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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4074-4083
Suppression of Hematopoietic Activity in Tenascin-C-Deficient Mice
By
Masatsugu Ohta,
Takao Sakai,
Yumiko Saga,
Shin-ichi Aizawa, and
Masaki Saito
From the Division of Biochemistry, Cancer Institute, Hokkaido
University, School of Medicine, Sapporo; the Division of Hemopoiesis,
Institute of Hematology, Department of Medicine, Jichi Medical School,
Tochigi; Cellular and Molecular Toxicology Division, National Institute
of Health Sciences, Tokyo; Laboratory of Morphogenesis, Institute of
Molecular Embryology and Genetics, Kumamoto University, School of
Medicine, Kumamoto, Japan; and the Departments of Medicine
and Biomolecular Chemistry, University of Wisconsin-Madison,
Madison, WI.
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ABSTRACT |
Tenascin-C (TN-C), a member of the extracellular matrix (ECM)
glycoprotein family, is expressed on the surface of stromal cells in
the hematopoietic system or lymphoid organs. Recently, TN-C-deficient
mutant mice produced by TN-C gene targeting through homologous
recombination were shown to develop normally, although TNs have been
reported to play important roles in organogenesis and carcinogenesis.
In the present study, we found that colony-forming capacity of bone
marrow (BM) cells was considerably lower in TN-C-deficient mice (a
decrease of ~35% from control), although their mononuclear cell
count and BM architecture showed no significant difference from those
of normal mice. Furthermore, in long-term BM culture in vitro,
hematopoietic cell production (a decrease of ~40% in Dexter's
condition and of ~65% in Whitlock-Witte's condition from control),
colony-forming capacity of the produced cells (a decrease of ~60%
from control), and longevity of the cultures were markedly lower in the
TN-C-deficient mice than in control mice, whereas hematopoiesis in the
TN-C-deficient mutant mice was sustained. The addition of TN-C
glycoprotein to long-term BM cultures of TN-C-deficient mice clearly
induced the recovery of hematopoietic cell production and
colony-forming capacity of hematopoietic progenitor cells. Thus, these
results provide direct evidence that an ECM glycoprotein
component, TN-C, plays a relevant role in hematopoiesis through
interactions between stromal cells and hematopoietic progenitor cells.
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INTRODUCTION |
THE HEMATOPOIETIC microenvironment plays
an essential role in hematopoiesis through cell-to-cell,
cell-to-extracellular matrix (ECM), and cell-to-growth factor
interactions.1,2 Hematopoietic microenvironment is a
complex structure in which stem cells, progenitor cells, stromal cells,
growth factors, and ECM molecules each interact to regulate
hematopoietic cell growth and differentiation. It remains a major quest
to determine how the components of mictoenvironment regulate
lineage-specific blood cell differentiation. Previous studies showed
direct evidence of interaction between hematopoietic stem cells and
stromal cells by the finding of the expression of tyrosine kinase
receptor family on hematopoietic stem cells and their ligands which
stimulate the proliferation of hematopoietic stem cells in molecular
level. The evidence in hereditarily anemic mice3 that
hematopoietic stem cells from heterozygote anemic Sl/Sld mouse4 can
repopulate the stroma and rescue a lethally irradiated wild-type
(+/+) host, but +/+ stem cells fail to rescue the
Sl/Sld anemia showed that the
defect of Sl locus resulted in the defective hematopoietic
microenvironment.5,6 White spotting (W) and steel
(Sl) mutations have been identified4 and it has
been shown that W encodes the c-kit proto-oncogene, a
tyrosine kinase membrane receptor,7,8 and Sl
encoded the mast cell growth factor (also known as Steel factor
[SLF], stem cell factor, or kit ligand).9,10 A
novel tyrosine kinase receptor specific to hematopoietic stem cells,
called flt3/flk-2, has been identified,11,12 and its
ligand, flk-ligand (FL), has recently been cloned and characterized as
proliferative factor for primitive hematopoietic cells.13
On the other hand, to clarify the function of each molecule related to
hematopoietic regulation, a series of the mouse model of the null
mutation of the gene has been generated by disruption of the gene using
homologous recombination in embryonic stem (ES) cells. The knockout
mice disrupting hematopoietic growth factor receptor genes and growth
factor genes have been reported. Mice deficient in flk2 showed
deficiencies in primitive B-lymphoid progenitors and in T-cell and
myeloid reconstitution by mutant stem cells using bone marrow
transplantation (BMT) experiments.14 Furthermore,
double-mutant mice of both flk2 and c-kit showed more severe
hematopoietic deficiency.14 Mice deficient in flk-1, the
