Stem Cells, Vol. 15, No. 5, 340-346,
September 1997
© 1997 AlphaMed Press
Conditions that Support Long-Term Production of Osteoclast Progenitors In Vitro
Minako Y. Lee,
Karen L. Fevold,
Yukari Muguruma,
Joan L. Lottsfeldt
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington, USA
Key Words. Osteoclast • Progenitor • CFU-O • Long-term bone marrow culture • G-CSF
Correspondence:
Dr. Minako Y. Lee, Department of Biological Structure, Box 357420, University of Washington School of Medicine, Seattle, WA 98195, USA.
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Abstract
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To better understand the mechanisms of osteoclast precursor development from hematopoietic stem cells, we examined the conditions that support the production of osteoclast progenitors, osteoclast colony-forming units (CFU-O), from long-term bone marrow cultures established under myeloid (Dexter's) and lymphoid (Whitlock and Witte's) conditions. Nonadherent cells harvested weekly from myeloid or lymphoid long-term cultures were assayed for CFU-O-derived colony formation in agar in the presence of a murine osteoclast colony-stimulating factor. The myeloid system supported CFU-O production for weeks, but the system produced many other types of myeloid colonies and cells as well, and quantification of CFU-O-derived colonies was difficult. The lymphoid long-term culture system also produced CFU-O; however, CFU-O production in the lymphoid system appeared more selective than in the myeloid system, but was transient. Interestingly, the addition of medium containing G-CSF to these cultures greatly enhanced (>200%) the CFU-O production. This enhanced CFU-O production was confirmed using bone marrow cultures established on a defined marrow stromal cell line under lymphoid conditions and supplemented with recombinant murine G-CSF. Thus, G-CSF facilitates the development of clonogenic osteoclast progenitors from hematopoietic stem cells in lymphoid long-term culture conditions. This culture system may serve as a useful model for ex vivo generation of osteoclast progenitors.
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Introduction
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It is well established that osteoclasts are derived from hematopoietic stem cells [1]; however, the mechanisms of immature osteoclast development from the complex hematopoietic system are still unclear. Since Testa et al. first demonstrated the formation of multinucleated osteoclast-like cells from feline bone marrow cells cultured in a modified Dexter's long-term bone marrow culture system [2], a number of investigators have used similar culture conditions and observed the formation of osteoclast-like cells from the bone marrow of various species [3-5]. Multinucleated osteoclast-like cells generated in such cultures in the presence of 1,25-dihydroxyvitamin D3 have been shown to fulfil the functional criteria of osteoclasts [1]. In these studies, short culture periods, e.g., five to six days for murine [5], and two to three weeks for primate bone marrow cells [3], were required to generate osteoclast-like cells, suggesting that these cells were formed from terminally differentiated osteoclast precursors, preosteoclasts. The long-term bone marrow culture conditions described by Dexter et al. [6] support myelopoiesis and generate myeloid progenitor colony-forming units (CFU-cs) for months. Culture conditions described by Whitlock and Witte [7], on the other hand, support long-term lymphopoiesis. These long-term culture systems are useful for in vitro studies of stromal-cell-dependent progenitor cell differentiation from relatively primitive stem cell populations. Long-term culture conditions that support the production of clonogenic osteoclast progenitors, the more immature precursor cell population than preosteoclasts, have not been described. Our recent discovery of an osteoclast colony-stimulating factor (O-CSF) has made it feasible to identify and quantify clonogenic osteoclast progenitors, osteoclast colony-forming units (CFU-O) [8-9]. In this study, we attempted to define favorable culture conditions for the recruitment and differentiation of osteoclast progenitors by examining the generation of CFU-O from long-term murine bone marrow cell cultures established under myeloid [6] or lymphoid [7] conditions.
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Materials and Methods
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Mice
Ten- to twelve-week-old (BALB/c x CE) F1 mice of both sexes were used as bone marrow cell donors [10]. These mice were bred in the vivarium of the University of Washington where they were fed and cared for according to the NIH guidelines. Parental male CE and female BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were sacrificed following the protocol approved by the University of Washington Animal Care Committee. Bone marrow cells were flushed from femurs and suspended in RPMI 1640 medium (GIBCO BRL; Grand Island, NY) containing 5% fetal calf serum (FCS) (Hyclone; Logan, UT).
