Erzeugung von iPSC^{ACVRL1(wt/mut)}-abgeleiteten Endothelzellen für In-vitro-Studien zur hereditären hämorrhagischen Teleangiektasie Typ 2 (HHT2)

Introduction. Hereditary hemorrhagic telangiectasia (HHT) type 2 is a vascular disease in which one allele of the ACVRL1 gene is mutant (mut). Clinically, HHT2 patients often present with severe nosebleeds and a reduced quality of life. Previously we reported about modifying an induced pluripotent stem cell (iPSC) line using CRISPR/Cas9, giving rise to a mutation in exon 8 of the ACVRL1 gene which is resembling the situation found in HHT2 patients. The aim of the study was to use this iPSC line together with its parental wildtype (wt) line to generate endothelial cells (ECs) to be used for HHT2 in vitro studies.

Material and methods. iPSC^{ACVRL1(wt/mut)} as well as iPSC^{ACVRL1(wt/wt)} cells were deployed in the study. Endothelial differentiation was performed according to a protocol by Patsch et al. (Nat Cell Biol, 2015, 994-1003). For this, cells were initially differentiated into mesodermal cells using CHIR-99021 (8 µmol/L) and BMP4 (25 ng/ml). For endothelial differentiation, 200 ng/ml VEGF-A and 2 µmol/L Forskolin were added. Flow cytometry was used to measure the fraction of CD31-positive ECs. Tube formation was analyzed with the ImageJ/Fiji Angiogenesis Analyzer.

Results. Between 10 to 25% of iPSC^{ACVRL1(wt/wt)} cells exhibited CD31-positive ECs, whereas iPSC^{ACVRL1(wt/mut)} yielded only between 6 and 10% ECs. After EC differentiation, both iPSC types but not the controls were capable of tube formation. Mutant ECs compared to wt ECs exhibited a diminished capacity for tube formation, which was attributed to a significant increase in the number of isolated segments, total length of isolated branches, and the mean mesh size.

Discussion. ECs could be efficiently derived from ACVRL1 wt and mut iPSCs, which can serve as valuable resources for in vitro studies of HHT type 2.

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