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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1272-1279
Isolation and Characterization of Murine Clonogenic Osteoclast
Progenitors by Cell Surface Phenotype Analysis
By
Yukari Muguruma and
Minako Y. Lee
From the Department of Biological Structure, University of Washington
School of Medicine, Seattle, WA.
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ABSTRACT |
Osteoclasts are bone resorbing cells of hematopoietic origin;
however, a progenitor cell population that gives rise to mature osteoclasts remains elusive. We have characterized a unique cell surface phenotype of clonogenic osteoclast progenitors (colony-forming unit-osteoclast [CFU-O]) and obtained a marrow cell
population selectively enriched for these progenitors. Whole bone
marrow cells were sequentially separated based on physical and cell
surface characteristics, and the presence of CFU-O and other
hematopoietic progenitors was examined. CFU-O was enriched in a
nonadherent, low-density, lineage-marker-negative
(Lin ), Thy1.2-negative (Thy1.2),
Sca1-negative (Sca1), and c-kit-positive
(c-kit+) population, as were the progenitors that were
responsive to macrophage-colony-stimulating factor(CSF; CFU-M),
granulocyte-macrophage-CSF (CFU-GM), and stem cell factor (CFU-SCF).
When the LinThy1.2Sca1
population was divided into c-kithigh and
c-kitlow populations based on c-kit fluorescence, over 88%
of CFU-M, CFU-GM, and CFU-SCF were found in the c-kithigh
population. In relation to the above mentioned hematopoietic progenitors, CFU-O was significantly higher in the c-kitlow
population: 80% of progenitors present in the c-kitlow
population were CFU-O. The CFU-O in both c-kithigh and
c-kitlow populations showed key features of the osteoclast:
multinucleated tartrate-resistant acid phosphatase-positive cell
formation, expressions of vitronectin receptors, c-src and calcitonin
receptors, and bone resorption. We have identified a progenitor cell
population in the earliest stage of the osteoclast lineage so far
described and developed a method to isolate it from other hematopoietic progenitors. This should help pave the way to understand the molecular mechanisms of osteoclast differentiation.
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INTRODUCTION |
T HE HEMATOPOIETIC ORIGIN of osteoclasts
has been clearly shown by a number of in vivo1-4 and in
vitro studies5,6; however, the precise identity of
osteoclast progenitors and their relationship to other hematopoietic
progenitors are still controversial.7-10 Therefore,
questions critical to the understanding of the cellular and molecular
mechanisms of differentiation from hematopoietic stem cells to the
osteoclast lineage still need to be addressed.
We have previously identified a distinctive population of
colony-forming cells that, after being stimulated by conditioned medium
of a bone-resorbing murine mammary carcinoma, gave rise to mononuclear
cells expressing several key features of osteoclasts in murine bone
marrow cultures.11 The factor responsible for this
osteoclast colony formation, osteoclast colony stimulating factor
(O-CSF), has been isolated and is currently undergoing further
molecular characterization.12 Our previous functional studies of O-CSF-responsive osteoclast progenitor cells suggested that
they were distinct from macrophage progenitors: O-CSF-responsive cells
were relatively resistant to 5 fluorouracil treatment,11 and they could survive for many days in a growth factor-free culture condition.13 These features were not observed for
macrophage progenitors. In this investigation, we attempted to further
characterize clonogenic osteoclast progenitors by studying their
physical properties and expressions of stem-cell-associated cell
surface molecules. The main purpose of this was to distinguish
osteoclast progenitors from the hematopoietic progenitors that are
responsive to macrophage colony-stimulating factor (M-CSF),
granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem
cell factor (SCF). We have succeeded in identifying a fraction of bone
marrow cells that are predominantly enriched for clonogenic osteoclast
progenitors.
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MATERIALS AND METHODS |
Mice.
Female C57Black6 mice obtained from Jackson Laboratory (Bar Harbor, ME)
were used as bone marrow donors at 8 to 13 weeks of age. They were
housed in the vivarium of the University of Washington, and both the
animal care and the experiments were conducted in accordance with the
institutional guidelines approved by the National Institute of Health.
