Development of genetically engineered human iPSC-derived endothelial cell models to study the effects of TIE2, KRIT1 and CCM2 mutations

Mutations in the TIE2, KRIT1, and CCM2 genes are known to cause venous malformations and cerebral cavernous malformations. The cellular and molecular mechanisms by which these genetic defects lead to disease remain largely unknown, and no curative medicinal treatments have yet been identified for vascular malformations. Endothelial cells (iECs) derived from induced pluripotent stem cells (iPSCs) may offer promising new tools to advance research in vascular biology and vascular diseases. The aim of this doctoral dissertation was to investigate whether iECs can model vascular malformations caused by genetic defects, and what advantages iEC technology may offer compared to traditional methods. Additionally, by utilizing iEC technology and modern genome-editing methods, the goal was to develop molecular switches that control cell cycle regulation to advance stem cell therapies.
Targeted mutations in the genes under study were introduced using the CRISPR-Cas9 gene-editing method. The effects of these mutations were examined in traditional cell culture dishes, advanced 3D cell models, organ-on-chip systems, and through cell transplants in mice, employing microscopy, transcriptomics, and epigenetic analyses. By comparing normal iEC control cells to genetically modified iECs, changes were observed in the key properties and functionalities of endothelial cells. The study also succeeded in optimizing methods for achieving efficient gene editing and in creating a molecular "kill switch" by which potentially harmful cells could be eliminated from the body after cell therapy.
Based on the results of this doctoral research, iEC models provide valuable new research methods that can deepen our understanding of vascular biology and pathophysiology, and potentially accelerate drug discovery for new treatments of vascular malformations.