Int.

J. Exp. Path. (1992), 73, 387-401

An assessment of the ability of human bone marrow cultures to generate osteoclasts A.M. Flanagan, M.A. Hortont, E.L. Doreyt, D.A. Collins, R.S. Evely*, J.M. Moseley*, F.C. Firkin*, T.J. Chambers, M.H. Helfricht and T.J. Martin* Department of Histopathology, St George's Hospital Medical School, London, tICRF Haemopoiesis Unit, St Bartholomew's Hospital Medical College, London, UK and *Department of Medicine, St Vincent's Hospital, University of Melbourne, Australia

Received for publication 30 October 1991 Accepted for publication 5 February 1992

Summary. Several groups have successfully generated osteoclasts in cultures of murine haemopoietic cells. This approach would clearly be useful in the analysis of mechanisms of regulation of human osteoclast formation if analogous results could be obtained in cultures of human bone marrow. This communication describes independent attempts by three groups to generate unequivocally defined osteoclasts from bone marrow obtained from human iliac crest, femoral neck, rib, and from foetuses. The haemopoietic tissue was incubated using techniques described by others for production of osteoclast-like cells, and with variants of this technique using strategies based on our experiences with murine osteoclastogenesis. Haemopoietic cells were incubated with calcium regulating hormones, cytokines, osteoblastic supernatants, and osteoblastic or bone marrow stromal cell layers. Formation of cells capable of excavation of bone slices was rarely seen. Despite the paucity of bone resorbing cells, multinucleate cells (MNCs) developed with similar characteristics to the MNCs that have been interpreted as osteoclast-like in human bone marrow cultures. The MNCs were, however, calcitonin-receptor (CTR) negative, and did not show the typical pattern of reactivity with osteoclast-specific antibodies. They possessed instead an antigenic profile characteristic of macrophage polykaryons. We conclude that the MNCs which consistently generate in human bone marrow cultures do not possess phenotypic characteristics specific for osteoclasts and appear to be macrophage polykaryons. The conditions required for osteoclast generation in cultures of human haemopoietic cells remain to be defined.

Keywords: osteoclast, macrophage, bone resorption, calcitonin receptor, monoclonal antibody, vitronectin receptor The osteoclast is the cell that resorbs bone. Cross-circulation experiments, quail-chick chimaeras, and experiments in which bone marrow transplantation cures rodent and

human osteopetrosis have established that osteoclasts derive from haemopoietic precursors (Gothlin & Ericsson 1973; Walker 1973; Kahn & Simmons 1975; Loutit & Nisbet

Correspondence: Dr A.M. Flanagan, Department of Histopathology, St George's Hospital Medical School, Cranmer Terrace, London SWi 7 ORE, UK.

