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Thromb Haemost 2015; 113(04): 792-805
DOI: 10.1160/TH14-06-0503
Cellular Haemostasis and Platelets
Schattauer GmbH

Wiskott-Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation

Praewphan Ingrungruanglert   *
1   Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
,
Pramuk Amarinthnukrowh   *
2   Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
3   Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
,
Ruttachuk Rungsiwiwut
4   Human Embryonic Stem Cell Research Center, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
,
Supang Maneesri-le Grand
5   Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
,
Darintr Sosothikul
6   Division of Pediatric Hematology/Oncology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
,
Kanya Suphapeetiporn
2   Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
3   Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
,
Nipan Israsena
1   Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
7   Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
,
Vorasuk Shotelersuk
2   Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
3   Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
› Author Affiliations
Further Information

Publication History

Received: 11 June 2014

Accepted after major revision: 04 November 2014

Publication Date:
24 November 2017 (online)

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Summary

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet production in vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.

Keywords

Wiskott-Aldrich syndrome - WASP - iPSCs - platelet - thrombopoiesis

* These authors contributed equally to this work.


 

  • References

  • 1 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663-676.
    CrossrefPubMedGoogle Scholar
  • 2 Lee G, Papapetrou EP, Kim H. et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 2009; 461: 402-406.
    CrossrefPubMedGoogle Scholar
  • 3 Marchetto MC, Carromeu C, Acab A. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 2010; 143: 527-539.
    CrossrefPubMedGoogle Scholar
  • 4 Moretti A, Bellin M, Welling A. et al. Patient-specific induced pluripotent stemcell models for long-QT syndrome. N Engl J Med 2010; 363: 1397-1409.
    CrossrefPubMedGoogle Scholar
  • 5 Grskovic M, Javaherian A, Strulovici B. et al. Induced pluripotent stem cells-- opportunities for disease modelling and drug discovery. Nat Rev Drug Discov 2011; 10: 915-929.
    PubMedGoogle Scholar
  • 6 Onder TT, Daley GQ. New lessons learned from disease modeling with induced pluripotent stem cells. Curr Opin Genetics Devel 2012; 22: 500-508.
    PubMedGoogle Scholar
  • 7 Orange JS, Stone KD, Turvey SE. et al. The Wiskott-Aldrich syndrome. Cell Mol Life Sci 2004; 61: 2361-2385.
    PubMedGoogle Scholar
  • 8 Thrasher AJ, Burns SO. WASP: a key immunological multitasker. Nat Rev Immunol 2010; 10: 182-192.
    CrossrefPubMedGoogle Scholar
  • 9 Snapper SB, Rosen FS. The Wiskott-Aldrich syndrome protein (WASP): roles in signaling and cytoskeletal organisation. Ann Rev Immunol 1999; 17: 905-929.
    CrossrefPubMedGoogle Scholar
  • 10 Taylor MD, Sadhukhan S, Kottangada P. et al. Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Sci Transl Med 2010; 02: 37ra44.
    PubMedGoogle Scholar
  • 11 Moulding DA, Blundell MP, Spiller DG. et al. Unregulated actin polymerisation by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp Med 2007; 204: 2213-2224.
    CrossrefPubMedGoogle Scholar
  • 12 Devriendt K, Kim AS, Mathijs G. et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat Genet 2001; 27: 313-317.
    CrossrefPubMedGoogle Scholar
  • 13 Snapper SB, Rosen FS, Mizoguchi E. et al. Wiskott-Aldrich syndrome proteindeficient mice reveal a role for WASP in T but not B cell activation. Immunity 1998; 09: 81-91.
    CrossrefPubMedGoogle Scholar
  • 14 Sabri S, Foudi A, Boukour S. et al. Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment. Blood 2006; 108: 134-140.
    CrossrefPubMedGoogle Scholar
  • 15 Kajiwara M, Nonoyama S, Eguchi M. et al. WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro. Br J Haematol 1999; 107: 254-262.
    CrossrefPubMedGoogle Scholar
  • 16 Haddad E, Cramer E, Riviere C. et al. The thrombocytopenia of Wiskott Aldrich syndrome is not related to a defect in proplatelet formation. Blood 1999; 94: 509-518.
    CrossrefPubMedGoogle Scholar
  • 17 Woods NB, Parker AS, Moraghebi R. et al. Brief report: efficient generation of haematopoietic precursors and progenitors from human pluripotent stem cell lines. Stem Cells 2011; 29: 1158-1164.
    CrossrefPubMedGoogle Scholar
  • 18 Vodyanik MA, Bork JA, Thomson JA. et al. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohaematopoietic potential. Blood 2005; 105: 617-626.
    CrossrefPubMedGoogle Scholar
  • 19 Kaufman DS, Hanson ET, Lewis RL. et al. Haematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001; 98: 10716-10721.
    CrossrefPubMedGoogle Scholar
  • 20 Takayama N, Nishimura S, Nakamura S. et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells. J Exp Med 2010; 207: 2817-2830.
    CrossrefPubMedGoogle Scholar
