Summary
This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two “clonings” and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of β-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by “budding” structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.
Similar content being viewed by others
References
Ackerman, G. A. Substituted naphthol AS phosphate derivatives for the localization of leukocyte alkaline phosphatase activity. Lab. Invest. 11:563–573; 1962.
Aronow, M. A.; Gerstenfeld, L. C.; Owen, T. A., et al. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells. J. Cell. Physiol. 143:213–221; 1990.
Ashton, B. A.; Allen, T. D.; Howlett, C. R., et al. Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin. Orthop. 151:294–307; 1980.
Ashton, B. A.; Abdullah, F.; Cave, J., et al. Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow. Preliminary assessment of their osteogenicity. Bone 6:313–319; 1985.
Aufmkolk, B.; Hauschka, P. V.; Schwartz, E. R. Characterization of human bone cells in culture. Calcif. Tissue Int. 37:228–235; 1985.
Bab, I.; Ashton, B.; Gazit, D., et al. Kinetics and differentiation of marrow stromal cells in diffusion chambers in vivo. J. Cell. Sci. 84:139–151; 1986.
Bab, I.; Passi-Even, L.; Gazit, D., et al. Osteogenesis in vivo diffusion chamber cultures of human marrow cells. Bone Miner. 4:373–386; 1988.
Battmann, A.; Kern, A.; Jundt, G., et al. Constant differentiation of a human osteosarcoma cell-line through nude mice passages and long term culture. 38th Annual meeting of the European tissue culture society. London; 1990:55.
Bellows, C. G.; Aubin, J. E.; Heersche, J. N. M. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 14:27–40; 1991.
Benayahu, D.; Kletter, Y.; Zipori, D., et al. Bone marrow-derived stromal cell line expressing osteoblastic phenotype in vitro an osteogenic capacity in vivo. J. Cell. Physiol. 140:1–7; 1989.
Bonadio, J.; Holbrook, K. A.; Gelinas, R. E., et al. Altered triple helical structure of type I procollagen in lethal perinatal osteogenesis imperfecta. J. Biol. Chem. 260:1734–1742; 1985.
Bruder, S. P.; Caplan, A. I. Cellular and molecular events during embryonic bone development. Connect. Tissue Res. 20:65–71; 1989a.
Bruder, S. P.; Caplan, A. I. First bone formation and the dissection of an osteogenic lineage in the embryonic chick tibia is revealed by monoclonal antibodies against osteoblasts. Bone 10:359–375; 1989b.
Bruder, S. P.; Gazit, D.; Passi-Even, L., et al. Osteochondral differentiation of avian bone marrow cells in diffusion chambers in vivo. J. Bone Miner. Res. 11:141–151; 1990.
Chung, C.; Golub, E. E.; Forbes, E., et al. Mechanism of action of β-glycerophosphate on bone cell mineralization. Calcif. Tissue Int. 51:305–311; 1992.
Crisp, A. J.; McGuire-Goldring, M. B.; Goldring, S. R. A system for culture of human trabecular bone and hormone response profiles of derived cells. Br. J. Exp. Pathol. 65:645–654; 1984.
Davies, J. E. Human bone marrow cells synthesize collagen, in diffusion chambers implanted into the normal rat. Cell Biol. Int. Rep. 11:12; 1987.
Friedenstein, A. J.; Chailakhjan, R. K.; Lalykina, K. S. The development of fibroblast colonies in monolayer cultures of guinea pig bone marrow and spleen cells. Cell Tissue Kinet. 3:393–403; 1970.
Friedenstein, A. J. Precursor cells of melanocytes. Int. Rev. Cytol. 47:327–355; 1976.
Friedenstein, A. J.; Chairlakhjan, R. K.; Gerasimow, U. V. Bonemarrow osteogenic stem-cells. In vitro cultivation and transplantation in diffusion-chambers. Cell Tissue Kinet. 20:263–272; 1987.
Goshima, J.; Goldberg, V.; Caplan, A. I. The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks. Clin. Orthop. 262:298–311; 1991.
Gotoh, Y.; Hiraiwa, K.; Nagayama, M. In vitro mineralization of osteoblastic cell derived from human bone. 8:239–250; 1990.
Harmand, M. F.; Thomasset, M.; Rouais, F., et al. In vitro stimulation of articular chondrocyte differentiated function by 1,25-dihydroxycholecalciferol or 24R,25-dihydroxycholecalciferol. J. Cell. Physiol. 119:359–365; 1984.
Harmand, M. F.; Bordenave, L.; Duphil, R., et al. Human “osteoblastlike” cells in culture: responsiveness to 1,25-(OH)2D3. In: Norman, A. W.; DeGruyter, W., et al., eds. Vitamin D. A chemical biochemical and clinic update. Walter de Gruyter & Co. 1985:197–198.
Haynesworth, S. E.; Goshima, J.; Goldberg, V. M., et al. Characterization of cells with osteogenic potential from human marrow. Bone 13:81–88; 1992.
Hekkelman, Ng, B.; Heersche, J. W. The effect of cortisol on the adenosine 3′,5′-monophosphate response to parathyroid hormone of bone in vitro. Endocrinology 104:1130–1135; 1979.
