualitatively similar effects to those shown in Fig 5A and B were seen in CD34 cells from fresh cord blood, although they showed a greatly increased migratory capacity on both H120 and H0 fragments (at least

10 times greater) compared with those from peripheral blood and included a minority of cells that could migrate on H120 in the absence of growth factors, which the peripheral blood cells were unable to do (Fig 5C). Blocking antibodies to a4 and b1 , added to cells at the start of a 4-hour migration assay, reduced the migration due to IL-3 by a mean of 74% (P .001) when both antibodies were present (Fig 5D). After 16 hours, virtually no effect of the antibodies was apparent. To address this nding, the CD34 cell adhesion assay was used to test the inhibitory activity of fresh antibodies (blocking a4 and b1 as previously described) and the same antibodies left for 16 hours at 37C. Adhesion to H120 was reduced by a mean of 88% with fresh antibodies, but only by a mean of 17% with the antibodies left for 16 hours. The loss of antibody activity during prolonged incubation would therefore account for the loss of inhibition of migration. The expression of VLA-4 on CD34 cells remained unchanged when analyzed by FACS using anti-VLA4/PE immediately after enrichment (83% CD34/ VLA-4/, mean uorescence of VLA-4 109) compared with cells left overnight at 37C in serum-free medium (87% CD34/ VLA-4/, mean uorescence of VLA-4 107). The possibility that the effect of IL-3 is due to maintenance of cell viability rather than through a direct action on migration was excluded by conrming viability of all cells by trypan blue exclusion after 16 hours in serum-free medium without IL-3. Furthermore, after incubation of cells overnight in serum-free medium at 37C followed by addition of IL-3 the next day, the cells were still able to migrate with the same efciency (data not shown). Finally, there was no difference in cell numbers when cells were counted before and after incubation for 16 hours in serum-free medium containing IL-3, thereby excluding proliferation as the cause of the increased numbers of cells in the lower chamber in the presence of IL-3.

DISCUSSION

Cell adhesion and migration are essential processes in the control of growth and differentiation of hematopoietic stem
Fig 3. FACS sorting of CD34 cells (collected from R2 gate) for migration experiments.
EFFECT OF IL-3 ON a4b1 ADHESION 1863
Fig 4. (A) Migration of CD34 cells from a peripheral blood stem cell harvest over 16 hours at 37C through lters coated with Hmg/mL and BSA with and without a mixture of SCF 100 ng/mL, IL-ng/mL, G-CSF 500 ng/mL, and IL-U/mL. Migration was increased with H120 compared with BSA (P !.01) in the presence of growth factors. (B) Migration of CD34 cells over 16 hours through lters coated with BSA, H120, and H0 at 15 mg/mL with the mixture of growth factors used in (A). Migration was increased with H120 compared with BSA (P .001) and H0 (P .001).

and progenitor cells, and the increasing use of peripheral blood stem cells for transplantation emphasizes the potential practical benets that may accrue from a deeper understanding of these fundamental cellular properties. The presence of integrins on the surface of hematopoietic cells and their signicance in controlling cell attachment to the extracellular matrix has been highlighted in a number of publications,7-11 but to our knowledge, the present study is the rst to examine the migration of CD34 hematopoietic progenitor cells in vitro on a single extracellular matrix ligand in response to growth factors. In one recent study CD34 cells were shown to migrate between endothelial cells grown on top of
Transwell inserts, but the role of individual growth factors was not addressed.27 In a separate study, SCF and other growth factors were shown to have chemotactic and chemokinetic activity for murine hematopoietic progenitor cells, but this study did not incorporate a ligand such as bronectin on which integrin-mediated adhesion and migration could occur.28 We have focused on the role of the strongly expressed a4b1 integrin on CD34 cells (Fig 1) using as substrate recombinant bronectin fragments (H120 and H0) that bind only to this integrin and not to the a5b1 integrin, which is also expressed by these cells and binds to the RGD sequence in the central cell-binding domain of bronectin. Fibronectin is a component of the marrow extracellular matrix,29 and we have found (unpublished data) that the alternatively spliced high-afnity CS1 sequence (present in H120) is expressed by human marrow stromal cells. Results shown in Fig 2 clearly demonstrate that CD34 cells from peripheral blood adhere to both H120 and H0 fragments and the use of specic antibodies indicated that attachment was mediated by the a4b1 integrin (Fig 2B). The H120 fragment, which contains the high-afnity LDV sequence, was the superior adhesive substrate enabling 65% of cells to attach in serum-free medium by comparison with only 24% attachment on H0 (Fig 2A). The combination of IL-3 with SCF and G-CSF caused a signicant reduction in adhesion to H120 and the addition of single growth factors indicated that the inhibition was due to the action of IL-3 (Fig 2D). The effect was quite rapid, as the cells were preincubated at room temperature for only 30 minutes with IL-3 before the 30-minute adhesion assay. Previous studies on adhesion of hematopoietic progenitor cells derived from bone marrow rather than peripheral blood or cord blood have suggested that cell surface integrins are activated by cytokines.30-32 In two of these studies,30,31 IL-3 was one of the cytokines that increased adhesion of CD34 cells to whole bronectin. However, the low levels of initial adhesion in the absence of growth factors are difcult to compare with our results using H120 and our nding that whole bronectin also gave low cell attachment values implies that adhesion to H120 is inherently stronger. Similar results obtained with FDCP-mix cells on H120 argue against the possibility of integrin activation by the CD34 cell-enrichment procedure, as does the reduced attachment of CD34 cells shown at 5 minutes. In addition, the cells in these studies were held at 4C overnight in serum-free medium to ensure that they were completely free of any growth factors, whereas the procedures involved in cell preparation used in our work took about 4 hours and were performed at room temperature; cells were extensively washed during this time and were unlikely to have any signicant contamination by growth factors. We have not yet looked at CD34 cells isolated directly from bone marrow, but we think it is unlikely that they would show qualitatively different responses. We considered that because IL-3 affected cell adhesion in a distinctive manner from other growth factors, it may be inuencing cell migration. A Transwell lter assay was established in which cells could migrate through 5-mm pore lters coated on both sides with 15 mg/mL of bronectin fragments. Results indicated that the H120 fragment could

