1. Art and Diseño

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Normal Human Peripheral Blood Mononuclear Cells Mobilized With
Granulocyte Colony-Stimulating Factor Have Increased Osteoclastogenic
Potential Compared To Nonmobilized Blood
By Louise E. Purton, Minako Y. Lee, and Beverly Torok-Storb
Single-cell suspensions of granulocyte colony-stimulating
factor (G-CSF)-mobilizedperipheral blood mononuclear cells
(G-PBMC) cultured in alpha minimal essential medium
((UMEM)containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared t o be dependent
on cell density per surface area, so that 5.8 ? 1.3 aggregates
formed per 1 x l o 5 cells when G-PBMC were cultured at
densities greater than or equal t o 1 x lo5 cells/cmz. The
frequency of aggregate formation was less than 1 per lo5
cells when G-PBMC were cultured at densities less than 1
x I O 5 cellslcm'. Once formed, aggregates became adherent
within 72 hours, and then, over the course of 21 days, released CD3lCD41CD25-positive cells into the supernatant.
This T-cell production peaked between days 7 and 14, reach-
ing a total of 1,269 rt 125.9 cells released per aggregate
by day 21. Between days 14 and 21, the aggregates also
generated macroscopic clusters of adherent mononuclear
and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the
macroscopic foci coalesced into monolayers. Multinucleated
TRAP-positive cells were distinguished from macrophage
polykaryons by the absence of CD14 expression and the
presence of osteoclast-specific membrane receptors for calcitonin and cu,p,-vitronectin. The osteoclast nature of these
cells was further demonstrated by their ability t o form resorption lacunae on dentine slices. Comparable osteoclast
formation was not detected in cultures of normal marrow
or normal nonmobilized peripheral blood.
0 1996 by The American Society of Hematology.
0
late progenitors to form colonies that demonstrate osteoclast
characteristics.".I4 However, in these systems, it has been
difficult to identify and isolate the osteoclast precursors and
to define precisely the factors involved i n the regulation of
osteoclast development.
In this study, we report the growth of osteoclast progenitors from granulocyte colony-stimulating factor (G-CSF)mobilized peripheral blood mononuclear cells from normal
human donors (G-PBMC). The macroscopic clusters of osteoclasts grown from G-PBMC were not detected in cultures
of normal marrow or nonmobilized peripheral blood.
STEOCLASTS are large, multinucleated cells involved
in bone resorption. They are derived from hematopoietic stem cells within the bone marrow, and their precursors
can be detected in low frequencies in the peripheral circulation.'.' Osteoclast progenitors are considered to proliferate
and differentiate into mononuclear preosteoclasts. Mature
osteoclasts form by the fusion of these preosteoclasts into
polykaryons, apparently due to local hormonal infl~ences.~.'
In vitro, studies of the development and function of osteoclasts have been limited to systems in which cells are grown
in the presence of osteoblasts, stroma, growth factors, or
hormones."' These studies have demonstrated that osteoclasts can be grown from granulocyte-macrophage colonyforming units, which also give rise to monocytes and
macrophages.'".' ' However, the factors influencing the
commitment of granulocyte-macrophage colony-forming
units into osteoclast progenitors are unclear. It appears that
la,2S-dihydroxyvitamin D3 has a role in increasing osteoclast differentiation,'* and osteoclasts are grown in the presence of this vitamin in most culture systems in which they
are currently studied. '.'" Furthermore, a murine osteoclastspecific colony-stimulating factor has been isolated from a
murine mammary carcinoma and shown to selectively stimu-
From the Program in Transplantution Bivlogy, Clinicuf Resetirch
Division, Fred Hutchinson Cancer Research Center, und the Departments of Biological Structure and Medicine, Universiiy vf Washington, Seattle, WA.
Submitted June 12, 1995; accepted October 16, 1995.
Supported by Grants No. DK34431, CA18221, CA18029, and
HL36444 from the National Institutes .;fHealth, Department of
Health and Human Services, Bethesda. MD.
Address reprint requests to Louise E. Purton, PhD, Fred Hutchinson Cancer Research Center, I124 Columbiu St, M-318, Seattle.
WA 98104.
