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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3263-3272
Growth-Supporting Activities of Fibronectin on Hematopoietic
Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for
Fibronectin Activities of CS1 and Cell-Binding Domains
By
Takafumi Yokota,
Kenji Oritani,
Hideki Mitsui,
Keisuke Aoyama,
Jun Ishikawa,
Hiroyuki Sugahara,
Itaru Matsumura,
Schickwann Tsai,
Yoshiaki Tomiyama,
Yuzuru Kanakura, and
Yuji Matsuzawa
From the Second Department of Internal Medicine and the Department of
Hematology/Oncology, Osaka University Medical School, Osaka, Japan; and
Fred Hutchinson Cancer Research Center, Seattle, WA.
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ABSTRACT |
Fibronectin (FN) is supposed to play important roles in various
aspects of hematopoiesis through binding to very late antigen 4 (VLA4)
and VLA5. However, effects of FN on hematopoietic stem cells are
largely unknown. In an effort to determine if FN had a
growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dull
hematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a
serum and growth-factor-deprived medium, and then subjected to
clonogenic assay in the presence of hematopoietic growth factors. The
pretreatment of the CD34+ cells with FN gave rise to
significantly increased numbers of granulocyte-macrophage
colony-forming units (CFU-GM), erythroid burst colony-forming units,
and mixed erythroid-myeloid colony-forming units. In addition, the
numbers of blast colony-forming units and CFU-GM that developed after
culture of EML-C1 cells with SCF and the combination of SCF and
interleukin-3, respectively, were augmented by the pretreatment with
FN. The augmented colony formation by FN was completely abrogated by
the addition of CS1 fragment, but not of GRGDSP peptide,
suggesting an essential role of FN-VLA4 interaction in the FN effects.
Furthermore, the effects of various FN fragments consisting of
RGDS-containing cell-binding domain (CBD), heparin-binding
domain (HBD), and/or CS1 portion were tested on clonogenic
growth of CD34+ cells. Increased colony formation was
induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other
fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD
of FN were required for the augmentation of clonogenic growth of
hematopoietic stem/progenitor cells in vitro. In addition to the in
vitro effects, the in vivo administration of CBD-CS1 fragment into mice
was found to increase the numbers of hematopoietic progenitor cells in
bone marrow and spleen in a dose-dependent manner. Thus, FN may
function on hematopoietic stem/progenitor cells as a growth-supporting
factor in vitro and in vivo.
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INTRODUCTION |
HEMATOPOIETIC CELLS receive critical
signals for proliferation and differentiation from bone marrow (BM)
microenvironment, including cytokines, stromal cells, and
extracellular matrix (ECM).1-3 In long-term BM cultures,
hematopoietic stem cells are preferentially found within the adherent
cell layers. Blood cell formation in these cultures was completely
blocked by inhibition or modulation of interactions between stromal and
hematopoietic cells.4-6
ECM is composed of a variety of molecules such as fibronectin (FN),
collagens, laminin, and proteoglycans. ECM in BM is not merely an inert
framework; it also mediates specialized functions.7-9 Some
components of ECM have been shown to bind to growth factors produced by
stromal cells and to immobilize them around cells, resulting in giving
spaces where hematopoietic cells and growth factors colocalize. For
example, heparan-sulfated proteoglycans can bind to tumor growth
factor- , basic fibroblast growth factor, interleukin-3 (IL-3),
granulocyte/macrophage colony-stimulating factor (GM-CSF), and
IL-7.10-13 Osteonectin can immobilize platelet-derived growth factor.14 In addition, ECM can bind to cell surface
glycoproteins. FN, collagens, and laminin are ligands of integrins
which not only control anchorage, spreading, and migration of
hematopoietic cells but also induce signal transduction pathway in
these cells.7,8,15,16 Hyaluronan is a ligand of CD44, and
in several systems, facilitates cell-cell adhesion and cell
migration.9,17,18
FN, one of the major ECM components, is a disulfide-linked dimeric
glycoprotein consisting of two similar but not identical 220-kD
monomers. Each monomer is composed of type I-III domains, identified as repeating amino acid motifs in the primary
structure.19 It contains several functional sites with
cell-binding properties.20,21 The CS1 portion which is
alternatively spliced and located near the COOH-terminal of
heparin-binding domain (HBD) is recognized by very late antigen 4 (VLA4). The central cell-binding domain (CBD) which includes the
minimal essential sequence, RGD(S), is recognized by VLA5. It has been
reported that hematopoietic cells change requirement of interactions
between FN and integrins along with their maturation. For example,
primitive hematopoietic stem cells, long-term BM culture initiating
cells (LTC-IC), and multi-lineage CFU-Mix progenitors adhere to the
CS-1 portion, but significantly less to the CBD of FN.22
Progenitors of erythroid or lymphoid lineages have been shown to attach
to FN, but differentiated cells of these lineages become unable to
adhere to FN because of their reduction of FN-receptor
expression.23-25 In addition, a number of studies have
shown that FN functions as not only an adhesion molecule but also a
signal inducer via its binding to integrins.15,16,26,27 Monoclonal antibodies (MoAbs) to VLA4 inhibited lympho-hematopoiesis in
long-term BM cultures5 and erythropoiesis in fetal
mice,28,29 suggesting that FN-VLA4 interaction has crucial
roles in hematopoiesis. Adhesion of FN to VLA5 induced apoptosis on
human myeloid leukemia cell lines and terminal differentiation on human
mature monocytes,30,31 suggesting possible involvement of
FN-VLA5 interaction in negative regulation of hematopoiesis.
