Advertisement

Megakaryocyte and Platelet Production from Human Cord Blood Stem Cells

  • Amélie Robert
  • Valérie Cortin
  • Alain Garnier
  • Nicolas Pineault
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 788)

Abstract

The cloning of thrombopoietin together with advances in the culture of hematopoietic stem cells have paved the way for the study of megakaryopoiesis, ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes, such as platelets. This chapter describes a 14-day culture protocol for the production of human megakaryocytes (MKs) and platelets, and assays that can be used to characterize the functional properties of the platelets produced ex vivo. CD34+ cells isolated from cord blood cells are grown in a serum-free medium supplemented with newly developed cytokine cocktails optimized for MK differentiation, expansion, and maturation. Detailed methodologies for flow cytometry analysis of MKs and platelets, for the purification of platelets and functional assays, are presented together with supporting figures. The chapter also provides a brief review on megakaryocytic differentiation and ex vivo MK cultures.

Key words

Megakaryocytes Platelets Hematopoietic stem cells Cord blood Flow cytometry Ex vivo cell culture Culture medium optimization Stem cells expansion Cytokines 

Notes

Acknowledgments

The authors wish to thank Lucie Boyer for helpful discussions and revision of this chapter. This work was supported in part by a Strategic Project grant from the Canadian Natural Science and Engineering Research Council (NSERC). A. Robert owns an Industrial R&D Fellowship from NSERC.

