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Giant cell formation through fusion of cells derived from a human giant cell tumor of tendon sheath
J Orthop Sci (2004) 9:581–584 DOI 10.1007/s00776-004-0825-0
Giant cell formation through fusion of cells derived from a human giant cell tumor of tendon sheath Masami Hosaka1, Masahito Hatori1, Richard Smith2, and Shoichi Kokubun1 1 2
Department of Orthopaedic Surgery, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan Department of Orthopaedic Surgery, University of Tennessee-Campbell Clinic, Memphis, TN 38163, USA
Abstract Although the mechanism of multinucleation in giant cell tumors of tendon sheath (GCTTS) remains unknown, two mechanisms have been proposed: one is cell fusion and the other amitotic division. The purpose of this study was to clarify the multinucleation process of cultured cells from GCTTS using an in vitro fluorescent cell membrane labeling technique. Cultured GCTTS cells obtained from a 7-year-old Japanese girl were divided into two groups, one for PKH-2 (green) staining and the other for PKH-26 (red) staining. After staining with the dyes, the cell populations were mixed and observed with a fluorescent microscope on the 4th and 14th days after mixing. On both the 4th and 14th days, the cultured GCTTS giant cells showed a mosaic of green and red colors, thus indicating cell membrane fusion. Images of double fluorescent labeled giant cells indicated cell fusion of mononucleated stromal cells that lead to multinucleated giant cells in these GCTTS cell cultures. These findings suggest that multinucleation in GCTTS results from the fusion of mononuclear stromal cells in vitro.
multinucleated giant cells are formed through the fusion of mononuclear cells in GCTTS.8 Based on the morphology, Mirra suspected that the multinucleated giant cells in bone GCT may also grow by syncytial fusion of mononuclear cells.6 Hosaka et al. established a cell line of GCTTS with the property of giant cell formation and observed microscopically that the nuclei of giant cells are identical to those of mononuclear cells and that the mononuclear cells fused into giant cells.4 The purpose of this study was to verify the cell fusion leading to multinucleation by using an in vitro fluorescent cell membrane labeling technique in this cell line.
Materials and methods Materials
Key words Cell fusion · GCT of tendon sheath · Fluorescent cell membrane labeling
Introduction The constituents of giant cell tumors of tendon sheath (GCTTS) are multinucleated giant cells, mononuclear cells, form cells, siderophages, and inflammatory cells. Multinucleated giant cells have a variable number of nuclei, ranging from as few as 3–4 to as many as 50–60. The exact mechanism of the multinucleation remains unclear. Mirra et al. proposed two mechanisms through which the multinucleation may occur: one is cell fusion and the other amitotic division.6 Enzinger noted that
Offprint requests to: M. Hatori Received: February 24, 2004 / Accepted: July 29, 2004
Cultured cells obtained from the GCTTS of a 7-year-old Japanese girl were used.4 The cultured cells were established from a xenografted tumor in the back of a nude mouse (Clea, Tokyo, Japan). Only mononuclear cells were observed in the primary culture, and these remained constant in growth (Fig. 1). Multinucleated giant cells appeared at passage 3 and were constantly observed thereafter. The fusion of mononuclear cells into giant cells was verified by light and phase-contrast microscopy (Figs. 2, 3). The cultured cells at passage 9 were inoculated subcutaneously into the back of the mouse at 1 ⫻ 107 cells per 0.5 ml modified Eagle medium (MEM; Invitrogen, San Diego, CA, USA). At 8 weeks after inoculation, a subcutaneous tumor appeared and became 10 ⫻ 10 mm in size. The xenografted tumors had the same histological findings of the primary tumor except for absence of multinucleated giant cells. The tumor was composed of mononuclear oval or spindle-shaped cells with moderate pleomorphism (Fig. 4).