Femurs and humeri were aseptically removed after the mice were
sacrificed, and bone marrow cells were extracted by grinding the femurs
and humeri as previously described.11 Whole bone marrow
cells were suspended in Medium 199 (Bio-Whittaker, Walkersville, MD)
containing 2% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT).
Adherent cell depletion by Sephadex G10 column.
An adherent cell-depleted bone marrow cell suspension was obtained by
passage through columns of Sephadex G10 based on a technique previously
described.14 A whole bone marrow cell suspension, containing 107 to 108 cells, was applied to a
10-mL syringe containing 8 mL of washed and autoclaved Sephadex G10
(Pharmacia Biotech, Piscataway, NJ). After 45 minutes of incubation at
37°C, nonadherent cells were eluted with prewarmed Medium 199.
Density gradient.
Percoll solutions with specific densities of 1.10, 1.09, 1.07, and 1.06 g/mL in 0.15 mol/L NaCl were prepared according to the manufacturer's
protocol (Pharmacia Biotech), and the gradient was formed by
sequentially layering 2 mL of each solution in round-bottom polypropylene tubes. On top of the gradient, 2 mL of the nonadherent cell suspension containing 107 to 108 cells was
layered. The gradient was centrifuged at 1,000g at 4°C for
25 minutes.15 Cells at the interfaces 1.06/1.07 g/mL and
1.07/1.09 g/mL were pooled and washed with cold 0.15 mol/L NaCl and
then with phosphate-buffered saline (PBS) containing 2% FCS and 0.1%
NaN3.
Magnetic bead depletion of lineage-positive cells.
Cells obtained with the previously explained procedures were first
incubated with a cocktail of rat monoclonal antibodies (MoAbs) specific
for murine B lymphocytes (B220), granulocytes (Gr-1; PharMingen, San
Diego, CA), macrophages (Mac-1), and erythroid cells
(YW25.12.716; a gift from Dr S.M. Watt, Imperial Cancer
Research Fund, London, UK). After a 30-minute incubation on ice, the
cells were washed with PBS containing 0.5% bovine serum albumin (BSA)
and 5 mmol/L EDTA, resuspended, and counted. Goat antirat IgG beads
(Miltenyi Biotech Inc, Sunnyvale, CA) were added at the
concentration of 20 µL/107 cells then incubated in the
refrigerator for 20 minutes, and finally washed twice with PBS/0.5%
BSA/5 mmol/L EDTA. Cells negative for the above lineage markers
(Lin) were selected using MACS magnetic
cell separation technology (Miltenyi Biotech Inc) according to the
manufacturer's protocol.
Immunofluorescent staining and cell sorting.
The following MoAbs were used for cell surface labeling and cell
sorting: anti-c-kit, anti-Thy1.2, and anti-Sca1 (PharMingen). The
MoAbs were used either biotinylated or fluoresceinated. Biotinylated MoAb was detected with streptavidin-conjugated phycoerythrin (Caltag Laboratories, South San Francisco, CA). After being labeled with the above MoAbs, Lin cells were sorted using
FACStarplus (Becton Dickinson, Mountain View, CA). The
following rat immunoglobulins were used as isotype controls:
biotinylated IgG2b, fluoresceinated IgG2a, and fluoresceinated IgG2b
(PharMingen).
Growth factor reagents.
A clonal cell line, CESJ3, derived from a hypercalcemia and
granulocytosis-inducing murine mammary carcinoma12 was
cultured in serum-free HL-1 medium (Bio-Whittaker) as a source of
O-CSF. CESJ3 cells also have been shown to produce G-CSF and M-CSF, but not GM-CSF or interleukin-3 (IL-3).17 The culture
supernatant was concentrated approximately 500-fold by ultrafiltration,
filtered (0.22 µm), and stored in aliquots at  70°C. The
conditioned medium of CESJ3 cells prepared in this manner was
designated as CESJ medium and was used to stimulate osteoclast
progenitors at the optimal concentration. Recombinant murine (rm) SCF
and M-CSF were kindly provided by Dr S. Lyman (Immunex, Seattle, WA)
and Dr L. Rohrschneider (Fred Hutchinson Cancer Research Center,
Seattle, WA), respectively. rm GM-CSF, IL-3, and IL-6 were purchased
from Genzyme (Cambridge, MA). The following concentrations of CSFs were
used throughout these experiments: CESJ medium 5% vol/vol, rmM-CSF 240 U/mL, rmGM-CSF 50 ng/mL, rmSCF 50 ng/mL, rmIL-3 20 ng/mL, and rmIL-6 20 ng/mL.
