(337j) Maturing iPSC-Derived Hepatocytes in a Multicellular Liver Organoid for Applications in Pharmacology and Toxicology

The liver performs an array of vital functions, including filtration, secretion, and biotransformation, or the metabolism of toxicants and xenobiotics. Thus, in vitro liver models are a valuable tool for investigating mechanisms of injury by toxins or identifying potential drugs for treatment prior to clinical studies. Currently, hepatic models ideally use primary human hepatocytes (PHHs), which are obtained directly from the liver and most appropriate for such studies. However, these cells are difficult to acquire, and the donor’s background can significantly impact data during experimentation. Induced pluripotent stem cell-hepatocyte like cells (iHLCs) exhibit significant potential for in vitro patient-specific studies since they maintain the host genotype, are retrieved non-invasively from skin cells, and are differentiated into hepatocytes. Although, widespread use is limited due to their fetal characteristics and lower liver functions. Current methods for maturation commonly require variations of chemical cocktails which has led to inconsistent results.

We have assembled an iHLC-based 3D liver organoid with Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs), other hepatic cells, that recapitulate the in vivo environment, cellular composition, and architecture. This method can provide a more systematic, repeatable, and reproducible method for maturing iHLCs that could be implemented across the liver research community.

Briefly, hepatocytes were seeded on Type 1 collagen hydrogels. To assemble organoids, LSECs and KCs were encapsulated in a hydrogel composed of Type 1 collagen and fibronectin above iHLCs or PHHs. Organoids were initially seeded in a ratio of 10 hepatocytes: 2 LSECs: 1 KCs, identical to in vivo. The control consisted of hepatocytes cultured as a collagen sandwich. iHLC-organoids were maintained up to 14 days. Two prototypic hepatotoxicants, acetaminophen (APAP) and ethanol (EtOH) were administered on time points and cultures were ended 24 hours later to determine mode of cell death.

Markers of hepatocyte maturation such as albumin expression, cytochrome P450, enzymes responsible for biotransformation, expression, and the ability to metabolize APAP and EtOH were significantly higher in iHLC organoids after 7 and 14 days in culture compared to controls and the equivalent PHH models. These trends were concomitant with a decrease in fetal markers, such as alphafetoprotein. The data indicates that iHLCs require further maturation before they can be potentially used as substitutes for PHHs. Multicellular organoids exhibit significant potential for efficient and sustainable applications in pharmacology and toxicology. Mature iHLC-organoids could individualize drug and environmental toxicant testing, opening new avenues for patient-specific medicine.