1864 SCHOFIELD ET AL

Fig 5. (A) Migration of CD34 cells over 16 hours at 37C through lters coated with H120 at 15 mg/mL with IL-ng/mL, SCF 100 ng/mL, and G-CSF 500 ng/mL separately and in combination. IL-3 alone stimulated more migration than SCF (P !.01) and G-CSF (P !.01) alone. (B) Migration of CD34 cells over 16 hours at 37C through lters coated with H120 at 15 mg/mL with different concentrations of SCF, IL-3, and GCSF. A total of 1 ng/mL IL-3 stimulated more migration than 10 ng/mL SCF (P .05). (C) Migration of CD34 cells from cord blood over 16 hours at 37C through lters coated with BSA and H120 and H0 coated at 15 mg/mL with and without IL-ng/mL. Migration on H120 is increased with IL-3 (P .005). (D) Migration of CD34 cells from cord blood over 4 hours at 37C through lters coated with H120 at 15 mg/mL with IL-ng/mL. Effect of blocking anti-a4 and anti-b1 antibodies on IL-3 stimulated migration. Signicant reduction occurred with anti-a4 (P .01), anti-b1 (P !.01), and the combination (P !.001).
not support migration of peripheral blood CD34 cells in the absence of growth factors (Fig 4A); cord blood CD34 cells were also nonmigratory on H0, but a small level of migration was observed on H120 (Fig 5C). Addition of a combination of growth factors provided a strong stimulus for both peripheral blood and cord blood CD34 cells to migrate on H120 and to a lesser degree on H0 (Figs 4B and 5C). When single growth factors were tested, IL-3 was clearly the most effec-
tive at inducing migration, giving an increase of approximately threefold over SCF or G-CSF (Fig 5A and B). IL-3 was effective at a concentration of 1 ng/mL with maximum activity occurring at 10 ng/mL (Fig 5B). Cells from cord blood were more responsive in the migration assay than those from peripheral blood, but in both cell populations, IL-3 was signicantly more active than SCF and G-CSF. Migration in a short-term assay (4 hours) was strongly inhib-
EFFECT OF IL-3 ON a4b1 ADHESION 1865
ited by combined use of antibodies to the a4 and b1 subunits indicating the importance of a4b1 integrin for migratory activity (Fig 5D). The mechanism of IL-3induced modulation of integrin function is unclear, but it would appear to be an example of inside-out signaling in which the IL-3 receptor elicits an intracellular signal (or signals) that modies a4b1 integrin binding to its H120 (or H0) substrate. This signal is distinct in some way from signals resulting from activation of the SCF or G-CSF receptors. The IL-3 signal affects both cell adhesion, which is reduced, and cell migration, which is enhanced. We do not know if the activation state of a4b1 integrin is affected during the course of the adhesion and migration assays, although surface expression is unchanged over a period of 16 hours. Reduction in adhesion may be a necessary rst step in weakening cell attachment to the substratum leading to increased motility and migration. However, the relationship between adhesion and migration is complex. Adhesion allows cells to generate the traction essential for movement, but adhesion complexes must rapidly dissociate and reform if motility is to be maintained. Several recent reports have shown a link between integrin-mediated migration and growth factor pathways, eg, carcinoma cells expressing avb5 require prior cellular activation with growth factors for migration on vitronectin33 and CS1-melanoma cells expressing avb5 or a recombinant avb5 /b3 only migrate when exposed to insulin-like growth factor or insulin.4 A candidate signaling molecule for mediating the IL-3 effects in CD34 cells is the rac guanosine triphosphate (GTP)-binding protein, which induces lamellipodia and may play a role in migration of broblasts and can be activated by growth factors, eg, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF).34 The capacity of IL-3 to reduce adhesion and promote migration of CD34 cells indicates a possible role for this cytokine in promoting movement of hematopoietic progenitor cells in vivo. However, despite the many effects of IL3 on hematopoiesis, including stimulation of the survival and proliferation of multipotent stem and progenitor cells, its normal physiologic role is unclear. Unlike other hematopoietic cell growth factors, such as SCF, GM-CSF, G-CSF, M-CSF, and IL-6, which are all produced by marrow stromal cells, IL-3 is produced by T lymphocytes.35-37 Although some T cells are found in the bone marrow, IL-3 is difcult to detect except by RT-PCR techniques.36 IL-3 has been shown in a mouse model to cause migration of both pluripotent and committed progenitors from bone marrow to spleen,38 and it has chemotactic and chemokinetic activity for murine hematopoietic progenitor cells, which is similar to SCF.28 The mobilization of peripheral blood progenitor cells by G-CSF is enhanced by IL-3,23 as is the in vitro chemotaxis of CD34 cells in response to the chemokine SDF-1.39 Thus IL-3, either directly or indirectly, can clearly inuence the local positioning of cells in the marrow stroma and also facilitate their migration. It is interesting to note, however, that although G-CSF is a weak activator of migration in our in vitro assay, it is much more potent than IL-3 in mobilizing hematopoietic progenitor cells from the marrow to the blood.24 This may

indicate that mobilization of peripheral blood stem cells is a reection not only of migration, but also of changes in adhesive properties of marrow stromal endothelial cells, which in turn may promote access of IL-3 bearing T lymphocytes.13 In summary, our ndings show that by modulating the function of a4b1 integrin, IL-3 is a potent regulator of the adhesion and migration of CD34 cells. Its effects can be clearly distinguished from those of SCF and G-CSF. The mechanisms that support the effects of IL-3 are unknown, but it seems probable that there is a distinctive signal from the IL-3 receptor to the a4b1 integrin. The nature of this signal and the manner in which it regulates integrin function are now being investigated.

ACKNOWLEDGMENT

We thank Nydia Testa and Erica de Wynter for helpful technical advice and discussion of data. We also acknowledge the excellent assistance of Suzanne Bridge in the preparation of this manuscript.