Although proliferation and maturation of hematopoietic cells are
suggested to be regulated in part by alterations in FN-integrin interaction, the precise role of the FN-integrin interaction in hematopoiesis remains unclear. Furthermore, little is known about effects of FN on the growth of primitive hematopoietic stem cells. In
this study, we have investigated the effects of FN on clonogenic growth
of human CD34+ cells purified from umbilical cord blood
(UCB) and a murine multipotent hematopoietic cell line. Here, we show
that FN has a growth-supporting activity on hematopoietic
stem/progenitor cells in vitro and in vivo, and that both CS1 portion
and CBD are structurally important for the growth-supporting effects of
FN, whereas the FN effects seem to be mediated mainly through the
interaction between FN(CS1) and VLA4 integrin.
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MATERIALS AND METHODS |
Reagents and antibodies.
Endotoxin-free human plasma fibronectin (FN) was purchased from
Collaborative Biomedical Products (Bedford, MA). Endotoxin-free bovine
serum albumin (BSA) and human serum albumin were purchased from Sigma
(St Louis, MO) and Fujisawa Pharmaceutical Co Ltd (Osaka, Japan),
respectively. FN fragments, C274, CH271, H296, C-CS1, and CH296 were
generous gifts of Takara Shuzo Co Ltd (Siga, Japan). The concentrations
of endotoxin of FN fragments were less than 0.1 endotoxin units/mL as
tested by an E-TOXATE kit (Sigma). Synthetic peptides GRGDSP and GRGESP
were purchased from Iwaki Glass (Tokyo, Japan), and CS1 from Sigma. A
synthetic peptide composed of 24 amino acids within the cytoplasmic
portion of human integrin 3 subunit (3 721-744;
IHDRKEFAKFEEERARAKWDTANN) was a generous gift from Toyobo Co Ltd
(Osaka, Japan) and used as a control peptide for CS1. Iron-saturated
human transferrin was purchased from Sigma. Human regular insulin was a
kind gift of Yamanouchi Pharmaceutical Co Ltd (Tokyo, Japan).
Recombinant murine (rm) stem cell factor (SCF) and rm IL-3 were kindly
provided by Kirin Brewery Co Ltd (Tokyo, Japan). Murine MoAbs reactive
with human integrin  4 subunit (SG17), 5 subunit (KH72), and 1
subunit (SG19) were kind gifts of Dr K. Miyake (Department of
Immunology, Saga Medical School, Japan). LM142, a murine MoAb
recognizing human v subunit, was a generous gift from Dr D. Cheresh
(The Scripps Research Institute, La Jolla, CA). M-KID2, recognizing
human 3 subunit, was purchased from Southern Biotec (Birmingham,
AL), and anti-human CD34 MoAb conjugated to phycoerythrin (PE) from
Becton Dickinson (Mountain View, CA).
Source of cells.
UCB, obtained from normal single deliveries after their parents'
informed consent, was used as a source of human hematopoietic progenitor cells. UCB mononuclear cells (MNC) were isolated by Ficoll-Hypaque density gradient centrifugation (Lymphoprep; Nycomed, Oslo, Norway). To enrich CD34+ cell population, UCB MNC
were negative-selected by immunomagnetic beads conjugated with anti-CD3
and anti-CD11b MoAbs, followed by positive-selection using
immunomagnetic beads conjugated with an anti-human CD34 MoAb (Miltenyi
Biotec, Berguisch Gladbach, Germany). In our series, more than 96% of
the purified cells always expressed CD34 and viability of them was
higher than 98%.
EML-C1 cells32 were routinely grown in Iscove's modified
Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with
20% heat-inactivated horse serum (HS; ICN Biomedical Inc, Costa Mesa,
CA) and 10 ng/mL rmSCF.
Flow cytometry.
Surface expression of integrins on human UCB CD34+ cells
was analyzed by two-color flow cytometry as previously
described.6 Briefly, UCB MNC suspended in
phosphate-buffered saline (PBS) containing 1% BSA and 0.1% sodium
azide were incubated with 0.2% human Ig for 15 minutes at 4°C to
reduce nonspecific binding. Cells were then stained with mouse
anti-human 3, 4, 5, v, or 1 MoAb, followed by incubation
with goat anti-mouse IgG conjugated with fluorescein isothiocyanate
(FITC). After washing, they were stained with PE-conjugated mouse
anti-human CD34 MoAb. Cell surface immunofluorescence was evaluated by
flow cytometer (FACScan; Becton Dickinson).
Sorted CD34+ cells were stained with PE-conjugated
anti-human CD34, FITC-conjugated anti-human 4, or FITC-conjugated
anti-human 5, and subsequently analyzed by single-color flow
cytometry.