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A. M. Flanagan et al. 1982). Cells in rabbit and murine haemo- ies, Irvine, Scotland and Sydney, NSW, Auspoietic tissue can also be induced to generate tralia) was used for isolation of human and osteoclasts in vitro (Fuller & Chambers 1987; murine bone marrow cells and the washing Takahashi et al. 1988; Hattersley & of cell lines. All media used for incubation of Chambers 1989a). Three essential criteria cells contained heat-inactivated serum are used to define these cells as osteoclasts: unless otherwise stated: foetal bovine serum their ability to resorb bone, to express the (FBS), neonatal calf serum (NCS), horse CTR, and to develop from haemopoietic cells serum (HoS) were obtained from Gibco, under the obligate influence of 1,2 5-dihy- Paisley, Scotland and Flow, Australia and droxyvitamin D3 (1,25(OH)2 vitamin D3) on human serum (HuS) was obtained from an appropriate bone marrow stromal micro- normal adult volunteers. HoS was also environment (Hattersley & Chambers kindly donated by Dr D. Roodman (San Antonio, Texas). L-Glutamine (2 mM) 1989b; Udagawa et al. 1989). It would assist in the analysis of the (Glaxo, Middlesex, UK and Flow, Australia) mechanisms of regulation of osteoclast pro- and antibiotics, either benzyl penicillin (100 duction in man if analogous techniques IU/ml) and streptomycin (100 Mug/ml) could be applied to human bone marrow (Glaxo) or gentamicin (40 Mg/ml) (Flow) cultures. Although there have already been supplemented all media. Haemopoietic cells many published reports of stimulatory effects were cultured in one of the following media: of calcium-regulating hormones and cyto- Eagle's Minimal Essential Medium with kines on the formation of osteoclast-like cells Earle's Salts (Flow) (MEM: Earle's), alpha from human bone marrow (MacDonald et al. modified MEM (Flow) (aMEM), RPMI (Flow) 1986, 1987; Chenu et al. 1988, 1990; and Fischer's medium (Gibco). Hughes et al. 1989; Kukita et al. 1989, The following were generous gifts: inter1990a, b; Kukita & Roodman 1989; Taka- leukin-la and ,B (IL-la and (B) from Ciba hashi et al. 1989; Kurihara et al. 1990a, b; Geigy, Basle, Switzerland; interleukin 3 (ILThavarajah et al. 1990), the osteoclastic 3) and interleukin 6 (IL-6) from Dr S. Clark nature of these cells remains controversial. (Genetics Institute, Cambridge, MA); transThis communication describes the combined forming growth factor ( (TGF-P) from experience of three independent laboratories Genentech, Inc. CA; granulocyte monocyte which have applied rigorous criteria to the colony stimulating factor (GM-CSF) from Dr identification of the osteoclast phenotype, N. Nicola (Walter & Eliza Hall Institute, including in particular the capacity to resorb Melbourne, Australia); parathyroid horbone and the possession of CTRs, in an mone (PTH) from Dr J. Zanelli (National attempt to devise a culture technique in Institute for Biological Standards, Hampwhich osteoclasts can be induced to develop stead, London, UK); 1,25(OH)2 vitamin D3 from human haemopoietic cells in vitro. Our from Leo Laboratories, Bucks, UK and Roche experience, and that of others (Birch et al. Laboratories, Welwyn Garden City, UK and 1990), has been that the multinucleated salmon calcitonin (sCT) from Sandoz, Basle, osteoclast-like cells which are consistently Switzerland. Prostaglandin E2 (PGE2) and generated in our cultures of human bone hydrocortisone were purchased from Sigma, marrow cannot be identified as osteoclasts. Poole, Dorset, UK. The human osteoblastic cell lines HOS, U20S and MG63 were donated by Dr N. Teich (ICRF, Lincoln's Inn Materials and methods Fields, London, UK); SaOS2 by Dr G. Rodan (Merck Sharp & Dohme, Pennsylvania) and a Materials human bladder carcinoma cell line 5637, a Serum-free Hepes-buffered Medium 199 or cell line which secretes factors that induce Minimal Essential Medium (Flow Laborator- the formation of mixed haemopoietic col-

388

Multinucleate bone marrow cells onies (Myers et al. 1984), from Dr R. Clutterbuck (Royal Marsden Hospital, Surrey, UK). Devitalized bone slices were prepared from bovine cortical bone as previously described (Chambers et al. 1984).

Antibodies. The antibodies used were either commercially available (Coulter, Florida) or formed part of the panel of the 4th International Workshop and Conference on Human Leucocyte Differentiation Antigens (Knapp et al. 1989). One or more antibodies to the following specificities were tested: HLA class 1,HLADR;CD1lb, lic, 13, 14,15,16, 18, 33, 34, 45, 64, 68; vitronectin receptor axV(13C2), ,B3(Y2/51) and cxV,3 complex (23C6). Their cellular specificity and biochemical features have been described previously (Knapp et al. 1989). The majority react with monocytes and/or tissue macrophages; CD1 3 and 68 react with both osteoclasts and macrophages; and vitronectin receptor antibodies react preferentially with osteoclasts (Horton et al. 1985; Davies et al. 1989).

Methods Liquid human bone marrow culture. Human bone marrow cells were obtained from the posterior iliac crest of healthy volunteers, from rib and sternal marrow of haematologically normal patients undergoing thoracic surgery, from femoral necks of patients with myeloma and Paget's disease of bone, from patients with lymphoma undergoing autologous bone marrow harvests with no detectable bone marrow involvement, and from the long bones of 18-26-week foetuses. Marrow mononuclear cells were obtained by centrifugation on Ficoll/Hypaque gradients (Sigma). The cells were plated into tissue culture wells containing the stated volumes of medium; two-chamber flasks (1 ml) (Lab Tek, Naperville, IL); 96 (200 ul) or 24-(1 ml) well plates (Flow) containing plastic coverslips (Thermanox, Lux, Flow) and/or bone slices. In all experiments, marrow was cultured

389

in the presence and absence of 1,25(OH)2 vitamin D3 with or without other calcium regulating hormones, cytokines alone or in combination, or with conditioned medium from osteosarcoma cell lines (see Table 1). All cultures were fed once or twice weekly by replacing half the medium containing supplements or vehicles, and were incubated at 37°C in a humidified atmosphere of 5% CO2.