Colony-forming unit (CFU) assays.
Progenitors were analyzed by colony formation in culture medium
containing agar.11 Bone marrow cells were cultured in 15 × 10 mm Linbro wells (ICN Biomedicals Inc, Costa Mesa, CA) at 103 to 105 cells/mL, depending on the degree of
fractionation, in supplemented Medium 199 containing 20% FCS and 0.3%
Bacto agar (Difco Laboratories, Detroit, MI) in the presence of defined
CSFs. In some experiments, agar was replaced by 0.25% agarose (FMC
BioProducts, Rockland, ME). Agar cultures were incubated at 37°C in
a humidified atmosphere with 5% CO2 for 14 days. The
colonies that developed during the culture period were stained for
tartrate-resistant acid phosphatase (TRAPase), a specific enzyme marker
for the murine osteoclast, and then counterstained with Hemal
blue.11 Colonies derived from osteoclast progenitors were
identified by their composition of cells stained for TRAPase. The cells
from these colonies expressing TRAPase activity were bright red,
distinctive from the blue color of negative cells. Colonies, defined as
groups of 50 or more cells, were examined under a microscope and were
classified into three categories based on the percentage of red stained
cells in a colony. TRAPase-positive colonies contained >90% positive
cells, mixed colonies had 10% to 90% positive cells, and negative
contained <10% positive cells.11 All colonies appearing
in an agar plate were scored, and the results were expressed as colony
numbers per unit of cell numbers. For practical reasons, progenitors
that formed colonies in response to CESJ medium, M-CSF, GM-CSF, and SCF
(plus IL-3 and IL-6) were designated as CFU-O, CFU-M, CFU-GM, and
CFU-SCF, respectively.
Chamber slide cultures of fractionated cells.
LinThy1.2Sca1c-kithigh
(c-kithigh) or
LinThy1.2Sca1c-kitlow
(c-kitlow) cells were cultured in Lab-Tek 4-well chamber
slides (Nunc Inc, Naperville, IL) at 500 cells/well in supplemented
Medium 199 containing 20% FCS in the presence of CESJ medium, M-CSF,
or GM-CSF. The cultures were fed with the above culture medium
containing CSFs twice a week and incubated for 14 days. On day 14, the
cells that had attached to the chamber slides were fixed and stained
for TRAPase.
Immunocytochemical analysis of osteoclast markers.
c-kithigh or c-kitlow cells were cultured in
the presence of CESJ medium as described previously in CFU assays at
103 cells/mL in agarose medium using 35-mm Petri dishes. On
day 14, individual colonies were sterilely lifted using an Eppendorf
pipet under an inverted microscope and dispersed in 100 µL
minimum essential medium- (-MEM; GIBCO, Grand
Island, NY) containing 15% FCS. Some colonies were cytospotted onto
glass slides. The other colonies were cocultured with ST2
cells,18 a bone marrow-derived stromal cell line (Riken
Cell Bank, Tsukuba, Japan), in 4-well chamber slides for up to 14 additional days in the presence of 10-8 mol/L
1,25(OH)2D3 (Calbiochem, San Diego, CA) and
10-6 mol/L hydrocortisone (Upjohn, Kalamazoo, MI),
replacing half of the culture medium with fresh medium twice a week.
The cytospot slides and chamber slides at the indicated time were fixed
with 1% formaldehyde for immunoperoxidase staining. A rabbit
polyclonal antibody that recognizes both the v and
 3 subunits of the vitronectin receptor complex (GIBCO)
was used at a 1:100 dilution (antivitronectin receptors [anti-VTR]).