The publication costs of this article were defrayed in part by page
charge puyment. This urticle must t h e r e j k be hereby marked
"advertisement" in uccordance with 18 U.S.C. section I734 solely to
indicate this ,fact.
0 1996 by The American Society of Hematology.
0006-497//96/8705-0018$3.00/0
1802
MATERIALS AND METHODS
Donors, marrow. and peripherul blood mononuclear celi processing. Marrow, nonmobilized PBMC, and G-PBMC were obtained from normal donors after provision of informed consent as
defined by the Internal Review Board at the Fred Hutchinson Cancer
Research Center (Seattle, WA). G-PBMC donors were mobilized
with recombinant human G-CSF at 16 wg/kg/d (Amgen Inc, Thousand Oaks. CA) by administering two subcutaneous injections per
day for 5 days, 4 days before G-PBMC collection and once after
the first collection. Leukapheresis was performed using a continuousflow blood cell separator (Cobe Laboratories, Lakewood, CO) on 2
consecutive days beginning on day S of rhG-CSF administration.
Samples for experiments were taken either from the first or second
day of collection. Cells were suspended in Hanks balanced salt
solution ([HBSS], GIBCO, Gaithersburg, MD) and centrifuged at
200 X g for 6 minutes to remove platelets before hemolysis in
hemolysis buffer (ammonium chloride I SO mmol/L, and sodium
bicarbonate I2 mmol/L).
Marrow was obtained from allogeneic donors by aspiration from
the iliac crest under general anesthesia. Samples were collected into
10% Normosol R (Abbott Laboratories, North Chicago, IL) supplemented with I O IU/mL preservative-free heparin. Cells were suspended in HBSS and centrifuged at 200 X g for 10 minutes to
remove platelets before hemolysis in hemolysis buffer.
Culture conditions. Hemolyzed huffy coat preparations of each
population were suspended in alpha minimal essential medium
([uMEM] GIBCO) supplemented with 10% fetal calf serum (FCS).
penicillin (100 U/mL), and streptomycin sulfate (100 p,g/mL) (complete aMEM). Viability of the cells was assessed by trypan blue
exclusion, The cultures were established at varying cell concentraBlood, Vol 87,No 5 (March l), 1996:pp 1802-1808
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OSTEOCLASTS IN G-PBMC
1803
t
Ir
Fig 1. Formation of osteoclasts from aggregatesderived from G-PBMC. Inverted phase-contrast micrographs show (AI attached aggregates
at 48 hours of culture after nonadherent cells were removed (original magnification ~ 1 0 0 )(BI
. release of T lymphocytes from aggregates at
10 days of culture (original magnification x 100). (C) resolution of aggregates and emergence of large multinucleated cells at 18 days (original
magnification x400). and (D) growing foci of adherent cells at day 28. Giant multinucleated cells are evident (original magnification ~ 4 0 0 ) .
tions, with optimal growth obtained at 5 X IO6 viable cells/well in
six-well plates (Costar, Cambridge, MA).
Cultures were placed in incubation at 37°C with 5% CO2 in air
atmosphere. After 3 days, nonadherent cells were removed from the
cultures. Adherent populations were washed three times with HBSS
and then fed with complete (uMEM and returned to incubation. Cultures were fed every 3 to 4 days with complete (uMEM.
Analysis of nonadherent cells produced bv the cultures. Adherent cells were cultured for 7 days, washed in HBSS, and refed with
complete (rMEM. Nonadherent cells were then retrieved from these
cultures at 3- to 4-day intervals. These cells were centrifuged (400
8 for IO minutes), supernatant was removed, and the cells were
incubated with normal mouse serum to block nonspecific binding
sites. Cells were then stained with Leu4 (phycoerythrin [PE]-conjugated mouse IgGl anti-CD3; Becton Dickinson, San Jose, CA),
fluorescein isothiocyanate (FITC)-conjugated mouse IgG 1 anti-CM
(Amac Inc. Westbrook, ME), FITC-conjugated mouse IgG 1 antiCD25 (Becton Dickinson), and PE-conjugated mouse IgG2a antiHLA-DR (Becton Dickinson). Staining was performed for 20 minutes on ice. Control staining was performed simultaneously using
isotype-matched, irrelevant antibodies directly conjugated to FITC
and PE. Cells were washed twice in HBSS/l% bovine serum albumin
(BSA) and analyzed on a FACSCAN flow cytometry system (Becton
Dickinson).
