REVIEW
Differentiation and function of osteoclasts
Takeshi Miyamoto and Toshio Suda
The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on September 9, 2002)

Abstract. Osteoclasts, which are responsible for bone resorption, are rare cells with only 2–3 cells
seen per 1 mm3 of bone. However, the loss of function in osteoclasts, problems with their differentiation and decrease in their number lead to bone osteosclerosis/osteopetrosis. On the other hand, anincrease in their number or function induces bone osteoporosis, indicating that osteoclasts play apivotal role in bone homeostasis. It has been demonstrated that bone destruction and hypercalcemia
induced by metastatic tumors are carried out by osteoclasts activated by the tumor cells, and the inhibition of osteoclast formation prevents the bone destruction and even bone metastasis. Abnormal
osteoclast function is closely related to various diseases. Furthermore, osteoclasts are indispensable informing bone marrow to produce blood cells, and the absence of osteoclasts causes osteopetrosis,
resulting in extramedullary hematopoiesis. Although the physiological roles of osteoclasts are welldescribed, the mechanisms of their differentiation remain to be elucidated. Recently, RANK (receptor
activator of nuclear factor kappaB) and its ligand (RANKL) have been identiﬁed and their essential
roles in osteoclastogenesis have been demonstrated, which has provided new insights into the osteoclast differentiation pathway. We have established an in vitro osteoclast culture system by isolating
osteoclast precursor cells and culturing them in the presence of macrophage colony stimulating factor(M-CSF) and soluble RANKL. This system has enabled us to analyze the regulation mechanisms inosteoclast formation. (Keio J Med 52 (1): 1–7, March 2003)
Key words: osteoclast, differentiation, bone resorption

with bone resorption). Therefore, osteoclasts are indispensable to maintain bone homeostasis.
Hematopoiesis shifts to the bone marrow concomitantly with the formation of the bone marrowcavity from the fetal liver. The bone is formed throughchondrocytes or the mesenchymal membrane. Proliferating chondrocytes prevent the vascular invasion
mediated by angiogenesis-inhibitory factors such as
chondromodulin in the early embryonic stage.2 In thelater stage, hypertrophic chondrocytes differentiatingfrom chondrocytes in the center of the bone induce
vascular invasion, mediated by angiogenic factors suchas vascular endothelial growth factors (VEGFs) (Fig.
2). The induced vascular system establishes the blood
supply to, and circulation in the bony structures, andit supplies osteoclasts, which resorb the bone to create
the bone marrow cavity ﬁlled with hypertrophic andmineralized chondrocytes. Hematopoietic stem cells

Role of Osteoclasts
In postnatal life, bone is continuously remodeledby osteoblasts and osteoclasts, which synthesize bonematrix and resorb bone, respectively. Osteoblasts arederived from mesenchymal stem cells with a potential
to differentiate to chondrocytes, adipocytes and stromalcells (Fig. 1).1 On the other hand, osteoclasts arederived from hematopoietic stem cells (Fig. 1). These
two kinds of cell, though of different origins, cooperatewith each other to maintain homeostasis of bony tissue.
Any imbalance between bone formation by osteoblasts
and bone resorption by osteoclasts causes bone abnormalities including osteopetrosis (due to a defect inosteoclastogenesis or the absence of functional osteoclasts), osteosclerosis (due to increased bone formation) and osteoporosis (due to increased bone resorption or a relative decrease in bone formation compared

Reprint requests to: Dr. Toshio Suda, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan, e-mail: sudato@sc.itc.keio.ac.jp1

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Miyamoto T and Suda T: Differentiation and function of osteoclasts

Characterization of Osteoclasts

Fig. 1 Differentiation pathway of mesenchymal stem cells and hematopoietic stem cells. Key players in bone formation, osteoblasts andosteoclasts belong to different cell lineages. Osteoclasts are derived
from hematopoietic stem cells, whereas osteoblasts are from mesenchymal stem cells.

The deﬁnition of osteoclasts is ‘‘bone resorbing
cells’’. Osteoclast differentiation has various characteristic features, such as multinucleation induced by thecell fusion of mononuclear osteoclasts to cover a larger
area, synthesis of the vacuolar proton pump and acid todissolve the bone mineral, formation of rufﬂed borders
to secret protons and acid, and formation of a sealingzone to prevent proton and acid leakage.
Mononuclear osteoclasts tightly adhere to bone, andfuse with each other to form multinuclear osteoclasts.
The fused cells are re-organized, polarized and construct osteoclast-speciﬁc structures. Transcytosis develops from the basal to the apical side of osteoclasts toenable the discharge of dissolved bone debris.3 Multinucleation tartrate-resistant acid phosphatase (TRAP)
activity and the calcitonin receptor (CTR) are available
markers to detect osteoclasts, other than their bone
resorbing activity.
Differentiation of Osteoclasts(HSCs) lodge in the stromal cells of the newly formed
bone marrow, whereafter medullary hematopoiesis
begins. In the absence of osteoclasts, the bone marrowcavity is not formed and extramedullary hematopoiesis
takes place in the spleen, liver and kidney.