'Recharge' and co-culture experiments. Human bone marrow cells (106 cells per ml) were used to 'recharge' a semi-confluent human bone marrow stroma which had been sedimented onto bone slices and plastic coverslips at a density of 5 x 105 cells/ml (Dexter et al. 1977). Human bone marrow cells (106 cells/ml) were also co-cultured with an adherent cell layer derived from an explant of an osteoclastoma (5 x 105 cells/ml), a murine calvarial cell line ts8 (5 x I05 cells/ml) (Hattersley & Chambers 1990b), and a murine bone marrow cell line ST2 (4 x I04 cells/ ml) (Udagawa et al. 1989), the last two of which are known to support osteoclast generation from murine spleen cells in vitro. All stromal layers were sedimented onto bone slices and coverslips at the stated densities 24 hours prior to the addition of human and murine haemopoietic cells. Human bone marrow cells were also co-cultured with rat osteosarcoma UMR 106 cells in studies in which the two cell types were separated by a 0 45,m pore size membrane. UMR 106 cells were plated at a density of 106/ml in Millicell wells (Millipore, Bedford, UK) and replaced every 3 days. Millicells were placed in tissue culture wells overlying normal human bone marrow cells. Conditioned medium (CM) experiments. Human osteosarcoma cell lines HOS, U20S, MG63, SaOS2, human bladder carcinoma cell line 5637, rat osteosarcoma cell line UMR 106 and a fibroblastic stroma derived from an osteoclastoma explant were cultured in MEM: Earle's in a T75 flask (Flow) until confluent. The medium was removed and the cells washed before replenishing the

390

A. M. Flanagan et al.

cultures with 10 ml of serum-free medium containing 10-8 M 1,25(OH)2 vitamin D3. This medium was removed after 24 hours and added to cultures of human bone marrow cells at concentrations of 10 or 20%. Leucocyte conditioned medium (LCM) was prepared by culturing (106/ml) human peripheral mononuclear cells isolated by Ficoll/ Hypaque centrifugation in 30 ml of cxMEM, 3 ml of autologous plasma and 1 jug/ml of purified phytohaemagglutinin (Sigma). After 5 days the cells were separated from the medium by centrifugation (400 g for 5 min). The LCM was tested in short-term human bone marrow culture to confirm colonyforming activity.

(TRAP) (Burstone 1958) and immunophenotyping using immunofluorescent and indirect immunoalkaline phosphatase method (Horton et a]. 1985) were performed by standard techniques.

Isolation of human osteoclasts. Human osteoclasts were isolated from the long bones of foetuses aged between 10 and 14 weeks without delay on receipt from the operating theatre, using techniques described previously (Chambers & Magnus 1982). The freshly isolated cells were incubated on bone slices to assess their ability to resorb bone, and on coverslips to compare the immunophenotype of isolated human osteoclasts with MNCs generated in human marrow cultures.

MNC formation in marrow culture A total of 87 studies were performed with human bone marrow to determine effects of different culture conditions on MNC formation (Tables 1 and 2). Assessment of morphology of cells on bone and/or plastic and of non-adherent cells at weekly intervals demonstrated progression towards a population of cells of the granulocyte series, mononuclear cells and macrophages. Presence of MNCs with typical morphological features described for osteoclast-like cells was detected in variable and relatively low numbers (< 2 5/cm2) at week 3, in the absence of 1,25(OH)2 vitamin D3.

Bone resorption. Bone slices were placed in culture wells before addition of haemopoietic cells, stromal cells and isolated human osteoclasts. At the end of the experiments, the presence of excavations, typical of osteoclastic functional activity, was sought in the scanning electron microscope (Chambers et a). 1984).

