(119e) Engineering an iPSC-Based 3D Liver Organoid for Hepatotoxicity Studies

Approximately 23% of Americans take five or more prescription drugs every day. Adverse reactions to medications can cause drug-induced liver injury (DILI), which is the leading cause of acute liver failure in the United States. The majority of DILI cases are idiosyncratic, where the effects of drug- related injury can vary amongst individuals and result in very different responses. When hepatic function deteriorates, the entire body is affected due to the array of functions that the liver performs, such as metabolism, filtration, synthesis, and biotransformation.

The four major cell types in the liver sinusoid, the region where biotransformation of toxicants and xenobiotics occurs, include hepatocytes (parenchymal cells), and three major non-parenchymal cells (NPCs) including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs). Hepatocytes secrete cytochrome P450 (CYP450) enzymes, which enable the biotransformation of drugs and other xenobiotics. Genetic variations in the transcriptome contribute to differences in the expression of various proteins that are involved in metabolism.

Currently, in vitro liver models use primary human hepatocytes (PHHs) or hepatocyte cell lines. While PHHs are ideal for such studies, they are difficult to obtain and are limited in availability. In efforts to address the issues surrounding PHHs, induced pluripotent stem cells (iPSCs) exhibit significant potential for organoid development. iPSCs can be retrieved non-invasively from dermal fibroblasts. iPSCs exhibit infinite proliferation, maintenance of the host genotype, and differentiation into various cell types, such as hepatocyte-like cells (iHLCs). One major concern when working with iHLCs is their fetal phenotype. iHLCs have been shown to exhibit similar morphology to PHHs, but express lower hepato-specific functions. Therefore, our goal is to investigate whether interactions with hepatic NPCs may improve liver functions in iHLCs. We have assembled 3D liver organoids that recapitulate the in vivo environment and architecture of the liver sinusoid for hepatotoxicity studies.

Methods

The 3D organoids were assembled using hepatic parenchymal cells and NPCs. The functions of iHLC-based organoids were compared to cultures containing PHHs. In general, iHLCs were seeded on Type 1 collagen hydrogels and allowed to mature for an additional seven days. To assemble organoids, LSECs were encapsulated in a hydrogel composed of Type 1 collagen and 1% (v/v) fibronectin above the iHLCs or PHHs. The control consisted of hepatocytes cultured as a collagen sandwich (CS). The initial ratio of hepatocytes : LSECs was approximately 5:1. Two prototypic hepatotoxicants, acetaminophen (APAP) and ethanol (EtOH), were administered 24h later at input concentrations of 2.5 mM (1⁄2 LC50, lethal concentration 50) and 5 mM (LC50) for APAP and 80 mM (1⁄2 LC50) and 160 mM (LC50) or EtOH. Cultures were ended either at 24h or 72h post-toxicant administration.

Results

Initial results show iHLC-based organoids produce approximately 76% lower urea than PHH-based organoids. PHH organoids exhibited a statistically significant 40% decrease in urea 24h after administering APAP at 1⁄2 LC50 APAP. Additionally, PHH organoids exhibited a 20% decrease in urea 24h after administering EtOH at LC50. The depletion of glutathione in iHLCs organoids was 1.6-fold and 1.7-fold after administration of EtOH at 1⁄2 LC50 and LC50, respectively, compared to iHLC organoid controls. PHH organoids also exhibited a 12% decrease in the secretion of a liver-specific enzyme, alanine aminotransferase, compared to PHHs in CS after administering EtOH at LC50.