Before 1980, the origin of multinuclear osteoclasts
was controversial, with arguments existing between
osteogenic precursors and HSCs, but the latter wereshown to be the origin of osteoclasts.4 Osteoclasts are

Fig. 2 Bone marrow formation. Bone is formed through chondrocytes. The hypertrophic chondrocytes give rise to mineralized or apoptotic
chondrocytes at the center of the bone, then vascular cells invade toward the mineralized/apoptotic chondrocytes. Subsequently, osteoclasts
develop in the bone marrow and enlarge the cavity through resorption, and hematopoiesis begins.

Keio J Med 2003; 52 (1): 1–7

Fig. 3 Development of osteoclasts from hematopoietic stem cells(HSCs). HSCs give rise to monocyte/macrophage progenitor cells,
and then cells differentiate into mononuclear osteoclasts, which
express various osteoclastic molecules such as vitronectin receptor(integrin a v b3 ), TRAP, CTR and MMP9. Mononuclear osteoclasts
already have bone resorbing activity, and they fuse with each other toform multinuclear osteoclasts. The cytoskeletal system is reorganizedin multinuclear osteoclasts to generate osteoclast-speciﬁc structures
such as the sealing zone, rufﬂed borders, and a transcytosis systemdevelops to discharge the resorbed bone debris.

derived from HSCs through monocyte lineage progenitor cells (Fig. 3).5,6 Mononuclear osteoclasts express
enzymatic activity of TRAP,7 CTR, vitronectin receptor (integrin a v b3 ) and matrix metalloproteinase(MMP) 9. Although mononuclear osteoclasts also resorb bone, multinucleation is brought about by cell fusion of mononuclear osteoclasts. This multinucleation is
the most characteristic feature of osteoclasts. Cell fusion is not an osteoclast-speciﬁc phenomenon, but isobserved in other tissues such as myotubules and placentas. However, amongst these cell types osteoclasts
are the largest, with more than 100 nuclei seen in someosteoclasts. The mechanism involved in the cell fusion
is not clariﬁed, but a mechanism similar to viral fusion ispostulated, since some inhibitors of viral fusion prevent
osteoclast fusion (unpublished data). Multinucleatedcells are highly polarized cells and well re-organize toform new cytoskeletal constructions such as a sealingzone and rufﬂed borders. The polarity is strictly established in osteoclasts. The sealing zone and rufﬂed
border develop in the basal side and the bone debris

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resorbed from the basal area is transferred via the cytoplasm to the apical side.
An in vitro study on osteoclastogenesis has been
performed in a co-culture system between osteoclast
precursor cells derived from bone marrow or spleenmononuclear cells with stroma cells in the presenceof osteotropic factors.8 In this system, bone marrowderived stroma cells (ST2, MC3T3E1 or MC3T3-G2PA6) or primary mouse calvariae-derived osteoblasts
were cultured as osteoclastogenesis-supporting cells,in the presence of osteotropic factors such as 1,25dihydroxy-vitamin D3 (1,25(OH)2 D3 ), parathyroid hormone (PTH), dexamethasone (DEX), prostaglandin
E2 , and gp130 family proteins such as interleukin (IL)6, IL-11, oncostatin M or IL-1. Cell-to-cell contact
between osteoclast precursor cells and stroma cells is
required in this co-culture system,9 and this evidence
provides the speculation that the osteoclastogenesis
inducible molecule might be expressed on the stroma
cells in the presence of osteotropic factors. This molecule was isolated from the stroma cells by the expression cloning technique and was named RANKL(also called osteoclast differentiation factor (ODF)/
osteoprotegerin ligand (OPGL)/TNF-related activation induced cytokine (TRANCE).10–14 RANKL is a
type 2 transmembrane protein and a member of
TNFa superfamily. RANKL has two receptors, one is
RANK initially isolated from the dendritic cell cDNA
library10 and the other is the osteoprotegerin (OPG)/
osteoclastogenesis inhibitory factor (OCIF). This soluble receptor15,16 inhibits osteoclastogenesis by preventing the RANK-RANKL interaction. The former
is expressed in osteoclast progenitors and the latter isexpressed in many tissues including osteoclast inducible
stroma cells. 1,25(OH)2 D3 and DEX induce expression of RANKL whereas they inhibit the expression of
OPG/OCIF in stroma cells. The balance of the expression level between RANKL and OPG/OCIF is criticalfor osteoclast formation.
In order to analyze the differentiation of osteoclasts
in detail, an in vitro culture system is crucial. Although
RANKL is a transmembrane protein, the extracellularregion has sufﬁcient activity to induce osteoclast differentiation.11,12 Thus osteoclasts are formed in the presence of macrophage colony stimulating factor (M-CSF)
and a soluble form of RANK (sRANKL) instead ofstroma cells.
Cell Density and Multinucleation
We have improved and established a more sophisticated culture system by isolating osteoclast precursor
cells from murine bone marrow.17 To isolate osteoclast precursor cells, we studied expressions of c-Fms,
a receptor of M-CSF and RANK, since M-CSF plus

