(486b) The Assembly of Novel Human 3D Liver Tissues Using Induced Pluripotent Stem Cells

Authors: 
Wills, L., Virginia Tech
Rajagopalan, P., Virginia Tech
Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body. Human iPSC-derived hepatocyte-like cells are a new source for hepatic cells. iPSC-derived hepatocyte-like cells are being evaluated on their expression of mature hepatic phenotypes and their functional capability. Since iPSC-derived hepatocyte-like cells can exhibit fetal characteristics, we have assembled 3D human liver models using iPSC-derived hepatocyte-like cells and hepatic non-parenchymal cells. We have assembled similar 3D models with primary human hepatocytes to compare the functional capability of these cultures.

iCell® Hepatocytes 2.0, (Cellular Dynamics International) and primary human hepatocytes were cultured as monolayers, collagen sandwich and 3D cultures. In the 3D cultures, liver sinusoidal endothelial cells were added to generate 3DHL cultures. Urea secretion was measured over an 18-day period to measure the hepatic function of iPSC-derived hepatocyte-like cells. The monolayer and CS models exhibited urea production for the duration of the culture and exhibited no significant decrease in urea production from the start of the culture to day 18. Albumin production was also observed in the iPSC-derived hepatocyte-like cell cultures until day 18. Since cytochrome P450 enzymes play a critical role in hepatic biotransformation, we measured the activity of cytochrome P450 2E1 (CYP2E1) at different time-points over the culture period. The iPSC-derived hepatocyte-like cells exhibited CYP2E1 activity at days 9 and 18, with no significant decrease occurring between the two time points. All of these results indicate iPSC-derived hepatocyte-like cells are capable of maintaining hepatocyte function for up to 18 days in culture.

Two well-studied hepatotoxicants, acetaminophen and ethanol, were administered at two different concentrations to investigate the drug metabolism of iPSC-derived hepatocyte-like cell cultures. Urea secretion was measured prior to and 24 hours after drug administration. iPSC-derived hepatocyte-like cell in CS cultures exhibited a significant decrease in urea production after the administration of 5 mM acetaminophen.

In the 3D cultures, the presence of liver sinusoidal endothelial cells resulted in a protective effect. The reduction in glutathione upon the administration of ethanol was lower in the presence of liver sinusoidal endothelial cells. The 3D cultures of iPSC-derived hepatocyte-like cells exhibited a decrease in mitochondrial membrane integrity upon the administration of 2.5 mM (LC50) of acetaminophen. We have conducted immunofluorescence studies to visualize bile canaliculi, actin cytoskeleton, intra-cellular albumin expression and other phenotypic markers.

Our results indicate that iPSC-derived hepatocyte-like cells exhibit several markers of adult hepatic phenotypes. Although, their functional capability may be lower than primary human hepatocytes, these cells exhibit significant potential to be used in hepatic tissue engineering. We have also evaluated the toxicant metabolism capacity of these cells. Ongoing investigations are focused on longer-term cultures as well as RNASeq measurements to understand how the inclusion of non-parenchymal cells alters the gene expression of iPSC-derived hepatocyte-like cells.