REFERENCES

1. Dexter TM, Allen TC, Lajtha LG: Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 91:335, 1977 2. Verfaille CM: Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood 82:2045, 1993 3. Clarke EA, Brugge JS: Integrins and signal transduction pathways: The road taken. Science 268:233, 1995 4. Filardo EJ, Deming SL, Cheresh DA: Regulation of cell migration by the integrin b subunit ectodomain. J Cell Sci 109:1615, 1996 5. Hynes RO: Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69:11, 1992 6. Roberts R, Gallagher JT, Spooncer E, Allen TD, Bloomeld F, Dexter TM: Heparan sulphate bound growth factors: A mechanism for stromal cell mediated haemopoiesis. Nature 332:376, 1988 7. Teixido J, Hemler ME, Greenberger JS, Anklesaria P: Role of b1 and b2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma. J Clin Invest 90:358, 1992 8. Kerst JM, Sanders JB, Slaper-Cortenbach ICM, Doorakkers M Ch, Hooibrink B, van Oers RHJ, von dem Borne AEG kr, van der Schoot CE: a4b1 and a5b are differentially expressed during myelopoiesis and mediate the adherence of human CD34/ cells to bronectin in an activation-dependent way. Blood 81:344, 1993 9. Simmons PJ, Masinovsky B, Longenecker BM, Berenson R, Torok-Storb B, Gallatin WM: Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 80:388, 1992 10. Verfaille CM, McCarthy JB, McGlave PB: Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors is accompanied by alterations in their interaction with bronectin. J Exp Med 174:693, 1991 11. Williams DA, Rios M, Stephens C, Patel VP: Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 352:438, 1991 12. Miyake K, Weissman I, Greenberger JS, Kincade PW: Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med 173:599, 1991 13. Chan P, Aruffo A: VLA-4 integrin mediates lymphocyte migration on the inducible endothelial cell ligand VCAM-1 and the extracellular matrix ligand bronectin. J Biol Chem 268:24655, 1993 14. Papayannopoulou T, Nakamoto B: Peripheralization of hemo-

1866 SCHOFIELD ET AL

poietic progenitors in primates treated with anti-VLA-4 integrin. Proc Natl Acad Sci USA 90:9374, 1993 15. Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R: Impaired migration but not differentiation of haematopoietic stem cells in the absence of b1 integrins. Nature 380:171, 1996 16. Chan BMC, Kassner PD, Schiro JA, Byers HR, Kopper TS, Hemler ME: Distinct cellular functions mediated by different VLA integrin a subunit cytoplasmic domains. Cell 68:1051, 1992 17. Humphries MJ, Akiyama SK, Komoriya A, Olden K, Yamada KM: Identication of an alternatively spliced site in human plasma bronectin that mediates cell type-specic adhesion. J Cell Biol 103:2637, 1986 18. Humphries MJ, Komoriya A, Akiyama SK, Olden K, Yamada KM: Identication of two distinct regions of the type III connecting segment of human plasma bronectin that promote cell type-specic adhesion. J Biol Chem 262:6886, 1987 19. Mould PA, Komoriya A, Yamada KM, Humphries MJ: The CS5 peptide is a second site in the IIICS region of bronectin recognised by the integrin a4b1. J Biol Chem 266:3579, 1991 20. Mould P, Humphries MJ: Identication of a novel recognition sequence for the integrin a4b1 in the COOH-terminal heparin-binding domain of bronectin. EMBO J 10:4089, 1991 21. Huang S, Terstappen LWMM: Lymphoid and myeloid differentiation of single human CD34/, HLA-DR/, CD380 haematopoietic stem cells. Blood 83:1515, 1994 22. Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM: A functional comparison of CD34/ CD380 cells in cord blood and bone marrow. Blood 86:3745, 1995 23. Huhn RD, Yurkow EJ, Tushinski R, Clarke L, Sturgill MG, Hoffman R, Sheay W, Cody R, Philipp C, Resta D, George M: Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rh G-CSF) in normal volunteers. Exp Hematol 24:839, 1996 24. Henon PR, Becker M: Cytokine enhancement of peripheral blood stem cells. Stem Cells 11:65, 1993 25. Weaver A, Ryder D, Crowther D, Dexter TM, Testa NG: Increased numbers of long-term culture-initiating cells in the apheresis product of patients randomized to receive increasing doses of stem cell factor administered in combination with chemotherapy and a standard dose of granulocyte colony-stimulating factor. Blood 88:3323, 1996 26. Mould PA, Askari JA, Craig SE, Garratt AN, Clements J, Humphries MJ: Integrin a4b1 -mediated melanoma cell adhesion and migration on vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced IIICS region of bronectin. J Biol Chem 269:27224, 1994 27. Mohle R, Moore MAS, Nachman RL, Rai S: Transendothe

lial migration of CD34/ and mature hematopoietic cells: An in vitro study using a human bone marrow endothelial cell line. Blood 89:72, 1997 28. Okumura N, Tsuji K, Ebihara Y, Tanaka I, Sawai N, Koike K, Komiyama A, Nakahata T: Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells. Blood 87:4100, 1996 29. Weiss R, Reddi A: Appearance of bronectin during the differentiation of cartilage, bone and bone marrow. J Cell Biol 88:630, 1981 30. Levesque JP, Leavesley DI, Niutta S, Vadas M, Simmons PJ: Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins. J Exp Med 181:1805, 1995 31. Levesque JP, Haylock DN, Simmons PJ: Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34/ hemopoietic progenitors. Blood 88:1168, 1996 32. Hardy CL, Minguell JJ: Modulation of the adhesion of hemopoietic progenitor cells to the RGD site of bronectin by interleukin 3. J Cell Physiol 164:315, 1995 33. Klemke RL, Yebra M, Bayna E, Cheresh DA: Receptor tyrosine kinase signalling required for integrin avb5 -directed cell motility but not adhesion on vitronectin. J Cell Biol 127:859, 1994 34. Nobes, CD, Hawkins P, Stephens L, Hall A: Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J Cell Sci 108:225, 1995 35. Kittler EL, McGrath H, Temeles D, Crittenden RB, Kister VK, Quesenberry PJ: Biological signicance of constitutive and subliminal growth factor production by bone marrow stroma. Blood 79:3168, 1992 36. Gibson FM, Scopes J, Daly S, Rizzo S, Ball SE, GordonSmith EC: IL-3 is produced by normal stroma in long-term bone marrow cultures. Br J Haematol 90:518, 1995 37. Eaves CJ, Cashman JD, Kay RJ, Dougherty J, Otsuka T, Gaboury A, Hogge DE, Lansdorp PM, Eaves AC, Humphries RK: Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 78:110, 1991 38. Lord BI, Molineux G, Testa NG, Kelly M, Spooncer E, Dexter TM: The kinetic response of haemopoietic precursor cells, in vivo, to highly puried, recombinant interleukin-3. Lymphokine Res 5:97, 1986 39. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC: The chemokine SDF-1 is a chemoattractant for human CD34/ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34/ progenitors to peripheral blood. J Exp Med 185:111, 1997

From bloodjournal.hematologylibrary.org by guest on June 8, 2011. For personal use only.