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PURTON, LEE, AND TOROK-STORB
1804
control FlTC
CD25 FlTC
..
CD4 FITC
CD25 FlTC
Fig 2. flow cytofluorometric analysis of viable nonadherent cells
releasedfrom adherent aggragates in G-PBMC cultures. Histograms
represent twocolor analysis of cells contained within a forward and
orthogonal light-scattering gate established to exclude dead cells
(not shown). Histograms as labeled indicate that nonadherent cells
express CD3, CD4, CD25, and HLA-DR vcontrol calls incubatedsimultaneously with irrelevant mouse IgGl-FITC, IgG1-PE, and IgG2a-PE.
Phenotypical analysis by cytochemical staining. Cultured cells
were stained for tartrate-resistant acid phosphatase (TRAP) activity.
Cell populations were washed three times in phosphate-buffered
saline (PBS), fixed in 10% (voVvol) neutral buffered formalin, and
air-dried. Populations were then preincubated for 1 hour at 37°C in
a 0.2-mom Tris-buffered solution (pH 9.0) and stained for TRAP
activity in the presence of 10 mmol/L L(+)-tartaric acid (Sigma
Chemical CO, St Louis, MO) as previously des~ribed.'~.''Cells containing TRAP activity stained a distinctive red color.
Analysis of calcitonin receptor expression. The presence of calcitonin receptors on the cells was investigated according to a previously described method.16Cells were grown on eight-well chamber
slides for 2 weeks. Populations were then incubated with 0.2 nmoVL
human '251-calcitonin(specific activity, 1,634 Ci/mmolL, Peninsula
Labs, Belmont, CA) in aMEM containing 0.1% BSA for 1 hour at
room temperature. Nonspecific binding was assessed by adding an
excess amount (300 nmol/L) of unlabeled human calcitonin (Sigma
Chemical CO) to some chambers. Background was estimated by
incubating some wells in medium alone. After labeling, the populations were washed in cold PBS and then fixed in 10% neutral buffered formalin. The dried slides were then coated with NBT-2 emulsion (Kodak, Rochester, NY), exposed in the dark at 4°C for 2
weeks, and developed. Cells were counterstained with Diff-Quik
Solution I1 (American Scientific Products, McGaw Park, IL) and
evaluated for calcitonin receptor expression using a Zeiss (Oberkochen, Germany) Axiovert 35 inverted light microscope.
Expression of the vitronecrin receptor. The monoclonal antibody, 23C6 (kindly provided by Dr Michael A. Horton, London,
England) recognizes the integrin a,/&vitronectin receptor that is
present on the membranes of osteoclasts."~'* The presence of this
antigen on each of the cell populations was determined by indirect
immunofluorescence using a biotin-streptavidin-FITC coupling
method. Cells were grown on eight-well chamber slides, washed in
PBS, and fixed in 2.5% paraformaldehyde in 0.1 m o m phosphate
buffer containing 4% sucrose.
The cells were first incubated in 1% BSA in PBS for 30 minutes,
and then incubated with mouse IgGl antihuman 23C6 diluted 1:50
or an isotype-matched irrelevant primary antibody for 40 minutes
at 4°C. The cells were washed in PBS three times and then incubated
at m m temperature with a biotinconjugated goat antimouse IgG
(Southern Biotechnology Associates, Inc. Birmingham, AL), diluted
1:20 in PBS/I% BSA for 30 minutes, followed by an FITC-conjugated streptavidin (Southern Biotechnology Associates, Inc), diluted
1:20 in PBS for 1 hour. Cell autofluorescence and nonspecific binding of the biotin-streptavidin-FITC were also investigated. Nuclei
were counterstained with 1 pg/mL 4', 6diamidine-2 phenylindoledihydrochloride (DAPI). Human foreskin fibroblasts were used as a
negative control.