Bone Marrow Stroma Adherent Cell Layer
Adherent stromal cell layers were established in 25-cm2 tissue culture flasks with vented filter caps (Costar; Cambridge, MA) as follows: in the Dexter's myeloid culture system, 6.5 x 106 bone marrow cells were placed in 8 ml Dexter's medium consisting of alpha minimal essential medium (MEM), 20% horse serum (Hyclone), 2 mM l-glutamine, 10–6 M hydrocortisone, and 0.5% antibiotic solution (GIBCO), and flasks were incubated at 33°C in 5% CO2 with weekly refeeding as described [6]. In the Whitlock and Witte's lymphoid culture system, 6.5 x 106 bone marrow cells were placed in 6.5 ml lymphoid medium consisting of RPMI 1640 (GIBCO), 5% heat-inactivated FCS (Hyclone), 2 mM l-glutamine, and 5 x 10–5 M 2-mercaptoethanol [7], incubated at 37°C in 5% CO2; twice a week, culture media were replenished using the established protocol [7]. In some experiments, stromal cell monolayers were established in lymphoid culture medium using the murine bone marrow stromal cell line, S17, which has been shown to support B lymphopoiesis [11].
Inoculation of Stromal Cultures with Recharge Cell Population
Bone marrow cells were obtained from femurs of two to three normal mice as described above. Adherent cells were depleted from the cell suspensions by passing 5 x 107 to 1 x 108 cells over a 10-ml column of Sephadex G-10 (Pharmacia; Piscataway, NJ) [10]. The eluted cells, enriched for immature mononuclear cells, were cultured on confluent stromal cell layers at a concentration of 1 x 106 cells/flask. To inoculate cultured monolayers of S17 cells, cells expressing B220 antigen were depleted from Sephadex G10 column passed marrow cell suspensions using a panning technique with 14.8 hybridoma supernatant as described [10]. Cultures were subsequently fed weekly with 4 ml of the appropriate media per flask according to the established protocol [6-7]. After the bone marrow cell inoculation, cultures were fed weekly and 4 ml samples containing nonadherent cells were retained. The number of viable cells produced per flask was determined by Trypan blue dye exclusion, and these cells were used for CFU-O assays (see below). In some experiments, flasks were supplemented with various conditioned media (see below).
CFU-O Assays
Clonogenic osteoclast progenitors in the nonadherent cell population from myeloid and lymphoid culture flasks were identified by their ability to form characteristic colonies in semisolid agar culture medium in response to O-CSF [8-9]. These colonies were composed of mononuclear cells strongly positive for tartrate-resistant acid phosphatase (TRAPase), a cytochemical enzyme marker for the osteoclast. Osteoclastic properties of these TRAPase-positive cells have been previously documented [8, 12]. Briefly, 5 x 104 viable cells/ml were cultured in 15 x 10 mm Linbro wells (0.5 ml/well) (Flow Laboratories; McLean, VA) with supplemented medium 199 (Whittaker BioProduct; Walkersville, MO) containing 20% FCS, 0.3% agar, and optimal concentrations of CESJ medium as a source of O-CSF (see below). After two weeks of incubation, the agar cultures were transferred to glass slides, stained for TRAPase activity, and counterstained [8]. TRAPase-positive cells were stained bright red, distinctive from the blue color of TRAPase-negative cells. Colonies, defined as groups of 50 or more cells, and clusters, 8 to 50 cells, were then categorized as TRAPase-positive (>90% of cells demonstrating TRAPase), TRAPase-mix (10%-90% positive), and TRAPase-negative (<10% positive) and then enumerated using light microscopy. For practical reasons, TRAPase-positive colony and cluster numbers were combined to assess the number of CFU-O. Macrophage progenitors were analyzed by their ability to form colonies of macrophages in response to macrophage CSF (M-CSF) (L-cell medium). Multilineage progenitors were analyzed by their colony formation in response to murine stem cell factor (SCF) (a gift from Dr. S. Lyman, Immunex).
