(460e) Maturation of Ihlcs through Intercellular Signaling in 3D Liver Organoids

The liver is the largest visceral organ in the body, performing an array of vital functions, including filtration, synthesis, and biotransformation. When liver function deteriorates, the entire body is affected. Currently, in vitro models to study the liver use primary human hepatocytes (PHHs) or hepatic cell lines. Although PHHs are most appropriate for such studies, these cells are difficult to obtain and can have significant donor variability, potentially affecting data during experimentation. Induced pluripotent stem cell-hepatocyte like cells (iHLCs) exhibit significant potential for in vitro studies. iHLCs are retrieved non-invasively from skin cells and maintain host genotype, making them ideal for patient-specific studies. Although, widespread use is limited due to their fetal phenotypic characteristics and expression of lower hepato-specific functions. Current methods most commonly require variations of chemical cocktails that need to be administered in specific temporal sequences, which has led to inconsistent results in maturation.

Therefore, our goal was to investigate whether interactions with hepatic non-parenchymal cells (NPCs) may improve liver functions in iHLCs. We have assembled 3D liver organoids with Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs), both NPCs, that recapitulate the in vivo hepatic cell ratios, environment, and architecture. This method can provide a more systematic, repeatable, and reproducible process for maturing iHLCs that could be used more consistently across the liver research community.

Methods

The 3D organoids were assembled using iHLCs or PHHs and NPCs. In general, hepatocytes were seeded on Type 1 collagen hydrogels. iHLCs were allowed to mature for seven days. PHHs were seeded similarly one day before organoid assembly. To assemble organoids, LSECs or LSECs and KCs were encapsulated in a hydrogel composed of Type 1 collagen and 1% (v/v) fibronectin above the iHLCs or PHHs. The initial ratio of hepatocytes: LSECs is 5:1. The initial ratio of hepatocytes: KCs is 10:1. Organoids assembled with KCs, LSECs and either iHLCs or PHHs are denoted henceforth as i3DHLK or p3DHLK. The control consisted of iHLCs or PHHs cultured as a collagen sandwich (iCS or pCS). iHLC-organoids were analyzed on Day 1, Day 7, and Day 14 after assembly. PHH organoids were analyzed on Day 1 and Day 7. Two prototypic hepatotoxicants, acetaminophen (APAP) and ethanol (EtOH), were administered at input concentrations of 2.5 mM (LC₅₀, lethal concentration 50) and 5 mM (2 X LC₅₀) for APAP and 80 mM (½ LC₅₀) and 160 mM (LC₅₀) or EtOH. Cultures were ended for analysis to determine mode of cell death 24 hours after toxicant administration.

Results

We observe that after either 7 or 14 days of co-culture, approximately 70% of cells exhibit expression of intracellular albumin in i3DHLK cultures, which is similar to p3DHLK organoids. Fluorescence intensity of CYP enzymes was significantly higher in i3DHLK organoids compared to iCS throughout the culture period. Additionally, the i3DHLK organoids exhibited a significant decrease in AFP fluorescence intensity by 37% at Day 7 compared to Day 1, showing similar expression compared to the equivalent PHH organoid. These results indicate that iHLCs are expressing mature markers in i3DHLK organoids similar to the equivalent PHH models.

When APAP was administered at 2.5 mM, i3DHLK organoids (after 14 days of co-culture) exhibited a statistically significant increases in apoptosis and necrosis, compared to iCS cultures. When EtOH was administered at 160 mM, the percentage of live cells in i3DHLK was significantly lower by 25% after 7 days of co-culture compared to iCS. These results indicate that i3DHLK organoids are exhibiting sensitivity upon toxicant administration.

Conclusions and Future Work

i3DHLK organoids exhibit increased hepatic mature markers and function compared to monocultures. The iHLCs also exhibited similar phenotypic markers compared to PHHs in 3DHLK models. Implementation of these organoids in the liver research community could serve as a more reproducible method to mature iHLCs. Quantification of specific signaling factors secreted by the NPCs is ongoing. Future work will include adding hepatic stellate cells (HSCs), to further recapitulate the liver environment.

References

1. Arias et. al. 6^th (2001).
2. Orbach et. al. In Vitro. (2018).