Characterisation of immortalised lymphatic endothelial cells to implement CRISPR-Cas9-mediated introduction of "PIK3CA" variants associated with lymphatic malformations

Details

Supervisors

Brouillard, Pascal ; Gillis, Annika

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enLymphatic malformations (LMs) are rare congenital vascular anomalies mainly caused by activating PIK3CA variants, leading to hyperactivation of the PI3K/AKT signalling pathway and abnormal lymphangiogenesis. Current experimental models remain limited, particularly due to the restricted lifespan of primary lymphatic endothelial cells (LECs). The development of immortalised lymphatic endothelial cells (imLECs) therefore represents a promising alternative for LM research. The aim of this thesis was to evaluate the feasibility of implementing CRISPR-Cas9 technology in imLECs to introduce PIK3CA variants associated with LMs. First, limiting dilution experiments demonstrated that imLECs were capable of generating proliferative single-cell clones while maintaining expression of lymphatic markers such as PROX1. Nucleofection experiments further showed that exogenous genetic material could be successfully introduced into these cells. The phenotype of imLECs was subsequently characterised over several passages. Although the cells initially maintained a typical endothelial morphology and lymphatic marker expression, progressive alterations were observed at higher passages, including reduced proliferation, decreased PROX1 expression, and altered sprouting capacities in functional assays. In parallel, LM patient-derived LECs harbouring a PIK3CA variant displayed increased AKT activation and disorganised sprouting behaviour consistent with the LM phenotype. Finally, preliminary CRISPR-Cas9 experiments aiming to introduce the PIK3CA-H1047R variant into imLECs were initiated. Although further validation is still required, this work demonstrates the potential of combining imLECs with CRISPR-Cas9 technology and provides a foundation for the future development of stable in vitro models for LM research.