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Fig. 1. Phase-contrast micrograph of primary cultured cells. The primary culture mainly consists of spindle cells and polygonal cells. No multinucleated giant cells are observed. ⫻100
M. Hosaka et al.: Cell fusion in GCTTS cell culture
Fig. 4. Light micrograph of tumors in nude mouse after inoculation of cultured cells. The xenografted tumors gave basically the same appearance of the primary tumor, but multinucleated giant cells were not observed. Hematoxylin and eosin, ⫻200
Cell fusion analysis with fluorescent staining
Fig. 2. Phase-contrast micrograph of passage 9 cultured cells. Multinucleated giant cells can be seen at passage 9. ⫻200
Fig. 3. Light micrograph of passage 9 cultured cells. Fusion of mononuclear cells into the giant cells was observed. Hematoxylin and eosin, ⫻200
At passage 9, the cultured cells were examined for cell fusion by staining with fluorescent cell membrane dyes PKH-2 and PKH-26 (Zynaxis Cell Science, Malvern, PA, USA). Cultured cells at passage 9 were suspended using a 0.25% trypsin–4 mM ethylenediaminetetraacetic acid (EDTA) solution, centrifuged at 400 g for 5 min, and washed twice with MEM without serum. After resuspension of a concentration of 1 ⫻ 107 cells/ ml, the cultured cells were divided into two groups. The cells of one group were suspended with PKH-2 dye solution (Zynaxis Cell Science) at 2 µM in the supplied Diluent A. The labeling conditions were 5 ⫻ 106 cells/ml in PKH-2 dye at 2 µM. The cells of the other group were suspended with PKH-26 dye (Zynaxis Cell Science) at 2 µM in the supplied Diluent C. The labeling conditions were 5 ⫻ 106 cells/ml in PKH-26 dye at 2 µM. After incubating the two groups of cells with their respective labels at room temperature for 5 min, the reaction was stopped by adding 6 ml MEM with 10% fetal bovine serum (FBS) to remove unbound dye. The labeled cells were then centrifuged at 400 g for 10 min and washed three times in 5 ml MEM with 10% FBS. The two groups of cells were then mixed. One day after mixing, the cells were allowed to grow and attach to the bottom of plastic Petri dishes (Falcon, Los Angeles, CA, USA). On the 4th and 14th days after mixing, they were observed and photographed with a fluorescent microscope (Leica, Wetzlar, Germany) provided with an epifluorescence attachment and a phase-contrast condenser.
M. Hosaka et al.: Cell fusion in GCTTS cell culture
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Discussion
Fig. 5. Double fluorescence photograph just after mixing of the labeled cell populations in vitro stain with PKH-2 and PKH-26. Mononucleated cells were labeled either green (PKH-2) or red (PKH-26). ⫻400
Fig. 6. Double fluorescence photograph on the fourth day after in vitro stain with PKH-2 and PKH-26. Multinucleated giant cells were labeled both green and red; double fluorescence photography demonstrated multinucleated giant cells stained orange. ⫻400
Results Cell fusion analysis by fluorescent staining The primary culture of the GCTTS cells consisted of spindle- and polygonal-shaped mononuclear cells. Multinucleated cells appeared by passage 3, and those are shown from passage 9. Under the fluorescence microscope, every mononucleated cell had a cell membrane labeled either green (PKH-2) or red (PKH-26) (Fig. 5). On the 4th and 14th days, after mixing of the labeled cell populations, multinucleated giant cells had a cell membrane labeled both green and red with each fluorescent filter. Double fluorescence photography revealed the multinucleated giant cell membrane as having a mosaic appearance with red and green (Fig. 6).
There have been few papers describing the potential for multinucleation of giant cell tumor (GCT) cells in culture.1,3,5 Gallardo et al. noted the disappearance of multinucleated cells from the primary culture with the persistence of a heterogeneous population of mononucleated cells.1 They were able to demonstrate new giant cell formation by placing cells from later passages in nutritive agar platforms.1 Huang et al. also noted that multinucleated giant cells persisted in culture up to passage 17.5 The present authors have previously reported a cell line with giant cell formation properties distinct from those of GCTTS.4 There have been no other reports about the establishment of a GCTTS cell culture. In our culture of GCTTS cells, it is noteworthy that multinucleated giant cells were continuously seen after passage 3. The mechanism of multinucleation remains unknown. These findings suggest that mononuclear stromal cells are the main tumor cells of GCTTS and have potential to fuse with each other, resulting in multinucleation. The exact mechanism, syncytial fusion or amitotic division, of the multinucleation of giant cell tumor cells remains unclear. Mirra described that in GCT of bone, the multinucleated giant cells must grow by syncytial fusion of the mononuclear cells because, in most cases, stromal cells can be seen within the cytoplasm of giant cells.6 Seki et al. reported that multinucleated giant cells were negative for proliferating cell nuclear antigen (PCNA), suggesting that multinucleated giant cells were nondividing.7 Horiuchi et al. reported that multinucleated giant cell syncytial formation could be seen morphologically in a cell culture of giant cell tumor of bone.3 We observed giant cell formation through the fusion of mononuclear cells in their established GCTTS cell lines microscopically.4 Horan et al. developed in vivo and in vitro fluorescent cell membrane labeling techniques.2 The great benefit of this technology is its ability to retain the probe in the cell. Little or no transfer of fluorescent dye from the labeled cells to nonlabeled cells was seen after several days in cultures.2 Horiuchi et al. applied in vitro fluorescent cell membrane labeling techniques to cultured cells from giant cell tumor of bone and observed giant cell formation by mononuclear cells.3 The present authors also applied this method to cultured GCTTS to confirm the multinucleation process of GCTTS by mononuclear cells. Double photography with this method clearly showed a mosaic pattern of red and green colors resulting from the mixture of red and green stain in stromal cells. These findings suggest that multinucleation in GCTTS occurs through the fusion of mononuclear stromal cells in vitro.