receptor tyrosine kinase, playing an important role in endothelial development, showed an early defect in the development of hematopoietic cells characterized by the absence of yolk-sac blood islands and vasculogenisis in the mouse embryo.15 Disruption of
erythropoietin (EPO) or EPO receptor gene resulted in reduced primitive
erythropoiesis in mouse embryo, but committed erythroid progenitors
were present in homozygous fetal liver,16 and disruption of
common  subunit of the receptors for
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-3 (IL-3) and IL-5 caused impaired signaling of growth
factors to hematopoietic progenitors.17 Transforming growth
factor- (TGF-) signal is mediated through two types of receptors,
type I (TGF-RI) and type II (TGF-RII ),18 and
TGF-1 has been shown as an inhibitor of hematopoietic stem cell
growth.18,19 TGF- receptor type II-deficient mice showed deficiency of yolk sac hematopoiesis and
vasculogenesis.20 TGF-1-knockout mice also showed
retardation of yolk-sac vasculature and hematopoietic
system.21 Macrophage inflammatory protein-1 (MIP-1)
is a chemokine that has pro-inflammatory activity. In the hematopoietic
system, MIP-1 enhance colony formation stimulated by GM-CSF and
M-CSF and suppress colony formation by more immature progenitor
cells.22 However, MIP-1 knockout mice resulted in no
overt hematopoietic abnormalities.23
Gene disruptions of cell-adhesion-related molecules have extensively
been studied. Integrin family are thought to be critical in controlling
differentiation and migration of blood cell precursors. Hirsch et
al24 reported that hematopoietic stem cells lacking 1
integrins could form and differentiate into different lineages but
could not colonize the fetal liver. Arroyo et al25 showed that disruption of 4 integrin gene resulted in impaired T and B
lymphopoiesis, but in normal monocyte and natural killer cell development. These reports indicate that the maintenance of
hematopoietic stem cells in BM may require unidentified molecule(s)
related to adhesive interaction other than 1 and 4 integrins.
Furthermore, p53 tumor suppressor gene-deficient mice showed that mice
homozygous for the null allele appeared normal but were
susceptible to the spontaneous development of a variety of neoplasms
including hematopoietic malignancy (20 malignant lymphoma cases
of 30 examined),26 suggesting that p53 has an important
role in the maintenance of normal hematopoiesis by the regulatory
effect of cell-cycle control.
Gene targeting of ECM component has not been fully examined yet. It is
reported that fibronectin-knockout mutant mice caused early embryonic
lethality, and blood island formation was impaired in endodermal
component of the yolk sac.27 We have previously shown that
the ECM glycoproteins TN, fibronectin, and laminin were expressed in
established stromal cells by murine BM adherent cell
cultures.28 Furthermore, when stromal cells were cocultured with nonadherent BM cells, which resulted in the formation of active
hematopoietic areas in culture, the expression of TN was markedly
enhanced during lymphoid differentiation under Whitlock-Witte's culture conditions.28 These findings suggest that the ECM
component might play a substantial role in lineage-specific
hematopoiesis. Tenascins are ECM glycoproteins that have been
postulated to be regulators of cell-to-cell interaction, embryogenesis,
and the process of malignant transformation.29-33 A recent
study on the establishment of TN-C gene-deficient mutant mice showed
that homozygous null mutant mice were born live and developed
normally.34 Forsberg et al35 also reported
normal development of newly generated TN-C knockout mice, and showed
that the healing process of skin wounds and severed nerves was not
diminished in the TN-C gene-deficient condition. However, little is
known about regulation of hematopoiesis in TN-C-deficient mice. In the
present study we examined the hematopoietic activity of mutant mice in
which TN-C gene expression was completely disrupted to clarify the
physiological roles of this molecule in hematopoiesis, using so-called
long-term BM cultures (LTBMCs) as a model system of in vitro
hematopoiesis. In this system, stromal cells and hematopoietic cells
derived from whole BM were maintained in culture, and hematopoietic
stem cells migrated within the adherent stromal layer via
microenvironmental homing, forming hematopoietic focus-like
cobblestones in appearance. In this culture, the relationship between
the stromal cells and stem cells is maintained, and in the presence of
the hematopoietic cell and stromal cell interactions, proliferation of
hematopoietic stem cells and specific progenitor cells can be
maintained over several weeks.36 Our results show that
TN-C-deficient mice retained their capacity for hematopoiesis; however, the extent of the colony-forming capacity of hematopoietic progenitor cells and production of hematopoietic cells in LTBMCs of
TN-C-deficient mice were significantly lower than in those of
TN-C-expressing control mice.
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MATERIALS AND METHODS |
Mice.