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Miyamoto T and Suda T: Differentiation and function of osteoclasts

Fig. 4 Stroma-free culture system. Osteoclast precursor cells were isolated from bone marrow mononuclear cells on the basis of the expressionof c-Fms and RANK (A), and cultured with M-CSF alone or M-CSF and sRANKL for 6 days in a stroma-free culture system (B). Cultured cells
were subjected to TRAP-solution assay (C) or TRAP-staining (D). Note that the TRAP-activity was greatest in c-Fmsþ RANK cells cultured
with M-CSF and sRANKL (C). Large numbers of multinuclear TRAP-positive cells were observed among c-Fmsþ RANK cells (Da), while
only mononuclear TRAP-positive cells developed from c-Fmsþ RANKþ cells (Db), and c-Fms RANKþ cells did not proliferate in this culture(Dc). Bar ¼ 100 mm.

RANKL induce osteoclast differentiation in a stroma
cell-free culture in vitro. Bone marrow mononuclear
cells were subdivided into three fractions by cell surface markers (c-Fmsþ RANK , c-Fmsþ RANKþ , cFms RANKþ ) (Fig. 4). Each fraction was sorted by
FACS and cultured in the presence of M-CSF andRANKL for 6 days and TRAP staining was performed
to analyze the osteoclastogenesis. Multinuclear TRAPpositive osteoclasts were contained in c-Fmsþ RANK
cells, while only mononuclear TRAP-positive cells
were detected in c-Fmsþ RANKþ cells and no TRAPpositive cells were seen in c-Fms RANKþ cells (Fig. 4).
The difference in multinuclear formation between cFmsþ RANK and c-Fmsþ RANKþ is caused by the celldensity, and the difference of cell density results fromthe proliferating activity between these fractions.17 cFmsþ RANK fraction showed colony-forming activity
in the presence of M-CSF alone or M-CSF andsRANKL, while c-Fmsþ RANKþ did not. Multinuclear
osteoclasts were formed from c-Fmsþ RANKþ cells
when ten times more cells were cultured suggesting

adequate cell density is required to fuse and form multinuclear osteoclasts.17
From the results above, c-Fmsþ RANK cells havethe highest multinuclear osteoclast formation activity,
however, these cells do not express RANK. We haveshown that M-CSF induced RANK expression in thec-Fmsþ RANK cells ﬁrst, and then RANKL induced
the differentiation signal (Fig. 5).18 In the absence ofRANKL, these cells differentiated into macrophages
through a default pathway.
Anchorage-dependent Osteoclastogenesis
Osteoclast formation is restricted to the bone surface, suggesting that bone provided a suitable environment for osteoclastogenesis. Although the expressionof RANKL is relatively limited in osteoblasts, it is also
expressed in other cells and tissues, such as T cells andspleen cells. We therefore studied the effect of adherent
or non-adherent culture conditions on osteoclast proliferation and differentiation. We prepared the adher-

Keio J Med 2003; 52 (1): 1–7

5

Fig. 5 Differentiation model of osteoclasts, macrophages and dendritic cells from common precursor cells. A determination of osteoclast anddendritic cell differentiation from common precursor cells is shown.

ent condition in plastic culture dishes with a liquid medium to permit cell adhesion, whereas the non-adherent
condition was prepared using a methylcellulose semisolid culture medium to inhibit cell adhesion to thesubstrate. Under adherent condition, osteoclastogenesis with multinucleation was induced.17 However,under non-adherent condition, osteoclast formation was
reduced to approximately 30% compared with the adherent condition and no multinuclear osteoclasts wereformed.17 The reduced differentiation activity to osteoclasts seen under non-adherent condition was recovered,
when the cells were transferred to the adherent condition, indicating that osteoclasts progenitors can differentiate into osteoclast through adhesion to the matrix.
Alternatively, osteoclastogenesis was stimulated byoligomerized RANKL under non-adherent conditions.
RANKL is a member of the TNFa superfamily known
to act as a trimer form. Actually, the trimerizedRANKL called leucin-zipper RANKL (LZ-RANKL)
effectively induced osteoclastogenesis compared withthe monomer form even under non-adherent conditions. Thus, adhesion might be required to transduce
the efﬁcient RANK signaling for osteoclastogenesis. On
the other hand, multinucleation was not fully induced
by LZ-RANKL. This suggests that the polarity ofcell adhesion is important for cell fusion. Anchorage-

