Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain; Current address: Amarna Therapeutics S.L., Instituto Cartuja, C/ Leonardo da Vinci 19ª, Seville, Spain.
Miguel G
MG
Toscano
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain; Current address: University College London-Institute of Child Health, London, UK.
Pilar
P
Muñoz
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Almudena
A
Sánchez-Gilabert
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Marién
M
Cobo
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Karim
K
Benabdellah
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Per
P
Anderson
Genomic Oncology Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Verónica
V
Ramos-Mejía
Genomic Oncology Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Pedro J
PJ
Real
Unidad de Enfermedades Infecciosas e Inmunopatologías Pediátricas, Hospitales Universitarios Virgen del Rocío, Instituto de Biomedicina de Sevilla, Sevilla, Spain.
Olaf
O
Neth
UGC Hematología y Hemoterapia, Hospital Infantil Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS)/CSIC/Universidad de Sevilla, Seville, Spain.
Agueda
A
Molinos-Quintana
Sangamo BioSciences, Inc., Pt. Richmond Tech Center, Richmond, California, USA.
Philip D
PD
Gregory
Sangamo BioSciences, Inc., Pt. Richmond Tech Center, Richmond, California, USA.
Michael C
MC
Holmes
Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalusian Regional Government, Parque Tecnológico Ciencias de la Salud, Granada, Spain. Electronic address: francisco.martin@genyo.es.
Francisco
F
Martin
D018952:Antigens, CD34 / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D055786:Gene Knockout Techniques; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology*; D008954:Models, Biological; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000172:deficiency*
D018952:Antigens, CD34; D040201:Platelet Membrane Glycoprotein IIb; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human