In vitro metabolic zonation through oxygen gradient on a chip

Abstract

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are differently affected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufficient to instruct a functional metabolic zonation during the differentiation of human embryonic stem cells (hESCs) from endoderm toward terminally differentiated hepatocytes, thus mimicking the in vivo situation. For this purpose, a microfluidic device was designed for the generation of a stable oxygen gradient. The oxygen gradient was applied to differentiating hESCs at the pre-hepatoblast stage. The definitive endoderm and hepatic endoderm cells were characterized by the expression of the transcription factor SOX-17 and alpha-fetoprotein (AFP). Immature and mature hepatocytes were characterized by hepatocyte nuclear factor 4-alpha (HNF-4α) and albumin (ALB) expression and also analyzed for cytochrome P450 (CYP3A4) zonation and glycogen accumulation through PAS staining. Metabolic zonated genes expression was assessed through quantitative real time PCR. Application of the oxygen gradient during differentiation induced zonated glycogen storage, which was higher in the hepatocytes grown in high pO2 compared to those grown in low pO2. The mRNA levels of glutamine synthetase (GLUL), beta-catenin (CTNNB) and its direct target cyclin D1 (CCND1) showed significantly higher expression in the cells grown in low pO2 compared to those grown in high pO2. On the contrary, carbamoyl-phosphate synthetase 1 (CPS1), ALB, the proliferative marker ki67 (MKI67) and cyclin A (CCNA) resulted to be significantly higher expressed in cells cultured in high pO2 compared to those cultured in low pO2. These results indicate that the oxygen gradient generated in our device can instruct the establishment of a functional metabolic zonation in differentiating hESCs. The possibility to obtain differentiated hepatocytes in vitro may allow in the future to deepen our knowledge about the physiology/pathology of hepatocytes in relation to the oxygen content.

Characterization of liver zonation‐like transcriptomic patterns in HLCs derived from hiPSCs in a microfluidic biochip environment

“…As an alternative to scarce primary hepatocytes, stem cells have appeared as a promising tool for in‐vitro reproduction of in‐vivo phenomena such as zonation. Recently, embryonic stem cells differentiated into hepatic lineage have been shown to assume zone‐specific functionality when exposed to oxygen gradients 16 . However, complete mature phenotypes are yet to be obtained, even though they appear as a vital option for a realistic representation of those phenomena.…”

“…Recently, embryonic stem cells differentiated into hepatic lineage have been shown to assume zone-specific functionality when exposed to oxygen gradients. 16 However, complete mature…”

96-Well Oxygen Control Using a 3D-Printed Device

“…This may be particularly useful for deconstructing complex physiologic phenomena such as liver zonation, in which metabolism is spatially separated according to the availability of oxygen, hormones, and metabolites. Current microfluidic models of this phenomena have ascribed a varying, and sometimes con-tradictory, effect of O 2 on enzymatic expression 28,29,30 . By isolating oxygen as a variable, our device may be used to elucidate the role of oxygen in complex physiologic or pathophysiologic phenomena.…”

Functionally Graded Biomaterials for Use as Model Systems and Replacement Tissues

“…For example, an oxygen gradient ranging from 2% to 21% was created to investigate the influence of oxygen tension on gene expression and metabolism on human ESC‐derived hepatocytes. [ 74 ] One side of a microfluidic device was exposed to a channel of a continuously flowing 95% N 2 /5% CO 2 gas mixture to generate the oxygen gradient. Oxygen gradients were also created by infusing sodium sulfite, an oxygen scavenger, through a microfluidic device.…”