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References 1. Gallardo H, De Lustig ES, Schajowicz F. Growth and maintenance of human giant-cell bone tumors (osteoclastomas) in continuous culture. Oncology 1975;24(2):146–59. 2. Horan PK, Slezak SE. Stable cell membrane labelling. Nature (Lond) 1989;340:167–8. 3. Horiuchi H, Kuroda M, Machinami M. In vitro formation of multinucleated giant cells in GCT of bone. J Jpn Orthop Assoc 1996; 70(6):S910 (in Japanese). 4. Hosaka M, Hatori M, Kokubun S. A cell line with multinucleated giant cell formation established from a human giant cell tumor of tendon sheath: preliminary report. J Orthop Sci 2001;6:414– 18.
M. Hosaka et al.: Cell fusion in GCTTS cell culture 5. Huang TSW, Green AD, Beattie CW, et al. Monocytemacrophage lineage of giant cell tumor of bone: establishment of a multinucleated cell line. Cancer (Phila) 1993;71:1751–60. 6. Mirra JM, Picci P, Gold RH. Theoretical considerations: do osteoclast-like giant cells grow by amitotic division or by syncytial fusion? In: Mirra JM, Picci P, Gold RH, editors. Bone tumors, vol 2. Philadelphia: Lea & Febiger; 1989. p. 991. 7. Seki K, Hirose T, Hasegawa T, et al. Giant cell tumor of tendon sheath: an immunohistochemical observation on the characteristics and the capacity of proliferation of tumor cells. Zentralbl Pathol 1993;139:287–94. 8. Weiss SW, Goldblum JR. Benign tumors and tumor-like lesions of synovial tissue. In: Weiss SW, Goldblum JR, editors. Enzinger and Weiss’s soft tissue tumors. 4th ed. St. Louis: Mosby; 2001. p. 1037– 62.
Giant cell formation through fusion of cells derived from a human giant cell tumor of tendon sheath Masami Hosaka1, Masahito Hatori1, Richard Smith2, and Shoichi Kokubun1 1 2
Department of Orthopaedic Surgery, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan Department of Orthopaedic Surgery, University of Tennessee-Campbell Clinic, Memphis, TN 38163, USA
Abstract Although the mechanism of multinucleation in giant cell tumors of tendon sheath (GCTTS) remains unknown, two mechanisms have been proposed: one is cell fusion and the other amitotic division. The purpose of this study was to clarify the multinucleation process of cultured cells from GCTTS using an in vitro fluorescent cell membrane labeling technique. Cultured GCTTS cells obtained from a 7-year-old Japanese girl were divided into two groups, one for PKH-2 (green) staining and the other for PKH-26 (red) staining. After staining with the dyes, the cell populations were mixed and observed with a fluorescent microscope on the 4th and 14th days after mixing. On both the 4th and 14th days, the cultured GCTTS giant cells showed a mosaic of green and red colors, thus indicating cell membrane fusion. Images of double fluorescent labeled giant cells indicated cell fusion of mononucleated stromal cells that lead to multinucleated giant cells in these GCTTS cell cultures. These findings suggest that multinucleation in GCTTS results from the fusion of mononuclear stromal cells in vitro.
multinucleated giant cells are formed through the fusion of mononuclear cells in GCTTS.8 Based on the morphology, Mirra suspected that the multinucleated giant cells in bone GCT may also grow by syncytial fusion of mononuclear cells.6 Hosaka et al. established a cell line of GCTTS with the property of giant cell formation and observed microscopically that the nuclei of giant cells are identical to those of mononuclear cells and that the mononuclear cells fused into giant cells.4 The purpose of this study was to verify the cell fusion leading to multinucleation by using an in vitro fluorescent cell membrane labeling technique in this cell line.
