Pathways of cell identity in liver organoids

Liver cancer represents a significant disease burden in Canada. While treatment of this disease can be challenging, understanding the mechanisms behind hepatic development provides valuable insight. A complex network of transcriptional regulators and epigenetic modifiers drives hepatic fate in both development and cancer progression, and the use of model systems allows for greater understanding of these processes and can inform novel treatment strategies. Organoids, defined as three-dimensional miniature versions of organs constructed from cultured cells, show great promise as a model system. Because they can recapitulate the structure and function of the organ they mimic, they provide greater physiological relevance and similarity to in vivo development and disease processes when compared to traditional two-dimensional cell culture methods. This project aims to develop and characterize a three-dimensional hepatic organoid model that can better recapitulate early human liver differentiation in vitro. We have used a novel protocol to differentiate human pluripotent stem cell (hPSC) aggregates into multilineage organoids by the stepwise addition of small molecules to three-dimensional hPSC culture. The expression of stage- and cell type-specific marker genes indicates differentiation into hepatic lineages, including definitive endoderm, liver bud, hepatoblast, and hepatic mesenchymal cells. In addition, there is evidence of non-hepatic lineages arising in these organoids, highlighting the issue of heterogeneity in organoid culture and the importance of careful protocol optimization and robust characterization. Future work with this hepatic organoid protocol will focus on further optimization of the differentiation protocol to ensure efficient and consistent differentiation, and use of the organoids as a model system to investigate hepatic transcriptional networks alongside our lab’s current models.