Hepatic Differentiation and Maturation of Human Embryonic Stem Cells Cultured in a Perfused Three-Dimensional Bioreactor

Abstract

Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems.

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

“…The first is the generation of 3D aggregates. As shown in previous studies (Miki et al, 2011;Sivertsson et al, 2013) and in the work reported here, culture of hPSC-derived hepatic cells as 3D aggregates leads to the upregulation of expression of a wide range of genes involved in different aspects of liver function. Aggregation alone, however, does not appear to promote maturation of the population to the stage at which the cells have functional levels of enzyme activity.…”

“…This study did not, however, provide any insights into the pathways that promote maturation or show that the approach was applicable to different cell lines. More recent studies have shown that culture on specific polymers (Hay et al, 2011), culture as 3D aggregates (Sivertsson et al, 2013) or the combination of enforced expression of key transcription factors together with 3D aggregation (Takayama et al, 2013) promote the development hPSC-derived hepatocytes that express CYP genes. Of these, only two showed inducible CYP activity.…”

“…As previous studies have shown that cell aggregation can promote some degree of maturation of hESC-derived hepatic cells (Miki et al, 2011;Nagamoto et al, 2012;Sivertsson et al, 2013;Takayama et al, 2013), we next generated aggregates from the day 26 population (Fig. 3A) and cultured them for 6 days in the presence of HGF, dexamethasone (Dex) and oncostatin M (OSM) to determine whether these conditions would affect maturation of the hepatoblast-like cells in our cultures.…”

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology

“…In addition, in these hollow-fibre bioreactors, the differentiation of human embryonic stem cells towards hepatic cells as a potential alternative cell source for studies on hepatic cells is demonstrated by a significant up-regulation of genes related to liver functions (Miki et al, 2011) and enhanced hepatic differentiation in the 3D four-compartment bioreactor system compared to conventional 2D cultures (Sivertsson et al, 2013).…”

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

“…The properties of hESC (capacity to differentiate to all major cell types) are also found in other human pluripotent stem cells, such as human induced pluripotent stem cells (hiPSC) or cells generated by nuclear transfer 5 . For instance, many different hESC lines have been differentiated into neurons 6 , renal cells 7 , neural crest cells 8 , cardiomyocytes [9][10][11][12] , or hepatocytes like cells 13,14 . Moreover, hESC can spontaneously differentiate into cells of all three germ layers [15][16][17][18] in embryoid bodies (EBs) specific signatures have been captured by transcriptomics profile using the Affymetrix microarray platform.…”