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

Abstract

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 productionin 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.

Platelets in Wiskott-Aldrich syndrome: Victims or executioners?

“…Moreover, CD34 + cells that were isolated from patients with WAS showed decreased formation of MK colonies compared with healthy donors, and MKs that were obtained in vitro had an abnormal morphology . Similar results were also obtained by using induced pluripotent stem cell lines from two patients with WAS . Dysfunctional MKs and PLTs have also been observed from normal embryonic stem cell lines in which the WAS locus was targeted by zinc finger nucleases.…”

“…103 Similar results were also obtained by using induced pluripotent stem cell lines from two patients with WAS. 104 Dysfunctional MKs and PLTs have also been observed from normal embryonic stem cell lines in which the WAS locus was targeted by zinc finger nucleases. Geneedited MKs and especially PLTs displayed altered phenotypes and defective responses to agonists.…”

Cytoskeletal regulation of platelet formation: Coordination of F-actin and microtubules

“…This, coupled with the use of MKs/platelets derived from patient induced-pluripotent stem (iPS) cells (e.g. Ingrungruanglert et al 2015), will enable the development of both patientspecific therapeutics and models for studying specific diseases. 1) Immature MKs residing in the osteoblastic niche undergo several rounds of endomitosis to increase their size, ploidy and membrane content whilst migrating to sinusoidal vessels in the vascular niche.…”

hiPSC‐derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine

“…Gaint axonal neuropathy (GAN) [136] Menkes disease (MD) [137] Frontotemporal dementia (FTD) [138,139] Spinal cerebral ataxia type2 (SCA2) [140] Ataxia telangiectasia (AT) [141] Dravet syndrome (DVS) [142] Hematological Swachman-Bodian-Diamond syndrome (SBD) [125] Adenosine deaminase deficiency (ADA) severe combined immunodeficiency (SCID) [125] Fanconi anemia (FA) [143] Sickle cell anaemia (SCA) [144] Beta-thalassaemia (BT) [145] Polycythaemia vera (PV) [146] Congenital amegakaryocytic thrombocytopenia (CAMT) [147] Paroxysmal nocturnal haemoglobinuria (PNH) [148] Dyskeratosis congenita (DC) [149] a-Thalassaemia (AT) [150] Aplastic anaemia (AA) [151] Myeloproliferative disorder (MPN) [152] Chronic myeloid leukaemia (CML) [153] Juveline myelomonocytic leukaemia (JMML) [154] Chronic infantile neurological, cutaneous and articular syndrome (CINCA) [155] X-linked chronic granulomatous disease (X-CGD) [156] Severe congenital neutropaenia (SCN) [157] Wiskott-Aldrich syndrome (WAS) [158] Metabolic Gaucher disease type III (GD) [125] Juvenile diabetes mellitus (JDM) [125] Lesch-Nyhan syndrome (LNS) [125] Aplha1-Antitrypsin deficiency (A1ATD) [159] Pompe disease (PomD) [160] Familial hypercholesterolaemia (FH) [161] Tyrosinaemia (TYS) [162] Glycogen storage disease type1 (GSD) [162] 1578…”