TN-C-deficient mutant mice were created by TN-C gene-targeting in
murine (ES) cells as described previously.34 In the present study, C57BL/6 mice were used as the control mice because ES cells used
in the targeting (TT2 cells) were derived from an F1 embryo of C57BL/6
and CBA mice. C57BL/6 mice were obtained from The Japan SLC Co
(Shizuoka, Japan). Mice were housed at the Department of Experimental
Medicine, Jichi Medical School, Tochigi, and at the Institute of Animal
Experimentation, Hokkaido University School of Medicine. This
experiment was performed in accordance with the Jichi Medical School
Guide for Laboratory Animals and with the Hokkaido University Guide for
the Care and Use of Laboratory Animals.
Basic data.
Both TN-C-deficient mice and control mice were weighed. Blood was
drawn from the tail vein for hematocrit, white blood cell count, and
hemogram. Spleen and liver weight was measured after killing of the
mice by cervical dislocation. BM obtained from a femur was fixed for
histological examination.
Continuous BM cultures.
LTBMCs were established as described previously.37 In each
experiment, age-matched 7-week-old (young) and 50-week-old (aged) TN-C-deficient mice and C57BL/6 mice were used. For investigation of
the myeloid and macrophage hematopoietic system, we used culture conditions based on Dexter's method36 with
modifications.38 Briefly, the contents of an adult mouse
femur and tibia were flushed into a 25-cm2 culture flask
(Falcon no. 3109; Becton Dickinson Labware, Franklin Lakes, NJ) using
an 18-gauge needle in Fisher's medium supplemented with 20% horse
serum (Life Technologies, Inc, Grand Island, NY) and
10 6 mol/L hydrodortisone sodium hemisuccinate (Sigma
Chemical Co, St Louis, MO). Cultures were incubated at 33°C in
humid air containing 7% CO2 and the medium was changed
weekly by removal of all nonadherent cells and medium and replacement
with 6.0 mL fresh medium. For investigation of the lymphoid
hematopoietic system, we used the method originally established by
Whitlock et al39 with some modification.
Briefly, the flushed BM cells were prepared at the concentration of 4 × 105 cells /mL and
seeded into a 25-cm2 culture flask in 5-mL RPMI-1640 medium
supplemented with 5% fetal calf serum (FCS; HyClone Lab Inc, Logan,
UT) and 5 × 105 mol/L 2-mercaptoethanol
(Sigma). Cultures were incubated at 37°C in humid air containing
5% CO2, medium was changed weekly, and BM nucleated cells
were recharged once at day 14. Nonadherent cells were counted weekly
and stained with Wright-Giemsa staining solution for morphological
assessment.
Crossover reconstitution of cocultures of stromal cells and
hematopoietic cells.
Stromal cell lines were established from adherent cells from LTBMCs of
TN-C-deficient and control mice. On day 30 after establishment of
LTBMCs, adherent cells were removed by treatment with 0.25% trypsin
(Life Technologies, Inc, Rockville, MD) and replated in tissue culture
plate (Falcon no. 3001) in Dulbecco's Modified Eagle Medium
(MEM) (Life Technologies, Inc) supplemented with 10% FCS.
Cultures were maintained at 37°C, 5% CO2 and passaged twice a week. After several passages, growing cells were plated at a
limiting dilution and cloned by using penicylinders to separate single
cell-derived colonies.40 To evaluate the supportive
ability of stromal cell lines for hematopoiesis, we used the technique of marrow transplantation in vitro. Donor cells were prepared by the
method described previously.40 Briefly, fresh BM cells obtained from age-matched 7-week-old mice were suspended in RPMI-1640 medium. To prepare the adherent cell-depleted BM fraction, BM cells
were passed through a fine mesh and were applied on a Sephadex G-10
(Pharmacia, Uppsala, Sweden) column as described
previously.41 The pass-through fraction was collected and
washed with the medium. Recipient stromal cells were plated in 12-well
plate (Falcon no. 3043) in 1.5-mL culture medium per well at the
density of 2 × 105 cells/well in Dulbecco's MEM
supplemented with 5% FCS. After 48 hours of cultures when the stromal
culture grew subconfluent, 8 × 105 donor cells/well
in Dexter's condition or 2 × 105 cells/well in
Whitlock-Witte's condition were engrafted in the specified medium as
described above.
Colony-forming assay.