  • 21 Takayama N, Nishikii H, Usui J. et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate haematopoietic progenitors. Blood 2008; 111: 5298-5306.
    CrossrefPubMedGoogle Scholar
  • 22 Lu SJ, Li F, Yin H. et al. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Cell Res 2011; 21: 530-545.
    CrossrefPubMedGoogle Scholar
  • 23 Takayama N, Eto K. Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application. Cell Mol Life Sci 2012; 69: 3419-3428.
    CrossrefPubMedGoogle Scholar
  • 24 Chatchatee P, Srichomthong C, Chewatavorn A. et al. A novel termination codon mutation of the WAS gene in a Thai family with Wiskott-Aldrich syndrome. Int J Mol Med 2003; 12: 939-941.
    PubMedGoogle Scholar
  • 25 Amarinthnukrowh P, Ittiporn S, Tongkobpetch S. et al. Clinical and molecular characterisation of thai patients with wiskott-Aldrich syndrome. Scand J Immunol 2013; 77: 69-74.
    CrossrefPubMedGoogle Scholar
  • 26 Fusaki N, Ban H, Nishiyama A. et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 2009; 85: 348-362.
    CrossrefPubMedGoogle Scholar
  • 27 Ban H, Nishishita N, Fusaki N. et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci USA 2011; 108: 14234-14239.
    CrossrefPubMedGoogle Scholar
  • 28 Nishishita N, Shikamura M, Takenaka C. et al. Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naive state. PLoS One 2012; 07: e38389.
    CrossrefPubMedGoogle Scholar
  • 29 Pruksananonda K, Rungsiwiwut R, Numchaisrika P. et al. Eighteen-year cryopreservation does not negatively affect the pluripotency of human embryos: evidence from embryonic stem cell derivation. Biores Open Access 2012; 01: 166-173.
    CrossrefPubMedGoogle Scholar
  • 30 Park IH, Zhao R, West JA. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008; 451: 141-146.
    CrossrefPubMedGoogle Scholar
  • 31 Reems JA, Pineault N, Sun S. In vitro megakaryocyte production and platelet biogenesis: state of the art. Transfus Med Rev 2010; 24: 33-43.
    CrossrefPubMedGoogle Scholar
  • 32 Gekas C, Graf T. Induced pluripotent stem cell-derived human platelets: one step closer to the clinic. J Exp Med 2010; 207: 2781-2784.
    CrossrefPubMedGoogle Scholar
  • 33 Grottum KA, Hovig T, Holmsen H. et al. Wiskott-Aldrich syndrome: qualitative platelet defects and short platelet survival. Br J Haematol 1969; 17: 373-388.
    CrossrefPubMedGoogle Scholar
  • 34 Luthi JN, Gandhi MJ, Drachman JG. X-linked thrombocytopenia caused by a mutation in the Wiskott-Aldrich syndrome (WAS) gene that disrupts interaction with the WAS protein (WASP)-interacting protein (WIP). Exp Hematol 2003; 31: 150-158.
    CrossrefPubMedGoogle Scholar
  • 35 Ancliff PJ, Blundell MP, Cory GO. et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006; 108: 2182-2189.
    CrossrefPubMedGoogle Scholar
  • 36 Leven RM, Yee MK. Megakaryocyte morphogenesis stimulated in vitro by whole and partially fractionated thrombocytopenic plasma: a model system for the study of platelet formation. Blood 1987; 69: 1046-1052.
    PubMedGoogle Scholar
  • 37 Tablin F, Castro M, Leven RM. Blood platelet formation in vitro. The role of the cytoskeleton in megakaryocyte fragmentation. J Cell Sci 1990; 97: 59-70.
    PubMedGoogle Scholar
  • 38 Italiano Jr. JE, Lecine P, Shivdasani RA. et al. Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes. J Cell Biol 1999; 147: 1299-1312.
    CrossrefPubMedGoogle Scholar
  • 39 Junt T, Schulze H, Chen Z. et al. Dynamic visualisation of thrombopoiesis within bone marrow. Science 2007; 317: 1767-1770.
    CrossrefPubMedGoogle Scholar
  • 40 Thon JN, Macleod H, Begonja AJ. et al. Microtubule and cortical forces determine platelet size during vascular platelet production. Nat Commun 2012; 03: 852.
    CrossrefPubMedGoogle Scholar
  • 41 Kim K, Doi A, Wen B. et al. Epigenetic memory in induced pluripotent stem cells. Nature 2010; 467: 285-290.
    CrossrefPubMedGoogle Scholar
  • 42 Thompson A, Zhao Z, Ladd D. et al. A new transgenic mouse model for the study of cell cycle control in megakaryocytes. Stem Cells 1996; 14 (Suppl. 01) 181-187.
    CrossrefPubMedGoogle Scholar
  • 43 Liu J, Levens D. Making myc. Curr Top Microbiol Immunol 2006; 302: 1-32.
    PubMedGoogle Scholar
  • 44 Astrakhan A, Sather BD, Ryu BY. et al. Ubiquitous high-level gene expression in haematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome. Blood 2012; 119: 4395-4407.
    CrossrefPubMedGoogle Scholar
  • 45 Bosticardo M, Draghici E, Schena F. et al. Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome. J Allergy Clin Immunol 2011; 127: 1376-1384 e1375.
    CrossrefPubMedGoogle Scholar
  • 46 Boztug K, Schmidt M, Schwarzer A. et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med 2010; 363: 1918-1927.
    CrossrefPubMedGoogle Scholar
  • 47 Zou J, Sweeney CL, Chou BK. et al. Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood 2011; 117: 5561-5572.
    CrossrefPubMedGoogle Scholar
  • 48 Suzuki N, Yamazaki S, Yamaguchi T. et al. Generation of Engraftable Haematopoietic Stem Cells From Induced Pluripotent Stem Cells by Way of Teratoma Formation. Mol Ther 2013; 21: 1424-1431.
    CrossrefPubMedGoogle Scholar