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Ex Vivo Differentiation of Cord Blood Stem Cells into Megakaryocytes and Platelets

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Basic Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 946))

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Abstract

Megakaryocytes (MK) are hematopoietic cells present in the bone marrow that are responsible for the production and release of platelets in the circulation. Given their very low frequency (<1%), human MK often need to be derived in culture to study their development or to generate sufficient material for biological studies. This chapter describes a simplified 14-day culture protocol that efficiently leads to the production of MK and platelets from cord blood enriched progenitor cells. A serum-free medium is suggested for the growth of the CB cells together with an optimized cytokine cocktail developed specifically for MK differentiation, expansion, and maturation. Methodologies for flow cytometry analysis, MK and platelets estimation, and MK progenitor assay are also presented.

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References

  1. Pang L, Weiss MJ, Poncz M (2005) Megakaryocyte biology and related disorders. J Clin Invest 115:3332–3338

    Article  PubMed  CAS  Google Scholar 

  2. Dolzhanskiy A, Basch RS, 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 89:426–434

    PubMed  CAS  Google Scholar 

  3. Cortin V, Garnier A, Pineault N, Lemieux R, Boyer L, Proulx C (2005) Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design. Exp Hematol 33:1182–1191

    Article  PubMed  CAS  Google Scholar 

  4. van den Oudenrijn S, von dem Borne AE, de Haas M (2000) Differences in megakaryocyte expansion potential between CD34(+) stem cells derived from cord blood, peripheral blood, and bone marrow from adults and children. Exp Hematol 28:1054–1061

    Article  PubMed  Google Scholar 

  5. Schattner M, Lefebvre P, Mingolelli SS, Goolsby CL, Rademaker A, White JG, Foster D, Green D, Cohen I (1996) Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes. Stem Cells 14:207–214

    Article  PubMed  CAS  Google Scholar 

  6. Proulx C, Boyer L, Hurnanen DR, 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 12:179–188

    Article  PubMed  CAS  Google Scholar 

  7. Williams JL, Pipia GG, Datta NS, Long MW (1998) Thrombopoietin requires additional megakaryocyte-active cytokines for optimal ex vivo expansion of megakaryocyte precursor cells. Blood 91:4118–4126

    PubMed  CAS  Google Scholar 

  8. Fujiki H, Kimura T, Minamiguchi H, Harada S, Wang J, Nakao M, Yokota S, Urata Y, Ueda Y, Yamagishi H, Sonoda Y (2002) Role of human interleukin-9 as a megakaryocyte potentiator in culture. Exp Hematol 30:1373–1380

    Article  PubMed  CAS  Google Scholar 

  9. De Bruyn C, Delforge A, Martiat P, Bron D (2005) Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood. Stem Cells Dev 14:415–424

    Article  PubMed  Google Scholar 

  10. Feng Y, Zhang L, **ao ZJ, Li B, Liu B, Fan CG, Yuan XF, Han ZC (2005) An effective and simple expansion system for megakaryocyte progenitor cells using a combination of heparin with thrombopoietin and interleukin-11. Exp Hematol 33:1537–1543

    Article  PubMed  CAS  Google Scholar 

  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, Niitsu Y (2006) Ex vivo large-scale generation of human platelets from cord blood CD34+ cells. Stem Cells 24:2877–2887

    Article  PubMed  CAS  Google Scholar 

  12. Shaw PH, Gilligan D, Wang XM, Thall PF, Corey SJ (2003) Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system. Biol Blood Marrow Transplant 9:151–156

    Article  PubMed  CAS  Google Scholar 

  13. Sigurjonsson OE, Gudmundsson KO, Haraldsdottir V, Rafnar T, Agnarsson BA, Gudmundsson S (2004) Flt3/Flk-2 ligand in combination with thrombopoietin decreases apoptosis in megakaryocyte development. Stem Cells Dev 13:183–191

    Article  PubMed  CAS  Google Scholar 

  14. Boyer L, Robert A, Proulx C, 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

    Article  PubMed  CAS  Google Scholar 

  15. Blair A, Baker CL, Pamphilon DH, Judson PA (2002) Ex vivo expansion of megakaryocyte progenitor cells from normal bone marrow and peripheral blood and from patients with haematological malignancies. Br J Haematol 116:912–919

    Article  PubMed  Google Scholar 

  16. Choi ES, Hokom MM, Nichol JL, Hornkohl A, Hunt P (1995) Functional human platelet generation in vitro and regulation of cytoplasmic process formation. C R Acad Sci III 318:387–393

    PubMed  CAS  Google Scholar 

  17. Ungerer M, Peluso M, Gillitzer A, Massberg S, Heinzmann U, Schulz C, Munch G, Gawaz M (2004) Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment. Circ Res 95:e36–e44

    Article  PubMed  CAS  Google Scholar 

  18. Lefebvre P, Winter JN, Meng Y, Cohen I (2000) Ex vivo expansion of early and late megakaryocyte progenitors. J Hematother Stem Cell Res 9:913–921

    Article  PubMed  CAS  Google Scholar 

  19. Sullenbarger B, Bahng JH, Gruner R, Kotov N, Lasky LC (2009) Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Exp Hematol 37:101–110

    Article  PubMed  CAS  Google Scholar 

  20. Gandhi MJ, Drachman JG, Reems JA, Thorning D, Lannutti BJ (2005) A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells. Blood Cells Mol Dis 35:70–73

    Article  PubMed  CAS  Google Scholar 

  21. Case J, Hicks C, Trickett A, Kwan YL, Manoharan A (2006) The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L. J Interferon Cytokine Res 26:76–82