Progenitor cell assays were performed with BM cells from the femur and
tibia before the initiation of LTBMCs and nonadherent cells produced by
murine LTBMCs. Previously described assay conditions required for
growth of multilineage colonies (colony-forming unit in
granulocyte-erythroid-megakaryocyte-macrophage,
CFU-GEMM)19,37 were used with some modifications. Briefly,
cells were plated in methylcellulose (Methocel A-4A, premium; Dow
Chemical Co, Midland, MI) cultures at a concentration of 5 × 104 cells/mL at 37°C in humid air containing 5%
CO2. One milliliter of a methylcellulose culture was added
to each 35-mm Petri dish. Cultures contained 1.0% methylcellulose, 1%
bovine serum albumin, 30% FCS, 40 µmol/L 2-mercaptoethanol, 2 U/mL
of erythropoietin (Kirin Brewery, Tokyo, Japan), and 1% pokeweed
mitogen-stimulated mouse spleen cell-conditioned medium (SCCM) as a
source of IL-3. Triplicate plates were cultured for 14 days, and
colonies (>50 cells) were counted using an inverted microscope.
Macroscopic hemoglobinized colonies were counted as erythroid bursts
(burst-forming unit-erythroid, BFU-E). Spleen colony-forming assay
(colony-forming unit in spleen, CFU-S) was performed as described
previously.42 Briefly, hematopoietic cells from BM or
LTBMCs were washed and resuspended at 2 × 106
cells/mL in phosphate-buffered saline (PBS)( ). Recipient C57BL/6 mice were exposed to 9-Gy total body irradiation and 0.25 mL of the
cell suspension was injected to recipient mice via the lateral tail
vein. At day 12, the macroscopic colonies were counted after fixation
of the spleens in Bouin's solution.
Addition of TN-C glycoprotein to LTBMCs and colony-forming assay.
To confirm the biological role of TN-C, TN-C purified from human
melanoma cells (A375) was added to the LTBMCs and colony assay system.
TN-C was purified by a series of biochemical procedures including
Sepharose CL4B (Pharmacia Biotech, Tokyo, Japan) gel filtration,
gelatin Sepharose 4B (Pharmacia) gel affinity chromatography, and
DEAE-5PW (Toyo Soda, Tokyo, Japan) ion-exchange high-performance liquid
chromatography using the method described by Oike et al.43 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions showed that finally purified TN-C consisted of a protein band with an apparent molecular weight of 250 kD with over 95% purity. No fibronectin immunogenicity
was detected (data not shown). LTBMCs were set in 12-well plates
(Falcon no. 3043) in 1.5 mL culture medium per well with an appropriate
concentration of TN-C, and medium with TN-C was changed weekly. In the
colony assay system, TN-C was added to the methylcellulose medium. TN-C derived from human glioma cell line U-251MG (Life Technologies, Inc)
was also used in a series of experiments. Mouse plasma fibronectin (FN)
(UCB-Bioproducts S.A., Brussels, Belgium) or heparan sulfate (HS)
derived from bovine kidney (Seikagaku Corp, Tokyo, Japan) was used as a
control for TN-C.
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RESULTS |
Hematologic parameters in vivo.
Hematologic parameters of both mutant and control mice were examined in
vivo at weeks 7 and 50. No statistical differences were observed
between TN-C-deficient mutant and control mice at week 7 in the data
of either parameter (P > .05, Student's t-test), body weight (22.0 ± 0.8 v 22.8 ± 1.0 g), hematocrit
(51.3% ± 1.3% v 48.4% ± 2.9%), white blood cell
count (7,300 ± 300 v 7,400 ± 200/µL), BM mononuclear cell count (39.2 ± 0.7 v 38.0 ± 1.1 × 106/femur and tibia),
spleen weight (0.1 v 0.1 g), and liver weight (1.1 ± 0.2 v 1.1 ± 0.1 g). No statistical differences were observed in
both mice group at week 50 (data not shown). However, colony-forming capacity of BM cells of TN-C-deficient mutant mice was considerably lower than that of control mice (Tables 1
and 2, week 0). Histological assessment
using hematoxylin-eosin staining showed that the microscopic architecture of BM, spleen, thymus, and liver was the same in mutant
and control mice (data not shown).
Longevity of LTBMCs.