The cells were washed thoroughly, mounted, and analyzed using
a Deltavision SA3 wide-field deconvolution microscope (Applied
Precision, Inc, Mercer Island, WA) coupled to a cooled-CCD camera
(Photometrics, Inc. Tucson, AZ)under a Nikon 60X objective (Chiyoda-Ku, Tokyo, Japan). Color images were transferred to the Macintosh-based program Photoshop 2.5 (Adobe Systems, Mountain
View, CA), from which they were printed as hard copies using a
Phaser IISD color printer (Tektronix, Beaverton, OR).
Analysis of myeloid and lymphoid antigens. Direct immunofluorescence was used to examine the presence of myeloid and
lymphoid antigens over the first 2 weeks of culture. The cells were
first incubated in 1% BSA in PBS for 30 minutes and then incubated
with a PE-conjugated LeuM3 mouse IgG2b anti-CDl4 (Becton
Dickinson) diluted 1:20 or an FITCconjugated IOT3b mouse IgGl
anti-CD3 (Amac, Westbrook, ME) diluted 1:20 for 30 minutes at
4°C. The cells were washed in PBS three times, and the nuclei
were stained with DAPI (1 pg/mL,). Controls included staining with
isotype-matched nonbinding irrelevant primary antibodies. The cells
were fixed in 2.5% paraformaldehyde in 0.1 mol/L PBS containing
4% sucrose, mounted, and viewed by confocal microscopy as described earlier.
Demonstration of resorption lacunae. Sterilized dentine slices
(8 x 8 x 0.1 nun) were prepared from cow teeth and placed at the
bottom of Costar 48-well plates. G-PBMC were plated over the
dentine slices at a density of 1 x IO6 cells/well, in complete aMEM.
These cultures were incubated, as already specified, for 21 days.
The supernatant was then removed, and dentine slices were washed
in PBS.
Cells were removed from the dentine by immersing the slices in
B
Fig 3. Osteoclast characteristics of mononuclaar and multinucleated cells grown from G-PBMC aggregates. (A and B) Expression of the
integrin a.&-vihonectin receptor by multinucleated cells. Specific binding of antivitronectln receptor monoclonal antibody 23C6 is revealed
by green fluorescence after incubation with goat antimouse IgG-biotin and avidin-FITC (A). Only DAPI-stained nuclei are apparent in control
G-PBMC populations incubated with an isotype-matched, nonbindingcontrol antibody plus biotintonjugated goat antimouse IgG and avidinFITC IB) (original magnification ~400).
(C) TRAP staining of adherent cells revealsthat many mononuclear precursors and large multinucleated
cells are TRAP-positive, as indicatedby the red color (original magnification ~ 4 0 0 )ID
. and E)Autoradiographyof ['"ll-human calcitonin binding
to cultures of G-PBMC. Many grains are seen localized over the multinucleated cell incubated with ['%human calcitonin in the absence of
excess unlabeled calcitonin (D). In the presence of excess unlabeled human calcitonin, only background grains are detected (E) (original
magnification x500).
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OSTEOCLASTS IN G-PBMC
L!
1805
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PURTON, LEE, AND TOROK-STORB
1806
50% (vollvol) sodium hypochlorite in distilled water for 30 minutes
at room temperature. Dentine slices were then washed in distilled
water, briefly sonicated, dehydrated in ethanol. and air-dried.
Mounted samples were sputter-coated with 15 nm gold and viewed
in a Jeol JSM-840A scanning microscope (Peabody, MA)."
RESULTS
Normal donor G-PBMC cultured in complete aMEM
formed nonadherent aggregates within 24 hours. Within 72
hours, the aggregates adhered to the culture plates (Fig IA).
In six experiments using different sources of G-PBMC, aggregate formation occurred at a frequency of 5.8 t 1.3/10s
cells when cells were plated at a density of 1 X IO' to I X
10" cells/cm'. At less than an initial plating density of 1 X
10" cellslcm', the frequency decreased to less than 1/10'
cells.
The aggregate formation observed with G-PBMC was not
detected in cultures of normal peripheral blood or marrow;
however, single large, multinucleated, TRAP-positive cells
could be grown from both of these cell sources. However,
these were rare events, occurring at a frequency of less than
1/10" cells plated.