References

  1. 1.
    Avecilla, S. T., Hattori, K., Heissig, B., Tejada, R., Liao, F., Shido, K., Jin, D. K., Dias, S., Zhang, F., Hartman, T. E., Hackett, N. R., Crystal, R. G., Witte, L., Hicklin, D. J., Bohlen, P., Eaton, D., Lyden, D., de Sauvage, F., and Rafii, S. (2004) Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis Nat Med 10, 64–71.Google Scholar
  2. 2.
    Junt, T., Schulze, H., Chen, Z., Massberg, S., Goerge, T., Krueger, A., Wagner, D. D., Graf, T., Italiano, J. E., Jr., Shivdasani, R. A., and von Andrian, U. H. (2007) Dynamic visualization of thrombopoiesis within bone marrow Science 317, 1767–70.Google Scholar
  3. 3.
    Dunois-Larde, C., Capron, C., Fichelson, S., Bauer, T., Cramer-Borde, E., and Baruch, D. (2009) Exposure of human megakaryocytes to high shear rates accelerates platelet production Blood 114, 1875–83.Google Scholar
  4. 4.
    Reems, J. A., Pineault, N., and Sun, S. (2010) In vitro megakaryocyte production and platelet biogenesis: state of the art. Transfus Med Rev 24, 33–43.Google Scholar
  5. 5.
    Gandhi, M. J., Drachman, J. G., Reems, J. A., Thorning, D., and Lannutti, B. J. (2005) A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells Blood Cells Mol Dis 35, 70–3.Google Scholar
  6. 6.
    Fujimoto, T. T., Kohata, S., Suzuki, H., Miyazaki, H., and Fujimura, K. (2003) Production of functional platelets by differentiated embryonic stem (ES) cells in vitro Blood 102, 4044–51.Google Scholar
  7. 7.
    Nishikii, H., Eto, K., Tamura, N., Hattori, K., Heissig, B., Kanaji, T., Sawaguchi, A., Goto, S., Ware, J., and Nakauchi, H. (2008) Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells J Exp Med 205, 1917–27.Google Scholar
  8. 8.
    Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., and Nakauchi, H. (2008) Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors Blood 111, 5298–306.Google Scholar
  9. 9.
    Choi, E. S., Nichol, J. L., Hokom, M. M., Hornkohl, A. C., and Hunt, P. (1995) Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional Blood 85, 402–13.Google Scholar
  10. 10.
    Ungerer, M., Peluso, M., Gillitzer, A., Massberg, S., Heinzmann, U., Schulz, C., Munch, G., and Gawaz, M. (2004) Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment Circ Res 95, e36–44.Google Scholar
  11. 11.
    Matsunaga, T., Tanaka, I., Kobune, M., Kawano, Y., Tanaka, M., Kuribayashi, K., Iyama, S., Sato, T., Sato, Y., Takimoto, R., Takayama, T., Kato, J., Ninomiya, T., Hamada, H., and Niitsu, Y. (2006) Ex vivo large-scale generation of human platelets from cord blood CD34+ cells Stem Cells 24, 2877–87.Google Scholar
  12. 12.
    Boyer, L., Robert, A., Proulx, C., and Pineault, N. (2008) Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system J Immunol Methods 332, 82–91.Google Scholar
  13. 13.
    Sullenbarger, B., Bahng, J. H., Gruner, R., Kotov, N., and Lasky, L. C. (2009) Prolonged continuous in vitro human platelet production using three-dimensional scaffolds Exp Hematol 37, 101–10.Google Scholar
  14. 14.
    Proulx, C., Dupuis, N., St-Amour, I., Boyer, L., and Lemieux, R. (2004) Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39°C Biotechnol Bioeng 88, 675–80.Google Scholar
  15. 15.
    Schipper, L. F., Brand, A., Reniers, N. C., Melief, C. J., Willemze, R., and Fibbe, W. E. (1998) Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood Br J Haematol 101, 425–35.Google Scholar
  16. 16.
    Mattia, G., Vulcano, F., Milazzo, L., Barca, A., Macioce, G., Giampaolo, A., and Hassan, H. J. (2002) Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release Blood 99, 888–97.Google Scholar
  17. 17.
    Pang, L., Weiss, M. J., and Poncz, M. (2005) Megakaryocyte biology and related disorders J Clin Invest 115, 3332–8.Google Scholar
  18. 18.
    Debili, N., Coulombel, L., Croisille, L., Katz, A., Guichard, J., Breton-Gorius, J., and Vainchenker, W. (1996) Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow Blood 88, 1284–96.Google Scholar
  19. 19.
    Italiano, J. E., Jr., Lecine, P., Shivdasani, R. A., and Hartwig, J. H. (1999) Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes J Cell Biol 147, 1299–312.Google Scholar
  20. 20.
    Clarke, M. C., Savill, J., Jones, D. B., Noble, B. S., and Brown, S. B. (2003) Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death J Cell Biol 160, 577–87.Google Scholar
  21. 21.
    Kikuchi, J., Furukawa, Y., Iwase, S., Terui, Y., Nakamura, M., Kitagawa, S., Kitagawa, M., Komatsu, N., and Miura, Y. (1997) Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: involvement of cyclin-dependent kinase inhibitor p21 in polyploidization Blood 89, 3980–90.Google Scholar
  22. 22.
