VASCERN HHT Reference Centre, Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Marburg, Philipps-Universität Marburg, 35033 Marburg, Germany.
Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps-Universität Marburg, 35037 Marburg, Germany.
Cells. 2023 Jun 10;12(12):1600. doi: 10.3390/cells12121600.
Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the 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 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 gene was carried out in previously characterized wild-type () iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout () in wild-type () iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the 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 and iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed 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.
遗传性出血性毛细血管扩张症 2 型（HHT2）是一种常染色体显性疾病，其中一个基因的等位基因发生突变。患者表现出 TGF-β/BMP 依赖性血管生成障碍，临床上常表现为严重鼻出血和生活质量下降。我们的研究目的是使用 CRISPR/Cas9 敲除正常诱导多能干细胞（iPSC）中的基因，并评估其对 TGF-β 和 BMP 相关基因表达以及血管生成的影响。该基因的 CRISPR/Cas9 敲除在之前特征明确的野生型（）iPSC 中进行。通过在野生型（）iPSC 中单等位基因敲除（）产生 HHT2 iPSC 系，导致基因中 17 个碱基对的框移缺失[NG_009549.1：g.13707_13723del; NM_000020.3：c.1137_1153del]。生成类胚体（EBs）后，通过添加 4ng/mL BMP4、2%B27 和 10ng/mL VEGF 诱导内皮细胞分化。通过免疫细胞化学监测内皮细胞分化。通过使用源自单个 iPSC 培养物的 mRNA 以及源自内皮细胞分化后的 EBs 的 RT-qPCR 分析了 151 个 TGF-β/BMP 相关基因。在基因和基因 iPSC 以及内皮细胞之间观察到差异 TGF-β/BMP 基因表达。源自 CRISPR/Cas9 设计的 HHT2 突变型 iPSC 的 EBs 及其同型野生型 iPSC 对照可作为 HHT2 体外研究的有价值资源。
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