Once the aggregates present in G-PBMC were attached
and it was possible to remove all nonadherent cells, it became apparent that small round nonadherent cells were being
generated by the aggregates (Fig 1B). The newly produced
nonadherent cells, collected and analyzed by immune cytochemistry and flow cytometry, were found to express CD3,
CD4, CD25, and HLA-DR, suggesting they were activated
T lymphocytes (Fig 2). The adherent aggregates continued to
generate T cells for approximately 4 weeks. In three separate
experiments using three different sources of G-PBMC, the
number of aggregates formed by 5 X lo6 cells plated in a
IO-cm2surface area was 327 2 45.42. In these experiments,
the number of T cells generated between days 7 and 14 of
culture was 877.6 +- 125.9 per aggregate. Between days 14
and 21, production decreased to 392 ? 31.5 per aggregate,
and from days 21 to 28 it was 20.3 2 6.8 per aggregate.
Between 1 and 2 weeks of culture, large multinucleated
cells with clear cytoplasm became visible under the aggregates (Fig 1C). Many of these cells appeared to be formed
by cell fusion. The number of these giant fused cells increased with time over a period of 4 weeks. During this
time, the aggregates resolved into complex monolayers (Fig
ID). These cells adhered strongly to the culture flasks, and
this adhesion was trypsin-resistant.
The osteoclast identity of the cultured cells was demonstrated by their expression of the vitronectin receptor (Figs
3A and 3B). cytoplasmic TRAP activity (Fig 3C), and the
presence of calcitonin receptors (Figs 3D and E). In contrast,
these cells were negative for the myeloid antigen CD14 (data
not shown). Finally, after 21 days of culture, the candidate
osteoclasts were capable of forming resorption lacunae on
dentine slices, hence demonstrating the essential features of
osteoclasts (Fig 4).
Different media were tested for their ability to support the
growth of the osteoclasts. The medium supporting optimal
growth of osteoclasts was aMEM containing 10% FCS. Formation of these cells was supported to a lesser degree by
Fig 4. Scanning electron microscopy of a dentine slice cocultured
with aggregates formed from G-PBMC, showing two resorption lacunae. The smaller patterned recesses are dentinal tubules (original
magnification ~ 8 5 0 ) .
RPMI (GIBCO) containing 10% FCS, but not by long-term
culture media that contains horse serum and calf serum."
medium 199 (GTBCO) containing 20% FCS, or serum-deprived medium containing 1% Nutridoma (Boehringer
Mannheim, Indianapolis, IN).
DISCUSSION
This study describes a method by which macroscopic
clusters of osteoclasts can be grown in culture from their
progenitors in the absence of stromal cells, cell lines,
growth factors, or la,25-dihydroxyvitamin D3. Growth
of large clusters of osteoclasts was associated with cell
aggregates, the formation of which was dependent on the
number of cells plated per surface area. Optimal growth
occurred in aMEM containing 10% FCS. with G-PBMC
plated at 5 x 10' cells/well in a six-well plate. Under these
conditions, 327 ? 45.2 macroscopic foci of osteoclasts
were detected per well.
Multinucleated osteoclasts were distinguished from
macrophage polykaryons by the presence of osteoclastspecific membrane receptors for calcitonin and the integrin
a,p,-vitronectin, and by expression of TRAP. Functionally, these cells were capable of forming resorption lacu-
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OSTEOCLASTS IN G-PBMC
1807
ACKNOWLEDGMENT
nae on dentine slices, hence confirming their osteoclast
nature. In addition, these cells did not express the myeloid
The authors sincerely thank Paul Goodwin and Tim Knight for
antigen, CD14, which has been shown to be present on
assistance with image analysis, Liz Caldwell for assistance with
giant macrophages but not on human osteoclasts.20Formascanning electron microscopy, and Bonnie Larson, Julie Sentkowski,
tion of mature, multinucleated osteoclasts appeared to ocand Harriet Childs for preparing the manuscript.
