Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells
Lazovic, Bojana; Nguyen, Hoang-Tuan; Ansarizadeh, Mohammadhassan; Wigge, Leif; Kohl, Franziska; Li, Songyuan; Carracedo, Miguel; Kettunen, Jere; Krimpenfort, Luc; Elgendy, Ramy; Richter, Kati; De Silva, Laknee; Bilican, Bilada; Singh, Prateek; Saxena, Pratik; Hong, Xuechong; Eklund, Lauri; Hicks, Ryan (2024-05-21)
Springer
21.05.2024
Lazovic, B., Nguyen, HT., Ansarizadeh, M. et al. Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells. Angiogenesis 27, 523–542 (2024). https://doi.org/10.1007/s10456-024-09925-9
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© The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
https://creativecommons.org/licenses/by/4.0/
© The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:oulu-202405294062
https://urn.fi/URN:NBN:fi:oulu-202405294062
Tiivistelmä
Abstract
Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.
Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.
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