Abstract
Differentiation and maturation of megakaryocytes occur in close association with cellular and extracellular components in the bone marrow. Thus, direct examination of these processes in the native environment provides important information regarding the development of megakaryocytes. In this chapter, we present methods applied to mouse bone marrow to (1) examine the ultrastructure of megakaryocytes and their state of maturation in situ in fixed bone marrow sections and (2) study the dynamics of proplatelet formation by real-time observation of fresh bone marrow explants where megakaryocytes have matured in their natural physiological context. Combining these two approaches allows detailed investigation of in situ megakaryocyte differentiation, including proplatelet formation, which is the final maturation step before platelet release.
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References
Kaushansky, K., 2005, Thrombopoietin and the hematopoietic stem cell. Ann N Y Acad Sci, 1044: p. 139–41.
Shivdasani, R.A., and H. Schuze., 2005, Culture, expansion, and differentiation of murine megakaryocytes. Curr Protoc Immunol, Chapter 22: p. Unit 22F 6.
Battinelli, E.M., et al., 2007, Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis. Curr Opin Hematol, 14(5): p. 419–26.
Liu, J., et al., 2008, Genetic manipulation of megakaryocytes to study platelet function. Curr Top Dev Biol, 80: p. 311–35.
Travlos, G.S., 2006, Normal structure, function, and histology of the bone marrow. Toxicol Pathol, 34(5): p. 548–65.
Kacena, M.A., et al., 2006, A reciprocal regulatory interaction between megakaryocytes, bone cells, and hematopoietic stem cells. Bone, 39(5): p. 978–84.
Debili, N., et al., 1993, Effects of the recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells. Blood, 82(1): p. 84–95.
Avraham, H., et al., 1992, Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells. Blood, 79(2): p. 365–71.
Avecilla, S.T., et al., 2004, Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med, 10(1): p. 64–71.
Leven, R.M., and F. Tablin., 1992, Extracellular matrix stimulation of guinea pig megakaryocyte proplatelet formation in vitro is mediated through the vitronectin receptor. Exp Hematol, 20(11): p. 1316–22.
Sabri, S., et al., 2004, Differential regulation of actin stress fiber assembly and proplatelet formation by alpha2beta1 integrin and GPVI in human megakaryocytes. Blood, 104(10): p. 3117–25.
Larson, M.K., and S.P. Watson., 2006, Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3. Blood, 108(5): p. 1509–14.
Behnke, O., 1968, An electron microscope study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J Ultrastruct Res, 24(5): p. 412–33.
Hunter, E., 1993, Practical Electron Microscopy. A Beginner’s Illustrated Guide, ed. C.U. Press. Vol. 2: Hunter E, Maloney P and Bendayan M. 1–170.
Zucker-Franklin, D., 1988, Megakaryocytes and platelets, ed. A.o.B.C.F.a. Pathology. Vol. 2: Zucker-Franklin D, Greaves MF, Grossi CE, Marmont AM, eds. 621–93.
Harker, L.A., and C.A. Finch., 1969, Thrombokinetics in man. J Clin Invest, 48(6): p. 963–74.
Thiery, J.P., and M. Bessis., 1956, (Mechanism of platelet genesis; in vitro study by cinemicrophotography.). Rev Hematol, 11(2): p. 162–74.
Thiery, J.P., and M. Bessis., 1956, (Genesis of blood platelets from the megakaryocytes in living cells.). C R Hebd Seances Acad Sci, 242(2): p. 290–2.
Italiano, J.E, Jr., et al., 1999, Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol, 147(6): p. 1299–312.
Strassel, C., et al., 2009, Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome. Haematologica, 94(6): p. 800–10.
Acknowledgments
The authors wish to thank Patricia Laeuffer, Fabienne Proamer, Monique Freund, Catherine Ziessel, and Jean-Yves Rinckel for technical assistance. This work was supported by INSERM, EFS-Alsace, ARMESA, and ANR (Agence Nationale pour la Recherche, Grant N ANR-07-MRAR-016-01).
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Eckly, A., Strassel, C., Cazenave, JP., Lanza, F., Léon, C., Gachet, C. (2012). Characterization of Megakaryocyte Development in the Native Bone Marrow Environment. In: Gibbins, J., Mahaut-Smith, M. (eds) Platelets and Megakaryocytes. Methods in Molecular Biology, vol 788. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-307-3_13
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DOI: https://doi.org/10.1007/978-1-61779-307-3_13
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