Substrate Stiffness Modulates the Maturation of Human Pluripotent Stem-Cell-Derived Hepatocytes

Obtaining functional hepatocytes from human pluripotent stem cells (hPSCs) holds great potential for applications in drug safety testing, as well in the field of regenerative medicine. However, developing functionally mature hPSC-derived hepatocytes (hPSC-Heps) remains a challenge. We hypothesized that the cellular microenvironment plays a vital role in the maturation of immature hepatocytes. In this study, we examined the role of mechanical stiffness, a key component of the cellular microenvironment, in the maturation of hPSC-Heps. We cultured hPSC-Heps on collagen-coated polyacrylamide hydrogels with varying elastic moduli. On softer substrates the hPSC-Heps formed compact colonies while on stiffer substrates they formed a diffuse monolayer. We observed an inverse correlation between albumin production and substrate stiffness. The expression of key cytochrome enzymes, which are expressed at higher levels in the adult liver compared to the fetal liver, also correlated inversely with substrate stiffness, whereas fetal markers such as Cyp3A7 and AFP showed no correlation with stiffness. Culture of hPSC-Heps on soft substrates for 12 days led to 10–30 fold increases in the expression of drug-metabolizing enzymes. These results demonstrate that substrate stiffness similar to that of the liver enables aspects of the maturation of hPSC-Heps

Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications