Bone Marrow Culture System Research Articles

Stimulatory effects of neopterin on hematopoiesis in vitro are mediated by activation of stromal cell function.

The pteridine neopterin (NP) was shown to be produced by monocytes and is known to be a useful marker of immunological activation, although, its biological activity is still unclear. Recently, we found that intravenous administration of NP increased the numbers of blood leukocytes, and granulocyte-macrophage progenitor cells (CFU-GM) in the bone marrow and spleens of mice. In order to elucidate the mechanism whereby NP stimulates hematopoiesis, the effects of NP on hematopoietic stem cell proliferation and differentiation in vitro were studied using a long-term bone marrow culture (LTMC) system with cloned stromal cell line, MS-5. Adding NP to the LTMC increased the numbers of cells in total, CFU-GM and colony-forming unit in spleen (CFU-S). NP also increased the number of CFU-GM in a soft agar culture system, but it did not enhance CFU-GM colony formation when target bone marrow cells were semi-purified (T, B and adherent cell-depleted bone marrow cells) and cultured in this system, suggesting that NP did not directly affect the proliferation of hematopoietic progenitors. Conditioned medium obtained from NP-treated stromal cells had much greater colony-stimulating activity than that obtained from untreated stromal cells. Furthermore, NP treatment stimulated the production of IL-6 and GM-CSF by stromal cells. All these findings suggest that NP stimulates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.

Inhibition of bone resorption by 17β‐estradiol in human bone marrow cultures

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17β-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17β-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17β-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17β-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17β-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17β-E2-induced inhibition of bone resorption. J. Cell. Physiol. 175:99–108, 1998. © 1998 Wiley-Liss, Inc.

Inhibition of bone resorption by 17beta-estradiol in human bone marrow cultures.

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17beta-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17beta-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17Beta-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17Beta-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17beta-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17beta-E2-induced inhibition of bone resorption.

23( S )25( R )-1,25-Dihydroxyvitamin D 3 -lactone, a naturally occurring metabolite of 1,25-dihydroxyvitamin D 3 , inhibits osteoclast-like cell formation in human bone marrow cultures

We have used a human bone marrow culture system that forms multinucleated cells (MNCs), 50% of which express the osteoclast phenotype, to examine the 23(S)25(R)-1,25-dihydroxyvitamin D3-26,23-lactone (1,25-D3-lactone) on osteoclast-like cell formation. The 1,25-D3-lactone is a vitamin D3 metabolite that has recently been detected in human serum under physiological conditions at concentrations of approximately 131 pg/ml (3 × 10−10 M) and can inhibit bone resorption induced by 1,25-dihydroxyvitamin D3 (1,25-D3) in vivo and in vitro. We examined the effects of the 1,25-D3-lactone on the formation of MNC that cross-reacted with 23C6 monoclonal antibody (23C6-positive MNC), which preferentially binds to osteoclasts. All metabolites of 1,25-D3 except the 1,25-D3-lactone increased both total and 23C6-positive MNC formation in a dose-dependent manner. In contrast, the 1,25-D3-lactone inhibited both total and 23C6-positive MNC formation, whether the cultures were treated with 1,25-D3, parathyroid hormone, or interleukin-1β, all potent stimulators of MNC formation. This inhibitory action of 1,25-D3-lactone on MNC formation was very similar to the inhibitory effects of calcitonin. These data suggest that (1) 1,25-D3-lactone is a potent natural inhibitor of formation of cells with the osteoclast phenotype at physiological concentrations and (2) the inhibition of these cells by 1,25-D3-lactone may not result solely from its competitive binding to the 1,25-D3 receptor.

Identification of an IL-7-Associated Pre-Pro-B Cell Growth-Stimulating Factor (PPBSF). II. PPBSF Is a Covalently Linked Heterodimer of IL-7 and aMr 30,000 Cofactor

AbstractEvidence is provided in a companion paper for an IL-7-associated molecular complex that selectively stimulates the proliferation and presumptive differentiation of pre-pro-B cells in our long-term bone marrow culture system and “primes” them to proliferate in response to monomeric IL-7. Here, Western immunoblot analysis reveals that this pre-pro-B cell growth-stimulating factor (PPBSF) is a self-assembling heterodimer of IL-7 and a cofactor with a Mr of 30,000. Thus, when developed with anti-IL-7 mAb, PPBSF migrates electrophoretically as a covalently bound ∼55-kDa molecule under nonreducing conditions but dissociates under reducing conditions. Furthermore, the addition of rIL-7 or native IL-7 to medium conditioned by stromal cells from IL-7 gene-deleted (−/−) mice results in the formation of active 45-kDa and 55-kDa molecular complexes, respectively. Antiserum prepared in IL-7(−/−) mice against affinity-purified PPBSF contained separable reactivities for IL-7 and the non-IL-7 component of PPBSF. The PPBSF cofactor detected by this antiserum migrates as an ∼30-kDa molecule and is able to maintain the viability, but not the proliferation, of pre-pro-B cells. Furthermore, the cofactor is produced constitutively by IL-7(−/−) and IL-7(+/+) bone marrow stromal cells under pro-B- but not pre-B-type culture conditions. Consequently, IL-7 appears to exist almost entirely as a heterodimer (i.e., PPBSF) in pro-B-type cultures, whereas it exists almost entirely as a monomer in pre-B-type cultures. Although the identity of the PPBSF cofactor remains to be determined, it does not appear to be stem cell factor, insulin-like growth factor-1, thymic stromal-derived lymphopoietin, flt3, stromal cell-derived factor-1, or IL-7R.

Engineering a human bone marrow model: a case study on ex vivo erythropoiesis.

Bone marrow, with its intricate, three-dimensional tissue structure facilitating cell-cell interactions, provides a microenvironment supporting the production of hundreds of billions of multilineal blood cells everyday. We have developed a three-dimensional bone marrow culture system in which marrow cells are cultured in a reactor packed with porous microspheres. The culture supports a three-dimensional growth configuration and multilineal hemopoiesis mimicking the bone marrow in vivo. We studied ex vivo human erythropoiesis using the three-dimensional culture system. The system sustained extensive erythropoiesis at low erythropoietin concentrations (0.2 U/mL), plus stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and insulin-like growth factor-I. Erythroid cell production lasted for more than 5 weeks, and the percentage of erythroid cells in the nonadherent cell population was approximately 60%. Flow cytometric analysis using cell surface markers specific for erythroid cells (CD71 and glycophorin-A) indicated that the culture produced early, intermediate, and late erythroid cells. As the culture progressed, the erythroid cell population shifted gradually toward mature cell types. When compared to the three-dimensional culture, the traditional flask cultures failed to support extensive erythropoiesis under the same conditions. This indicates that the three-dimensional bone marrow culture system provides a microenvironment conducive to erythropoiesis under more physiological conditions and is a better bone marrow model.

ONCOSTATIN M STIMULATES MACROPHAGE-POLYKARYON FORMATION IN LONG-TERM HUMAN BONE-MARROW CULTURES

Though oncostatin M (OSM) is a potent mediator of the inflammatory reaction, its role in inflammation and bone resorption is still unclear. A long-term bone-marrow culture system is usually developed to allow the formation of multinucleated cells (MNC) and was used here to define the effects of human recombinant OSM on human MNC formation. OSM significantly upregulated (1.9- to 5.6-fold) the number of MNC in these cultures in a dose- and time-dependent manner. Cell nucleation and tartrate-resistant acid phosphatase activity were also increased. MNC did not display osteoclast characteristics, such as response to calcitonin and failure to resorb dentin surface. However, they expressed a non-specific α-naphthyl acetate esterase as well as macrophage differentiation antigens (CD11b, CD13 and CD33) and were able to perform phagocytosis. Similar effects were observed after addition of 1α,25-dihydroxyvitamin D3. Moreover, in these culture conditions, human bone-marrow mononuclear cells were capable of low-grade resportion in the presence of bone-marrow stromal cells. This low grade resorption was significantly inhibited by addition of 25 ng/ml OSM. Our data demonstrate for the first time that human recombinant OSM significantly stimulates the formation of MNC and could be involved in the inflammatory process via macrophage-polykaryon formation from human bone marrow.

Regulation of osteoclastogenesis by antisense oligodeoxynucleotides specific to zinc finger nuclear transcription factors Egr-1 and WT1 in rat bone marrow culture system.

