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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3230-3238
The Effect of  4 1-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
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.
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ABSTRACT |
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
 4 1-integrin-mediated 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 CD34+38+DR+
and CD34+38 DR+ 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 (P .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 .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.
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INTRODUCTION |
HEMATOPOIETIC 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 specific matrix proteins and that can
mediate transfer of information to the intracellular compartment. Recent findings 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 fibroblasts 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 insulin-mediated
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 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 fibronectin facilitates
CD3-mediated cell growth.9,10 Fibronectin has also been
shown to promote a twofold enhancement of proliferation of erythroid
colony-forming 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-, CFU-E-, and macrophage
colony-forming unit (CFU-M)-derived colonies from CD34+
cells, an effect that was reversed by blocking the fibronectin 51-integrin interaction with
RGD-containing 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
fibronectin substrate.13 However, adhesion of hematopoietic
progenitors to stroma is associated with inhibition of proliferation
that occurs through fibronectin 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 fibronectin.15,16 Long-term
culture-initiating cells and CFU-mix progenitors adhere to the
fibronectin 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 fibronectin.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 fibronectin
(the specific 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 finding 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 fibronectin. 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 defined 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,
CD34+38+DR+ (more mature) and
CD34+38DR+ (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.
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MATERIALS AND METHODS |
Cloning and expression of recombinant HepII/IIICS variants.
cDNA clones for the five different variants of the HepII /IIICS
region of human fibronectin were synthesised using reverse transcription polymerase chain reaction (RT-PCR) amplification of
primary human skin fibroblast 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. Individual clones containing each of
the variants were identified 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-S-transferase 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
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| 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.
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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 anti-mouse antibody conjugated to
magnetic beads according to the manufacturer's instructions (mini-MACS
CD34 isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). For
maximum purity the isolated cells were also sorted on a
fluorescence-activated cell sorter (FACS) flow cytometer
(FACS Vantage flow cytometer; Becton Dickinson, San Jose, CA) using a
second fluorochrome-conjugated (fluorescein isothiocyanate [FITC] or
phycoerythrin [PE]) CD34 antibody (anti-HPCA-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 fluorochrome-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 CD34+38+DR+ and
CD34+CD38DR+ populations
after first gating by size on the lymphocyte population and by
fluorescence (f11) on the CD34+ cell population (R2)
(Fig 2A). The two additional fluorescence parameters, fl2 (CD38-PE) and fl3 (HLA-DR-tricolor), were used to
analyze subpopulations of CD34+ cells that were
CD38+DR+ 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.
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| 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
CD38+DR+ and
CD38DR+.
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Migration of CD34+38+DR+ and
CD34+38DR+ cells within
U-shaped wells: Effect of 41 binding
sequences.
Single cells of CD34+38+DR+ and
CD34+38DR+ 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-µL aliquots of
serum-free medium and that had been precoated with the fibronectin
fragments H120 and H0 at 15 µg/mL (3.75 µg/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 37°C in a humidified 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-four-well plates (Costar, Cambridge, MA) were
coated for 60 minutes at room temperature with 1 mL aliquots of
recombinant H120 or H0 proteins diluted with Dulbecco's PBS to a
concentration of 15 µg/mL (7.5 µg/cm2). Nonspecific
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 left uncoated. A total of 103 to 2.5 × 104 purified 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 37°C in a humidified 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-Grünwald-Giemsa after cytocentrifugation.
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RESULTS |
Adhesion and migration patterns of single
CD34+38+DR+ and
CD34+38DR+ 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 defined phenotype isolated by FACS (Fig 2).
Microscopic examination revealed that single cells of
CD34+38+DR+ 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
CD34+38DR+ 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 fibronectin
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 CD34+38+DR+ and
CD34+38DR+ 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 significantly increased in H120 wells compared with
control wells. The mean number of wells in which migratory growth on
H120 occurred from cells of
34+38+DR+ phenotype did not change
between weeks 1 and 2. Migration and growth occurred less frequently in
the more immature 34+38DR+
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 significantly 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.
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| Fig 3.
Single cells of
CD34+38+DR+ and
CD34+38DR+ 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
CD34+38+DR+ cell at 1 week in
a BSA-coated well. Cells are growing in the base of the well. (B)
Proliferation from a
CD34+38DR+ 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 CD34+38+DR+ cell at 2 weeks
in a well coated with H120. Cells have proliferated in focal sites
around and up the sides of the well.
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| Fig 4.
Single cells of
CD34+38+DR+
(represented as +) and
CD34+38DR+ (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 = 2,3, or 4) ± SD in which a
migratory pattern was seen expressed as a percentage of the result with
H120 at week 1 for the
CD34+38+DR+ (+) phenotype.
Migration was defined as movement of cells against gravity up and
around the sides of the wells.
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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 2.5 × 104
CD34+ cells were placed in triplicate into wells that had
been precoated with H120 and H0 fragments and contained serum-free
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 = 43 × 104 ± 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, 1.0 ± 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 significant in these relatively short-term
cultures (Fig 5A).
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| Fig 5.
(A) A total of 2.5 × 104
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 5 × 103
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.
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In a similar experiment to determine the effects of H120 on the
maintenance of CD34+ cells in culture, 5 × 103 FACS-purified 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 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 significantly 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-Grünwald-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.
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| Fig 6.
(A) Adherent layer of cells in H120-coated wells stained
in situ with May-Grünwald-Giemsa after removing cells in
suspension. (B) Cells adherent to uncoated wells stained in situ with
May-Grünwald-Giemsa after removal of suspension cells. (C)
Photograph of cytospin of suspension cells stained with
May-Grünwald-Giemsa. For differential cell count see text.
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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, 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 serum-free medium
was added. Figure 7 shows the results from one
experiment in which the cells proliferating from 2.5 × 103 input CD34+ cells were counted on day 13. Higher total cell counts were seen with H120 compared with H0 and BSA
controls at all 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. Significant increases in proliferation occurred
with H120 compared with H0 at all growth factor dilutions, with the greatest 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-sixteenth-growth factor dilution.
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| Fig 7.
A total of 2.5 × 103 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.
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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 2.5 ×103
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 first 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
significantly 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
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).
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| Fig 8.
A total of 2.5 × 103 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.
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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 identified 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 fibronectin 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 fixed 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 confirm 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
CD34+38DR+ cell
population,26 and cord blood
CD34+DR+ cells appear to contain the majority
of primitive hematopoietic progenitor cells.27 Our use of
the CD34+38DR+ population
from cord blood is thus representative of an immature subset. However,
it has been shown more recently that the
CD34+38DR population
in cord blood is enriched in long-term culture-initiating cells
(LTC-ICs),28 and our use of
CD34+38DR+ cells may
therefore not reflect properties of the most primitive subset. Single
cells from the CD34+38DR+
subset showed a migratory pattern of growth on an H120 substrate from
the first 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 first 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 fibronectin 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 fibronectin 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 amplification 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 fibronectin may provide a more physiological
environment in which to induce expansion. In this respect,
stromal-conditioned media have been found to significantly 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-E-derived 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 findings show that ligation of
41 integrin by the IIICS region of
fibronectin 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.
| |
FOOTNOTES |
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. Schofield, 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.
| |
ACKNOWLEDGMENT |
The authors thank M Hughes and J Barry for technical assistance with
FACS sorting, Suzanne Bridge for excellent assistance in the
preparation of this manuscript, and Professor M Dexter for helpful
advice and valuable discussions.
| |
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Abstract
Introduction
Abstract