A MoAb against chicken pp60src (Oncogene Science Inc,
Manhasset, NY) diluted in PBS containing 0.1% BSA and 0.05% Tween 20 was used at a 1:100 dilution (anti-src). B220 at a 1:10 dilution was
used as a negative control. The slides were incubated with anti-VTR,
anti-src, or anti-B220 overnight at 4°C followed by a 30-minute
incubation with an appropriate biotinylated secondary antibody:
antirabbit IgG, antimouse IgG, or antirat IgG (Southern Biotechnology,
Birmingham, AL) at a 1:200 dilution. Expressions of VTR and c-src
were detected using a Vectastain ABC kit (Vector Laboratories,
Burlingame, CA) and Sigma Fast DAB (Sigma Chemical Co, St Louis, MO).
Expression of calcitonin receptors.
The cells from individual CFU-O-derived colonies in
c-kithigh and c-kitlow populations were
cocultured with ST2 cells in chamber slides in the presence of
1,25(OH)2D3 and hydrocortisone as described previously. At the indicated time, the cells in the chamber slides were
exposed to 0.2 nmol/L 125I-calcitonin (Peninsula
Laboratories, Belmont, CA) in Medium 199 containing 0.1% BSA for 1 hour at 22°C following the method previously described.19 Nonspecific binding was assessed by including
an excess amount (300 nmol/L) of unlabeled calcitonin in certain chambers. After labeling, the slides were washed with PBS, fixed, and
stained for TRAPase. Slides were then coated with NBT-2 nuclear emulsion (Kodak, Rochester, NY), exposed in the dark for 2 weeks at
4°C, developed, and counterstained. Cells were evaluated for TRAPase and calcitonin receptor expression using a light microscope.
Formation of resorption pits on dentine slices.
Sperm whale dentine slices (150 µm thick), prepared with a low speed
diamond saw, were sterilized by ultraviolet irradiation for 30 min/side
and placed into Lab-Tek 8-well chamber slides in which monolayers of
ST2 cells were previously established. Slices were soaked overnight in
-MEM at 37°C. c-kithigh or c-kitlow
cells were cultured as described previously in the presence of CESJ
medium in agarose medium. On day 14, individual colonies were sterilely
lifted, and the cell suspensions representing individual colonies were
overlaid onto the dentine slices in chamber slides. Cells and dentine
slices were incubated for 14 days in the presence of 10-8
mol/L 1,25 (OH)2D3 and 10-6 mol/L
hydrocortisone, replacing half of the culture medium with fresh medium
twice a week. Resorption pits were visualized using a modification of a
previously described method.20 The dentine slices were
soaked in 50% bleach for 10 minutes and ultrasonicated for 1 minute to release cells attached to the slices. After being washed
sequentially with deionized water and methanol, the slices were stained
with 2% Coomassie brilliant blue (Sigma Chemical Co) in methanol and
air dried. Excess Coomassie blue was removed by moistening the dentine
slices with 1 mol/L NaOH and then scrubbing them with a smooth-surfaced
paper. The dentine slices were examined for resorption pits using a
dissecting microscope.
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RESULTS |
Stepwise enrichment of osteoclast progenitors from adult mouse bone
marrow.
To determine whether osteoclast progenitors can be enriched in a
selected cell population, we have separated whole bone marrow into
different cell populations by sequential cell fractionation and
examined the incidence of osteoclast, as well as other hematopoietic progenitors, in each population. Consistent with our previous observations,11,13 over 95% of CFU-O-derived colonies
formed in response to CESJ medium were intensely TRAPase-positive
whereas >99% of M-CSF-stimulated colonies were TRAPase-negative
macrophage colonies. Approximately, 15% to 50% of GM-CSF-stimulated
colonies were of TRAPase-positive and TRAPase-mixed types; the others
were TRAPase-negative macrophage and/or granulocyte colonies.