The binding of soluble FN to sorted CD34+ cells was also
assessed by single-color flow cytometry. In brief, sorted
CD34+ cells were incubated with 100 µL of Hanks'
balanced salt solution (Ca2+, 1.26 mmol/mL;
Mg2+, 0.90 mmol/mL) containing 50 µg/mL biotin-labeled
soluble FN for 15 minutes in the presence or absence of a 20-fold
excess of unlabeled FN. Subsequently, they were stained with
FITC-conjugated avidin (Vector Laboratories, Inc, Burlingame, CA) and
analyzed by flow cytometer. All staining and washing steps were
performed in Hanks' balanced salt solution at 37°C.
Clonal cell cultures.
To assess the clonogenic ability, methylcellulose progenitor assays was
performed as previously described.33 Briefly, human CD34+ cells preincubated in diverse conditions were plated
at low cell density (3-5 × 102/mL) in methylcellulose
media (Veritas, Vancouver, Canada) consisting of IMDM, 0.9%
methylcellulose, 10 4 mol/L 2-mercaptoethanol, 2 mmol/L
L-glutamine, 30% fetal calf serum (FCS), 1% deionized crystallized
BSA, 3 U/mL recombinant human (rh) erythropoietin, 50 ng/mL rh SCF, 10 ng/mL rh GM-CSF, 10 ng/mL rh G-CSF, and 10 ng/mL rh IL-3. Cultures were
set up in duplicate or triplicate and incubated in a humidified
atmosphere at 37°C and 5% CO2 for 16 days. The numbers
of granulocyte-macrophage colony-forming units (CFU-GM), erythroid
burst colony-forming units (BFU-E), and mixed erythroid-myeloid
colony-forming units (CFU-GEMM) were scored on days 12 to 16 of culture
with an inverted microscope according to established
criteria.33 In some experiments, types of colonies were
determined by differential counting of May-Grunwald-Giemsa-stained
preparations.
The clonogenic ability of EML-C1 cells was also assessed in
methylcellulose culture. The cells were preincubated with 100 µg/mL
of BSA, FN, or FN fragments in IMDM at 37°C for 1 hour and subsequently cultured at the density of 1.0 × 103/mL in
methylcellulose media containing 50 ng/mL rmSCF alone (for blast
colony-forming units [CFU-Blast]) or 50 ng/mL rmSCF + 2 ng/mL rmIL-3
(for CFU-GM). Cultures were established in triplicate or quardricate in
12-well plates. The number of CFU-Blast and CFU-GM were assessed on day
14 and day 7 of cultures, respectively.
Cell proliferation assay.
To quantitate the proliferation of cells, MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
(Sigma) rapid colorimetric assay was used as previously
reported.34 In brief, quadricate aliquots of EML-C1 cells
(3.0 × 104 cells suspended in serum-free IMDM
containing 20 ng/mL rmSCF) were cultured in 96-well flat-bottom
microtiter plates for an indicated period at 37°C in the presence of
100 µg/mL BSA or FN. MTT (10 µL of 5 mg/mL solution in PBS) was
added for the final 4 hours of cultures, and 100 µL of acid
isopropanol (0.04 N HCL in isopropanol) was added and mixed. The
optical density was then measured on the Microelisa plate reader
(Corona Electric, Ibaragi, Japan) with a test wavelength of 540 nm.
Cell adhesion assay.
Adhesion assays were performed as previously described with minor
modification.35 Briefly, 50 µg/mL FN and FN fragments in
PBS were adsorbed to wells of 24-well plates (Iwaki Glass) overnight at
4°C. Nonspecific bindings were blocked with 1 mg/mL BSA in PBS for
the following 2 hours at 37°C. EML-C1 cells were labeled with FITC,
suspended in IMDM, and then added to the coated wells
(2.5 × 105 cells/well). After 2 hours of incubation at
37°C, the nonadherent cells were removed by three gentle washings
with warm IMDM, and then 1.0 mL lysate buffer (10 mmol/L Tris, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2% NP-40) was added to
each well (fluorescence intensity [FI] Adhesion). Lysate
buffer (1 mL) was also added to 2.5 × 105 FITC-labeled
EML-C1 cells (FI Whole cell). Cell lysates were centrifuged for 10 minutes to remove nuclei and insoluble debris, and then FI of the
supernatants was assessed on a fluorescence spectrophotometer (Hitachi
F3000, Tokyo, Japan). To calculate the percentage of adhesion, the
following formula was used: % Adhesion = FI Adhesion/FI Whole cell × 100.
Mice and in vivo treatment with FN fragments.
Female BALB/C mice at the age of 8 to 10 weeks were purchased from
Nippon SLC (Shizuoka, Japan). The mice were injected intravenously with
the indicated doses of FN fragments daily for 4 days. On day 5, 24 hours after the last injection, peripheral blood, BM, and spleens of
the mice were obtained and subjected to methylcellulose colony assay.
The methylcellulose media for murine cells consisted of IMDM, 0.9%
methylcellulose, 30% FCS, 104 mol/L 2-mercaptoethanol,
2 mmol/L L-glutamine, 1% deionized crystallized BSA, 3 U/mL rh
erythropoietin, 100 ng/mL rm SCF, 3 ng/mL rm IL-3, and 10 ng/mL rh
IL-6. On days 10 to 16 of culture, colonies were counted on the basis
of morphological criteria as previously described.33
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RESULTS |
Expression of integrins on human cord blood CD34+
cells.