dependent osteoclastogenesis might be involved in theprevention of ectopic osteoclast formation.
Dendritic Cell Differentiation from
Osteoclast Precursor Cells
Although the osteoclast differentiation is reduced
under non-adherent conditions, it is not completely
inhibited, suggesting that some other osteoclastogenesis
inhibitory system might be involved in tissues otherthan bone. Lymphoid tissues including the spleen andlymph nodes express the molecules for osteoclastogenesis in vitro such as M-CSF and RANKL. However,no osteoclast formation is induced in these tissues.
We analyzed the effect of GM-CSF expressed inlymphoid tissues on osteoclast differentiation.5 Osteoclastogenesis was completely inhibited by GM-CSF at
an early stage of differentiation in a dose-dependent
manner, and cells were committed to a dendritc celllineage, judging from the fact that CD11c and DEC205
were expressed by GM-CSF. Therefore, osteoclast precursor cells have the potency to differentiate into
osteoclasts, macrophages and dendritic cells (Fig. 5).
GM-CSF did not inhibit expression of c-Fms and
RANK, suggesting that the signals of M-CSF andRANKL might be transduced even in the presence of6Miyamoto T and Suda T: Differentiation and function of osteoclasts

GM-CSF. However, among the intracellular molecules
critical for osteoclast differentiation, c-fos expression isstrongly inhibited by GM-CSF. Overexpression of c-fos
overcomes the inhibitory effect of GM-CSF on osteoclastogenesis. It is suggested that c-fos suppression is an
important event for inhibition of osteoclast differentiation. In dendritic cell differentiation, GM-CSF induces
RANK expression in common precursor cells, andRANKL induces the terminal differentiation. On thecontrary to osteoclastogenesis, dendritic cell differentiation is inhibited by M-CSF or c-fos expression.17
Most recently, interferon-beta (IFN-b) was reported
to inhibit osteoclastogenesis in a RANKL-induced c-fos
dependent manner, which is a different mechanism
from viral infection.19 RANKL induces the c-fos expression in osteoclast precursor cells, and c-fos induces
IFN-b which in turn inhibits the c-fos expression in anegative-feedback regulation. Thus various molecules
effectively affect the osteoclast/dendritic cell/macrophage common precursor cells and regulate their differentiation.

of daily living. Recently, bisphosphonates have beenused therapeutically to prevent osteoclast-induced bone
fractures and hypercalcemia.

Malignant Tumor Cells Induce Osteoclasts
via RANKLIn adult T-cell leukemia (ATL) patients, hypercalcemia is induced concomitantly with an increased
number of osteoclasts and accelerated bone resorption. Several cytokines, such as PTHrP, IL-1 and TGFbeta are implicated in ATL associated hypercalcemia.
These cytokines indirectly induce osteoclastogenesis via
stroma cells. However, from the aspect that increased
osteoclastogenesis is mainly observed in the local areaof tumor invasion, we speculated that ATL cells might
express a membrane associated protein and directly
induce osteoclastogenesis. Among the molecules, westudied the expression of RANKL. ATL cells frompatients with hypercalcemia induced osteoclast differentiation, whereas ATL cells from non-hypercalcemic
patients did not.20 ATL cells from hypercalcemic
patients expressed high levels of RANKL, whereas cells
from normocalcemic patients did not. A soluble receptor for RANKL inhibited osteoclastogenesis, indicating that RANKL expressed by ATL directly induced
osteoclast differentiation. As observed in ATL, otherbone metastatic tumors such as breast cancer andmultiple myeloma cells express RANKL, and induce
osteoclastogenesis. These lines of evidence indicate that
pathological bone fractures and hypercalcemia causedby bone metastasis are induced by osteoclasts activatedby tumor cells. These fractures sometime cause paralysis, and the presence of hypercalcemia is associatedwith a poor prognosis. The regulation of osteoclastogenesis in the metastatic bone tumor is an important
problem when trying to maintain the patient’s activities

Future Aspects
There are several diseases which cause bone destruction such as osteoporosis, bone metastases andbone marrow invasion from malignant tumors. If we areable to regulate osteoclastogenesis, bone destruction
might be prevented and the quality of life of the patientis improved. We intend to regulate bone diseases, andnow we are trying to isolate osteoclast-speciﬁc molecules by the DNA subtraction method between osteoclasts and macrophages derived from common precursor cells. Since these two types of cells express quite
similar molecules, osteoclast-speciﬁc molecules havebeen successfully identiﬁed. We have already cloned
several molecules and investigated their function on
osteoclastogenesis. These osteoclast-speciﬁc molecules
are strong candidates to target the regulation of osteoclast function. In the near future, we will hopefully beable to regulate osteoclast activity to prevent bone destruction by osteoclasts.
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