Date of Award
Spring 4-4-2023
Embargo Period
6-1-2028
Document Type
Dissertation - MUSC Only
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular and Cellular Biology and Pathobiology
Additional Department
Regenerative Medicine and Cell Biology
College
College of Graduate Studies
First Advisor
Stephen Duncan
Abstract
Background and Aims: The incidence of Nonalcoholic Fatty Liver Disease (NAFLD) is dramatically increasing in adults and children, while effective pharmacological treatments remain unavailable. NAFLD can progress from lipid accumulation in the liver to more severe inflammation, cirrhosis, and cancer. It is the most common cause of chronic liver disease and is projected to be the leading cause of end-stage liver disease in the next decade. Many etiologies contribute to NAFLD. A single nucleotide polymorphism (SNP) in the Patatin-like Phospholipase Domain Containing Protein (PNPLA3 I148M) has the most significant genetic association with the disease and all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the scarcity of a cellular platform that recapitulates PNPLA3 I148Mmediated onset of lipid accumulation in human hepatocytes. We used hepatocytes generated from PNPLA3 I148M induced Pluripotent Stem Cells (iPSCs) to model the effect of the polymorphism on lipid content and lipid droplet accumulation, providing a platform to identify small molecules with potential therapeutic value using an established high-throughput screening platform.
Approach and Results: We generated isogenic PNPLA3 I148M/M and PNPLA3 ∆1/∆2 iPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9. Using established protocols, we differentiated the resulting cells into hepatocytes and measured the efficiency of differentiation. Using BODIPY 493/503 staining, we determined lipid accumulation in our iPSC- x derived hepatocytes. We then performed a small molecule screen to identify compounds that reduce lipid accumulation in the PNPLA3 variant cells. Both the PNPLA3 I148M/M and PNPLA3 ∆1/∆2 iPSC-induced hepatocytes revealed a significant increase in lipid content compared to control iPSC-induced hepatocytes. Our small molecule screen identified lead compounds that target specific pathways, including Src/PI3K/Akt signaling, that limit lipid accumulation in PNPLA3- deficient iPSC-hepatocytes. We showed that drugs that are currently in clinical trials and that target the same pathways are promising treatments for PNPLA3-derived NAFLD.
Conclusions: We conclude that human iPSC-derived hepatocytes with the PNPLA3 I148M variant or PNPLA3 loss-of-function alleles, can be used to effectively model the onset of NAFLD. We demonstrate that the model also provides a platform to identify molecular pathways with potential therapeutic value and that off-label use of therapeutics that target Src/PI3K/Akt signaling pathways can limit lipid accumulation in PNPLA3 mutant cells.
Recommended Citation
Doueiry, Caren, "A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Drugs to Potentially Reduce Steatosis in Nonalcoholic Fatty Liver Disease" (2023). MUSC Theses and Dissertations. 803.
https://medica-musc.researchcommons.org/theses/803
Rights
Copyright is held by the author. All rights reserved.