The large integrin superfamily includes many potential ligands for
components of ECM. The expression of integrins on CD34+
populations of UCB MNC was examined by two-color flow cytometry analysis. As shown in Fig 1A, 0.53% of UCB
MNC expressed CD34, a marker of human hematopoietic stem cells. These
CD34+ populations were stained strongly with anti-human
4 MoAb (mean fluorescence intensity [MFI], 28.15) and anti-human
1 MoAb (MFI, 40.87), and faintly with anti-human 5 MoAb (MFI,
8.81). In contrast, 3 and v subunits were not detected on their
surface.
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| Fig 1.
Expression of integrins on human CD34+
cells. (A) UCB MNC were stained with MoAbs specific for human integrin
3, 4, 5, v, or 1 subunit and FITC-conjugated goat
anti-mouse IgG, and then with PE-conjugated anti-human CD34 MoAb. The
stained cells were analyzed by flow cytometry. Quadrants are indicated
to show levels of background staining observed with appropriate
irrelevant control antibodies. (B) Purified CD34+ cells
were stained with anti-CD34-PE, anti-4-FITC or anti-5-FITC (shaded histograms). The levels of background staining with negative control antibodies (open histograms) are shown in each panel for direct
comparison.
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The purity of sorted CD34+ cells was 98.0% (Fig 1B). These
sorted cells expressed almost the same levels of 4 and 5 subunits (VLA4brightVLA5dull) as original
CD34+ cells, indicating that the levels of VLA4 and VLA5
expression on CD34+ cells were scarcely influenced by our
sorting method.
FN augments the clonogenic efficiency of human CD34+
cells.
To determine if human
CD34+/VLA4bright/VLA5dull cells had
adhesive interaction with FN, human purified CD34+ cells
were incubated with biotin-labeled soluble FN in the presence or
absence of a 20-fold excess of unlabeled FN, and then stained with
FITC-conjugated avidin. Flow cytometric analysis showed that CD34+ cells were stained with biotin-labeled soluble FN and
FITC-conjugated avidin (MFI, 13.72; MFI of negative control, 2.84), and
the addition of excessive unlabeled FN significantly blocked the
staining (MFI, 3.83; Fig 2),suggesting the specific binding of FN to CD34+ cells.
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| Fig 2.
Binding of soluble FN to human CD34+ cells.
Purified CD34+ cells were incubated with biotin-labeled
soluble FN in the presence (right panel, shaded histogram) or absence
(left panel, shaded histogram) of a 20-fold excess of unlabeled FN, and
then stained with FITC-conjugated avidin. The stained cells were
analyzed by a single-color flow cytometry. The level of background
staining with unlabeled FN and FITC-conjugated avidin (open histogram) is shown in each panel.
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We next investigated the effects of FN on the clonogenic growth of
human purified CD34+ cells. The cells were preincubated
with FN or BSA (100 µg/mL) for the indicated periods in IMDM without
iron-saturated human transferrin and human insulin, and then the cells
were subjected to methylcellulose colony assay (Fig
3A). CD34+ cells
rapidly lost their clonogenic ability in the absence of FN.
Preincubation with BSA alone for 4 hours led to approximately 50% of
decrease in the number of colonies, whereas trypan blue dye-exclusion
revealed that approximately all of cells were alive for at least 8 hours. In the presence of FN, by contrast, CD34+ cells
generated increased number of colonies after 1 hour incubation, and
maintained their clonogenic ability to the same level of fresh cells
even after 2 hours incubation.
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| Fig 3.
Effect of FN on the clonogenic capacity of human
hematopoietic progenitor cells. (A) The human CD34+ cells
were incubated with 100 µg/mL of BSA ( ,  ) or FN ( ,  ) in
IMDM (, ) or in IMDM containing 200 µg/mL transferrin and 10 µg/mL insulin (, ) at 37°C for the indicated periods. The preincubated cells (5.0 × 102) were subsequently
cultured in methylcellulose media to assess their clonogenic growth.
All of the results are shown as mean ± SD of triplicate cultures. (B)
Human CD34+ cells were incubated with 100 µg/mL of BSA,
human serum albumin, collagen type I, laminin, or FN in serum-free IMDM
with no other proteins at 37°C for 1 hour and subsequently were
cultured in methylcellulose media at 5.0 × 102/mL to
assess their clonogenic growth. The data represent mean ± SD of
triplicate cultures. Statistically significant difference from a
control (before incubation) value is indicated by two
(P < .01) asterisks. Similar results were obtained in three
independent experiments. ( ), BFU-E; (), CFU-GM.
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When CD34+ cells were preincubated with BSA in IMDM
containing iron-saturated human transferrin and human insulin, they
maintained their clonogenic ability to the same level of fresh cells
for at least 8 hours even in the absence of FN. When preincubated with
FN in the medium, CD34+ cells generated approximately
1.3-fold increased number of colonies after 2 hours of incubation and
preserved their clonogenic ability for the following 6 hours. Although
the difference between the two medium conditions in the numbers of
colonies implied the importance of transferrin and/or insulin
in supporting clonogenic ability of CD34+ cells, FN had the
enhancing effects on the clonogenic growth of CD34+ cells
in both conditions. In subsequent assays, preincubation with FN, its
fragments, or other ECM molecules was performed in serum-free IMDM
without transferrin and insulin to examine their direct effects on
hematopoietic stem/progenitor cells without influence of any serum
components or growth factors.
