Abstract
The generation of hepatocytes that are derived from human adipose stem cells (hASCs) represents an alternative to human hepatocytes for individualized therapeutic and pharmaceutical applications. However, the mechanisms facilitating hepatocyte differentiation from hASCs are not well understood. Here, we show that upon exposure to glycogen synthase kinase 3 (GSK3) inhibitors alone, the expression of definitive endoderm specific genes GATA4, FOXA2, and SOX17 in hASCs significantly increased in a manner with activation of Wnt/β-catenin signalling. Down regulation of the β-catenin expression attenuates the effect of GSK3 inhibitors on the induction of these specific genes. The cells induced using GSK3 inhibitors were directed to differentiate synchronously into hepatocyte-like cells (HLCs) after further combinations of soluble factors by a reproducible three-stage method. Moreover, hASC-HLCs induced using GSK3 inhibitors possess low-density lipoprotein uptake, albumin secretion, and glycogen synthesis ability, express important drug-metabolizing cytochrome P450 (CYP450) enzymes, and demonstrate CYP450 activity. Therefore, our findings suggest that activation of Wnt/β-catenin signalling via GSK3 inhibitors in definitive endoderm specification may represent an important mechanism mediating hASCs differentiated to functional hepatocyte. Furthermore, development of similar compounds may be useful for robust, potentially scalable and cost-effective generation of functional hepatocytes for drug screening and predictive toxicology platforms.
Highlights
Wnt/JNK signalling that coordinate endoderm fate, proliferation and morphogenesis[6,9]
To examine whether inhibition of glycogen synthase kinase 3 (GSK3) would mimic Wnt signalling through direct stabilization of β-catenin in human adipose stem cells (hASCs), the cells after 48 hours of serum starvation were treated with 50 ng/mL Wnt3a, 0.2 μM Chir98014 and 2 μM Chir99021 for 24 hours separately
These results suggest that the canonical Wnt/β-catenin axis was activated in Wnt3a, Chir98014 and Chir99021 treated hASCs
Summary
Wnt/JNK signalling that coordinate endoderm fate, proliferation and morphogenesis[6,9]. We demonstrate that the high concentration (100 ng/mL) of activin A signalling, which mimics the Nodal pathway, induces definitive endoderm specific transcription factors, including HEX, GATA4, FOXA2, and SOX17, expression in hASCs10. GSK3 is a serine/threonine kinase that plays a central role in the regulation of the Wnt/β-catenin signalling pathway, an important pathway for hepatic specification, hepatoblast proliferation, differentiation, and hepatocyte maturation[18,19,20]. We compared the effect of Wnt3a and two GSK3 inhibitors, Chir98014 and Chir99021 on the activation of Wnt/β-catenin signalling pathway, and the regulation of expression of definitive endoderm specific transcription factors GATA4, FOXA2, and SOX17 in hASCs. Thereafter, we investigated whether the cells inducing by GSK3 inhibitors may show equivalent developmental potential as activin A-induced definitive endoderm in their differentiation into functional hepatocytes from hASCs in vitro. This study may provide a useful approach for more cost-effective hepatocyte production for drug discovery and cell therapy
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