Generation of a Syngeneic Heterozygous <i>ACVRL1<sup>(wt/mut)</sup></i> Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
Li Xiang-Tischhauser,
Michael Bette,
Johanna R. Rusche,
Katrin Roth,
Norio Kasahara,
Boris A. Stuck,
Udo Bakowsky,
Maria Wartenberg,
Heinrich Sauer,
Urban W. Geisthoff,
Robert Mandic
Affiliations
Li Xiang-Tischhauser
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Michael Bette
Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-Universität Marburg, 35037 Marburg, Germany
Johanna R. Rusche
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Katrin Roth
Cellular Imaging Core Facility, Center for Tumor Biology and Immunology (ZTI), Philipps-Universität Marburg, 35043 Marburg, Germany
Norio Kasahara
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Boris A. Stuck
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Udo Bakowsky
Department of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, 35037 Marburg, Germany
Maria Wartenberg
Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, Friedrich Schiller University, 07747 Jena, Germany
Heinrich Sauer
Department of Physiology, Justus-Liebig University Giessen, 35392 Giessen, Germany
Urban W. Geisthoff
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Robert Mandic
VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany
Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.