BM cells from 7-week-old mice were subjected to LTBMCs in vitro using
two culture conditions: Dexter's culture system for myeloid-lineage
cell growth and differentiation, and Whitlock-Witte's for B-lymphoid
lineage. In the initiation of Dexter's condition, cultures from the
mutant and control mice showed stable hematopoietic focus formations
(cobblestone islands) consisting of hematopoietic progenitor cells
adhering to stromal layers. LTBMCs started to produce hematopoietic
cells from the foci 7 days after culturing. The number of hematopoietic
cells produced was more markedly decreased in the course of LTBMCs of
TN-C-deficient mice than in those of controls
(Fig 1a). No significant difference was
observed in the hemograms of the cells produced in each group during
active hematopoiesis (produced hematopoietic cells consisted of 3% to
5% immature blasts, 10% immature myeloid, 85% mature
myeloid, and ~5% monocytoid cells in Wright-Giemsa-stained
preparations). As the LTBMCs of TN-C-deficient mice got older, the
formation of hematopoietic foci diminished in comparison with control
mice. The cells produced from LTBMCs predominantly consisted of
monocytoid cells with few mature myeloid cells, and the adherent
stromal cell layers gradually became sparse (Fig 2a). After 20 weeks of culture, almost
all hematopoietic foci disappeared in the LTBMCs of TN-C-deficient
mice (Fig 2a), whereas the LTBMCs of control mice continued to maintain
active hematopoiesis (Fig 2b). Under the Whitlock-Witte culture
condition, after the stable formation of hematopoietic foci 4 weeks
after culturing, prominent decreases of hematopoietic foci and cell production (Figs 1b and 2c) was observed in comparison with control cultures (Figs 1b and 2d), as in Dexter's culture system. The cells
produced by the LTBMCs of the mutant and control mice conditioned according to the Whitlock-Witte method showed similar hemograms (produced cells consisted of approximately 5% to 10% immature blasts,
~2% myeloid, 5% to 8% monocytoid, and 70% to 80% small lymphocytes). Cell-surface marker analysis of the predominant small
lymphocytes showed positive reactivity against mouse B-cell marker,
anti-mouse antibody RA3-6B2 (B220; Caltag La. Inc, South San Francisco,
CA), using the indirect immunofluorescence method with a FACScan
instrument (Becton Dickinson, Lincoln Park, NJ) (data not shown).
Fifty-week-old mice were used to examine the effect of aging. The
longevity of TN-C-deficient mutant cultures were also lower than that
of control cultures. In both culture systems after 20 weeks of culture,
adherent stromal cell number did not differ from LTBMCs of control and
TN-C-deficient mice (15.6 ± 1.8 v 16.2 ± 2.0 × 105 cells/flask in Dexter's condition, and 10.3 ± 2.0 v 9.6 ± 2.5 × 105 cells/flask in
Whitlock-Witte's condition, P > .05, Student's t-test).
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| Fig 1.
Weekly nonadherent cell production by LTBMCs in Dexter's
culture condition (a) and in Whitlock-Witte's culture condition (b) from BM cells of TN-C-deficient mutant mice ( ) and of control mice
( ) obtained at 7 weeks of age. In Dexter's culture condition, hematopoietic foci formation and cell production became active 1 week
after culturing, and in Whitlock-Witte's culture condition, active
hematopoiesis began 4 weeks after the crisis phase.39 Results represented as mean ± SD of nonadherent cells produced weekly
by at least six cultures per group per week. Results were significantly
different between TN-deficient mouse group and controls (P < .01) in both culture systems. Two other identical experiments gave
comparable results.
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| Fig 2.
Morphology of LTBMCs obtained from 7-week-old mice.
Dexter's cultures of TN-C-deficient mice (a) and of control mice (b)
were photographed under an inverted microscope at week 25 after
culturing. Large hematopoietic foci were seen in the control culture.
Whitlock-Witte's cultures of TN-C-deficient mice (c) and of control
mice (d) were photographed at week 15. Bar, 100 µm. Significant
decrease in hematopoietic activity was observed in LTBMCs of
TN-C-deficient mutant mice.
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Crossover reconstitution of cocultures of stromal cells and
nonadherent hematopoietic cells.
Stromal cells in LTBMCs are not clonal ones because they are derived
from whole contents of BM of mice. We established stromal cell lines
from LTBMCs of control and TN-C-deficient mice and constituted
crossover cocultures of stromal cells and nonadherent BM hematopoietic
cells to confirm the decreased supporting activity of hematopoiesis in
TN-C-deficient condition. As shown in Fig 3, when stromal cells derived from TN-C-deficient mice were cocultured with hematopoietic cells from control or TN-C-deficient hematopoietic cells, cell production from formed hematopoietic foci was markedly decreased compared to that from cocultures with stromal cells from
control mice and hematopoietic cells from control or TN-C-deficient mice. Again it was clearly shown that cell production from coculture of
stromal cells from control mice and hematopoietic cells from TN-C-deficient mice was lower than that of both stromal cells and
hematopoietic cells from control mice.
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| Fig 3.
Crossover reconstitution of cocultures of stromal cells
and nonadherent hematopoietic cells derived from BM cells of
TN-C-deficient mutant and control mice obtained at 7 weeks of age.
Results represented as the percentage of the value obtained in
cumulative mean ± SD of nonadherent cells produced weekly for 4 weeks
by triplicate cultures per group, which gave 11.8 × 105
cells/well in Dexter's condition (a) and 1.7 × 105
cells/well in Whitlock-Witte's condition (b) in coculture of stromal
cells and hematopoietic cells from control mice. Two other identical
experiments gave comparable results.