cur by cell fusion, in agreement with previous investigat i o n ~ . ~ .These
’~
mononuclear progenitors were also
REFERENCES
positive for all of the osteoclast markers examined, in
1. Suda T, Takahashi N, Martin TJ: Modulation of osteoclast
accordance with other reports.l6,”
differentiation. Endocr Rev 13:66, 1992
Osteoclasts could also be grown from normal peripheral
2. Scheven BAA, Visser JWM, Nijweide PJ: In vitro osteoclast
blood and marrow under the given culture conditions;
generation from different bone marrow fractions, including a highly
enriched haematopoietic stem cell population. Nature 321:79, 1986
however, they occurred as single, relatively rare osteo3. Walker DG: Osteopetrosis cured by temporary parabiosis. Sciclasts, not as large clusters of osteoclasts as seen with Gence 180:875, 1973
PBMC. In the current study, osteoclasts in all cultures
4. Ash P, Loutit JF, Townsend KMS: Osteoclasts derive from
formed in the absence of l a , 25dihydroxyvitamin D3,
hematopoietic stem cells according to marker, giant lysosomes of
contradicting previous observation^.^,^ Our results indicate
beige mice. Clin Orthop Re1 Res 155:249, 1981
that vitamin D3 is not required for osteoclast formation.
5. Baron R, Neff L, Tran Van P, Nefussi JR, Vignery A: Kinetic
Osteoclast precursors arise in the marrow and migrate in
and cytochemical identification of osteoclast precursors and their
the body via the circulation. It is not surprising therefore
differentiationinto multinucleated osteoclasts. Am J Path01 122:363,
that these cells can be grown from peripheral blood and
1986
marrow. However, it would appear that osteoclast precursors
6. Roodman GD, Ibbotson KJ, MacDonald BR, Kuehl TJ, Mundy
in G-PBMC differ either qualitatively or quantitatively from
GR: 1,25-Dihydroxyvitamin D3 causes formation of multinucleated
those in marrow or normal blood, since the aggregate formacells with several osteoclast characteristics in cultures of primate
marrow. Proc Natl Acad Sci USA 82:8213, 1985
tion that precedes development of large clusters of osteo7. Fuller K, Chambers TJ: Generation of osteoclasts in cultures
clasts did not occur with cells from these other sources. The
of rabbit bone marrow and spleen cells. J Cell Physiol 132:441,
mechanisms responsible for increased detection of osteoclast
1987
progenitors in G-PBMC are unknown. However, it is reason8. Takahashi N, Akatsu T, Udagawa N, Sasaki T, Yamaguchi A,
able to speculate that the marrow hypercellularity induced
Moseley JM, Martin TJ, Suda T: Osteoblastic cells are involved in
by G-CSF may stimulate production of osteoclasts in an
osteoclast formation. Endocrinology 123:26OO, 1988
effort to remodel bone and accommodate hematopoiesis. The
9. Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T,
reported increase in osteoporosis seen in some chronic neuNishihara T, Koga T, Martin TJ, Suda T: Origin of osteoclasts:
tropenic patients receiving G-CSF therapy supports this hyMature monocytes and macrophages are capable of differentiating
pothesis.**
into osteoclasts under a suitable microenvironment prepared by bone
Although mobilization by rhG-CSF appears to be crucial
marrow-derived stromal cells. Proc Natl Acad Sci USA 87:7260,
for the formation of these osteoclast progenitors, it is un1990
10. Kurihara N, Suda T, Miura Y, Nakauchi H, Kodama H, Hiura
likely that G-CSF has a direct effect on the formation of
K, Hakeda Y,Kumegawa M: Generation of osteoclastsfrom isolated
these clusters, since a previous study has indicated that it
hematopoietic progenitor cells. Blood 7 4 1295, 1989
does not stimulate formation of TRAP-positive colonies
1 1. Hattersley G, Kerby JA, Chambers TJ: Identification of osteofrom murine hematopoietic ~ e l 1 s . lHowever,
~
it is possible
clast precursors in multilineage hemopoietic colonies.Endocrinology
that the different cellular composition of G-PBMC versus
128:259, 1991
normal peripheral bloodz3 is a critical factor. G-PBMC could
12. Suda T, Takahashi N, Abe E: Role of vitamin D in bone
contain a greater number of osteoclast precursors, making
resorption. J Cell Biochem 4953, 1992
the likelihood of aggregate formation greater. Alternatively,
13. Lee MY, Eyre DR, Osbome WRA: Isolation of a murine
there could be qualitative differences among G-PBMC that
osteoclast colony-stimulating factor. Proc Natl Acad Sci USA
favor osteoclast development.