1998 91: 3230-3238

The Effect of 41-Integrin Binding Sequences of Fibronectin on Growth of Cells From Human Hematopoietic Progenitors
Karen P. Schofield, Martin J. Humphries, Erika de Wynter, Nydia Testa and John T. Gallagher
Updated information and services can be found at: http://bloodjournal.hematologylibrary.org/content/91/9/3230.full.html Articles on similar topics can be found in the following Blood collections Hematopoiesis and Stem Cells (2845 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
The Effect of -Integrin Binding Sequences of Fibronectin on Growth of Cells From Human Hematopoietic Progenitors
By Karen P. Schofield, Martin J. Humphries, Erika de Wynter, Nydia Testa, and John T. Gallagher
Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between 41integrinmediated adhesion and growth of CD34 cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/ IIICS region of fibronectin. CD34 cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three 41-integrin binding sites, or the H0 fragment, which lacks the two highest affinity 41 binding sequences. Proliferation of single cells of CD3438DR and CD3438DR phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (PI.0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P I.001). The increase in total cell numbers was not at the expense of CD34 cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma. 1998 by The American Society of Hematology.
EMATOPOIETIC CELL development occurs within the bone marrow stroma where interactions between progenitor cells and their extracellular matrix (ECM) ligands are recognized to be of importance not only for normal differentiation and proliferation but also for maintenance of the hematopoietic stem cell.1 It is likely that highly regulated interactions between adhesion receptors on the progenitor cells and their ECM ligands play key roles in these processes. Foremost among adhesion receptors are the integrins that control cell attachment to specic matrix proteins and that can mediate transfer of information to the intracellular compartment. Recent ndings linking signaling pathways from integrins and growth factor receptors suggest that cooperation between these pathways is essential for normal cell development.2,3 Most cells require attachment to a substrate to enter the cell cycle and grow normally. For example, in broblasts cell anchorage is essential for cyclin E-CDK2 kinase activity,4 and the synthesis of cyclin A is enhanced by adhesion-dependent signals.5 Additional links between growth and adhesion in nonhematopoietic cells include an association between insulinmediated pathways and the v3-integrin receptor, which results in a 2.5-fold increase in DNA synthesis when cells are plated on vitronectin6 and between integrin-mediated signal transduction and the Ras pathway that is likely to impact on cell

From the Departments of Medical Oncology and Experimental Haematology, Paterson Institute for Cancer Research; and the Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK. Submitted August 11, 1997; accepted December 18, 1997. K.P.S. is a clinical research fellow of the Cancer Research Campaign. Address reprint requests to Karen P. Schoeld, MD, CRC Department of Medical Oncology, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 1998 by The American Society of Hematology. 0006-4971/98/9109-0029$3.00/0
growth.7 Although hematopoietic cells will grow in culture without adherence, a link between adhesion and growth has been shown in T cells where costimulation of the T-cell receptor and the 2-integrin receptor lymphocyte function antigen-1 (LFA-1) causes a synergistic enhancement of T-cell proliferation,8 and in the CD4 subset of T cells the interaction of 41 and 51 integrins with bronectin facilitates CD3-mediated cell growth.9,10 Fibronectin has also been shown to promote a twofold enhancement of proliferation of erythroid colonyforming units (CFU-Es) and erythroid burst-forming units (BFU-Es) and of granulocyte, erythroid, monocyte, megakaryocyte colony-forming unit (CFU-GEMM)derived colonies from human bone marrow11 and to potentiate the effect of interleukin-3 (IL-3) on the growth of CFU-GEMM, BFU-E, CFUE, and macrophage colony-forming unit (CFU-M)derived colonies from CD34 cells, an effect that was reversed by blocking the bronectin 51-integrin interaction with RGDcontaining peptides.12 More recently it has been reported that cytokines that stimulate the proliferation of CD34 cells enhance the adhesion of these cells to a bronectin substrate.13 However, adhesion of hematopoietic progenitors to stroma is associated with inhibition of proliferation that occurs through bronectin receptors.14 The integrins 41 (very late activation antigen-4 [VLA-4]) and 51 (VLA-5), which are present on CD34 cells, mediate adhesion to different domains of bronectin.15,16 Long-term culture-initiating cells and CFU-mix progenitors adhere to the bronectin COOH-terminal heparin-binding domain,17 and primitive murine spleen colony-forming unit (CFU-S) day-12 colony-forming cells adhere to the 41 binding sequence in bronectin.18 We have recently shown that IL-3 modulates 41-integrin function on CD34 cells, resulting in a reduction in cell adhesion to surfaces coated with the HepII/IIICS domain of bronectin (the specic ligand for 41 integrin) and an increase in cell migration on the same substrate.19 The importance of 41 integrins in hematopoiesis is further highlighted by a study that showed that the addition of anti-41 antibodies to long-term bone marrow cultures abrogated the production of lymphoid cells and retarded myelopoiesis20 and by the nding

Blood, Vol 91, No 9 (May 1), 1998: pp 3230-3238
EFFECT OF 41 ON PROGENITOR CELL GROWTH
that antibodies to 41 selectively mobilized hematopoietic progenitors into the blood.21 In the present study we set out to explore the adhesive and migratory patterns of growth of CD34 progenitor cells isolated from cord blood by assessing their proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant proteins of the IIICS region of bronectin. The 41 integrin binds to two main sites in the alternatively spliced IIICS region that are represented by the CS1 sequence, the highest affinity 41 binding site, and the CS5 sequence, which binds more weakly.22-24 The lowest affinity 41 binding site resides in the adjacent HepII domain and is represented by the peptide sequence designated H1.25 Minimal active peptide sequences have been dened for the three sites: LDV for CS1, REDV for CS5, and IDAPS for H1. We used two different recombinant proteins from the IIICS region as ligands for 41: H120, which contains all three binding sites, and HO, which contains H1 alone thus enabling assessment of the effect of strong and weak 41 binding activity on adhesion and proliferation. We initially assessed adhesive and migratory patterns of growth with H120 and H0 for two different subsets of CD34 cells representing different stages of maturity, CD3438DR (more mature) and CD3438DR (less mature), which were isolated as single cells.26-28 For comparison of proliferative responses, larger numbers of CD34 cells were expanded in wells coated with the H120 and H0 fragments.
MATERIALS AND METHODS Cloning and expression of recombinant HepII/IIICS variants. cDNA clones for the ve different variants of the HepII /IIICS region of human bronectin were synthesised using reverse transcription polymerase chain reaction (RT-PCR) amplication of primary human skin broblast mRNA as described by Mould et al.29 The PCR cloning strategy and the resulting recombinant proteins are shown in Fig 1. The PCR products were ligated into pUC119 and transformed into Escherichia coli JM109.
Fig 1. Diagram of recombinant proteins containing the five different splice variants of the HepII/IIICS region obtained by RT-PCR expression cloning. The 3 primer was complementary to the end of the 15th type III repeat and the 5 primer to the start of the 12th type III repeat of human fibronectin. The full-length fibronectin subunit is shown together with the alternatively spliced IIICS region, which is represented as the open box. The numbers assigned to the H variants refer to the number of amino acids in the IIICS. The locations of the three recognition sequences for 41, HI, CSI, and CS5, are indicated. Note that H120 contains all three sites and H0 contains HI alone.

