Mittal Nikhil, Tasnim Farah, Yue Cao, Qu Yinghua, Phan Derek, Choudhury Yukti, Tan Min-Han, Yu Hanry
Institute of Bioengineering and Nanotechnology, Singapore 138669.
Mechanobiology Institute, 10-01 T-Lab, National University of Singapore, 5A Engineering Drive, Singapore 117411.
ACS Biomater Sci Eng. 2016 Sep 12;2(9):1649-1657. doi: 10.1021/acsbiomaterials.6b00475. Epub 2016 Sep 1.
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.
从人类多能干细胞（hPSC）中获取功能性肝细胞在药物安全性测试以及再生医学领域具有巨大的应用潜力。然而，培养功能成熟的hPSC来源的肝细胞（hPSC-Heps）仍然是一项挑战。我们推测细胞微环境在未成熟肝细胞的成熟过程中起着至关重要的作用。在本研究中，我们研究了细胞微环境的关键组成部分——机械硬度在hPSC-Heps成熟中的作用。我们将hPSC-Heps培养在具有不同弹性模量的胶原包被的聚丙烯酰胺水凝胶上。在较软的基质上，hPSC-Heps形成紧密的集落，而在较硬的基质上，它们形成分散的单层。我们观察到白蛋白产生与基质硬度呈负相关。与胎儿肝脏相比，在成体肝脏中表达水平较高的关键细胞色素酶的表达也与基质硬度呈负相关，而诸如Cyp3A7和AFP等胎儿标志物与硬度无关。在软基质上培养hPSC-Heps 12天导致药物代谢酶的表达增加10至30倍。这些结果表明，与肝脏相似的基质硬度能够促进hPSC-Heps的成熟。
