Abstract
Hepatic development requires multiple sequential physicochemical environmental changes in an embryo, and human pluripotent stem cells (hPSCs) allow for the elucidation of this embryonic developmental process. However, the current in vitro methods for hPSC-hepatic differentiation, which employ various biochemical substances, produce hPSC-derived hepatocytes with less functionality than primary hepatocytes, due to a lack of physical stimuli, such as heart beating. Here, we developed a microfluidic platform that recapitulates the beating of a human embryonic heart to improve the functionality of hepatoblasts derived from hepatic endoderm (HE) in vitro. This microfluidic platform facilitates the application of multiple mechanical stretching forces, to mimic heart beating, to cultured hepatic endoderm cells to identify the optimal stimuli. Results show that stimulated HE-derived hepatoblasts increased cytochrome P450 3A (CYP3A) metabolic activity, as well as the expression of hepatoblast functional markers (albumin, cytokeratin 19 and CYP3A7), compared to unstimulated hepatoblasts. This approach of hepatic differentiation from hPSCs with the application of mechanical stimuli will facilitate improved methods for studying human embryonic liver development, as well as accurate pharmacological testing with functional liver cells.
Highlights
Hepatocytes are major components of the liver and have essential physiological roles, including protein and glucose synthesis and storage, detoxification, and excretion of exogenous molecules
We showed that hPSCderived hepatoblasts differentiated under the optimal stretching condition and expressed enzymes of drug metabolism and proteins specific to hepatoblasts
These findings demonstrate that dynamic mechanical forces are critical for differentiation of hepatic endoderm (HE) to hepatoblasts and must, be incorporated into differentiation models for human pluripotent stem cells (hPSCs)
Summary
Hepatocytes are major components of the liver and have essential physiological roles, including protein and glucose synthesis and storage, detoxification, and excretion of exogenous molecules. Identification of suitable healthy donors for liver transplantation, or hepatocytes for drug testing, remains challenging, warranting the development of alternative strategies for drug design and development One such strategy includes the use of human pluripotent stem cells (hPSCs), such as human embryonic and induced pluripotent stem cells [hESCs (Thomson et al, 1998) and hiPSCs (Takahashi et al, 2007; Yu et al, 2007), respectively], which show high potential due to their capacity for unlimited self-renewal and differentiation to almost any tissue cell type. Many studies have been performed to obtain hPSC-derived hepatocytes, the resulting cells remain as immature hepatocytes with fewer functional properties The cause of this limited development stems from the use of various biochemical factors, such as fibroblast growth factor (FGF), and bone morphogenesis protein (BMP) in the current differentiation protocols. As mechanical forces regulate a variety of biological factors, including molecules, cells, tissues, and organs (Sakamoto et al, 2010; Ren et al, 2015), their effects must be considered to provide optimal hepatic differentiation methods from hPSCs
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