Differentiation of osteoclasts is defined by the transcription factors expressed in response to bone microenvironments. In this work, we examined the effects of an expressional blockage of Egr-1 and/or WT1 on the differentiation of osteoclasts using specific antisense oligodeoxynucleotides (ODN). In a culture system forming preosteoclast-like cells (POC) from rat bone marrow cells depleted of marrow stromal cells, POC formation was markedly stimulated by the addition of Egr-1 antisense ODN compared to that in cultures in which sense ODN was added, whereas Egr-1 antisense ODN inhibited the formation of macrophage-like cells. The formation of multinucleated osteoclast-like cells was also stimulated by the addition of Egr-1 antisense ODN in whole bone marrow cultures. In contrast, WT1 antisense ODN did not affect POC formation induced by the treatment with Egr-1 antisense ODN; however, WT1 antisense ODN dramatically suppressed the formation of osteoclast-like multinucleated cells induced by the blockage of Egr-1 expression using Egr-1 antisense ODN. These data suggest that Egr-1 acts as the suppressor, not as the inducer, in osteoclastogenesis. The findings also suggested that WT1 could be involved in the multinucleation step of osteoclastogenesis, at least when Egr-1 expression was blocked.

Regulation of Osteoclastogenesis by Antisense Oligodeoxynucleotides Specific to Zinc Finger Nuclear Transcription Factors Egr-1 and WT1 in Rat Bone Marrow Culture System

Regulation of Osteoclastogenesis by Antisense Oligodeoxynucleotides Specific to Zinc Finger Nuclear Transcription Factors Egr-1 and WT1 in Rat Bone Marrow Culture System

Reduction of bone loss by denbufylline, an inhibitor of phosphodiesterase 4

The effects of denbufylline, a xanthine derivative with selective inhibitory activity on the phosphodiesterase (PDE) 4 isoenzyme, on bone loss in Walker 256/S-bearing rats and on mineralized nodule formation and osteoclastlike cell formation in bone marrow culture systems were examined. Serial oral administrations of denbufylline inhibited the decrease in the bone mineral density of femurs from Walker 256/S-bearing rats, without influence on the healthy rats. Denbufylline restored the bone mass and the number of osteoclasts and osteoblasts per trabecular surface in the femur metaphysis. Among PDE inhibitors, only PDE4-selective inhibitors increased the number of mineralized nodules and decreased the number of osteoclastlike cells in the in vitro bone marrow culture systems, and dibutyryl cyclic AMP mimicked these effects in the in vitro systems. These results suggest that the PDE4 isoenzyme may play an important role in bone turnover through cyclic AMP and that its inhibitors are candidates for therapeutic drugs for the bone loss diseases.

Importance of parenchymal:stromal cell ratio for the ex vivo reconstitution of human hematopoiesis.

Many new developments in tissue engineering rely on the culture of primary tissues which is composed of parenchymal and mesenchymal (stromal) cell populations. Because stroma regulates parenchymal function, the parenchymal:stromal cell (P:S) ratio will likely influence culture behavior. To investigate parenchymal-stromal cell interactions, the P:S ratio was systematically varied in a human bone marrow (BM) culture system, measuring the output of mature cells, immature progenitors (colony forming units-granulocyte/macrophage [CFU-GM]), and primitive stem cells (long-term culture-initiating cells [LTC-IC]). When parenchymal CD34-enriched cells were grown without stroma, cell and CFU-GM output increased linearly as inoculum density was increased, resulting in constant cell and CFU-GM expansion ratios. On irradiated preformed stroma (IPFS), culture output was significantly higher and less dependent on CD34-enriched cell inoculum density, resulting in greater expansion ratios at lower inoculum densities. The number of IPFS cells required to support CD34-enriched cells was independent of the CD34-enriched cell number, suggesting that IPFS did not provide discrete niches, but instead acted through soluble signals. Experiments using conditioned medium (CM) from IPFS confirmed the presence of soluble signals, but CM did not completely substitute for direct contact between CD34-enriched cells and IPFS. Because of known differences between IPFS and stroma growing within BM mononuclear cell (MNC) cultures, experiments were next performed using mixtures of CD34-enriched and CD34-depleted fractions of MNC. When inoculated with a fixed CD34+lin- cell number, culture output was optimal near the P:S ratio of the unmanipulated MNC sample and declined as CD34- cell number was increased or decreased. In cultures inoculated with a fixed total cell number, CFU-GM output increased as CD34+lin- cell number was increased, whereas LTC-IC output reached a plateau. These data suggest that a limited number of LTC-IC supportive niches were present in MNC stroma, whereas IPFS lacks these niches and acts predominantly through a less potent soluble mechanism. These studies underscore the importance of parenchymal-stromal cell interactions in the ex vivo reconstitution of tissue function and offer insight into the nature of these interactions in the human BM culture system.

Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells.

The question of whether a retroviral vector is expressed in vivo after infection of hematopoietic stem cells remains unpredictable. In this study, we show that a switch off of retroviral sequences can be tested in vitro using the long-term bone marrow culture system. The time kinetics of the down-regulation of retroviral expression in vitro and in vivo have been demonstrated to be similar. The correlation of switch off events in both systems allows for evaluation of retroviral expression in vitro provided the silencing is caused by viral sequences. This approach could be useful for testing vectors for gene therapeutical applications of the hematopoietic system.

Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors.

The process of B lymphopoiesis changes with increasing age, resulting in a severe drop in the number of pre-B cells in old mice. We have shown that the ability of freshly isolated pro-B cells to proliferate on stromal cells declines with age. In this study, we wanted to determine the reason for the diminished response of the aged pro-B cells. The functional alterations could arise from changes intrinsic to the pro-B cells or to composition of the precursor population. Changes in the composition of pro-B cells in marrow or long-term bone marrow culture system for B lymphocytes (LTBMC-B) could not account for the functional losses. We then examined the hypothesis that the diminished amount of proliferation on stromal cells was due to changes in the responsiveness to stroma-derived cytokines. The proliferation to IL-7 but not to stem cell factor (SCF) or insulin-like growth factor 1 (IGF-1) was severely impaired by 24 mo of age, independently of the concentration of IL-7 and length of culture time. The reduced IL-7 response could not be explained by an increase in the percentage of cells undergoing apoptosis; rather, a greater frequency of aged cells remained in G0/G1 after IL-7 stimulation. Furthermore, the impaired responsiveness of the aged pro-B cells is not due to diminished expression of the IL-7R alpha-chain or the common gamma-chain. Since only IL-7 responsiveness is impaired, we believe that the underlying molecular mechanism for the cellular alterations is specific to signaling through the IL-7 receptor complex.

Long-term B-lymphoid cultures from murine bone marrow establishment and cloning by using stromal cell line AC 6.21.

Nearly all hematopoietic cells in mammals derive from precursors that undergo much or all of their development in the bone marrow. In vitro models for many lineages are available and represent modifications of the original bone marrow culture system designed by Dexter and Lajtha (1) and described elsewhere in this book. In this chapter, we describe a second bone marrow culture system, first reported in 1982 (2), that provides an in vitro environment selectively supporting long-term proliferation and differentiation of early B lymphocyte lineage cells. This method can be used to obtain heterogeneous populations of immature precursors of the B cell lineage greatly enriched from other hematopoietic cell types. Clonal populations can also be obtained by extension of this method to limiting dilution culture.

Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast formation induced by parathyroid hormone.

Prostaglandin (PG)E2 is one of the most important endogenous bone resorbing factors. In the previous study, we demonstrated that osteoclast formation induced by IL-1 beta was mediated by PGE2 produced by induced prostaglandin endoperoxide H synthase-2 (PGHS-2) in osteoclastic cells. In the same bone marrow culture system, indomethacin also suppressed the osteoclast formation induced by PTH. The inhibition was abolished by exogenously added PGE2 at dose as low as 3 x 10(-9) M, which was too low to elevate the intracellular cAMP and calcium levels and also it was too low to cause osteoclast formation by itself. In order to estimate what kind of cell produced such small amount of PGE2 in the PTH treatment, we carried out antibody staining of PGHS-1&PGHS-2 and PGHS activity in the intact bone marrow cells. PTH was found to induce PGHS activity in tartrate-resistant acid phosphatase (TRACP) positive mononuclear cells and the PGHS activity was inhibited by NS-398, a specific inhibitor of PGHS-2. Immunocytochemical staining supported the expression of PGHS-2 in TRACP-positive mononuclear cells. These findings suggest that PGHS-2 induced by PTH may regulate osteoclast formation by different mechanism from that induced by IL-1 beta.

