(M-497) Primary and Pluripotent Stem Cell derived Multicellular Human Hepatic Spheroids reveals complex inter- and intra- cellular interactions and metabolic phenotypes

Introduction:: Introduction: Human metabolic liver diseases such as nonalcoholic fatty liver disease impact forty percent of American adults with over one-quarter of patients at risk of developing progressive liver disease and hepatic fibrosis. Unfortunately there are currently no FDA-approved drugs for the treatment of metabolic liver diseases. Moreover current model systems to study metabolic liver diseases are lacking as they do not meaningfully phenocopy the human disease and as a result putative liver disease targets identified using such models have not been ultimately validated in clinical trials. The development of more relevant and robust models of human liver disease is sorely needed. The application of human pluripotent stem cell (PSC) derived hepatocyte-like cells and primary human hepatocytes for clinical or research uses is dependent on the ability to generate hepatocytes or hepatocyte-containing constructs that maintain function long-term. We have established a scalable multi-well platform that enables the rapid generation of three-dimensional (3D) multicellular spheroids composed of primary and stem cell derived human liver parenchymal cell types including hepatocytes, liver sinusoidal endothelial cells (LSEC), stellate cells (HSC), and Kupffer cells. This 3D system is able to maintain hepatocyte function long-term and can also model the interactions between hepatocytes and nonparenchymal cells (LSEC, HSC, and Kupffer cells). In this study we demonstrate that this platform maintains human multicellular function long-term and is an enabling technology that can help to phenocopy complex disease phenotypes.


Materials and Methods:: Materials and Methods: Multicellular spheroids containing hepatocytes and nonparenchymal cells alone were generated from primary human liver tissue or from human PSCs. In order to stabilize hepatocyte differentiated function for extended experiments required the addition of nonparenchymal cell types which facilitated long-term maintenance of albumin, urea, and cytochrome P450 function for up to 56 days. In addition we found that liver-specific endothelial cell function (as evidenced by coagulation factor production), the capacity for Kupffer cell (liver-specific) activation (as evidenced by cytokine production), and the capacity for stellate cell activation (as evidenced by marker upregulation) was maintained over this time. Given the longevity and stability of the platform we examined whether such a platform could be used to model the complex interactions between hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells in vitro while modeling human disease states. We focused on nonalcoholic fatty liver disease (NAFLD) which is associated with chronic hyperglycemia and hyperlipidemia.


Results, Conclusions, and Discussions:: Results and Discussion: We generated multicellular spheroids and examined how chronic hyperglycemia and hyperlipidemia impacted multicellular spheroids. We found that multicellular spheroids maintain insulin-sensitive glucose metabolism in contrast to traditional hepatocyte-culture systems. Moreover, we found that chronic hyperglycemia and hyperlipidemia led to decreased insulin sensitivity, increased hepatocyte fat accumulation, hepatocyte injury, macrophage and stellate cell activation. We only saw these impacts in multicellular spheroids containing all of the varied cell types of the liver which was not seen in multicellular spheroids that are missing some of the different cell types. We added drug compounds currently in early development in clinical studies and found that some are able to mitigate these effects.


Conclusions: NAFLD impacts over forty percent of the American adult population (120+ million people) and thirty-five percent of the worldwide adult population (1.82+ billion people). Improved model systems that better phenocopy this disease are critically needed. Our work demonstrates that multicellular spheroids composed of primary and stem cell derived human liver parenchymal cell types including hepatocytes, liver sinusoidal endothelial cells, stellate cells, and Kupffer cells have stable function and can model complex disease phenotypes in vitro. Human model platforms that better phenocopy human diseases has the potential to better identify clinically relevant targets. Leveraging these technologies focused on metabolic phenotypes enabled studies of metabolic injury and can be leveraged in NAFLD drug discovery.


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