Conditioned Media
Conditioned medium (CM) containing O-CSF was prepared by growing a clone (CESJ) of a bone resorbing murine mammary tumor [8] in serum-free HL-1 medium (Ventrex, Hycor Biomedical; Portland, ME) to confluence. A large batch of CESJ culture supernatant was concentrated 500-fold over 10,000-molecular weight cutoff membranes, filtered (.22 µm, Durapore filters, Millipore; Marlborough, MA) and stored in aliquots at -70°C. This concentrated medium, defined as CESJ medium, was used at optimal concentrations to stimulate CFU-O-derived colonies in assays. For supplementing lymphoid long-term culture medium, CESJ cells, clonal Bc66 mammary tumor cells [8-9], C127 (MG-2) cells [13], or NIH 3T3 fibroblasts were cultured in lymphoid medium described above, and conditioned media were concentrated 10-fold over 10,000 molecular weight cutoff membranes. CESJ cells produce G-CSF and M-CSF in addition to O-CSF but not GM-CSF, or IL-3 [9, 14]. Bc66 cells produce M-CSF, but not O-CSF, G-CSF, GM-CSF, or IL-3 [9, 14]. C127 (MG2) cells produce recombinant murine (rm) G-CSF [13]. Conditioned medium of NIH 3T3 cells was used as a control. Recombinant mG-CSF and polyclonal antibody to mG-CSF were kind gifts from Dr. Shigekazu Nagata (Osaka BioScience Institute).
Data Analysis
Cell and colony data from long-term bone marrow cultures were obtained from two to three repeated experiments containing at least three flasks per experiment. Agar colony assays were performed in duplicate. Means, standard deviations (SD), and standard errors of mean (SE) were calculated, and the Student's t-test was used for mean comparisons. Differences were considered significant for p values of 0.05 or less.
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Results
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Production of CFU-O in Myeloid Long-Term Culture Conditions
Nonadherent cells harvested weekly after recharge from myeloid long-term culture flasks for up to eight weeks were examined for CFU-O. Progenitors that form TRAPase-positive colonies or clusters in response to CESJ medium were present among the harvested cells throughout the observation period ( Fig. 1). The incidence as well as the number of progenitors per flask reached a plateau at three to six weeks post-recharge. The morphology of TRAPase-positive colonies and clusters formed by CFU-O from myeloid culture flasks was spreading and diffuse with a high background of TRAPase-negative granulocytes and macrophages present. The colony types and numbers formed by progenitors produced from representative myeloid and lymphoid cultures are shown in Table 1. As expected, M-CSF or SCF responsive progenitors were also produced from myeloid cultures. CFU-O colonies became smaller and clusters predominated as the myeloid cultures were kept longer. Modest numbers of mixed colonies containing TRAPase-positive and -negative cells were also observed throughout the culture period (data not shown). Thus, the myeloid long-term marrow culture system can support the production of CFU-O for weeks after inoculation with bone marrow cells containing hematopoietic stem cells.
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Figure 1. CFU-O production from myeloid long-term bone marrow cultures: stromal cell culture flasks were established under myeloid conditions and recharged with normal bone marrow cells depleted of stromal cells. Nonadherent cells generated in culture flasks were analyzed weekly for CFU-O content. The incidence of CFU-O in harvested cells (open bars) and the absolute number of CFU-O per flask harvest (closed triangles) are shown. Values are means ± SD of data obtained from four flasks at each time point.
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Table 1. Osteoclast and hematopoietic progenitors produced from myeloid and lymphoid long-term bone marrow cultures
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Production of CFU-O in Lymphoid Long-Term Culture Conditions
Clonogenic osteoclast progenitors were also present in the nonadherent cell population generated under lymphoid culture conditions ( Fig. 2, Table 1). In contrast to the colonies formed from progenitors cultured under myeloid conditions, the progenitors from lymphoid culture flasks formed discrete TRAPase-positive colonies or clusters in agar assays in response to O-CSF; there were very few mixed colonies or other background cells present. Fewer CFU-O were generated in lymphoid cultures than in myeloid cultures. M-CSF responding macrophage progenitors were also produced from lymphoid cultures, but only a small number of SCF responding progenitors were produced.