Because FN was found to influence colony formation of CD34+
cells, we further examined the effects of FN as well as other ECM molecules on clonal growth of myeloid and erythroid progenitor cells.
Purified CD34+ cells were preincubated with BSA, human
serum albumin, collagen type I, laminin, or FN in IMDM for 1 hour, and
then cells were subjected to methylcellulose colony assay. As shown in
Fig 3B, 109 colonies consisting of 53 BFU-E and 56 CFU-GM were
generated from freshly isolated 500 CD34+ cells (before
incubation). When CD34+ cells were preincubated with each
type of ECM molecule for 1 hour, collagen type I or laminin did not
significantly affect the numbers and types of colonies. By contrast,
treatment with FN resulted in increased number of total colonies up to
153, and this supportive effect of FN was observed in both BFU-E and
CFU-GM colonies (elevation by 25% in BFU-E and 55% in CFU-GM).
Moreover, although the proportion of CFU-GEMM was very low, the number
of CFU-GEMM colonies was also increased by FN treatment (the mean number of CFU-GEMM: before incubation, 3.3; 1 hour treatment, 5.3). In
addition to colony numbers, size of colonies generated with FN
treatment was bigger than that of colonies developed without FN
treatment when scored on days 7 to 10, although FN itself could not
support colony formation in the absence of any added growth factors
(data not shown). These effects of FN were observed at a concentration
of 50 µg/mL as well as 100 µg/mL, but not statistically obvious at
a concentration of 10 µg/mL (data not shown).
Influence of GRGDSP and CS1 peptides on the effect of FN.
Human CD34+ cells of UCB MNC expressed both VLA4 and VLA5
as FN receptors (Fig 1). To determine which integrin was involved in
the supportive effects of FN, we evaluated the effects of
GRGDSP and CS1 peptides, which respectively block the
FN-VLA5 and FN-VLA4 interactions,20,21 on the numbers of
colonies developed after treatment with or without FN. In this series
of experiments, CD34+ cells that were preincubated with FN
for 1 hour generated 1.3-fold of colonies in number as compared with
those preincubated with BSA. The supportive effect of FN was completely
abrogated by CS1 peptide, whereas GRGDSP peptide had little effect (Fig
4). These results suggested that the
enhancing effect of FN on the clonogenic efficiency of human
CD34+ cells was exhibited mainly via the attachment of CS1
portion, but not GRGDSP region, of FN to cell surface integrins.
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| Fig 4.
Involvement of FN-integrin interaction in augmentation of
clonogenic growth of human hematopoietic progenitor cells. The human CD34+ cells were incubated with 100 µg/mL of BSA or FN
in serum-free IMDM at 37°C for 1 hour in the presence or absence of
the indicated peptides (10 µg/mL) and subsequently cultured in
methylcellulose media at 5.0 × 102/mL to assess their
clonogenic growth. GRGESP and 3 721-744 were used as control
peptides for GRGDSP and CS1, respectively. The reduction in colony
numbers was used to calculate the percentage inhibition relative to the
FN-increased colony number. The data represent mean percent inhibition ± SD of triplicate cultures. Statistically significant differences
from respective control values are indicated by one
(P < .05) or two (P < .01) asterisks. Similar
results were obtained in three independent experiments and when the
peptides were used at 100 µg/mL.
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Effects of FN fragments on the clonogenic efficiency of human
CD34+ cells.
To understand structural and minimal sequence requirement of FN for the
supportive effects on progenitor cells, several fragments of FN
(illustrated in Fig 5A) as well as native
form of FN were added to preincubations of CD34+ cells. As
shown in Fig 5B, CH296 (CBD-HBD-CS1) increased the number of colonies
at an almost similar level to native form of FN, and C-CS1 (CBD-CS1)
was also effective (39% elevation with FN, 34% elevation with CH296,
and 20% elevation with C-CS1). In contrast, C274 (CBD alone), CH271
(CBD-HBD), and H296 (HBD-CS1) did not increase the number of colonies,
but rather suppressed colony formation. It was therefore suggested that
both CS1 and CBD may be required for augmenting clonogenic growth of
hematopoietic progenitor cells, and the combination of three domains,
CBD-HBD-CS1, may substitute for native FN.
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| Fig 5.
Effects of FN fragments on the clonogenic capacity of
human hematopoietic progenitor cells. (A) Schematic representation of FN molecules and FN fragments. FN is made up of a series of repeats termed Type I, II, and III. Regions of FN with proved cell-binding activity are shown as the RGD-containing CBD, the nonintegrin-dependent HBD, and the LDV-containing CS1 region (CS1). The portions
adhere to VLA4 () and VLA5 ( ) are also indicated. (B) The human
CD34+ cells were incubated with 0.5 nmol/mL of FN or each
FN fragment in serum-free IMDM at 37°C for 1 hour and subsequently
cultured in methylcellulose media at 3.0 × 102/mL to
assess their clonogenic capacity. BSA was used as a control protein.
