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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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. |
format | Online Article Text |
id | pubmed-10297623 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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