Individual clones containing each of the variants were identied by restriction analysis and sequenced. Inserts were subcloned into the EcoRI site of the pGEX-2T expression vector and used to transform E coli strain BLR. Recombinant clones were expressed as glutathione-Stransferase fusion proteins. These were isolated by glutathione affinity chromatography, thrombin digestion, and heparin-agarose affinity chromatography. The H120 (containing H1, CS1, and CS5 sequences) and HO (containing the H1 sequence only) variants were used to coat the wells in CD34 expansion experiments. Enzyme-linked immunosorbent assay (ELISA) experiments using a monoclonal antibody to the HepII domain showed that each of the variants bound equally well to microtiter plates used for adhesion assays.29 Isolation and analysis of CD34 cells. Umbilical cord blood collected into heparin was layered over Ficoll-Hypaque (Lymphoprep; Nycomed, Birmingham, UK), and the interface mononuclear cells were collected following density gradient centrifugation. CD34 cells were isolated using a CD34 antibody (QBEND/10) and a secondary antimouse antibody conjugated to magnetic beads according to the manufacturers instructions (mini-MACS CD34 isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). For maximum purity the isolated cells were also sorted on a uorescence-activated cell sorter (FACS) ow cytometer (FACS Vantage ow cytometer; Becton Dickinson, San Jose, CA) using a second uorochrome-conjugated (uorescein isothiocyanate [FITC] or phycoerythrin [PE]) CD34 antibody (antiHPCA-2) and an isotype-matched IgG control (Becton Dickinson). For three-color FACS analysis and single-cell sorting, a CD38 antibody conjugated to PE (Becton Dickinson) and an HLA-DR antibody conjugated to tricolor (CALTAG; San Francisco, CA) were added to the cells with the CD34 FITC-conjugated antibody, and control cells were labeled with the corresponding uorochrome-conjugated isotype-matched IgGs. The cells were washed twice with phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA) before sorting. CD34 cells were subdivided into CD3438DR and CD34CD38DR populations after rst gating by size on the lymphocyte population and by uorescence (f11) on the CD34 cell population (R2) (Fig 2A). The two additional uorescence parameters, 2 (CD38-PE) and 3 (HLA-DR tricolor), were used to analyze subpopulations of CD34 cells that were CD38DR and CD38DR (Fig 2B).28 Single cells of each of the two subtypes were deposited into single wells containing serum-free medium (X-vivo 10; Bio-Whittaker, UK) and growth factors. Migration of CD3438DR and CD3438DR cells within U-shaped wells: Effect of 41 binding sequences. Single cells of CD3438DR and CD3438DR phenotype were deposited by the automated cell deposition unit (ACDU) of the FACS Vantage cell sorter into individual U-bottomed wells of a 96-well plate (Falcon, Oxford, UK) that contained 100-L aliquots of serum-free medium and that had been precoated with the bronectin fragments H120 and H0 at 15 g/mL (3.75 g/cm2) as described later. A combination of growth factors was added to the cultures: recombinant human IL-3 (rhIL-3; Sandoz, Basel, Switzerland) at 10 ng/mL, rhIL-6 (Sandoz) at 200 U/mL, rh granulocyte colony-stimulating factor (G-CSF; Amgen, Thousand Oaks, CA) at 500 ng/mL, rh stem cell factor (SCF; Amgen) at 100 ng/mL, and rh erythropoietin at 2 U/mL (Boehringer, Mannheim, Germany). These growth factor concentrations were established by determining plateau levels of growth in colony assays in our laboratory. The cells were incubated at 37C in a humidied atmosphere of 5% CO2 and 5% O2 in air, and growth and migratory patterns were assessed after 1 week and 2 weeks. Expansion of CD34 cells in liquid culture: Effect of 41 binding sequences. Twenty-fourwell plates (Costar, Cambridge, MA) were coated for 60 minutes at room temperature with 1 mL aliquots of recombinant H120 or H0 proteins diluted with Dulbeccos PBS to a concentration of 15 g/mL (7.5 g/cm2). Nonspecic binding sites were blocked for 30 minutes at room temperature with 1 mL of 10 mg/mL heat-denatured BSA. Control plates were prepared with BSA only or

