Engineering a three-dimensional culture system for the directed differentiation of pluripotent stem cells toward a hepatocyte-like cell fate

Abstract

Because stem cells have the ability to self-renew and differentiate into more specialized cell types, they hold enormous potential in the fields of regenerative and personalized medicine as well as providing a model system for studying development in vitro. Stem cells with the capacity to differentiate to hepatocytes, the functional cells of the liver, have potential applications in the pharmaceutical industry in high-throughput drug toxicity screening and in clinical settings in bioartificial liver devices or as candidates for transplantation to treat end-stage liver disease. However, these applications rely on the ability to generate and differentiate the stem cells to functionally mature hepatocytes in a robust and reproducible manner. We have recently optimized a multistage directed differentiation protocol for guiding human embryonic stem (hES) cells and rat multipotent adult progenitor cells (rMAPCs), among other stem cell types, toward a hepatic fate. We also recently showed that rMAPCs, which are isolated from the bone marrow of post-natal rats and exhibit the ability to self-renew and differentiate to all three lineages, can be cultured as three-dimensional aggregates without losing their potency or self-renewal capacity. In this study, we report three-dimensional aggregate-based culture systems that enhance the differentiation of rMAPCs and hES cells to hepatocyte-like cells. rMAPCs were allowed to self-assemble into undifferentiated aggregates before being differentiated via the four-step directed differentiation protocol. Compared to adherent monolayer cultures, differentiation as aggregates resulted in significantly higher expression of liver-specific transcripts, including albumin, and increased secretion of albumin and urea. The differentiated cell aggregates also demonstrated functional activities of primary hepatocytes, as demonstrated by pentoxyresorufin O-dealkylation (PROD) and ethoxyresorufin O-dealkylation (EROD), and ultrastructural features of hepatocytes by electron microscopy. A similar three-dimensional culture system likewise enhanced the differentiation of hES cells. HSF6 cells differentiated as a monolayer culture were dissociated and allowed to self-assemble into three-dimensional spheroids in an extended differentiation culture. Compared cells maintained in the monolayer culture, cells within the spheroids exhibited significantly higher expression levels of liver-enriched transcripts and proteins, including Albuming, PEPCK, and ASGPR-1. Cells in the spheroids demonstrated hepatic functions EROD, PROD, and biliary accumulation of fluorescein diacetate metabolite and ultrastructural characteristics of hepatocytes by electron microscopy. Finally, whole-genome transcriptome analysis was performed to investigate the expression profile of liver-specific sets of genes, including the hepatocyte nuclear factors (HNFs), cytochrome P450s (CYP450s), and UDP-glucuronosyltransferases (UGTs), during differentiation. Cells in the spheroid were shown to have overall increased expression levels of most of the genes in these families, although the expression levels were still lower than in adult liver. The transcriptome analysis was also used to identify genes that change during establishment of the spheroid culture that may play a role in the enhanced differentiation status of the cells; multiple members of the aldo/keto reductase (AKR) and metallothionein (MT) families were found to have much higher expression in spheroids than in monolayer culture. These studies demonstrate the ability of three-dimensional, scalable culture systems to enhance the differentiation of pluripotent stem cells toward a hepatic fate and to maintain the differentiated phenotype for extended culture. With modifications to further enhance the maturity of stem cell-derived hepatocyte-like cells, these systems may facilitate the translation of stem cell generated tissues to technology.