Kaplow, L. S. Leucocyte alkaline phosphatase cytochemistry. Applications and methods. NY Acad. Sci. 155:911–916; 1968.
Kassem, M.; Risteli, L.; Mosekilde, L., et al. Formation of osteoblastlike cells from human mononuclear bone marrow culture. APMIS Acta Pathol. Microbiol. Immunol. Scand. 99:269–274; 1991.
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.
Leboy, P. S.; Beresford, J. N.; Devlin, C., et al. Dexamethasone induction of osteoblast mRNA in rat marrow stromal cell cultures. J. Cell. Physiol. 146:370–378; 1991.
Luria, E. A.; Owen, M. E.; Friedenstein, A. J., et al. Bone formation in organ cultures of bone marrow. Cell Tissue Res. 248:449–454; 1987.
Mahonen, A.; Pirskanen, A.; Keinänen, R., et al. Effect of 1,25(OH)2D3 on its receptor mRNA levels and osteocalcin synthesis in human osteosarcoma cells. Biochim. Biophys. Acta 1048:30–37; 1990.
Majeska, R. J.; Rodan, G. A. Alkaline phosphatase inhibition by parathyroid hormone and isoproterenol in a clonal rat osteosarcoma cell line. Possible mediation by cyclic AMP. Calcif. Tissue Int. 34:59–66; 1982a.
Majeska, R. J.; Rodan, G. A. The effect of 1,25-(OH)2D3 on alkaline phosphatase in osteoblastic osteosarcoma cells. J. Biol. Chem. 257:3362–3365; 1982b.
Majeska, R. J.; Rodan, G. A. Culture and activity of osteoblasts and osteoblast-like cells. In: Butler, W. T., ed. Chemistry and biology of mineralized tissues. Birmingham: Ebsco Media; 1985:279–285.
Maniatopoulos, C.; Sodek, J.; Melcher, A. H. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res. 254:317–330; 1988.
McCulloch, C. A. G.; Strugurescu, M.; Hughes, F., et al. Osteogenic progenitor cells in rat bone marrow stromal populations exhibit selfrenewal in culture. Blood 77:1906–1911; 1991.
Michel, D.; Harmand, M. F. Fibrin seal in wound healing. Effect of thrombin and calcium on human skin fibroblast growth and collagen production. J. Dermatol. Sci. 1:325–334; 1990.
Nakahara, H.; Goldberg, V. M.; Caplan, A. I. Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J. Orthop. Res. 9:465–476; 1991.
Ohgushi, H.; Goldberg, V. M.; Caplan, A. I. Repair of bone defects with marrow cells and porous ceramic. Acta Orthop. Scand. 60(3):334–339; 1989.
Owen, M. Lineage of osteogenic cells and their relationship to the stromal system. In: Peck, W. A., ed. Bone and mineral research. Amsterdam: Elsevier Science Publishers BV; 1985:3:1–25.
Owen, M. E.; Friedenstein, A. J. Stromal stem cells: marrow-derived osteogenic precursors. In: Evered, D.; Harnett, S., eds. Cell and molecular biology of vertebrate hard tissues. New York: John Wiley & Sons; 1988:42–53.
Patt, A. M.; Maloney, M. A.; Flannery, M. L. Hematopoietic microenvironment transfer by stromal fibroblasts derived from bone marrow varying in cellularity. Exp. Hematol. 10:738–742; 1982.
Reddi, A. H. Regulation of bone differentiation by local and systemic factors. In: Peck, W. A., ed. Bone and mineral research, 3. Amsterdam: Elsevier Science Publishers; 1985:27–47.
Robey, P. G.; Termine, J. D. Human bone cells in vitro. Calcif. Tissue Int. 37:453–460; 1985.
Rodan, G. A.; Rodan, S. B. Expression of the osteoblastic phenotype. In: Peck, W. A., ed. Bone and mineral research, vol. 2. Amsterdam: Elsevier Science Publishers; 1983:244–285.
Rodan, S. B.; Fisher, M. K.; Egan, J. J., et al. The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells. Endocrinology 115:951–958; 1984.
Rodan, G. A.; Heath, J. K.; Yoon, K., et al. Diversity of the osteoblastic phenotype. In: Evered, D.; Harnett, S., eds. Cell and molecular biology of vertebrate hard tissues. New York: John Wiley & Sons, 1988:78–85.
Satomura, K.; Hiraiwa, K.; Nagayama, M. Mineralized nodule formation in rat bone marrow stromal cell culture without β-glycerophosphate. Bone Miner. 14:41–54; 1991.
Satomura, K.; Nagayama, M. Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro. Acta Anat. 142:97–104; 1991.
Tsuji, T.; Hughes, F. J.; McCulloch, C. A. G., et al. Effects of donor age on osteogenic cells of rat bone marrow in vitro. Mech. Aging Dev. 51:121–132; 1990.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Vilamitjana-Amedee, J., Bareille, R., Rouais, F. et al. Human bone marrow stromal cells express an osteoblastic phenotype in culture. In Vitro Cell Dev Biol - Animal 29, 699–707 (1993). https://doi.org/10.1007/BF02631426
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02631426