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Colony formation by the produced hematopoietic cells from LTBMCs.
Multi-lineage colony-forming capacity of nonadherent cells produced in
Dexter's cultures was evaluated using the colony-formation assay.
Nonadherent cells formed multilineage colonies as well as macroscopic
hemoglobinized erythroid bursts in the presence of IL-3 and EPO in
semisolid medium. In comparison with control mice, significantly
diminished colony formation was observed in TN-C-deficient mice
characterized by a lower number of multilineage colonies and erythroid
bursts (Table 1). To exclude the possibility that there may be a shift
of progenitor cell compartment between nonadherent progenitor cells
produced by cobblestone islands and adherent progenitors in
cobblestones, we tested the colony formation of adherent progenitors.
Adherent hematopoietic progenitors were obtained by procuring the
adherent cell layer of LTBMCs at 4 weeks of culture followed by passing
through the Sephadex G-10 column as described in Materials and Methods.
Nonadherent cells formed 81 ± 4 and 58 ± 2/5 × 104 total colonies in control and in TN-C-deficient
condition, respectively (P < .001, Student's t-test)
and adherent hematopoietic cells formed 35 ± 4 and 26 ± 2/5 × 104 in control and in TN-C-deficient
condition, respectively (P < .01, Student's t-test).
Thus, decreased colony-forming ability in LTBMCs of TN-C was not simply
a shift in progenitor cell compartments. Spleen colony formation (day
12 CFU-S), another assay that indicates colony formation of more
primitive progenitor cells, was significantly lower in TN-C-deficient
mice (Table 2). These results indicate that colony-forming capacity of
nonadherent cells produced in the cultures was lower in TN-C-deficient
conditions.
Addition of TN-C to LTBMCs of TN-deficient mice.
To confirm the biological role of TN-C on hematopoietic activity in
TN-C deficient mice, we performed a series of experiments. When 4 to
100 ng/mL TN-C derived from a melanoma cell line was added to the
LTBMCs of TN-C-deficient mice both in the Dexter's condition and
Whitlock-Witte's condition, hematopoietic focus formations,
cobblestone islands, recovered rapidly in a TN-C dose-dependent manner. The number of hematopoietic cells produced was
markedly higher in the TN-C-added LTBMCs of TN-C-deficient mice than
in the LTBMCs without TN-C (Figs 4a and
5a). The level of cell production by LTBMCs
in TN-C-deficient mice supplemented with 20 to 100 ng/mL TN-C
recovered to the level of control mice without TN-C addition (Fig 4a
v 4b and Fig 5a v 5b). Furthermore, TN-C-added LTBMCs of control mice also showed enhanced production of hematopoietic cells
(Figs 4b and 5b). However, addition of TN-C to LTBMCs at the
concentration over 500 ng/mL resulted in disruption of the stromal
layer and hematopoietic focus followed by decreased production of
hematopoietic cells. Glioma-derived TN-C had the same effect on the
production of hematopoietic cells produced by TN-C-deficient LTBMCs
(data not shown). These results indicated that the addition of TN-C to
the LTBMCs of TN-C-deficient mice induces a recovery from the impaired
hematopoiesis that is observed in TN-C-deficient mice. In crossover
reconstitution of coculture systems, it was also shown that TN-C
addition had same effect of recovery from the impaired hematopoiesis in
TN-C-deficient mice (Fig 3).
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| Fig 4.
Cumulative nonadherent cell production by LTBMCs treated
with TN-C glycoprotein in Dexter's culture condition from BM cells of
TN-C-deficient mutant mice (a) and control mice (b) obtained at 7 weeks of age. Results represented as cumulative mean ± SD of
nonadherent cells produced weekly by at least four cultures per group
per week. TN-C was added to LTBMCs at final concentration of 4 ng/mL
( ), 20 ng/mL (), 100 ng/mL ( ), and without TN-C ( ). Results
were significantly different between TN-C-treated and TN-C-nontreated
cultures (P < .01) in both culture systems. Two other
identical experiments yielded comparable results.
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| Fig 5.
Cumulative nonadherent cell production by LTBMCs treated
with TN-C glycoprotein in Whitlock-Witte's culture condition from BM
cells of TN-C-deficient mutant mice (a) and control mice (b) obtained
at 7 weeks of age. Results represented as cumulative mean ± SD of
nonadherent cells produced weekly by at least four cultures per group
per week. TN-C was added to LTBMCs at final concentration of 4 ng/mL
(), 20 ng/mL (), 100 ng/mL (), and without TN-C (). Results
were significantly different between TN-C-treated and TN-C-nontreated
cultures (P < .01) in both culture systems. Two other
identical experiments gave comparable results.
