2. Academic Validation
  3. Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells

Human iPSC and CRISPR targeted gene knock-in strategy for studying the somatic TIE2L914F mutation in endothelial cells

  • Angiogenesis. 2024 May 21. doi: 10.1007/s10456-024-09925-9.
Bojana Lazovic 1 2 3,  Hoang-Tuan Nguyen 3 4,  Mohammadhassan Ansarizadeh 3,  Leif Wigge 5,  Franziska Kohl 2 6,  Songyuan Li 2,  Miguel Carracedo 7,  Jere Kettunen 4,  Luc Krimpenfort 6,  Ramy Elgendy 2,  Kati Richter 3,  Laknee De Silva 3,  Bilada Bilican 2,  Prateek Singh 4,  Pratik Saxena 1,  Lars Jakobsson 6,  Xuechong Hong 1,  Lauri Eklund 3,  Ryan Hicks 8 9
Affiliations

Affiliations

  • 1 BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 2 Translational Genomics, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 3 Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland.
  • 4 Finnadvance Ltd., Oulu, Finland.
  • 5 Data Sciences and Quantitative Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 6 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
  • 7 Bioscience Renal, Research and Early Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
  • 8 BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden. ryan.hicks@astrazeneca.com.
  • 9 School of Cardiovascular and Metabolic Medicine & Sciences, King's College London, London, UK. ryan.hicks@astrazeneca.com.
PMID: 38771392 DOI: 10.1007/s10456-024-09925-9
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.

Keywords

Endothelial cells; Gene editing; TIE2; Venous malformations; iPSC.

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