The data are shown as mean ± SD of duplicate cultures. Statistically
significant differences from a control (before incubation) value are
indicated by one (P < .05) or two (P < .01)
asterisks. Similar results were obtained in three independent
experiments. (), BFU-E; (), CFU-GM.
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Effects of FN on a murine multipotent cell line, EML-C1.
In the experiments using human CD34+ cells, FN treatment
resulted in increased numbers of committed progenitors like CFU-GM and
BFU-E. However, it was difficult to evaluate colony formation of more
primitive and self-renewing progenitors, CFU-Blast, because of their
rarity. EML-C1 cells, a murine multipotent cell line,32 was
used for this purpose because they generated CFU-Blast colonies responding to SCF and generated CFU-GM colonies by
costimulation with SCF and IL-3 in methylcellulose media. These
observations were also reproducible when EML-C1 cells were preincubated
with or without FN (Fig 6A). Flow cytometry
analysis revealed that EML-C1 cells showed
VLA4bright/VLA5dull phenotype in the same
manner as human CD34+ cells (data not shown). In accordance
with the findings on human CD34+ cells, EML-C1 cells
preincubated with FN generated more colonies of CFU-Blast and CFU-GM
than those preincubated with BSA (2-fold elevation in CFU-Blast and
1.5-fold elevation in CFU-GM; Fig 6B).
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| Fig 6.
Effects of FN on the clonogenic efficiency of EML-C1
cells. (A) Cytocentrifuged preparations of the colonies generated in the presence of SCF alone or SCF + IL-3, with or without
FN, respectively, were photographed after Giemsa staining. (B) After
exposure to FN, EML-C1 cells (1.0 × 103) were plated
in methylcellulose media containing SCF alone or SCF + IL-3. The
number of CFU-Blast and CFU-GM were assessed on day 14 and day 7 of
culture. The data represent mean ± SD of triplicate cultures and statistically significant differences from control (BSA)
values are indicated by two (P < .01) asterisks. Similar results were obtained in three independent experiments.
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To further clarify the effects of FN on proliferation of EML-C1 cells,
they were cultured with or without 100 µg/mL FN in serum-free IMDM
containing SCF for the indicated periods, followed by measurement of
cell proliferation by MTT rapid colorimetric assay. In IMDM without FN,
EML-C1 cells could not respond to SCF and reduced their viable cell
number (Fig 7). In contrast, EML-C1 cells
could respond to SCF and grew gradually for at least 60 hours in the
presence of FN. EML-C1 cells did not grow in the absence of SCF,
regardless of treatment with FN (data not shown).
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| Fig 7.
Effects of FN on the proliferation of EML-C1 cells.
EML-C1 cells (3 × 104) were incubated with or without
FN in IMDM containing SCF (20 ng/mL) for the indicated times. Cell
proliferation was measured using an MTT colormetric assay. The results
are shown as mean ± SD of quardricate cultures. Statistically
significant differences from control (BSA) values are indicated by two
(P < .01) or three (P < .001) asterisks. ( ),
Medium; (), BSA; (), FN.
|
|
Effects of FN and its fragments on the clonogenic ability and
adhesion function of EML-C1 cells.
In several cell systems, cell attachment and cell spreading have been
proved to be critical in survival and division of
cells.36,37 To investigate whether the growth-supporting
activity of FN paralleled the adhesion function, the effects of FN as
well as its fragments on the adhesion and growth of EML-C1 cells were
examined. As shown in Table 1, FN, CH296
(CBD + HBD + CS1), and C-CS1 (CBD + CS1) supported the CFU-GM
production of EML-C1 cells, whereas other tested fragments did not have
supporting function. These findings were comparable with those obtained
from human CD34+ cells. In adhesion assay, EML-C1 cells
were found to adhere to CH296 and H296 (HBD + CS1) at the highest
level; the cells showed the lesser level of adhesion to FN, C274 (CBD
alone), CH271 (CBD + HBD), and C-CS1, and the percents of adhesion to
these molecules were almost similar. In cell-shape observation using an
inverted-contrast microscope, EML-C1 cells were found to attach and
spread on the growth-supporting FN fragments as well as the FN
fragments that did not stimulate CFU-GM production (data not shown).
Thus, there seemed to be no apparent correlation between the
growth-supporting activity and the adhesion function of the tested FN
molecules.
Effects of in vivo treatment of FN fragments on the number of
hematopoietic progenitors.
As all of the above results were obtained from in vitro experiments, we
examined whether FN truly acts on progenitor cells in vivo. FN
fragments, C274 and C-CS1, were injected intravenously to mice daily
for 4 days, and then peripheral blood, BM, and spleen cells of the mice
were obtained and subjected to methylcellulose colony assay. No
significant changes were observed in total cell numbers of BM and
spleen among mice after injection of these fragments (data not shown).
