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Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis

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...

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Autores principales: Xiang-Tischhauser, Li, Bette, Michael, Rusche, Johanna R., Roth, Katrin, Kasahara, Norio, Stuck, Boris A., Bakowsky, Udo, Wartenberg, Maria, Sauer, Heinrich, Geisthoff, Urban W., Mandic, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10297623/
https://www.ncbi.nlm.nih.gov/pubmed/37371070
http://dx.doi.org/10.3390/cells12121600
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author Xiang-Tischhauser, Li
Bette, Michael
Rusche, Johanna R.
Roth, Katrin
Kasahara, Norio
Stuck, Boris A.
Bakowsky, Udo
Wartenberg, Maria
Sauer, Heinrich
Geisthoff, Urban W.
Mandic, Robert
author_facet Xiang-Tischhauser, Li
Bette, Michael
Rusche, Johanna R.
Roth, Katrin
Kasahara, Norio
Stuck, Boris A.
Bakowsky, Udo
Wartenberg, Maria
Sauer, Heinrich
Geisthoff, Urban W.
Mandic, Robert
author_sort Xiang-Tischhauser, Li
collection PubMed
description 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 (ACVRL1(wt/wt)) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1(wt/mut)) in wild-type (ACVRL1(wt/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 ACVRL1(wt/wt) and ACVRL1(wt/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.
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spelling pubmed-102976232023-06-28 Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis Xiang-Tischhauser, Li Bette, Michael Rusche, Johanna R. Roth, Katrin Kasahara, Norio Stuck, Boris A. Bakowsky, Udo Wartenberg, Maria Sauer, Heinrich Geisthoff, Urban W. Mandic, Robert Cells Article 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 (ACVRL1(wt/wt)) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1(wt/mut)) in wild-type (ACVRL1(wt/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 ACVRL1(wt/wt) and ACVRL1(wt/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. MDPI 2023-06-10 /pmc/articles/PMC10297623/ /pubmed/37371070 http://dx.doi.org/10.3390/cells12121600 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xiang-Tischhauser, Li
Bette, Michael
Rusche, Johanna R.
Roth, Katrin
Kasahara, Norio
Stuck, Boris A.
Bakowsky, Udo
Wartenberg, Maria
Sauer, Heinrich
Geisthoff, Urban W.
Mandic, Robert
Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title_full Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title_fullStr Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title_full_unstemmed Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title_short Generation of a Syngeneic Heterozygous ACVRL1((wt/mut)) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis
title_sort generation of a syngeneic heterozygous acvrl1((wt/mut)) knockout ips cell line for the in vitro study of hht2-associated angiogenesis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10297623/
https://www.ncbi.nlm.nih.gov/pubmed/37371070
http://dx.doi.org/10.3390/cells12121600
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