Possible involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone treatment.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Influence of Photofrin® on the Hematopoietic Accessory Function of Murine Peritoneal Cells

The porphyrin photochemotherapeutic agent Photofrin® stimulates hematopoietic activity within the bone marrow (BM) and spleens of normal mice. We found that the intraperitoneal (i.p.) administration of Photofrin® also caused a 3–4 fold increase in peritoneal cell (PC) numbers, particularly cells bearing granulocyte surface antigens. Little granulocyte-macrophage progenitor activity was detectable within the PC population of control and Photofrin®-injected mice suggesting that Photofrin® had elicited an influx of inflammatory cells into the region. In contrast to cells from control animals, PC obtained 6 h to 168 h after the i.p. injection of Photofrin® exhibited a consistently inferior capacity to support the growth of BM colony forming units granulocyte-macrophage (CFU-GM). When PC from control or Photofrin®-treated mice were added to the BM culture system in the presence of defined growth factors there was no effect on the number of colonies formed. This finding indicated that negative regulatory elements were not responsible for the reduced hematopoietic accessory activity exhibited by PC from Photofrin®-treated mice. Supernatants conditioned by PC from Photofrin®-injected mice poorly supported BM CFU-GM growth in vitro and in contrast to PC supernatants prepared from control mice did not contain detectable amounts of granulocyte-macrophage colony stimulating factor (GM-CSF). The cellular changes which occur within the peritoneal cavity represent bystander events not directly related to the hematostimulatory action of Photofrin®.

Long-term bone marrow cultures in aplastic anaemia.

The long-term bone marrow culture (LTBMC) system provides an in vitro physiological model for the study of stromal cell mediated haemopoiesis in patients with aplastic anaemia. The two aspects of haemopoiesis--stromal and stem cell function--can be analysed separately using a modification of LTBMC with cross-over studies. Patients with aplastic anaemia universally demonstrate defective stem cell function in terms of reduced or absent marrow repopulating ability, reflecting a deficiency of long-term culture initiating cells. Defects in stromal cell function, as assessed by the ability of aplastic anaemia stroma to support normal generation of haemopoietic progenitors, are not common, but may conceal an isolated deficiency of a particular growth factor in some patients due to the overlapping nature of haemopoietic growth factor activities. The stem cell abnormality in aplastic anaemia reflects a deficiency in cell numbers, as well as dysfunction in certain cases. An increased level of apoptosis in aplastic anaemia marrow CD34+ cells exists, and this correlates well with disease severity. LTBMC studies demonstrate that more of the haemopoietic cells are nonviable (apoptotic and dead) compared with normal controls, and this correlates with reduced colony (CFU-GM) generation. An increase in apoptosis among primitive haemopoietic cells may contribute to the stem cell defect in aplastic anaemia. Haemopoietic growth factors such as G-CSF, when given after immunosuppressive therapy such as antilymphocyte globulin and cyclosporin for aplastic anaemia, may act partly by reducing the increased level of apoptosis, resulting in improved stem cell survival.

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Interleukin‐10 selectively inhibits osteoclastogenesis by inhibiting differentiation of osteoclast progenitors into preosteoclast‐like cells in rat bone marrow culture system

Recombinant human interleukin-10 (hIL-10) inhibited the formation of osteoclast-like multinucleated cells in rat whole bone marrow cultures. The effect of hIL-10 on the process of osteoclast formation was further examined, since the process of osteoclast formation includes the proliferation and the differentiation of osteoclast progenitors into mononuclear preosteoclasts and the fusion of preosteoclasts into multinucleated osteoclasts. In the nonadherent bone marrow cell culture system, which was free of stromal cells and formed preosteoclast-like cells, hIL-10 significantly inhibited the formation of preosteoclast-like cells even at a very low concentration (0.5 U/ml). The strong inhibition appeared even after treatment with hIL-10 for only the first 24 h of the culture. However, hIL-10 did not affect the fusion process of preosteoclast-like cells to form osteoclast-like multinucleated cells in the rat coculture system of preosteoclast-like cells with primary osteoblasts. Furthermore, hIL-10 completely inhibited the colony formation induced by granulocyte macrophage colony-stimulating factor (GM-CSF). These findings suggest that the inhibition of osteoclastogenesis by hIL-10 started at the early stage of the differentiation of osteoclast progenitors to preosteoclasts.