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Furthermore, when 2 to 10 µg/mL soluble fragment of FN or 5 to 50 µg/mL HS was added to the LTBMCs of TN-C-deficient mice both in the
Dexter's condition and Whitlock-Witte's condition, the number of
hematopoietic cells produced was markedly higher in the FN- or HS-added
LTBMCs of TN-C-deficient mice than in the LTBMCs without FN or HS.
This increment of hematopoietic cell production was less than that
observed in TN-C-added LTBMCs of TN-C-deficient mice (data not shown).
Addition of FN to LTBMCs at the concentration over 50 µg/mL and of HS
over 200 µg/mL also resulted in disruption of the stromal layer and
hematopoietic focus.
Effect of TN-C-added LTBMCs on colony formation by the produced cells
from LTBMCs.
In comparison with TN-C deficient mice, significantly greater colony
formation was observed in nonadherent cells produced by TN-C-added
LTMBCs of TN-C-deficient mice, characterized by an increased number of
multilineage colonies and erythroid bursts (Fig 6). As with cell production from
LTBMCs, the level of colony formation of nonadherent cells from LTBMCs
of TN-C-deficient mice added with 20 to 100 ng/mL TN-C recovered to
the level of control mice without the addition of TN-C to the LTBMCs
(Fig 6). Furthermore, colony formation of nonadherent cells from
control LTBMCs treated with TN-C also increased (Fig 6). In addition to
TN-C effect to LTBMCs on colony formation, FN or HS was added to LTBMCs
to see the effects of these molecules on colony formation. As shown in Fig 7, nonadherent cells produced by FN- or
HS-added LTBMCs of TN-C-deficient mice formed greater colonies
compared with those by control LTBMCs of TN-C-deficient mice. Again,
colony formation of nonadherent cells from control LTBMCs treated with
FN or HS also increased. To exclude the possibility that ECM molecule
itself acts on the progenitor cells to enhance colony-forming capacity of hematopoietic progenitor cells, we evaluated the influence of TN-C
on the colony-forming assay using BM cells or nonadherent cells from
LTMBCs of both control and TN-C-deficient mice. As shown in
Table 3, TN-C did not affect the number of
colonies in terms of the formation of multilineage colonies or
erythroid bursts in the presence or absence of the growth factors, IL-3 and EPO. Furthermore, other ECM molecules examined, FN or HS as control
to TN-C, also did not affect the colony formations
(Table 4). These results clearly show that
the exogenous addition of TN-C to LTBMCs changes stromal cell-mediated
hematopoiesis, but that TN-C did not directly act on hematopoietic
progenitor cells. Glioma-derived TN-C had the same effect on the
formation of hematopoietic colonies in a series of experiments
described above (data not shown).
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| Fig 6.
Effect of TN-C-added LTBMCs on colony formation by the
produced cells from LTBMCs of TN-C-deficient mice and of control mice. At weeks 3 and 6 after induction of LTBMCs with TN-C at the final concentration of 0, 4, 20, and 100 ng/mL, nonadherent cells were obtained and cultured in methylcellulose medium as described in Materials and Methods. Colonies (>50 cells) were counted at day 14. Visualized hemoglobinized colonies were counted as erythroid bursts
(BFU-E). Results are mean ± SD of triplicate plates and are expressed
as the percentage of the value obtained in nonadherent cells from
control LTBMCs without TN-C, which formed 134 ± 4 colonies including
14 ± 1 erythroid bursts per 5 × 104 nonadherent cells
at week 3, and 103 ± 7 colonies including 11 ± 1 erythroid bursts
at week 6 of LTBMCs. TN-C DM, tenascin-C-deficient mutant mice.
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View larger version (54K):
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| Fig 7.
Effect of FN- and HS-added LTBMCs on colony formation by
the produced cells from LTBMCs of TN-C-deficient mice and of control mice. At week 3 after induction of LTBMCs with FN at the final concentration of 0, 2, and 10 µg/mL or HS at 0, 5, and 50 µg/mL, nonadherent cells were obtained and cultured in methylcellulose medium.
Results are mean ± SD of triplicate plates and are expressed as the
percentage of the value obtained in nonadherent cells from control
LTBMCs without FN, which formed 122 ± 3 colonies including 7 ± 1 erythroid bursts per 5 × 104 nonadherent cells at week 3, and from control LTBMCs without HS, which formed 122 ± 2 colonies
including 9 ± 2 erythroid bursts per 5 × 104
nonadherent cells at week 3. TN-C DM, tenascin-C-deficient mutant mice.
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|
View this table:
[in this window]
[in a new window]
|
Table 3.