The combination of SCF, IL-3, and IL-6 stimulated a variety of
multilineage hematopoietic colonies including a few (<10%)
TRAPase-positive colonies. However, in contrast to the strong TRAPase
positivity exhibited by CFU-O-derived colonies, the TRAPase reaction
of colonies formed in response to GM-CSF or SCF cocktail was weak and
appeared pale red. As the sequential separation steps proceeded, the
incidence of CFU-O, as well as other hematopoietic progenitors,
increased in fractionated bone marrow samples
(Table 1). Approximately a 26-fold
enrichment of CFU-O was achieved in a nonadherent, low-density,
Lin population that represented less than 1%
(0.55%) of the original bone marrow sample. CFU-M, CFU-GM, and CFU-SCF
were also enriched in this population, 29-fold, 35-fold, and 42-fold,
respectively.
Analysis of stem cell-associated cell-surface markers expressed by
progenitors in the Lin population.
In our attempt to distinguish osteoclast progenitors from other
hematopoietic progenitors, we examined the expressions of cell surface
markers, c-kit, Thy1.2, and Sca1, on osteoclast progenitors. Lin cells were stained with MoAbs to either c-kit,
Thy1.2, or Sca1, and then each antigen positive or negative cell was
sterilely sorted using FACStarplus. Fractionated cells were
analyzed for progenitors. As shown in Fig
1, over 99% of CFU-O were c-kit+,
Thy1.2, and Sca1. CFU-M and
CFU-GM showed similar cell surface phenotypes to CFU-O whereas CFU-SCF
was c-kit+, Thy1.2, but heterogeneous
regarding Sca1 expression.
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| Fig 1.
Incidence of CFU-O, CFU-M, CFU-GM, and CFU-SCF in FACS
fractionated cell populations. Flow cytometric histograms of
Lin cells stained for (A) c-kit, (B) Thy1.2, and (C)
Sca1 are shown on the left. The dotted lines indicate isotype controls.
N and P indicate the negative and positive gates for cell collection. One or 2 × 103cells/mL from each cell fraction were
stimulated with CESJ medium, M-CSF, GM-CSF, or SCF + IL-3 + IL-6
for progenitor analysis. Bar graphs on the right (D-F) represent colony
formation from each selected cell population. Means and standard
deviations (SDs) from duplicate wells of three independent experiments
are shown. ( ) CFU-O; ( ) CFU-M; ( ) CFU-GM; ( ) CFU-SCF.
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Enrichment of osteoclast progenitors based on the level of c-kit
expression.
Because CFU-O appeared in the same populations as CFU-M, CFU-GM, and
CFU-SCF, we attempted to isolate osteoclast progenitors by different
degrees of c-kit expression. Lin cells were again
stained with c-kit, Thy1.2, and Sca-1 and analyzed by
FACStarplus (Fig 2A). Thy1.2
and Sca1 negative
(LinThy1.2Sca1)
cells were selected, and then the c-kit+ cells were
arbitrarily divided into c-kithigh and c-kitlow
groups as shown in Fig 2B. c-kithigh cells represented
28.6% and c-kitlow cells 29.1% of the
LinThy1.2Sca1
cell population. The two populations of cells were sorted and analyzed
for progenitors. The majority (>88%) of CFU-M, CFU-GM, and CFU-SCF
were found in the c-kithigh population. Interestingly, the
c-kit expression in CFU-O was heterogeneous
(Fig 3). Fifty seven percent of CFU-O was
in the c-kitlow population and 43% in the
c-kithigh population. As a result, CFU-O was further and
selectively enriched in the c-kitlow population; 80% of
the progenitors we examined in this population were osteoclast
progenitors. In contrast, CFU-O constituted 23% of total progenitors
in the c-kithigh population. Morphologically, colonies
derived from CFU-O in the c-kitlow population were smaller
than those derived from CFU-O in the c-kithigh population.
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| Fig 2.
Expression of cell surface markers (c-kit, Thy1.2, and
Sca1) on Lin cells. (A) Two-color flow cytometric
analysis of lin cells stained for c-kit, Thy1.2, and
Sca-1. Numbers represent percent of cells in the given areas. (B)
Fluorescence histogram of
LinThySca cells (boxed
area in A) analyzed for c-kit expression. The dotted line indicates an
isotype control. Lines indicate the gate for the collection of
c-kitlow and c-kithigh cells.
