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

Abstract

SUMMARYHuman pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.

Expression of the Clustered NeuAcα2–3Galβ O-Glycan Determines the Cell Differentiation State of the Cells

“…For neural differentiation of hESCs, the quick serum-free culture of embryoid body-like aggregates (SFEBq) method was used as previously described (9). The combination of three-dimensional cell aggregation and cAMP signaling induced the hepatic differentiation of hESCs (10). Human cancer cell lines and human umbilical vein endothelial cells (HUVECs) were obtained from Health Science Research Resources Bank (Osaka, Japan) and maintained in accordance with the supplier's protocol.…”

New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

“…Of the different cell types that can be generated from hPSCs, substantial effort in recent years has been directed at the generation of endoderm-derived lineages, specifically pancreatic β-cells for transplantation for the treatment of type 1 diabetes and hepatocytes for predictive toxicology and drug metabolism studies (Holditch et al, 2014;Sun et al, 2013). This effort has led to advances in our basic understanding of human development and the generation of protocols for directed differentiation of hPSCs to pancreatic and hepatic fates that yield what seem to be highly enriched end-stage populations (Chen et al, 2012;Ogawa et al, 2013;Pagliuca et al, 2014;Rezania et al, 2014;Si-Tayeb et al, 2010). Despite this progress, efficiencies of differentiation vary considerably between different hPSC lines (Toivonen et al, 2013b;Vitale et al, 2012), even with the most advanced protocols, and end-stage populations are immature and, in some instances, contaminated with other cell types.…”

“…The latter approach provides a rapid quantitative read-out but is dependent on the availability of antibodies against surface markers that should ideally be found only on the cells of interest. Currently, the markers most commonly used for monitoring endoderm induction from hPSCs are CD184 (CXCR4), CD117 (KIT) and EPCAM (D'Amour et al, 2005;Green et al, 2011;Jiang et al, 2013;Loh et al, 2014;Nostro et al, 2011;Ogawa et al, 2013). When used in combination, they do provide a reasonable assessment of the efficiency of endoderm development.…”

Pharmacogenetic profiling and metabolic activity of human embryonic stem cell derived hepatocytes: focus on CYP450-mediated oxidation

“…This has made them some of the most widely used hESC lines in research, and in particular the most commonly used hESC lines in SCDH research, specifically the H1 and H9 lines [11,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. To be useful in preclinical drug metabolism and toxicity screening, SCDHs must demonstrate consistent and reproducible activity of drug metabolism proteins, in particular the oxidative CYP enzymes.…”