Molecular markers. sCT was iodinated by a modification of the chloramine T method (Hunter & Greenwood 1962). Bone marrow cultures and isolated human osteoclasts were incubated in radioiodinated sCT, with or without excess unlabelled sCT, washed, fixed, and processed for autoradiography as previously described (Nicholson et a]. 1986; Hattersley & Chambers 1989a). Histochemistry for tartrate-resistant acid phosphatase

Calcitonin-induced shape change. Human and rat osteoclasts were sedimented onto a plastic substrate and allowed to spread for 1 hour. Five ng or 50 ng of sCT (final concentration) was added to the cultures. The cells were inspected for change in shape by phase contrast microscopy for 2 hours. Results

Modulation of MNCformation in marrow cultures Results of effects of additions to the cultures shown in Table 2 demonstrate formation of MNCs was increased by 1,2 5(OH)2 vitamin D3, IL-3 and IL-6, and the effect of 1,25(OH)2 vitamin D3 was augmented by the simultaneous presence of IL-6 and IL-3 (Figs 1 and 2). The greatest number of MNCs generated in these experiments occurred when haemopoietic cells were harvested from the (nonPagetic) head of femur of a Pagetic patient undergoing hip replacement. Formation of MNCs in the presence of 1,2 5(OH)2 vitamin D3 was reduced in the presence of TGF-,B and

Table 1. Summary of culture conditions for 58 liquid human bone marrow experiments Serum type (No. of batches) FBS 10% (7)

NCS 10% (3)

HuS 20% (2)

HuS (Autologous) 10% (2) + FBS 10% (1) HuS (Paget's) 10% (4)+FBS 10% (1) HuS (Hypercalcaemia of malignancy) 10% (3)+FBS 10% (1) HuS (Hyperparathyroidism) 10% (2) +FBC 10% HuS (Myeloma) 10%+FBS 10% FBS 10%+NCS 10% FBS 10% (1)+HoS 10% (1) HoS 10%+HuS 10% FBS 10%+HuS 10%

Experimental conditions BM BM: aspirate from myeloma patient fBM

No. of Tissue culture medium experiments I

1

2

MEM: Earle's MEM: Earle's

BM: PTH BM: PTH +ILI o BM: ILlI BM: osteoclastoma cm BM: 5637 cm BM: HOS cm BM: U20S cm BM: MG63 cm BM: SaOS-2 cm BM: PGE, BM co-cultured with osteoclastoma stroma BM: PTH

3 2 2

MEM: Earle's Fischer's MEM: Earle's MEM: Earle's MEM: Earle's

2

MEM: Earle's

3 2

MEM: Earle's MEM: Earle's

3

BM co-cultured with killed MBM stroma BM co-cultured with ts8 cells BM: PGE, BM: tPA BM: LIF BM: PTH BM: UMR 106 CM (50%) BM co-cultured with ST2 cells BM co-cultured with killed IJMR cells BM co-cultured with live UMR cells

2

MEM: Earle's Fischer's MEM: Earle's

4 2 2 2

1 2

MEM: Earle's MEM: Earle's MEM: Earle's MEM: Earle's MEM: Earle's aiMEM aMEM

2 2

axMEM axMEM

BM: recharge BM: recharge BM: recharge

2 4 3

MEM: Earle's MEM: Earle's MEM: Earle's

BM: recharge

1

MEM: Earle's

BM: recharge BM BM: PTH

I

BM co-cultured with osteoclastoma stroma BM

2

1

MEM: Earle's MEM: Earle's RPMI cxMEM

1

MEM: Earle's

I

Adult and foetal human bone marrow cells (BM + fBM respectively) (106-2 x 106 cells/ml) were cultured in the presence and absence of 1,2 5-dihydroxyvitamin D3 (10-I M) using 2 5 different batches of serum, with supplements or additional stroma where stated. The following agents were added at the stated concentrations: IL-la (10 ng/ml); IL-I/# (10 ng/ml); PTH (1-10 ng/ml); tPA (10 ng/ml); LIF (1100 IU/ml); TGF-fI (0.01-0.1 ng/ml); PGE2 (10-8 M). Six to 12 replicate cultures were present in each experiment. MBM stroma, murine bone marrow stroma; CM, conditioned medium.

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Multinucleate bone marrow cells 393 PGE2. Experiments which failed to generate were detected on one or two slices out of the MNCs in the presence of 1,25(OH)2 vitamin six to 12 slices examined in nine of the 64 D3 were not included in this study. experiments (maximum number of pits per Apart from these conditions (see Table 2), experiment, 10; maximum total area of bone we did not show any demonstrable effect on surface resorbed per experiment, 5 x 103 MNC number when human bone marrow Mm2). Bone resorption occurred only in the cells were cultured under a wide variety of presence of 1,2 5(OH)2 vitamin D3; there was conditions (Table 1). In all these experi- no other constant association with bone ments, MNCs were present in low numbers resorption. Pit formation was not detected in 55 experiments, which included many culwhen cultured in the absence of 1,25(OH)2 vitamin D3 (