Laryngorhinootologie 2023; 102(S 02): S194
DOI: 10.1055/s-0043-1767073
Abstracts | DGHNOKHC
Biomaterials/Tissue Engineering/Stem Cells

Generation of an ACVRL1 knockout iPS cell line for the in vitro study of HHT type 2 associated angiogenesis

Robert Mandic
1   Klinik für Hals-, Nasen- und Ohrenheilkunde, Kopf- und Hals-Chirurgie
,
Michael Bette
2   Institut für Anatomie und Zellbiologie
,
R. Johanna Rusche
1   Klinik für Hals-, Nasen- und Ohrenheilkunde, Kopf- und Hals-Chirurgie
,
A. Boris Stuck
1   Klinik für Hals-, Nasen- und Ohrenheilkunde, Kopf- und Hals-Chirurgie
,
Udo Bakowsky
3   Institut für Pharmazeutische Technologie und Biopharmazie
,
Boris Greber
4   Catalent Cell and Gene Therapy
,
Maria Wartenberg
5   Klinik für Innere Medizin I
,
Heinrich Sauer
6   Institut für Physiologie
,
W. Urban Geisthoff
1   Klinik für Hals-, Nasen- und Ohrenheilkunde, Kopf- und Hals-Chirurgie
,
Li Xiang-Tischhauser
1   Klinik für Hals-, Nasen- und Ohrenheilkunde, Kopf- und Hals-Chirurgie
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    Background Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutant. Patients exhibit severe disturbances in TGF-beta/BMP dependent angiogenesis and, clinically, present with severe nosebleeds and a highly reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem (iPS) cells and evaluate the effects on TGF-beta and BMP related gene expression and angiogenesis.

    Methods CRISPR/Cas9 knockout of the ACVRL1 gene was performed using wildtype iPS (ACVRL1wt/wt) cells. Embryoid bodies (EBs) were generated from iPS cells by changing the regular medium (Essential 8TM, Gibco) to RPMI 1640 medium (Gibco) supplemented with 5 ng/ml activin A, 4 ng/ml BMP4, and 2 μmol/L CHIR99021. EBs were induced to endothelial differentiation by adding 4 ng/ml BMP4 and 10 ng/ml VEGF. EB-associated angiogenesis was monitored by immunocytochemistry using a CD31-specific antibody. Analysis of 151 TGF-beta/BMP related genes was performed by RT-qPCR analysis.

    Results A HHT type 2 iPS cell line was generated by single knockout (ACVRL1wt/mut) of wild type (wt) (ACVRL1wt/wt) iPS cells resulting in a frameshift deletion in the ACVRL1 gene [NM_000020.3(ACVRL1):c.1137-1153del]. Compared to wildtype EBs, EBs derived from mutant iPS cells demonstrated an earlier but major reduction of endothelial differentiation as visualized after CD31 staining. Differential TGF-beta/BMP gene expression was observed between ACVRL1 wt and mutant iPS cell lines.

    Conclusion EBs derived from CRISPR/Cas9 designed HHT type 2 iPS cells together with their isogenic wt iPS counterpart can serve as valuable resources for HHT type 2 in vitro studies.

    Projektförderung durch "Forschungscampus Mittelhessen"


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    Conflict of Interest

    The authors declare that they have no conflict of interest.

    Publication History

    Article published online:
    12 May 2023

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