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Figure 2. CFU-O production from lymphoid long-term bone marrow cultures: stromal cell cultures were established in lymphoid conditions and recharged with normal bone marrow cells depleted of adherent cells. Nonadherent cells generated in culture flasks were weekly examined for CFU-O. The CFU-O incidence (open bars) and the absolute number of CFU-O per flask harvest (closed triangles) are shown. Values are means ± SD of data obtained from four flasks at each time point.
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Supplementation of Lymphoid Culture Medium
We speculated that the lymphoid culture conditions may be less favorable for supporting the survival and recruitment of osteoclast progenitors than the myeloid conditions. To evaluate this further, lymphoid culture medium was supplemented with CESJ medium that contains O-CSF, G-CSF, and M-CSF [8, 14]. Starting at four weeks post-recharge when the production of CFU-O had almost ceased, cultures were fed weekly with the lymphoid culture medium supplemented with CESJ medium or control HL-1 medium at 10% (v/v). To our interest, the production of CFU-O increased remarkably and lasted for weeks in flasks supplemented with CESJ medium ( Fig. 3). This suggested that a component or components in CESJ medium supported continuous generation of CFU-O from a more immature progenitor compartment in these cultures.
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Figure 3. Continued generation of CFU-O from lymphoid long-term cultures supplemented with CESJ medium: long-term bone marrow cultures were established under lymphoid conditions. Starting at four weeks post-recharge when the CFU-O production had almost ceased, cultures were fed weekly with lymphoid medium supplemented with CESJ medium or control HL-1 medium at 10% (v/v). The CFU-O incidence in the harvested nonadherent cell population is shown. Values are mean ± SD from three to five flasks at each time point. Closed circles: supplemented with CESJ medium; open circles: supplemented with HL-1 medium; open triangles: no supplementation.
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Production of CFU-O in Lymphoid Cultures Established on Defined Marrow Stromal Cells
To examine the possible effects of growth factors on production of CFU-O in culture, a defined stromal cell line, S17 [11], was used to establish the stromal monolayer under lymphoid culture conditions. The established stromal cell monolayers were recharged with normal bone marrow cells depleted of adherent cells and B220+ cells. Culture flasks were fed twice a week with lymphoid culture medium supplemented with medium conditioned with CESJ (O-CSF, G-CSF, M-CSF), Bc66 (M-CSF), C127 (MG2)(rmG-CSF), or NIH 3T3 cells. Two weeks later, nonadherent cells were harvested and analyzed for CFU-O in agar colony assays. Data are summarized in Figure 4. A significant increase in the number of CFU-O was observed in cultures supplemented with CESJ or MG2 medium but not in flasks supplemented with Bc66 or 3T3 medium. These data suggested that G-CSF, but not M-CSF, could be the factor enhancing the CFU-O generation in lymphoid culture conditions.
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Figure 4. Effect of growth factor-containing medium on CFU-O production from bone marrow cultures supported by S17 stromal cells: established monolayers of S17 stromal cells in lymphoid culture condition were inoculated with normal bone marrow cells depleted of adherent cells and B220+ cells. Cultures were fed twice a week with lymphoid culture medium supplemented with the following: CESJ (O-CSF, G-CSF, M-CSF), Bc66 (M-CSF), MG2 (rmG-CSF), or NIH 3T3 (irrelevant control) conditioned medium at 10% (v/v) each. Nonadherent cells were harvested two weeks later and examined for CFU-O production. Values are means ± SE obtained from four separate experiments. * p < 0.05 when compared with no CM control.
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Effect of G-CSF on CFU-O Production
To test the effect of G-CSF in CESJ medium, a pre-tested neutralizing titer of rabbit polyclonal antibody to murine G-CSF [10] or normal rabbit serum (NRS) was added to the lymphoid medium supplemented with CESJ medium and this medium was used for twice-weekly feeding of cultures established on S17 monolayers as described above. The incidence of CFU-O significantly decreased from 32.0 ± 2.8/5 x 105 cells in flasks supplemented with CESJ plus NRS to 18.0 ± 5.6/5 x 105 cells with CESJ plus anti-G-CSF, and the absolute number of CFU-O decreased from 10.1 x 103/flask with CESJ plus NRS to 2.3 x 103/flask with CESJ plus anti-G-CSF. Furthermore, when recombinant murine G-CSF was added to lymphoid culture medium, the production of CFU-O significantly increased compared with the medium control ( Table 2). These findings confirmed that G-CSF had the ability to enhance the production of CFU-O from long-term bone marrow cultures under lymphoid conditions.