Direct Effect of TN-C on Multilineage Colony Formation
Assay of BM Cells and Nonadherent Cells Produced by LTBMCs
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View this table:
[in this window]
[in a new window]
|
Table 4.
Direct Effect of ECM Component on Multilineage Colony
Formation Assay of BM Cells and Nonadherent Cells Produced by
LTBMCs
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DISCUSSION |
In hematopoietic organs, stromal cells such as fibroblasts, epithelial
cells, and macrophage-like cells develop networks to maintain
hematopoiesis, ie, hematopoietic stem cell self-renewal, proliferation,
and growth, by interaction with hematopoietic progenitor cells. ECM
glycoproteins produced by the stromal cells are known to play a
critical role in the regulation of cell growth and
differentiation.1,2 Recent evidence that TN-C is expressed
in the stromal cells of the hematopoietic system1,44,45 or
lymphoid organs46,47 and that anti-TN-C antibody blocks
the attachment of hematopoietic progenitor cells to the stromal
layer45 suggests the involvement of TN-C in the regulation
of hematopoietic progenitor cells by interaction with the stromal
cells. By the assessments of colony-forming capacity and longevity of
continuous BM cultures and crossover reconstitution assay, we showed
that hematopoietic activity in TN-C-deficient mutant mice is markedly
lower than in control mice in which TN-C gene is normally expressed.
The addition of TN-C glycoprotein to the LTBMCs of TN-C-deficient mice
clearly induced the recovery of hematopoietic cell production and
colony-forming capacity. In the LTBMCs of TN-C-deficient mice both in
Dexter's and Whitlock-Witte's condition, TN-C glycoprotein was not
detected at all in the stromal layer or conditioned media using
metabolic labeling and immunoprecipitation methods (data not shown).
This finding confirmed that TN-C was not induced in the LTBMCs of
TN-C-deficient mice by exogenous stimuli such as culture conditions
and concomitant hematopoietic progenitor cells. Furthermore, TN-C did
not induce the hematopoietic progenitor cells to enhance colony-forming
capacity indicated by the colony-forming assay coexistent with TN-C.
Thus, the impaired hematopoiesis in TN-C-deficient mice might be
affected by stromal cell-mediated hematopoiesis. We showed direct
evidence of the critical involvement of ECM in the maintenance of
multipotent hematopoietic progenitor cells in TN-C-deficient mutant
mice. As mentioned by Forsberg et al,35 indicating that a
number of ECM proteins might be compensatory and exchangeable during
the processes of tissue repair in TN-C-deficient mice, we also
demonstrated that the addition of FN or HS to LTBMCs induced a recovery
from the impaired hematopoiesis that was observed in TN-C-deficient mice. FN, one of the components of ECM molecules, plays an important role in the regulation of hematopoietic
differentiation,48,49 and glycosaminoglycan side chains,
especially HS, has been shown to be a key molecule as selective
compartmentalization of growth factors to regulate hematopoiesis
through stroma-stem interaction.1,50 The mechanism(s) of
action of TN-C on hematopoietic stem cell and microenvironment remain
to be determined; however, TN-C may possibly act by increasing binding
of hematopoietic cells to stromal cells and by increasing the
utilization of hematopoietic growth factors by the stem cells
cooperating with other ECM molecules. Our results imply that the
redundant supplementary ECM components other than TN-C such as FN
influence the interaction between the hematopoiesis-supportive
microenvironment and hematopoietic stem cells under TN-C-deficient
condition by the modification of behavior of stroma-stem cell and
stroma-growth factor-stem cell networks. Further studies are required
to clarify the function of the TN families in vivo with special
reference to organogenesis of hematopoietic tissues during development
and are also required to generate specific knockout mutant mice of
other ECM genes to evaluate the effects of defective gene(s) on
concordant hematopoietic regulation.
| |
FOOTNOTES |
Submitted August 13, 1997;
accepted January 15, 1998.
Supported in part by the Grants-in-Aid for Scientific Research on
Priority Areas No. 05274106 from the Education, Science and Culture of
Japan, and a grant from the Yamanouchi Foundation for Research on
Metabolic Disorders.
Address reprint requests to Masatsugu Ohta, MD, Division
of Biochemistry, Cancer Institute, Hokkaido University School of Medicine, N-15, W-7, Kita-Ku, Sapporo 060 Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs T. Osanai and J. Arikawa (Institute of Animal
Experimentation, Hokkaido University School of Medicine), and Drs Y. Hakamata and M. Murata (Laboratory of Experimental Medicine, Jichi
Medical School) for their valuable advice on animal care and handling.
We are also grateful to J. Yamanoi-Saito, Y. Fukuda, and M. Yamane for
excellent technical assistance.
| |
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Abstract
Introduction
Abstract