3 boyutlu hepatik organoidlerde lizozomal asit lipaz eksikliği modelinin geliştirilmesi

In the last decade, organoids have become the most powerful cell culture tool to study human biology in health and disease. Organoids are three-dimensional structures grown in an extracellular matrix that resemble organ structure and show organ-specific functions. Recently established protocols for liver-specific organoids enables the understanding of disease pathology and improvement of patient-specific therapies. Our aim in this work is to utilize organoid technology to model Lysosomal acid lipase deficiency (LALD). Lysosomal acid lipase (LAL) is an enzyme, mainly found in the liver, that breaks down cholesterol esters into free cholesterol. In the absence of the LAL enzyme, cholesterol esters begin to accumulate in liver cells and a spectrum of symptoms can be observed. The mild form with 3%-5% enzyme activity is called cholesterol ester storage disease (CESD) and it can go undiagnosed for years but the severe form is Wolman's disease with less than 1% LAL enzyme activity. Unless treated, WD patients have a life span of maximum a year. LALD models are limited to animal models. These models are completely inadequate when a personalized approach is desired to be developed. Until recently, there were no cell lines that model LALD, however, a recent study has established Wolman Disease organoids from patient-derived iPSCs and thus, paved the way for the understanding of molecular mechanism and treatment options for the disease. In this thesis, first, we have established iPSC clones stably expressing an inducible shRNA vector for the LIPA gene, then we have created an iPSC derived 2D hepatocyte and 3D endodermal hepatic organoid (eHEPO) model for the LALD. LAL deficient 2D hepatocytes have shown to have decreased levels of LAL enzyme activity in comparison with healthy counterparts. On the contrary, our eHEPO model has not been successful to show LAL enzyme activity impairment after a 40% reduction in gene expression. Thus, we conclude that a more dramatic decrease in the gene expression level is needed to mimic the disease phenotype. This suggests that strict regulation of the enzyme activity is present in the 3D organoid model and further investigation of molecular changes and regulation pathways is needed to understand the disease mechanism.