SCHOFIELD ET AL

Fig 2. (A) Initial gating of CD34 cells by fluorescence (fl1) shown as R2. (B) Two additional fluorescence parameters, fl2 (CD38-PE) and fl3 (HLA-DR-tricolor), were used to analyze subpopulations of CD34 cells that were CD38DR and CD38DR.
left uncoated. A total of 103 to 2.puried CD34 cells were added to individual wells in serum-free medium together with the standard concentration of growth factors described previously. Cells were incubated at 37C in a humidied atmosphere of 5% CO2 and 5% O2 in air for varying lengths of time. Viable cells were counted with a hematocytometer after staining with Trypan blue. Where appropriate, adherent and nonadherent fractions were separated for counting; nonadherent cells were removed by shaking the wells and washing twice with 1 mL PBS, and bound cells were removed by aspiration with PBS. Cell morphology was assessed by staining with May-Grunwald Giemsa after cytocentrifugation. RESULTS
Adhesion and migration patterns of single CD3438DR and CD3438DR cells within U-shaped wells: Effect of 41 binding sequences. The initial purpose of the study was to assess the pattern of cell adhesion and migration with different coating conditions using single cells of dened phenotype isolated by FACS (Fig 2). Microscopic examination revealed that single cells of CD3438DR phenotype started to divide in most wells after 2 or 3 days, and a proportion of these continued to proliferate to reach maximum growth at 2 to 3 weeks without a change in medium. Cells of the more immature CD3438DR phenotype took longer to divide, in some cases up to 2 weeks. Deposition of single cells into a U-shaped well enabled individual cells to be followed as they divided in the presence or absence of the H120 or H0 bronectin fragments.
Cell division in wells coated with H0 or BSA or in uncoated wells took place at the base of the well where cells settled by gravity (Fig 3A), whereas cells dividing in wells coated with the H120 fragment could be seen to have migrated around the base and up the sides of the well (Fig 3B). This pattern of single-cell migration and growth could easily be observed from the initial cell divisions, and as proliferation increased growth took place in focal sites around the well (Fig 3C). In the H0, BSA or uncoated-well proliferation continued to occur at the base of the well. Both CD3438DR and CD3438DR phenotypes showed the same migratory pattern of behavior in the presence of H120 from the earliest cell divisions. Figure 4 shows the mean number of wells in which a migratory pattern was seen for the two phenotypes with different coating conditions at weeks 1 and 2. Migration was signicantly increased in H120 wells compared with control wells. The mean number of wells in which migratory growth on H120 occurred from cells of 3438DR phenotype did not change between weeks 1 and 2. Migration and growth occurred less frequently in the more immature 3438DR cells after 1 week when overall growth was minimal but had increased by week 2 (Fig 4). The proliferation efficiency of single cells (ie, number of wells in which growth occurred) did not vary signicantly with coating conditions. Wells in which migration did not occur with H120 were usually those in which proliferation did not subsequently increase beyond a few cells. Occasionally a migratory pattern was seen in cells grown in uncoated wells, but this took the form of streaks or lines of cells rather than the spreading pattern seen with H120. Virtually no migration occurred in wells coated with BSA or H0. Effect of 41 binding sequences on CD34 cell growth and adhesion. To quantitate the proliferative responses of CD34 cells to the H120 and H0 fragments, larger numbers of cells were cultured initially for a mean of 7 days. A total of 2.CD34 cells were placed in triplicate into wells that had been precoated with H120 and H0 fragments and contained serumfree medium and the standard growth factor concentrations. Control wells were coated with BSA. When the cells were counted the adherent cells were counted separately from the nonadherent cells in suspension. A 10-fold increase in numbers of adherent cells (mean 6 104) was found in H120 wells compared with H0 wells (mean, 4.7 1.4 104; P .001). Only a small number of adherent cells were present in wells coated with BSA (mean, 1.104; P .001). At the end of the culture period total cell numbers were higher in H120 wells (mean, 90.3 10.3 104) compared with H0 wells (mean, 79.6 4.9 104) and BSA wells (mean, 72.0 6.6 104), but the differences were not statistically signicant in these relatively short-term cultures (Fig 5A). In a similar experiment to determine the effects of H120 on the maintenance of CD34 cells in culture, FACSpuried CD34 cells were placed in precoated wells in triplicate. Control wells were left uncoated. After expansion in culture cells in both, nonadherent and adherent layers were counted, resuspended in 0.5% BSA/PBS, and labeled with a CD34 antibody for FACS analysis. H120 wells again contained increased total numbers of cells (mean, 40.3 7.2 104) compared with uncoated wells (mean, 30.8 5.6 104). There

Fig 3. Single cells of CD3438DR and CD3438DR phenotypes were deposited into single U-shaped wells into serum-free medium and standard growth factors. The wells had been precoated with H120, H0, or BSA or left uncoated. Wells were photographed at 1 and 2 weeks. (A) Proliferation from a CD3438DR cell at 1 week in a BSA-coated well. Cells are growing in the base of the well. (B) Proliferation from a CD3438DR cell at 1 week in a well coated with H120. Cells have migrated around and up the sides of the well. (C) Proliferation from a CD3438DR cell at 2 weeks in a well coated with H120. Cells have proliferated in focal sites around and up the sides of the well.
Fig 6. (A) Adherent layer of cells in H120-coated wells stained in situ with May-Grunwald-Giemsa after removing cells in suspension. (B) Cells adherent to uncoated wells stained in situ with MayGrunwald-Giemsa after removal of suspension cells. (C) Photograph of cytospin of suspension cells stained with May-Grunwald-Giemsa. For differential cell count see text.
migration, and focal patterns of growth of CD34 cells. To assess the relative contribution of 41-integrin engagement by H120 on cell proliferation, a range of growth factor concentrations was used for cell expansion and cells cultured for periods of 9 to 13 days. CD34 cells (2.5 to 6.5 103) were added to wells precoated with H120 or H0 fragments to which a mixture of growth factors in serially diluted concentrations in serumfree medium was added. Figure 7 shows the results from one experiment in which the cells proliferating from 2.input CD34 cells were counted on day 13. Higher total cell counts were seen with H120 compared with H0 and BSA controls at all
Fig 4. Single cells of CD3438DR (represented as ) and CD3438DR (represented as ) phenotypes were deposited into single U-shaped wells into serum-free medium and standard growth factors. The wells had been precoated with H120, H0, or BSA or left uncoated. The figure shows the mean number of wells (n ,3,2 or 4) SD in which a migratory pattern was seen expressed as a percentage of the result with H120 at week 1 for the CD3438DR () phenotype. Migration was defined as movement of cells against gravity up and around the sides of the wells.
were increased numbers of cells in the adherent layer of H120 wells (mean, 11.1 2.5 104) compared with the uncoated wells (mean, 0.4 0.32 104; P .001). The percentage of CD34 cells analyzed by FACS was expressed as total numbers of cells. The mean total number of CD34 cells was similar in both H120 wells (16.2 3.0 103) and uncoated wells (14.3 1.2 103), and again the adherent layer in H120 wells contained signicantly increased numbers of cells (3.8 1.4 103) compared with noncoated wells (0.07 0.06 103; P .001; Fig 5B). To investigate the effects of H120 on the morphological phenotype of cells during a longer period in culture, CD34 cells (103) were added to wells containing serum-free medium and growth factors with and without the H120 fragment. After 2 weeks, the nonadherent cells growing in suspension were removed, counted, and stained with May-Grunwald-Giemsa and the adherent cells stained in situ on the well bases (Fig 6A through C). The adherent layers were of the same appearance in all H120 wells and contained aggregates of mainly blasts and promyelocytes with more mature myelocytes and metamyelocytes clearly seen at the edge of the main cell mass (Fig 6A). Cells grown on uncoated surfaces did not form an adherent layer, although a few cells were attached (Fig 6B). A differential count of the nonadherent cells was the same from all wells (Fig 6C). The majority of cells (mean of 58%) was predominantly of blast or promyelocyte morphology together with 16% myelocytes, 12% metamyelocytes, 7% neutrophils, and 7% erythroblasts. Effect of 41 binding sequences on CD34 cell growth with reducing concentration of growth factors. The foregoing results indicated that the H120 substrate supported adhesion,