Materials and methods Materials
Key words Cell fusion · GCT of tendon sheath · Fluorescent cell membrane labeling
Introduction The constituents of giant cell tumors of tendon sheath (GCTTS) are multinucleated giant cells, mononuclear cells, form cells, siderophages, and inflammatory cells. Multinucleated giant cells have a variable number of nuclei, ranging from as few as 3–4 to as many as 50–60. The exact mechanism of the multinucleation remains unclear. Mirra et al. proposed two mechanisms through which the multinucleation may occur: one is cell fusion and the other amitotic division.6 Enzinger noted that
Offprint requests to: M. Hatori Received: February 24, 2004 / Accepted: July 29, 2004
Cultured cells obtained from the GCTTS of a 7-year-old Japanese girl were used.4 The cultured cells were established from a xenografted tumor in the back of a nude mouse (Clea, Tokyo, Japan). Only mononuclear cells were observed in the primary culture, and these remained constant in growth (Fig. 1). Multinucleated giant cells appeared at passage 3 and were constantly observed thereafter. The fusion of mononuclear cells into giant cells was verified by light and phase-contrast microscopy (Figs. 2, 3). The cultured cells at passage 9 were inoculated subcutaneously into the back of the mouse at 1 ⫻ 107 cells per 0.5 ml modified Eagle medium (MEM; Invitrogen, San Diego, CA, USA). At 8 weeks after inoculation, a subcutaneous tumor appeared and became 10 ⫻ 10 mm in size. The xenografted tumors had the same histological findings of the primary tumor except for absence of multinucleated giant cells. The tumor was composed of mononuclear oval or spindle-shaped cells with moderate pleomorphism (Fig. 4).
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Fig. 1. Phase-contrast micrograph of primary cultured cells. The primary culture mainly consists of spindle cells and polygonal cells. No multinucleated giant cells are observed. ⫻100
M. Hosaka et al.: Cell fusion in GCTTS cell culture
Fig. 4. Light micrograph of tumors in nude mouse after inoculation of cultured cells. The xenografted tumors gave basically the same appearance of the primary tumor, but multinucleated giant cells were not observed. Hematoxylin and eosin, ⫻200
Cell fusion analysis with fluorescent staining
Fig. 2. Phase-contrast micrograph of passage 9 cultured cells. Multinucleated giant cells can be seen at passage 9. ⫻200
Fig. 3. Light micrograph of passage 9 cultured cells. Fusion of mononuclear cells into the giant cells was observed. Hematoxylin and eosin, ⫻200
At passage 9, the cultured cells were examined for cell fusion by staining with fluorescent cell membrane dyes PKH-2 and PKH-26 (Zynaxis Cell Science, Malvern, PA, USA). Cultured cells at passage 9 were suspended using a 0.25% trypsin–4 mM ethylenediaminetetraacetic acid (EDTA) solution, centrifuged at 400 g for 5 min, and washed twice with MEM without serum. After resuspension of a concentration of 1 ⫻ 107 cells/ ml, the cultured cells were divided into two groups. The cells of one group were suspended with PKH-2 dye solution (Zynaxis Cell Science) at 2 µM in the supplied Diluent A. The labeling conditions were 5 ⫻ 106 cells/ml in PKH-2 dye at 2 µM. The cells of the other group were suspended with PKH-26 dye (Zynaxis Cell Science) at 2 µM in the supplied Diluent C. The labeling conditions were 5 ⫻ 106 cells/ml in PKH-26 dye at 2 µM. After incubating the two groups of cells with their respective labels at room temperature for 5 min, the reaction was stopped by adding 6 ml MEM with 10% fetal bovine serum (FBS) to remove unbound dye. The labeled cells were then centrifuged at 400 g for 10 min and washed three times in 5 ml MEM with 10% FBS. The two groups of cells were then mixed. One day after mixing, the cells were allowed to grow and attach to the bottom of plastic Petri dishes (Falcon, Los Angeles, CA, USA). On the 4th and 14th days after mixing, they were observed and photographed with a fluorescent microscope (Leica, Wetzlar, Germany) provided with an epifluorescence attachment and a phase-contrast condenser.
M. Hosaka et al.: Cell fusion in GCTTS cell culture
583
Discussion
Fig. 5. Double fluorescence photograph just after mixing of the labeled cell populations in vitro stain with PKH-2 and PKH-26. Mononucleated cells were labeled either green (PKH-2) or red (PKH-26). ⫻400
Fig. 6. Double fluorescence photograph on the fourth day after in vitro stain with PKH-2 and PKH-26. Multinucleated giant cells were labeled both green and red; double fluorescence photography demonstrated multinucleated giant cells stained orange. ⫻400
Results Cell fusion analysis by fluorescent staining The primary culture of the GCTTS cells consisted of spindle- and polygonal-shaped mononuclear cells. Multinucleated cells appeared by passage 3, and those are shown from passage 9. Under the fluorescence microscope, every mononucleated cell had a cell membrane labeled either green (PKH-2) or red (PKH-26) (Fig. 5). On the 4th and 14th days, after mixing of the labeled cell populations, multinucleated giant cells had a cell membrane labeled both green and red with each fluorescent filter. Double fluorescence photography revealed the multinucleated giant cell membrane as having a mosaic appearance with red and green (Fig. 6).