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| Fig 3.
Incidence of CFU-O, CFU-M, CFU-GM, and CFU-SCF in
c-kithigh and c-kitlow populations. One × 103cells/mL of c-kithigh and
c-kitlow cells were plated in agar medium in the presence
of CESJ medium, M-CSF, GM-CSF, and SCF + IL-3 + IL-6. Colonies
developed during a 14-day culture period were evaluated. Values
represent means and SDs of quadruple wells in a representative
experiment. () CFU-O; () CFU-M; () CFU-GM; () CFU-SCF.
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Liquid culture of CFU-O-enriched populations.
c-kithigh or c-kitlow cells were cultured in
chamber slides in the presence of CESJ medium, M-CSF, or GM-CSF for 14 days, and the morphology of the cells attached to the slides was
examined after being stained for TRAPase. TRAPase-positive
multinucleated cells (more than three nuclei) were formed when either
c-kithigh or c-kitlow cells were cultured in
the presence of CESJ medium. However, cells from these same fractions
cultured in the presence of M-CSF or GM-CSF did not yield any
TRAPase-positive multinucleated cells (Table 2).
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Table 2.
TRAPase-Positive Cell and Multinucleated Cell Formation
From Fractionated Cells in the Presence of CESJ Medium, M-CSF, and GM-CSF
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Expression of osteoclast markers.
The cell surface phenotype of CFU-O-derived colonies from
c-kithigh and c-kitlow populations was examined
first on cytospot samples prepared on day 14 of culture in agarose. All
CFU-O-derived colonies examined so far expressed
v3 vitronectin receptors
(Fig 4A). The cells from individual
CFU-O-derived colonies expressed additional osteoclast markers, c-src
and calcitonin receptors, when these cells were further cultured on ST2
cells in the presence of 1,25(OH)2D3 and hydrocortisone for more than 7 days (Fig 4C and E).
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| Fig 4.
Expression of osteoclast markers on CFU-O derived colony
cells in the c-kitlow population. (A) Cytospot samples
prepared from day-14 agarose cultures were stained for
v3 vitronectin receptors. Note a large multinucleated cell showing positive reaction to anti-VTR. (B) Negative
control stained for B220. (C) Cells from individual CFU-O-derived colonies cocultured for 7 days with ST2 cells in the presence of
1,25(OH)2D3 and hydrocortisone and then stained
with anti-src. Note a group of cells expressing c-src (arrowheads) over
the monolayer of ST2 cells in the background. (D) No positive staining
on ST2 cells that were reacted with anti-src. (E) Autoradiography of cells from individual CFU-O-derived colonies cocultured for 14 days
with ST2 cells in the presence of 1,25(OH)2D3
and hydrocortisone and then exposed to 125I-CT. Note a
group of TRAPase-positive cells covered with dense silver grains
(arrowheads) indicating the expression of calcitonin receptors. (F)
Absence of 125I-CT binding to cells incubated with an
excess amount of cold CT. Scale bars represent 50 µm (A and B), 100 µm (C, E, and F), and 150 µm (D).
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Formation of resorption pits by CFU-O-enriched populations.
Cell suspensions from individual CFU-O-derived colonies formed in
response to CESJ medium from c-kithigh or
c-kitlow cells were cocultured with dentine slices in the
presence of 1,25(OH)2D3 and hydrocortisone. As
shown in Fig 5, resorption lacunae were
observed when dentine slices were cocultured with cell suspensions from
CFU-O-derived colonies in the presence of ST2 cells. Thus, it was
confirmed that the isolated CFU-O could indeed give rise to functional
osteoclasts.
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| Fig 5.
Resorption pits formed by CFU-O-derived colonies in the
c-kithigh cell population. (A) Cells from a colony
developed in the presence of CESJ medium were cocultured with a dentine
slice on ST2 cells for 14 days as described in the Materials and
Methods section. Pits, indicated by arrowheads, were visualized by
staining with Coomassie blue. (B) A control dentine slice incubated
only with ST2 cells for 14 days. Multiple dark dots and lines seen
throughout the specimen are dentinal tubules, whereas parallel regular
lines are saw marks. Scale bars represent 100 µm (A and B).