    Lordier, L., Jalil, A., Aurade, F., Larbret, F., Larghero, J., Debili, N., Vainchenker, W., and Chang, Y. (2008) Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling Blood 112, 3164–74.Google Scholar
  23. 23.
    Schulze, H., Korpal, M., Hurov, J., Kim, S. W., Zhang, J., Cantley, L. C., Graf, T., and Shivdasani, R. A. (2006) Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis Blood 107, 3868–75.Google Scholar
  24. 24.
    Richardson, J. L., Shivdasani, R. A., Boers, C., Hartwig, J. H., and Italiano, J. E., Jr. (2005) Mechanisms of organelle transport and capture along proplatelets during platelet production Blood 106, 4066–75.Google Scholar
  25. 25.
    Italiano, J. E., Jr., Patel-Hett, S., and Hartwig, J. H. (2007) Mechanics of proplatelet elaboration J Thromb Haemost 5 Suppl 1, 18–23.Google Scholar
  26. 26.
    Cortin, V., Garnier, A., Pineault, N., Lemieux, R., Boyer, L., and Proulx, C. (2005) Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design Exp Hematol 33, 1182–91.Google Scholar
  27. 27.
    De Bruyn, C., Delforge, A., Martiat, P., and Bron, D. (2005) Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood Stem Cells Dev 14, 415–24.Google Scholar
  28. 28.
    Bruno, S., Gunetti, M., Gammaitoni, L., Dane, A., Cavalloni, G., Sanavio, F., Fagioli, F., Aglietta, M., and Piacibello, W. (2003) In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells: rapid and transient megakaryocyte reconstitution Haematologica 88, 379–87.Google Scholar
  29. 29.
    Williams, J. L., Pipia, G. G., Datta, N. S., and Long, M. W. (1998) Thrombopoietin requires additional megakaryocyte-active cytokines for optimal ex vivo expansion of megakaryocyte precursor cells Blood 91, 4118–26.Google Scholar
  30. 30.
    Shaw, P. H., Gilligan, D., Wang, X. M., Thall, P. F., and Corey, S. J. (2003) Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system Biol Blood Marrow Transplant 9, 151–6.Google Scholar
  31. 31.
    Dolzhanskiy, A., Basch, R. S., and Karpatkin, S. (1997) The development of human megakaryocytes: III. Development of mature megakaryocytes from highly purified committed progenitors in synthetic culture media and inhibition of thrombopoietin-induced polyploidization by interleukin-3 Blood 89, 426–34.Google Scholar
  32. 32.
    Tajika, K., Ikebuchi, K., Inokuchi, K., Hasegawa, S., Dan, K., Sekiguchi, S., Nakahata, T., and Asano, S. (1998) IL-6 and SCF exert different effects on megakaryocyte maturation Br J Haematol 100, 105–11.Google Scholar
  33. 33.
    Proulx, C., Boyer, L., Hurnanen, D. R., and Lemieux, R. (2003) Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand J Hematother Stem Cell Res 12, 179–88.Google Scholar
  34. 34.
    Fox, N. E., and Kaushansky, K. (2005) Engagement of integrin α4β1 enhances thrombopoietin-induced megakaryopoiesis Exp Hematol 33, 94–9.Google Scholar
  35. 35.
    Bertolini, F., Battaglia, M., Pedrazzoli, P., Da Prada, G. A., Lanza, A., Soligo, D., Caneva, L., Sarina, B., Murphy, S., Thomas, T., and della Cuna, G. R. (1997) Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients Blood 89, 2679–88.Google Scholar
  36. 36.
    Robert A, B. L., Pineault N (2010) Stem Cells Dev (TITLE TO BE CONFIRMED). Submitted publication.Google Scholar
  37. 37.
    Harrison, P., Briggs, C., and Machin, S. J. (2004) Platelet counting. Methods Mol Biol 2004 272, 29–46.Google Scholar
  38. 38.
    Debili, N., Louache, F., and Vainchenker, W. (2004) Isolation and culture of megakaryocyte precursors Methods Mol Biol 272, 293–308.Google Scholar
  39. 39.
    Cazenave, J.-P., Ohlmann, P., Cassel, D., Eckly, A., Hechler, B., and Gachet, C. (2004) Prep-aration of washed platelet suspensions from human and rodent blood Methods Mol Biol 272, 13–28.Google Scholar
  40. 40.
    Walsh, P. N. (1972) Platelet washing by albumin density gradient separation (ADGS) Adv Exp Med Biol 34, 245–56.Google Scholar
  41. 41.
    Pineault, N., Boucher, J. F., Cayer, M. P., Palmqvist, L., Boyer, L., Lemieux, R., and Proulx, C. (2008) Characterization of the effects and potential mechanisms leading to increased megakaryocytic differentiation under mild hyperthermia Stem Cells Dev 17, 483–93.Google Scholar
  42. 42.
    Cortin, V., Pineault, N., and Garnier, A. (2009) Ex vivo megakaryocyte expansion and platelet production from human cord blood stem cells Methods Mol Biol 482, 109–26.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Amélie Robert
    • 1
  • Valérie Cortin
    • 1
  • Alain Garnier
    • 2
  • Nicolas Pineault
    • 1
    • 3
  1. 1.Département de Recherche et DéveloppementHéma-QuébecQuébec CityCanada
  2. 2.Department of Chemical EngineeringUniversité LavalQuébec CityCanada
  3. 3.Department of Biochemistry and MicrobiologyUniversité LavalQuébec CityCanada

Personalised recommendations