However, C-CS1 injection for 4 days led to an increase in the numbers
of colony-forming cells (CFC) in both BM (Fig 8A) and spleen (Fig
8B) in a dose-dependent manner; the effects
on CFC numbers were evident at 3 nmol/body/d and maximal at 30 to 100 nmol/body/d. These enhancing effects were not observed after injection
of C274 (10 nmol/body/d) in either organs. The increased numbers of
colonies were observed in both BFU-E and CFU-GM (at a dose of 100 nmol/body/d of C-CS1; 1.5- and 1.4-fold elevation in BM BFU-E and
CFU-GM, 2.6- and 1.4-fold elevations in spleen BFU-E and CFU-GM,
respectively). Unexpectedly, the number of CFC in peripheral blood did
not increase, but rather decreased 24 hours after injection of C-CS1
(normal control, 120 ± 13/mL of blood; C274 10 nmol/body/d,
99 ± 11/mL; C-CS1 10 nmol/body/d, 51 ± 6/mL; C-CS1 100 nmol/body/d, 46 ± 6/mL). However, because the proportion of CFC in
the peripheral blood is very small in the whole body, C-CS1 injection
at a dose of 100 nmol/body led to an approximately 1.3-fold increase in
the estimated number of total CFC. These results indicate that
particular structure of FN may not only influence the distribution of
hematopoietic progenitor cells but also support their growth in vivo.
View larger version (31K):
[in this window]
[in a new window]
| Fig 8.
Influences of FN fragments on hematopoiesis in vivo.
BALB/C mice were intravenously injected with C274 or C-CS1 fragment of FN daily for 4 days at the indicated doses (n = 5 in each dose). The mice were killed at day 5, and subsequently the numbers of CFC in
BM cells (1 × 104) (A) and spleen cells
(2 × 105) (B) were assessed. The data are shown as
mean ± SD. Control mice received an equal volume of PBS.
Statistically significant differences from control values (shown at the
left sides in each panel) are indicated by one (P < .05) or
two (P < .01) asterisks. The figure shows one of two
similar experiments. (), BFU-E; (), CFU-GM.
|
|
| |
DISCUSSION |
Blood cell formation is controlled by a complex set of events,
including interactions between ECM and hematopoietic
cells.1-3 Multiple cell types interact within a confined
space in hematopoietic organs to deliver and receive critical signals
for proliferation and differentiation. In this report, we have shown
that FN augments in vitro and in vivo growth of hematopoietic stem
cells. Attachment of FN to VLA-4, not to VLA-5, is essential, while
both CBD and CS1 domains of FN are structurally required for its
optimal effects.
Treatment of purified human CD34+ cells with FN increased
the number of colonies of CFU-GEMM, CFU-GM, and BFU-E. Increased number
of CFU-Blast colonies was also observed when EML-C1 cells were
pretreated with FN. Therefore, FN seemed to promote colony formation of
both committed and uncommitted progenitors, and these effects of FN
were seen on a wider stage of differentiation than that reported by
Weinstein et al.38 In addition to the number of colonies,
FN treatment increased the sizes of colonies when observed on days 7 to
10 of cultures. FN also augmented proliferation of EML-C1 cells in the
presence of SCF. These results suggested that FN might collaborate or
synergize functionally with cytokines to induce proliferation of
progenitor cells.
Verfaillie39 has previously shown that BM stromal cells
produce molecules that augment the cytokine-induced proliferation and
maturation of hematopoietic progenitors. Our results suggest that FN
may be one of such stromal cell molecules. Regarding the cooperation of
integrins and cytokine receptors, Lévesque et al40,41
have reported that signals mediated through cytokine receptors modulate
the activation of 1 integrins, thereby leading to generation of
secondary signals from FN-integrin interaction, which may synergize
and/or complete the initial signals from cytokines. Our results
provide additional support for signal coordination of integrin and
cytokine receptors, and raise the possibility that because pretreatment
of FN could augment cytokine-induced clonogenic growth of hematopoietic
progenitor cells in our system, FN may function to trigger progenitor
cells to become more responsive to cytokines by changing expression of
cytokine receptors, and/or by modifying signal pathway through
cytokine receptors.
VLA4 has been known to play pivotal roles in interactions between
hematopoietic stem/progenitor cells and stromal cells.42,43 Antibodies against VLA4 completely blocked production of hematopoietic cells in long-term BM cultures.5 The primitive
hematopoietic stem cells can adhere to CS1 site in the C-terminal
fragment of FN.22 In this study, CS1 fragments could almost
completely abrogate the effects of FN, suggesting that attachment of FN
to VLA4 may be essential for the supportive effects of FN on
hematopoietic progenitors. By contrast, the supportive effects of FN
were not significantly affected by the addition of GRGDSP peptides that could inhibit the interaction between CBD and VLA5, suggesting little
involvement of CBD/VLA5 interaction. However, the
CBD-HBD-CS1-connected or CBD-CS1-connected fragments had
growth-supporting activities almost similar to native FN, whereas
little or no activity was observed in HBD-CS1 that had approximately
comparable size with CBD-CS1. Furthermore, it was noted that there was
no significant correlation between the adhesion function and
growth-supporting activity of the FN molecules tested. It has been
shown that signal transduction in response to several growth factors is
affected by integrins when they are both aggregated and occupied by
ligands.44 It was therefore possible that the aggregation
and occupancy of integrins on hematopoietic stem/progenitor cells may
require both CBD and CS1 as a minimal functional structure of FN, and
also that the adhesion function of the FN fragments may not necessarily correlate with their proliferative activity. Furthermore, because the
structural importance of FN has been reported for VLA5-mediated cell
adhesion and/or the downstream signal
transduction,45 structural requirement may also exist in
the FN-VLA4 interaction.
