(L-464) Modeling Non-Alcoholic Fatty Liver Disease in vitro Derived from Human Induced Pluripotent Stem Cells

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 464

Presenting Author(s)

YW

Yao Wang

PhD Student
University of California, Irvine
Irvine, California, United States

Co-Author(s)

ZQ

Zhenzhu Qi

Undergraduate student
University of California, Irvine, United States
CC

Christopher Clark

PhD student
University of California, Irvine, United States

Last Author(s)

Quinton Smith, PhD

Assistant Professor
University of California, Irvine, United States

Introduction:: Non-alcoholic fatty liver disease (NAFLD) is one of the most common hepatic diseases in the United States, affecting nearly a quarter of US adults [1]. The high correlation of NAFLD to other health conditions such as obesity and type 2 diabetes suggests the potential cause of NAFLD development, whereas the pathogenesis of NAFLD has not been comprehensively revealed. Recent genome-wide association studies (GWAS) show genes such as patatin-like phospholipase domain-containing 3 (PNPLA3), contain ancestral specific single-nucleotide polymorphism (SNP). These SNPs impact the risk for NAFLD, unveiling the role of metabolic pathways in pathogenesis [2]. Non-alcoholic steatohepatitis (NASH), a more severe stage of NAFLD with increased inflammation, is susceptible to the scarring of the liver, thereby leaving permanent damage. Weight loss at the NAFLD stage has been recommended by the medical community to circumvent the progression to cirrhosis, yet there is no FDA-approved medication targeting NAFLD. Therefore, it is imperative that a model recapitulating critical features of NAFLD shall be developed for better elucidation of disease pathogenesis and evaluation of drug screening.

Materials and Methods:: Aiming at the establishment of the NAFLD model, human induced pluripotent stem cells (hiPSCs) were first differentiated to definitive endoderm. Endoderm cells were then differentiated toward hepatocyte-like cells (HLCs) with our optimized culture protocol based on hepatic growth factor. During the maturation phase, cells underwent either 2D or 3D culture to generate cell sheets or hepatic spheroids. Characterization of HLC was carried out with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence, and functional assays, including Periodic Acid-Schiff (PAS) staining, urea detection, cytochrome P450 3A4 (CYP3A4) activity, and albumin enzyme-linked immunoassays (ELISA). HLC was induced with various dietary factors to elicit steatosis, and the dosage was optimized based on the fluorescence intensity of lipid staining with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY). Triglyceride secretion was also measured to corroborate the steatotic induction. Further functional assays including bile canaliculi staining, albumin ELISA, and CYP3A4 activity, were performed to assess NAFLD-like morphological characteristics upon induction. Activation of fibroblasts was evaluated by qRT-PCR, immunofluorescence, and ELISA. Knockdown of PNPLA3 was achieved in the HLC maturation phase with 2-deoxy-2-fluoro-D-arabinonucleic acid antisense oligonucleotides. PNPLA3 knockdown cells were compared with scramble control in terms of functionalities.

Results, Conclusions, and Discussions:: We first showed the successful differentiation of HLCs with upregulated gene expression of ALB, SERPINA, and SREBF1. Immunofluorescence of ALB, A1AT, and EpCam also indicated that the cells we derived via 2D or 3D culture were of hepatic lineage. The HLCs were proved functional given that the PAS staining showed glycogen storage, and active urea biosynthesis was detected. After the induction of dietary factors, lipid accumulation was observed in HLCs. Quantifying BODIPY staining revealed optimal induction by 100 μΜ oleic acid (OA) treatment, resulting in cells with hepatocellular ballooning phenotype. Disrupted bile canaliculi were identified after OA treatment, along with lower albumin secretion. In contrast, OA led to increased triglyceride secretion and CYP3A4 activity. In order to study the paracrine effect of hepatocytes on hepatic stellate cells, which is a main contributor to liver fibrosis, human epidermal fibroblasts were either co-cultured with HLCs in Transwell or cultured with conditioned media from HLC (HLC-CM). Both HLC-CM and transwell culture aggravated extracellular matrix deposition, which was corroborated by the immunofluorescence intensity of fibronectin. Moreover, gene upregulation of ACTA2, COL1A1, and MYH11 was detected in fibroblasts cultured with HLC-CM. This also gave rise to amplified cytokine secretion, such as IL-6, suggesting the onset of inflammation. HLCs subject to PNPLA3 knockdown facilitated lipid formation, which was independent of OA induction.

Acknowledgements (Optional): :

References (Optional): :

Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease—meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64(1):73–84. doi:10.1002/hep.28431

Anstee, Q., Day, C. The genetics of NAFLD. Nat Rev Gastroenterol Hepatol 10, 645–655 (2013). https://doi.org/10.1038/nrgastro.2013.182