Fig 5. (A) A total of 2.CD34 cells isolated from cord blood were placed in triplicate into wells precoated with H120, H0, or BSA containing serum-free medium and standard growth factors. Adherent and nonadherent cells were counted after 6 days. Hatched areas represent the adherent cell component. (B) A total of CD34 cells were placed in triplicate into wells precoated with H120 fragment or left uncoated, containing serum-free medium and the standard concentration of growth factors. Adherent and nonadherent cells were counted after 8 days and analyzed by FACS for the proportion of CD34 cells in each fraction. Hatched areas represent the adherent cell component.
Fig 7. A total of 2.CD34 cells were added to wells precoated with H120 or H0 fragments to which the standard mixture of growth factors in serially diluted concentrations was added. Control wells were coated with BSA. Total cells were counted after 13 days. The figure shows the result of one of three similar experiments whose combined results are given in Table 1.
dilutions of growth factors. In the presence of H120 cell growth was not affected until growth factors were diluted by 1 in 16, whereas in the H0- and BSA-coated wells growth was reduced at one-eighth dilution of growth factors (Fig 7). At this dilution growth in H120 cultures yielded a 413-fold expansion of cells, whereas growth was 40% to 50% below this value in BSA- and H0-coated wells (265-fold and 217-fold, respectively). Cell numbers with H0 were expressed as a percentage of expansion with H120 for each of three separate experiments, and the relative fold expansion with H120 is shown in Table 1. Signicant increases in proliferation occurred with H120 compared with H0 at all growth factor dilutions, with the greatest
Table 1. CD34 Cell Growth With H120 and H0 Fragments: Effect of Reducing Growth Factor Concentrations
Growth Factor Concentration
mean difference in expansion (2.25-fold) occurring at a dilution of one sixteenth (P .001). A similar increase occurred when growth with H120 was compared with that in control wells coated with BSA in two experiments, where the greatest differential expansion (2.5-fold) between H120 and BSA cultures also occurred at a one-sixteenthgrowth factor dilution. Effect of 41 binding sequences on CD34 cell growth with time in culture. CD34 cells were isolated as previously described and added in duplicate to wells precoated with H120 or H0 fragments to which a mixture of growth factors at one fourth of the standard concentration in serum-free medium was added. The reduced growth factor concentration was shown to maintain plateau growth in all conditions and was used to facilitate any contribution to long-term growth provided by the H120 fragment. The medium was not renewed during the period of culture. In three separate experiments, total cells were counted at three or four time points between 5 and 14 days of culture. As previously shown, the H120-coated wells supported an adherent cell component throughout the growth period, and total cell counts from these wells were the combined adherent and nonadherent fractions. An example of the course of growth over 13 days from an input of 2.CD34 cells is shown in the experiment described in Fig 8. Cell expansion was 10-fold at day 5 for all wells, and this was followed by marked proliferation between days 5 and 8 with differences between H120, H0, and BSA wells rst appearing on day 8. On day 13 mean cell expansion was 450-fold with H120, 254-fold with H0, and 324-fold with BSA. Mean total cell numbers were signicantly increased on day 8 (P .05) and on day 13 (P .01) in the H120 wells compared with the H0 wells. In the absence of any change in culture medium, cell numbers

P Value

Fold Expansion With H120 Compared With H0

1/1 1/2 1/4 1/8 1/16

.01.05.01.01.001

1.53 1.63 1.57 1.69 2.25

In three separate experiments CD34 cells were cultured in serumfree medium and a serially diluted mixture of growth factors in wells coated with H120 or H0 fragments. Growth factor concentrations ranged from standard concentration (IL-ng/mL, IL-U/mL, G-CSF 500 ng/mL, SCF 100 ng/mL, erythropoietin 2 U/mL) to 116 concentration (IL-3 0.625 ng/mL, IL-6 12.5 U/mL, G-CSF 31.25 ng/mL, SCF 6.25 ng/mL, erythropoeitin 0.125 U/mL). Cells were counted at a mean of 11 days. Cell numbers with H0 were expressed as the mean percentage of expansion with H120 and the overall fold expansion with H120 compared with H0 is shown. Students t-test was used to calculate P values.
Fig 8. A total of 2.CD34 cells were added in duplicate to wells precoated with H120 or H0 fragments to which the standard mixture of growth factors at one fourth of the standard concentration in serum-free medium was added. Control wells were coated with BSA. Total cells were counted on different days of culture. The figure shows one of three similar experiments whose combined results are given in Table 2.
declined after this time in two experiments. The results of three time course experiments are shown in Table 2. The mean increase in cell expansion is shown for H120 compared with H0 (mean of 1.80-fold expansion, P .0001) and for H120 compared with BSA (mean of 1.45-fold expansion, P .001).