Recent studies have shown that ligation of FN to integrins stimulate a
variety of signaling events, including tyrosine phosphorylation, cytoplasmic alkalization, calcium influx, accumulation of cytoskeletal molecules at sites of cell adhesion, and altered gene
expression.15,16,26,27,46 Attachment of epithelial and
endothelial cells to ECM is essential for their survival both in vitro
and in vivo because they undergo apoptosis by the inhibition of these
interactions.47 In addition, several lines of evidence
suggest that signal transduction events through integrin-ligand
engagement are involved in the suppression of apoptosis in anchored
cells. For example, attachment of epithelium to ECM via integrins
regulated expression of IL-1 converting enzyme, a protein associated
with apoptosis.48 Apoptosis of detached endothelial cells
was suppressed by the addition of sodium vanadate, a protein tyrosine
phosphatase inhibitor.49 Among the signals via integrins,
pp125 focal adhesion kinase (pp125FAK) is particularly of
interest because constitutive activation of pp125FAK was
sufficient to rescue a epithelial cell line, MDCK, from
apoptosis.50 Moreover, injection of an
anti-pp125FAK antibody into rounded fibroblasts resulted in
the rapid onset of apoptosis.51 It is also known that
pp125FAK directly associates with subunit of integrins,
and its tyrosine phosphorylation and kinase activity are upregulated by
the binding of FN to integrins.52-54 It seems it would be
of interest to evaluate roles of pp125FAK in apoptosis of
EML-C1 cells, a multipotent hematopoietic cell line. Further analysis
will be necessary to reveal the signal transduction pathway and
structural basis of FN for promoting cell survival and growth.
The system to expand CFC in vitro is currently being investigated by
many research groups.55-57 Successful in vitro expansion of
primitive stem cells would promise invaluable contributions to clinical
medicine such as stem cell transplantation and gene therapy. A number
of studies have shown that multipotential hematopoietic progenitors
like CFU-GM, CFU-GEMM, and LTC-IC can be expanded in vitro after
exposure to combinations of various cytokines, including SCF, IL-1,
IL-3, IL-6, IL-11, GM-CSF, erythropoietin, interferon, and leukemia
inhibitory factor.55-57 However, transplantation experiments have shown that hematopoietic progenitors expanded by
cytokines do not have potential to support the long-term hematopoietic reconstitution.58,59 Because our results show that FN can
promote proliferation of self-renewing CFU-Blast stem cells in
cooperation with cytokines, this glycoprotein may be practicable for
the in vitro expansion of stem cells without impairing their long-term reconstituting ability.
In addition to the in vitro effect, the intravenous administration of
CBD-CS1 fragment of FN into mice was found to cause an increase of CFC
in the BM and spleen. The mechanism underlying the growth-supporting
activity of CBD-CS1 fragment in vivo may not be simple because ample
growth factors and serum proteins are present in vivo. However, our
data suggest that FN may be functional in terms of supporting growth of
hematopoietic progenitors in vivo, possibly in cooperation with growth
factors and/or serum components. Interestingly, in agreement
with our preliminary observation, it has recently been shown that the
number of CFC in peripheral blood and hematopoietic organs is not
influenced by CS1 treatment.60 These findings suggest that
although CS1 peptide could abrogate the FN effect in vitro, other VLA4
ligands like VCAM1 may substitute for FN in vivo. Recently, the FN-,
VLA4-, VLA5-, or 1-deficient mice have been generated by gene
targeting.61-65 However, precise roles of the FN-integrin
interactions in the in vivo hematopoiesis remain unclear because of
embryonic lethality of the gene-targeted mice. Because mechanisms of
hematopoiesis have been explored using in vitro embryonic stem cell
differentiation systems that may mimic early hematopoiesis in
vivo,66,67 it will be interesting to clarify when and how
FN-integrin interaction acts on early events of hematopoiesis by using
these systems. Clear and decisive information from such studies should
not only lead to a more precise understanding of mechanisms underlying
regulation of hematopoiesis, but also suggest novel therapeutic
procedures for patients with hematologic disorders.
| |
FOOTNOTES |
Submitted July 24, 1997;
accepted December 24, 1997.
Supported in part by grants from the Ministry of Education, Science,
and Culture; the Inamori Foundation; Senri Life Science Foundation; and
Mochida Memorial Foundation.
Address reprint requests to Takafumi Yokota, MD, The Second Department
of Internal Medicine, Osaka University Medical School, 2-2 Yamada-oka,
Suita, Osaka 565, Japan.
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 |
We thank Drs Tohru Kanzaki, Hajime Ogata, and Shinji Sogo for their
help in collecting fresh cord blood and sorting CD34+
cells. We also thank Dr Ikunoshin Kato for generously providing FN
fragments.
| |
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Abstract
Introduction
Abstract