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DISCUSSION |
Several studies involving bone marrow or spleen cell
transplantation4,21 have indicated that osteoclasts are
derived from hematopoietic cells. This concept was later confirmed by
in vitro experiments in which periosteum-free bone rudiments were
cocultured with a variety of hematopoietic cells as a source of
osteoclasts.5,6 A study by Scheven et al22
further showed the generation of osteoclasts from bone marrow fractions
enriched for hematopoietic stem cells. Other investigators have
supported the concept that osteoclasts are derived from immature
hematopoietic cells capable of colony formation7,23-25 and
from hematopoietic stem cell lines.26-28 However, the
precise lineage of osteoclasts and their relationship to other
hematopoietic cells are controversial. Studies have shown that CFU-G-
and CFU-GM-, but not CFU-M-enriched bone marrow fractions, have given
rise to osteoclasts in vivo and in vitro8,9; yet, these
investigators did not distinguish whether CFU-G and CFU-GM themselves
gave rise to osteoclasts or cells that were copurified with them were
the progenitors of osteoclasts. Although the identification of
osteoclast progenitors from granulocyte-macrophage7 or
multilineage hematopoietic colony-forming cells23-25 has
been attempted, only a fraction of cells from primary colonies gave rise to osteoclasts, and the identification of specific osteoclast progenitors was exceedingly difficult because the primary colonies did
not exhibit any identifiable osteoclast characteristics.
Precise identification of the osteoclast progenitor has been difficult
because of the lack of an osteoclast-specific growth factor. We have
previously isolated a murine O-CSF, a soluble product of a bone
resorbing tumor,12 and have established an in vitro colony
assay system to identify clonogenic osteoclast progenitors.11 In this system, the great majority of the
colonies formed in response to O-CSF are intensely TRAPase positive: a phenomenon not observed with any other hematopoietic growth factors, either alone or combined, so far examined. This osteoclast colony assay
system allows us to examine previously unrevealed progenitors of the
osteoclast in their early stages of development. Our
recent13 and present studies have unveiled several unique
characteristics of osteoclast progenitors.
In this study we have delineated osteoclast progenitors by their
physical properties and expressions of cell surface molecules associated with hematopoietic stem cells. First, consistent with previous studies,22,26 osteoclast progenitors were found in a marrow cell population that had been enriched in immature
hematopoietic progenitors: the nonadherent, low-density, and lineage
marker-negative cell population.
Second, osteoclast progenitors expressed c-kit but did not have Thy1.2
and Sca1 on their cell surface. This is in contrast to hematopoietic
stem cells that are c-kit- and Sca1-positive and do not express or
express a low level of Thy1.2.29 Although c-kit was found
in both stem cells and progenitors,30 cells that also
expressed Sca1 are considered more primitive. In this regard our
results were compatible: O-CSF-responsive osteoclast progenitors along
with other hematopoietic progenitors were immature but not as primitive
as Sca1-positive stem cells, which responded to SCF, IL-3, and IL-6
(Fig 1F). c-kit is a tyrosine kinase receptor for SCF, and c-kit-SCF
interaction is essential for the survival and proliferation of
hematopoietic stem cells and immature progenitors.31,32 However, our previous study has shown that SCF is not essential for the
differentiation of our osteoclast progenitors.13
Third, osteoclast progenitors were selectively enriched in the
c-kitlow cell population in relation to other hematopoietic
progenitors, the majority of which were found in the
c-kithigh population. Previously, a dissection study of
murine hematopoietic progenitors by c-kit expression has described
c-kitlow cells as primitive, dormant, multipotent
progenitors; c-kithigh cells as actively cycling
progenitors; and c-kit very low or undetectable as most of the mature
blood cells.33 Human primitive hematopoietic progenitors
have also been enriched in c-kitlow cells.34 In
this study, O-CSF responsive osteoclast progenitors were found in both
c-kithigh and c-kitlow cell populations;
however, they formed morphologically different colonies:
c-kithigh CFU-O was more inclined to form large macroscopic
TRAPase-positive colonies, whereas c-kitlow CFU-O formed
small colonies (50 to 100 cells) and clusters (8 to 50 cells). One
possible explanation for this is that c-kitlow cells are
dormant and require additional factor(s) or a longer incubation period
to form larger colonies. Alternatively, these cells may be more mature
and may be losing their ability to proliferate. These questions need to
be addressed in future studies.
