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

Praewphan Ingrungruanglert; Pramuk Amarinthnukrowh; Ruttachuk Rungsiwiwut; Supang Maneesri-le Grand; Darintr Sosothikul; Kanya Suphapeetiporn; Nipan Israsena; Vorasuk Shotelersuk

doi:10.1160/TH14-06-0503

Thrombosis and Haemostasis, Table of Contents

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^*

¹Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

,

Pramuk Amarinthnukrowh^*

²Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

³Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand

,

Ruttachuk Rungsiwiwut

⁴Human Embryonic Stem Cell Research Center, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

,

Supang Maneesri-le Grand

⁵Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

,

Darintr Sosothikul

⁶Division of Pediatric Hematology/Oncology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

,

Kanya Suphapeetiporn

²Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

³Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand

,

Nipan Israsena

¹Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

⁷Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

,

Vorasuk Shotelersuk

²Center of Excellence for Medical Genetics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

³Excellence Center for Medical Genetics, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand

› Author Affiliations

Abstract

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

Full Text

References

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