    Article  PubMed  CAS  Google Scholar 

  22. Shaw PH, Olszewski M, Kletzel M (2001) Expansion of megakaryocyte precursors and stem cells from umbilical cord blood CD34+ cells in collagen and liquid culture media. J Hematother Stem Cell Res 10:391–403

    Article  PubMed  CAS  Google Scholar 

  23. Nishikii H, Eto K, Tamura N, Hattori K, Heissig B, Kanaji T, Sawaguchi A, Goto S, Ware J, Nakauchi H (2008) Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells. J Exp Med 205:1917–1927

    Article  PubMed  CAS  Google Scholar 

  24. Reems JA, Pineault N, Sun S (2010) In vitro megakaryocyte production and platelet biogenesis: state of the art. Transfus Med Rev 24:33–43

    Article  PubMed  Google Scholar 

  25. Battinelli EM, Hartwig JH, Italiano JE Jr (2007) Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis. Curr Opin Hematol 14:419–426

    Article  PubMed  Google Scholar 

  26. Bluteau D, Lordier L, Di Stefano A, Chang Y, Raslova H, Debili N, Vainchenker W (2009) Regulation of megakaryocyte maturation and platelet formation. J Thromb Haemost 7(Suppl 1):227–234

    Article  PubMed  CAS  Google Scholar 

  27. Junt T, Schulze H, Chen Z, Massberg S, Goerge T, Krueger A, Wagner DD, Graf T, Italiano JE Jr, Shivdasani RA, von Andrian UH (2007) Dynamic visualization of thrombopoiesis within bone marrow. Science 317:1767–1770

    Article  PubMed  CAS  Google Scholar 

  28. Lordier L, Jalil A, Aurade F, Larbret F, Larghero J, Debili N, Vainchenker W, Chang Y (2008) Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling. Blood 112:3164–3174

    Article  PubMed  CAS  Google Scholar 

  29. Schulze H, Korpal M, Hurov J, Kim SW, Zhang J, Cantley LC, Graf T, Shivdasani RA (2006) Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis. Blood 107:3868–3875

    Article  PubMed  CAS  Google Scholar 

  30. Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig JH (1999) Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol 147:1299–1312

    Article  PubMed  CAS  Google Scholar 

  31. Robert A, Boyer L, Pineault N (2011) Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture. Stem Cells Dev 20:379–390

    Article  PubMed  CAS  Google Scholar 

  32. Cortin V, Pineault N, Garnier A (2009) Ex vivo megakaryocyte expansion and platelet production from human cord blood stem cells. Methods Mol Biol 482:109–126

    Article  PubMed  CAS  Google Scholar 

  33. Darzynkiewick Z, Huang X (2004) In: Collingan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W (eds) Current protocols in immunology. Wiley, New York, pp 5.7.1–5.7.18

    Google Scholar 

  34. Bornstein R, Garcia-Vela J, Gilsanz F, Auray C, Cales C (2001) Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation. Br J Haematol 114:458–465

    Article  PubMed  CAS  Google Scholar 

  35. Mattia G, Vulcano F, Milazzo L, Barca A, Macioce G, Giampaolo A, Hassan HJ (2002) Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release. Blood 99:888–897

    Article  PubMed  CAS  Google Scholar 

  36. Proulx C, Dupuis N, St-Amour I, Boyer L, Lemieux R (2004) Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C. Biotechnol Bioeng 88:675–680

    Article  PubMed  CAS  Google Scholar 

  37. Pineault N, Boucher J-F, Cayer M-P, Palmqvist L, Boyer L, Lemieux R, Proulx C (2008) Characterization of the effects and potential mechanisms leading to increased megakaryocytic differentiation under mild hyperthermia. Stem Cells Dev 17:483–494

    Article  PubMed  CAS  Google Scholar 

  38. Bergmeier W, Burger PC, Piffath CL, Hoffmeister KM, Hartwig JH, Nieswandt B, Wagner DD (2003) Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets. Blood 102:4229–4235

    Article  PubMed  CAS  Google Scholar 

  39. Muntean AG, Pang L, Poncz M, Dowdy SF, Blobel GA, Crispino JD (2007) Cyclin D-Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization. Blood 109:5199–5207

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by a National Blood Foundation Grant. A. Robert was the recipient of an Industrial R&D Fellowships from the Canadian Natural Science and Engineering Research Council.

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Authors and Affiliations

  1. Département de Recherche et Développement, Héma-Québec, Université Laval, Québec City, QC, Canada

    Nicolas Pineault

  2. Département de Recherche et Développement, Héma-Québec, Québec City, QC, Canada

    Amélie Robert, Valérie Cortin & Lucie Boyer

Authors
  1. Nicolas Pineault
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  2. Amélie Robert
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  3. Valérie Cortin
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  4. Lucie Boyer
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Corresponding author

Correspondence to Nicolas Pineault .

Editor information

Editors and Affiliations

  1. , Experimental Therapeutics, BC Cancer Agency, West 10th Ave. 675, Vancouver, V5Z 1L3, British Columbia, Canada

    Cheryl D. Helgason

  2. STEMCELL Technologies, Inc., W. Seventh Avenue 570, Vancouver, V5Z 1B3, British Columbia, Canada

    Cindy L. Miller

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Pineault, N., Robert, A., Cortin, V., Boyer, L. (2013). Ex Vivo Differentiation of Cord Blood Stem Cells into Megakaryocytes and Platelets. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8_13

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  • DOI: https://doi.org/10.1007/978-1-62703-128-8_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-127-1

  • Online ISBN: 978-1-62703-128-8

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