Fourth, both c-kithigh and c-kitlow cell
populations gave rise to TRAPase-positive multinucleated cells when
cultured in the presence of CESJ medium without the addition of
1,25(OH)2D3. Cells from these same populations
cultured in the presence of M-CSF or GM-CSF did not form such
TRAPase-positive multinucleated cells. In a previous
report,23 1,25(OH)2D3 was essential
for induction of multinucleated cell formation and TRAPase activity
from a population enriched for hematopoietic stem cells in the presence
of IL-3 or GM-CSF. On the other hand, another study35
suggested that 1,25(OH)2D3 was not required
and, in fact, was inhibitory in the formation of TRAPase-positive cells
from progenitors derived from murine long-term bone marrow cultures
that were stimulated with M-CSF or GM-CSF. O-CSF-responsive
progenitors do not require osteotropic hormones, such as
1,25(OH)2D3, for TRAPase-positive
multinucleated cell formation. Furthermore, in our culture system,
addition of 1,25(OH)2D3 did not have a
significant effect on the formation of TRAPase-positive cells in the
presence of CESJ medium, M-CSF, or GM-CSF (unpublished data). Such
differences in the necessity of 1,25(OH)2D3
could be explained by several factors, including culture conditions,
cell sources, and/or differentiation stages of progenitors used
for the studies.
Fifth, TRAPase-positive cells derived from individual CFU-O expressed
other osteoclast markers, namely VTRs, c-src, and calcitonin receptors.
VTR expression was observed in cells cultured in the presence of CESJ
medium for 14 days; however, c-src and calcitonin receptor expression
required an additional culture period on ST2 cells in the presence of
1,25(OH)2D3 and hydrocortisone. These results
are consistent with current understandings that VTR expression precedes
calcitonin receptor expression,36 and c-src expression is
required in relatively late stages of osteoclast
differentiation.37 The results also support the view that
our osteoclast progenitor populations are still in an immature stage of
osteoclast differentiation.
Finally, cells derived from isolated, individual CFU-O were able to
form resorption pits, the definitive identification of the functional
osteoclast, when they were cocultured with dentine slices in the
presence of 1,25(OH)2D3, hydrocortisone, and
stromal cells. These pits were relatively small and shallow, probably reflecting the immature nature of our osteoclasts. This finding confirms that our isolated CFU-O is, indeed, the osteoclast progenitor. Other studies conducted using multilineage colony-derived cells also
have shown that small pits can be formed by
osteoclasts.7,24 CFU-O-derived cells may require
additional factors, such as osteoblastic cells or factors derived from
osteoblastic cells, to fully differentiate into active functional
osteoclasts as suggested by others.38,39
In conclusion, using a sequential cell separation, we have shown that
osteoclast progenitors can be distinguished and isolated from other
hematopoietic progenitors, thus strongly indicating the presence of
previously unknown and unique progenitors of the osteoclast lineage.
O-CSF-responsive progenitors appear to be distinct and belong to an
earlier stage of osteoclast differentiation than those described
previously. The method for osteoclast progenitor isolation established
here will be useful for analyzing underlying molecular mechanisms of
early stages of osteoclast differentiation.
| |
FOOTNOTES |
Submitted June 17, 1997;
accepted October 15, 1997.
Supported in parts by grants from the National Institute of Health
(AR-42657) and Ostex International Inc, Seattle, WA.
Address reprint requests to Minako Y. Lee, MD, Department of Biological
Structure, 357420, University of Washington School of Medicine,
Seattle, WA 98195-7420.
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 Kathy Allen for her excellent technical assistance with the
cell sorting and Jody Lottsfeldt and Lynn Ferguson for their critical
reading of this manuscript.
| |
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Abstract
Introduction
Abstract