There have been few papers describing the potential for multinucleation of giant cell tumor (GCT) cells in culture.1,3,5 Gallardo et al. noted the disappearance of multinucleated cells from the primary culture with the persistence of a heterogeneous population of mononucleated cells.1 They were able to demonstrate new giant cell formation by placing cells from later passages in nutritive agar platforms.1 Huang et al. also noted that multinucleated giant cells persisted in culture up to passage 17.5 The present authors have previously reported a cell line with giant cell formation properties distinct from those of GCTTS.4 There have been no other reports about the establishment of a GCTTS cell culture. In our culture of GCTTS cells, it is noteworthy that multinucleated giant cells were continuously seen after passage 3. The mechanism of multinucleation remains unknown. These findings suggest that mononuclear stromal cells are the main tumor cells of GCTTS and have potential to fuse with each other, resulting in multinucleation. The exact mechanism, syncytial fusion or amitotic division, of the multinucleation of giant cell tumor cells remains unclear. Mirra described that in GCT of bone, the multinucleated giant cells must grow by syncytial fusion of the mononuclear cells because, in most cases, stromal cells can be seen within the cytoplasm of giant cells.6 Seki et al. reported that multinucleated giant cells were negative for proliferating cell nuclear antigen (PCNA), suggesting that multinucleated giant cells were nondividing.7 Horiuchi et al. reported that multinucleated giant cell syncytial formation could be seen morphologically in a cell culture of giant cell tumor of bone.3 We observed giant cell formation through the fusion of mononuclear cells in their established GCTTS cell lines microscopically.4 Horan et al. developed in vivo and in vitro fluorescent cell membrane labeling techniques.2 The great benefit of this technology is its ability to retain the probe in the cell. Little or no transfer of fluorescent dye from the labeled cells to nonlabeled cells was seen after several days in cultures.2 Horiuchi et al. applied in vitro fluorescent cell membrane labeling techniques to cultured cells from giant cell tumor of bone and observed giant cell formation by mononuclear cells.3 The present authors also applied this method to cultured GCTTS to confirm the multinucleation process of GCTTS by mononuclear cells. Double photography with this method clearly showed a mosaic pattern of red and green colors resulting from the mixture of red and green stain in stromal cells. These findings suggest that multinucleation in GCTTS occurs through the fusion of mononuclear stromal cells in vitro.
584
References 1. Gallardo H, De Lustig ES, Schajowicz F. Growth and maintenance of human giant-cell bone tumors (osteoclastomas) in continuous culture. Oncology 1975;24(2):146–59. 2. Horan PK, Slezak SE. Stable cell membrane labelling. Nature (Lond) 1989;340:167–8. 3. Horiuchi H, Kuroda M, Machinami M. In vitro formation of multinucleated giant cells in GCT of bone. J Jpn Orthop Assoc 1996; 70(6):S910 (in Japanese). 4. Hosaka M, Hatori M, Kokubun S. A cell line with multinucleated giant cell formation established from a human giant cell tumor of tendon sheath: preliminary report. J Orthop Sci 2001;6:414– 18.
M. Hosaka et al.: Cell fusion in GCTTS cell culture 5. Huang TSW, Green AD, Beattie CW, et al. Monocytemacrophage lineage of giant cell tumor of bone: establishment of a multinucleated cell line. Cancer (Phila) 1993;71:1751–60. 6. Mirra JM, Picci P, Gold RH. Theoretical considerations: do osteoclast-like giant cells grow by amitotic division or by syncytial fusion? In: Mirra JM, Picci P, Gold RH, editors. Bone tumors, vol 2. Philadelphia: Lea & Febiger; 1989. p. 991. 7. Seki K, Hirose T, Hasegawa T, et al. Giant cell tumor of tendon sheath: an immunohistochemical observation on the characteristics and the capacity of proliferation of tumor cells. Zentralbl Pathol 1993;139:287–94. 8. Weiss SW, Goldblum JR. Benign tumors and tumor-like lesions of synovial tissue. In: Weiss SW, Goldblum JR, editors. Enzinger and Weiss’s soft tissue tumors. 4th ed. St. Louis: Mosby; 2001. p. 1037– 62.