Abstract
Bone marrow megakaryocytes were first described well over one hundred years ago (1,2) and it is almost one hundred years since their role in platelet formation was first appreciated (3,4). Prior to the development of megakaryocyte isolation techniques, a great deal had been learned about megakaryocyte structure and megakaryocytopoiesis using electron microscopy and in vivo experimentation. However, the development of isolation techniques opened up entirely new areas of research on megakaryocytes. Megakaryocyte isolation has been a challenge due to the fragile nature of megakaryocytes and their relative rarity, constituting less than 1% of the nucleated cells in mammalian bone marrow. Nevertheless, techniques including density gradient centrifugation, centrifugal elutriation, selective aggregation, and immunomagnetic bead isolation have been described for megakaryocyte isolation from murine, rat, guinea pig, bovine, and human tissue (5–23).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Howell, W. H. (1890) Observations upon the occurrence, structure, and funtion of the giant cells of the marrow. J. Morphol. 4, 117–130.
Heidenhan, M. (1894) Neve Ontersuchungen Über die zentralkolrper udn ihre Beziehungen zum kern-und Zellerprotoplasma. Arch. Mikrosk. Anat. 43, 423–758.
Wright, J. H. (1906) The origin and nature of the blood plates. Boston Med. Surg. J. 154, 643–645.
Wright. J. H. (1910) The histogenesis of the blood platelets. J. Morphol. 21, 265–278.
Levine, R. F. and Fedorko, M. (1976) Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitors of platelet aggregation; enrichment achieved with a two-step separation technique. J. Cell Biol. 69, 159–172.
Levine, R. F. (1980) Isolation and characterization of normal human megakaryocytes. Br. J. Haematol. 45, 487–497.
Seitz, R. and Wesemann, W. (1980) Studies on megakaryocytes: isolation from rat and guinea pig and incorporation of 5-hydroxytryptamine. Eur. J. Cell Biol. 21, 183–187.
Leven R. M. and Nachmias, V. T. (1982) Cultured megakaryocytes: Changes in the cytoskeleton after ADP-induced spreading. J. Cell Biol. 92, 313–323.
Sitar, G. (1984) Isolation of normal human megakaryocytes. Br. J. Haematol. 58, 465–472.
Berkow, R. L., Straneva J. E., Bruno, E., Beyer, G. S., Burgess, J. S., and Hoffman, R. (1984) Isolation of human megakaryocytes by density centrifugation and counterflow centrifugal elutriation. J. Lab. Clin. Med. 103, 811–818.
Weseman, W., Raha, S., and McDonald, T. P. (1985) Isolation of mouse megakaryocytes. II. Functional and metabolic aspects of two different maturational stages. Eur. J. Cell. Biol. 37, 117–121.
Saigo, K., Ryo, R., Nakaya, Y., and Yamaguchi, N. (1985) Isolation of megakaryocytes by a combination of density gradient centrifugation and velocity sedimentation, and centrifugal elutriation. Kobe J. Med. Sci. 31, 251–261.
Tanaka, H., Ishida, Y., Kaneko, T., and Matsumoto, N. (1989) Isolation of human megakaryocytes by immunomagnetic beads. Br. J. Haematol. 73, 18–22.
Shoff, K. and Levine, R. F. (1989) Elutriation for isolation of megakaryocytes. Blood Cells 15, 285–305.
Mazur, E. M., Basilico, D., Newton, J. L., Cohen, J. L., Charland, C., Sohl, P. A., et al. (1990) Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells. Blood 76, 1771–1782.
Shikama, Y. (1990) Isolation of rat megakaryocytes by immunomagnetic beads. Fukushima J. Med. Sci. 36, 59–70.
Leven, R. M. and Rodriguez, A. (1991) Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow: effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation. Br. J. Haematol. 77, 267–273.
Van Pampus, E. C. M., van Geel, B. J. M., Huijgens, P. C., Wijermans, P. W., Ossenkoppele, G. J., Rodriguez, F., et al. (1991) Combining counterflow centrifugal elutriation and glycoprotein Ib-dependent purification of human megakaryocytes: Efficacy and selectivity. Eur. J. Haematol. 47, 299–304.
Kuter, D. J., Gminski, D., and Rosenberg, R. D. (1992) Botrocetin agglutination of rat megakaryocytes: a rapid method for megakarycoyte isolation. Exp. Hematol. 20, 1085–1089.
Schmitz, B., Radbruch, A., Kümmel, T., Wickenhauser, C., Korb, H., Hansmann, M. L., et al. (1994) Magnetic activated cell sorting (MACS)—a new immunomagnetic method for megakaryocytic cell isolation: Comparison of different separation techniques. Eur. J. Haematol. 52, 267–275.
Park, S. K., Olson, T. A., Ercal, N., Summers, M., and O’Dorsio, M. S. (1996) Characterization of vasoactive intestinal peptide receptors on human megakaryocytes and platelets. Blood 87, 4629–4635.
Miyazaki, R., Ogata, H., Iguchi, T., Sogo, S., Kushida T., Ito, T., et al. (2000) Comparative analyses of megakaryocytes derived from cord blood and bone marrow. Br. J. Haematol. 108, 602–609.
Nagata Y., Oda, M., Nakata, H., Shozaki, Y., Kozasa, T., and Todokoro, K. (2001) A novel regulator of G-protein signaling bearing GAP activity for Gαi and Gαq in megakaryocytes. Blood 97, 3051–3060.
Radley, J. M. and Scurfield, G. (1980) The mechanism of platelet release. Blood 56, 996–999.
Radley, J. M. and Haller, C. J. (1982) The demarcation membrane system of the megakaryocyte: A misnomer? Blood 60, 213–219.
Leven R. M. and Yee, M. K. (1987) Megakaryocyte morphogenesis stimulated in vitro by whole and partially fractionated thrombocytopenic plasma: A model system for the study of platelet formation. Blood 69, 1046–1052.
Leven, R. M. (1987) Megakaryocyte motility and platelet formation. Scanning Microscopy 1, 1701–1709.
Hunt, P., Hokom, M. M., Wiemann, B., Leven, R. M., and Arakawa, T. (1993) Megakaryocyte proplatelet-like process formation in vitro is inhibited by serum prothrombin, a process which is blocked by matrix-bound glycosaminoglycans. Exp. Hematol. 21, 372–381.
Ishida, Y., Yano, K., Ito, T., Shigematsu, H., Sadaki, K., Kondo, S., et al. (2001) Purification of proplatelet formation (PPF) stimulating factor: Thrombin/antithrombin III complex stimulates PPF of megakaryocytes in vitro and platelet production in vivo. Thromb. Haemost. 85, 349–355.
Lecine, P., Villeval, J. L., Vyas, P., Swencki, B., Xu, Y., and Shivdasani, R. A. (1998) Mice lacking transcription factor NF-E2 provide in vivo validation of the proplatelet model of thrombocytopoiesis and show a platelet production defect that is intrinsic to megakaryocytes. Blood 92, 1608–1616.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Leven, R.M. (2004). Isolation of Primary Megakaryocytes and Studies of Proplatelet Formation. In: Gibbins, J.M., Mahaut-Smith, M.P. (eds) Platelets and Megakaryocytes. Methods In Molecular Biology™, vol 272. Humana Press. https://doi.org/10.1385/1-59259-782-3:281
Download citation
DOI: https://doi.org/10.1385/1-59259-782-3:281
Publisher Name: Humana Press
Print ISBN: 978-1-58829-101-1
Online ISBN: 978-1-59259-782-6
eBook Packages: Springer Protocols