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Table 2. Effect of recombinant murine G-CSF on CFU-O production from cultures established on S17 cells under lymphoid culture conditions
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Discussion
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This study documents for the first time that O-CSF-responsive clonogenic osteoclast progenitors, CFU-O, can be generated in long-term cultures of murine bone marrow cells established either under myeloid or lymphoid conditions. Although long-term cultures established under lymphoid culture conditions could support progenitor production, it was transient and did not last as long as the production of osteoclast progenitors from myeloid culture conditions. Osteoclast progenitors may enter a dormant stage in lymphoid cultures, or possibly the recruitment of the osteoclast progenitor from a more primitive stem cell compartment is not favorable under these culture conditions. When such lymphoid cultures were supplemented with conditioned media containing G-CSF but not M-CSF, the production of osteoclast progenitors increased. This phenomenon is most likely attributed to the effect of G-CSF added in the lymphoid cultures. G-CSF regulates proliferation and maturation of neutrophilic granulocyte progenitors, but it also has the capacity to activate cell cycle dormant primitive progenitors [15]. Thus, the G-CSF supplement, in synergy with other stromal cell-derived factors, might have triggered cell division of the dormant primitive stem cells and recruited more progenitors into the O-CSF-responsive progenitor cell pool. Our long-term culture system established in a lymphoid condition and supplemented with G-CSF may serve as a useful model for studies of osteoclast progenitor generation in vitro. In addition, a recent report that treatment of normal individuals with human G-CSF causes the appearance of osteoclast precursors in circulation [16] suggests that our findings on the effect of G-CSF on osteoclast production may have clinical relevance.
A number of studies have documented that osteoclasts are derived from precursor cells present in the hematopoietic tissue; however, the precise mechanisms involved in the steps of osteoclast development from hematopoietic stem cells remain controversial [1]. Even the identity of osteoclast progenitors is uncertain. While studies by Kurihara et al. [17] have suggested a subpopulation of CFU-GM contained osteoclast precursors, others have claimed differentiation of osteoclasts from macrophage progenitors, and even from mature macrophages [18]. Others have suggested that osteoclast progenitors are in a primitive hematopoietic stem cell population [19]. Our studies of O-CSF-responsive progenitors [9, 20] have advocated that there is a distinct progenitor population for the osteoclast lineage. Studies in this report further confirm the presence of such an osteoclast progenitor population among the descendants of inoculated hematopoietic stem cells.
The long-term bone marrow culture systems we employed here support the production of hematopoietic progenitor cells and stem cells over long periods of time. Our study has demonstrated that osteoclast progenitors can also be produced for weeks once appropriate culture conditions are provided. It is conceivable that immature cells in the osteoclast lineage require different factors than those required for osteoclasts in more mature stages. For instance, the production of clonogenic osteoclast progenitors in our system does not require 1,25-dihydroxyvitamin D3, which is required for the production of apparently more mature, multinucleated, osteoclast-like cells in cultures of bone marrow cells [1]. It is interesting to note that G-CSF suppressed the production of B lymphocytes under similar experimental culture conditions in our previous study [10]. There is evidence for a murine progenitor that has the capacity for differentiation into the myeloid as well as B-lymphoid lineages [21]. G-CSF may interact with other factors in the marrow stromal environment and shift the pattern of cell production from the lymphoid to the myeloid type which may include osteoclast progenitors.
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Acknowledgments
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We thank Dr. Shigekazu Nagata (Osaka Bioscience Institute; Osaka, Japan) for providing recombinant murine G-CSF and antibodies to murine G-CSF, and Dr. Kenneth Dorshkind (University of California; Riverside, CA) for providing S17 stromal cells. This work was supported in part from NIH grants, CA-38189 and AR-42657.
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References
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accepted for publication on May 27, 1997.
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Abstract
Introduction