88:8500, 1991
In this regard, the production of CD4-positive T lympho14. Lee MY, Lottsfeldt JL, Fevold KL: Identification and characcytes in G-PBMC cultures is of interest, since other investiterization of osteoclast progenitors by clonal analysis of hematopoigations have shown that lymphocytes are capable of supportetic cells. Blood 80:1710, 1992
15. Liu C, Sanghvi R, Bumell JM, Howard GA: Simultaneous
ing the differentiation of osteoclasts. Lymphoid culture
demonstration of bone alkaline and acid phosphatase activities in
conditions have been shown to support the production of
plastic-embedded sections and differential inhibition of the activities.
murine colony-forming unit osteoclast^,^^ and interleukin-3
Histochemistry 86559, 1987
secreted by activated T lymphocytes has been implicated as
16. Takahashi N, Akatsu T, Sasaki T, Nicholson GC, Moseley
being involved in both increasing the number of marrow
JM,
Martin TJ, Suda T: Induction of calcitonin receptors by l a , 25cells that become TRAP-positive and inducing the fusion of
dihydroxyvitamin D, in osteoclast-like multinucleated cells formed
TRAP-positive mononuclear cells into p o l y k a r y o n ~ . ~ ~
from mouse bone marrow cells. Endocrinology 123:1504, 1988
Whether G-PBMC have a greater number of osteoclast pro17. Horton MA, Lewis D, McNulty K, Pringle JAS, Chambers TJ:
genitors, a more favorable accessory function, or both, reMonoclonal antibodies to osteoclastomas (giant cell bone tumors):
mains to be determined. Nevertheless, these studies show
Definition of osteoclast-specific cellular antigens. Cancer Res
that relatively pure cultures of osteoclasts can be obtained
455663, 1985
from G-PBMC.
18. David J, Warwick J, Horton M: Biochemical characterization
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1808
of human osteoclast-specific antigens defined by monoclonal antibodies 13c2 and 23c6. J Bone Miner Res 1:4, 1986 (abstr)
19. Roecklein BA, Torok-Storb B: Functionally distinct human
marrow stromal cell lines immortalized by transduction with the
human papilloma virus E6E7 genes. Blood 85:997, 1995
20. Athanasou NA, Alvarez JI, Ross FP, Quinn JM, Teitelbaum
SL: Species differences in the immunophenotype of osteoclasts and
mononuclear phagocytes. Calcif Tiss Int 50:427, 1992
21. Kurihara N, Chenu C, Miller M, Civin C, Roodman GD:
Identification of committed mononuclear precursors for osteoclastlike cells formed in long term human marrow cultures. Endocrinology 126:2733, 1990
22. Bishop NJ, Williams DM, Compston JC, Stirling DM, Pren-
PURTON.
LEE, AND
TOROK-STORB
rice A: Osteoporosis in severe congenital neutropenia treated with
granulocyte colony-stimulating factor. Br J Haematol 89:927, 1995
23. Dreger P, Haferlach T, Eckstein V, Jacobs S, Suttorp M,
Loffler H, Muller-Ruchholtz W, Schmitz N: G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: Safety,
kinetics of mobilization, and composition of the graft. Br J Haematol
87:609, 1994
24. Lee TH, Fevold KL, Muguruma Y, Lottsfeldt JL, Lee MY:
Relative roles of osteoclast colony-stimulating factor and macrophage colony-stimulating factor in the course of osteoclast development. Exp Hematol 22:66, 1994
25. Barton BE, Mayer R: E-3 induces differentiation of bone marrow precursor cells to osteoclast-like cells. J Immunol 143:321 l , 1989
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1996 87: 1802-1808
Normal human peripheral blood mononuclear cells mobilized with
granulocyte colony-stimulating factor have increased
osteoclastogenic potential compared to nonmobilized blood
LE Purton, MY Lee and B Torok-Storb
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