DISCUSSION

In most cells, adhesion is an essential process in the control of growth and differentiation and, although hematopoietic progenitor cells can grow in suspension and therefore can bypass this requirement, they have a close association with their stromal extracellular matrix which, under physiological conditions, may regulate interactions with locally available growth factors.30 Fibronectin is a component of the marrow extracellular matrix,31 and the CS1 sequence has been identied previously in a mouse stromal cell line.18 We have found (unpublished data, October 1997) that the alternatively spliced high-affinity CS1 sequence, present in H120, is expressed by human bone marrow stromal cells as is the sequence equivalent to H0. We have previously shown that CD34 cell adhesion and migration on bronectin through 41 integrins is regulated by hematopoietic growth factors, probably by an inside-out signaling mechanism, and we show here that similar ligation of 41 integrins can also synergize with signaling pathways from growth factors resulting in increased cell proliferation that is associated with cell attachment and migration during the growth period. The proliferation of cells in contact with H120 was associated with their adhesion during growth (Figs 5A and B), but this attachment was not xed as studies with single cells showed that cell migration around and up the sides of the wells also occurred (Figs 3B and C). We have recently shown that IL-3 can provide a stimulus to CD34 cells to transiently reduce adhesion and promote migration on H120 through 41 integrins. Here we conrm that a migratory stimulus occurs with H120 but not H0 during a period of growth from single cells originating from different subsets of CD34 progenitor cells. IL-3 is a component of the growth factor mixture and may provide the major stimulus responsible for the migration patterns seen. It has been suggested that hematopoietic stem cells in fetal bone marrow reside within the CD3438DR cell population,26 and cord blood CD34DR cells appear to contain the majority of primitive hematopoietic progenitor cells.27 Our use of the CD3438DR population from cord

Table 2. Growth of CD34 Cells With H120, H0, and BSA: Effect of Time in Culture
Day of Maximum Expansion Fold Expansion With H120 Compared With H0 Fold Expansion With H120 Compared With BSA
1.77 1.89 1.76 Mean, 1.80 0.05 P .0001
1.38 1.45 1.52 Mean, 1.45 0.05 P .001
In three separate experiments CD34 cells were cultured up to 14 days in serum-free medium and a mixture of growth factors at 14 the standard concentration in wells coated with H120, H0, or BSA. Cells were counted on different days of culture. The day of maximum fold expansion with H120 compared with H0 and BSA is shown. Students t-test was used to calculate P values.
blood is thus representative of an immature subset. However, it has been shown more recently that the CD3438DR population in cord blood is enriched in long-term culture-initiating cells (LTC-ICs),28 and our use of CD3438DR cells may therefore not reect properties of the most primitive subset. Single cells from the CD3438DR subset showed a migratory pattern of growth on an H120 substrate from the rst cell division implying a prerequisite degree of adhesion to H120 and suggesting that mobilization of at least this progenitor subset in response to growth factors may occur using the same cellular mechanisms. CD34 cells grown in liquid culture adhere to the H120 fragment during the period of growth, and only a few cells were attached to the H0 fragment or to BSA-coated or BSA-uncoated wells (Figs 5 and 6A and B). An increased number of cells proliferating from input CD34 cells was consistently found in wells coated with H120 compared with H0 and BSA (Fig 7 and 8; Tables 1 and 2). In time-course experiments this difference was rst apparent at day 8 and reached a maximum at days 11 to 13 (Fig 8 and Table 2) when expansion with H120 was a mean of 1.8-fold that seen with H0 and 1.45-fold over that with BSA. The greatest mean relative expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentration was reduced to one sixteenth of the standard mixture (Table 1). Thus, with limiting availability of growth factors, the contribution of H120 to proliferation is increased. The above results show that cells growing in contact with H120 received an additional proliferative stimulus from its highest affinity 41 binding site. Wells coated with H0 were a good control as this fragment is the same as the H120 fragment, apart from lacking the two highest affinity (CS1 and CS5) 41 sequences. The increase in cell proliferation between H120- and BSA-coated control wells was not as marked as between H120 and H0 wells (Fig 8 and Table 2). The reasons for this are not clear, although it is possible that BSA has no effect on growth, whereas the H1 sequence of H0 could have an inhibitory effect in the absence of the CS1 sequence. This possibility has been discussed previously.25 A previous report of the inhibitory roles of bronectin and stroma for hematopoietic progenitors14 is difficult to reconcile with our results with H120, but the methods used by this group differ considerably to those of our study, eg, the degree of inhibition of proliferation due to bronectin was asssessed over a relatively short time period by a thymidine suicide technique that contrasts with our direct measurement of longer term growth (up to 14 days) of the progeny of CD34 cells with a mixture of growth factors in contact with H120 and H0. Most cell amplication in cultures of CD34 cells is associated with differentiation of progenitors and stem cells in response to growth factors,32 and the number of the more primitive LTC-ICs usually declines,33,34 although more recent studies have shown that LTC-ICs can undergo expansion in liquid cultures.35 Growth of total nucleated cells from CD34 cells varies with the source of input cells,36 the number of input cells,37 and the combination of growth factors used. Increases in cell numbers from cord blood CD34 cells vary from an 85-fold increase at 10 days36 to a 791-fold total expansion.37 We showed an overall expansion in cell numbers using cord blood CD34 cells of 160-fold with H0 to 260-fold with H120 (means of six

experiments with one-fourth growth factor concentrations) over 11 to 13 days. Loss of stem cells has been attributed to removal of stromal cells,38 and the addition of components of the marrow stroma such as bronectin may provide a more physiological environment in which to induce expansion. In this respect, stromal-conditioned media have been found to signicantly enhance expansion of primitive hematopoietic stem cells and progenitor cells from CD34 cells taken from mobilized peripheral blood,39 and stromal-derived heparan sulphate has recently been shown to have a role in the maintenance of LTC-ICs.40 There was no difference in the morphological maturation stage of cells taken from cultures grown with or without H120, and the increase in total cell numbers with H120 was not at the expense of loss of CD34 cells as numbers of these were similar in all culture conditions (Fig 5B). Preliminary results (not shown) also indicate that numbers of granulocyte-macrophage colony-forming cell and BFU-Ederived colonies are maintained with H120. We have shown here that ligation of 41 integrins provides a stimulus to CD34 cell growth, and more information is now needed about the effect of H120 on the self-renewal versus differentiation decisions of earlier progenitors, together with any effects on cell survival and cell cycling. The results shown in Table 1 suggest that this probably needs to be performed with limiting concentrations of growth factors to enable the effects of H120 to be clearly expressed. Under such circumstances, which may more closely resemble steady-state conditions in the marrow, integrins may play a part in maintaining survival of stem cells as they do in epithelial and endothelial cells.42,42 In conclusion our ndings show that ligation of 41 integrin by the IIICS region of bronectin can synergize with growth factors resulting in an enhanced growth of CD34 cells occurring over a prolonged period in liquid culture. Growth occurs in an adhesion-related manner and is accompanied by cell migration. The use of H120 substrata in the ex vivo expansion of CD34 cells provides a model for understanding the role of stromal control of hematopoiesis, and in future studies we will hope to perform a detailed analysis of the effects of H120 on early progenitor and stem cells.
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