Cyclic Stretching Enhances Angiocrine Signals at Liver Bud Stage from Human Pluripotent Stem Cells in Two-Dimensional Culture

Angiocrine signals during the development and growth of organs, including the liver, intestine, lung, and bone, are essential components of intercellular communication. The signals elicited during the liver bud stage are critical for vascularization and enhanced during the intercellular communication between the cells negative for kinase insert domain receptor (KDR) (KDR- cells) and the cells positive for KDR (KDR+ cells), which constitute the liver bud. However, the use of a human pluripotent stem cell (hPSC)-derived system has not facilitated the generation of a perfusable vascularized liver organoid that allows elucidation of liver development and has great potential for liver transplantation. This is largely owing to the lack of fundamental understanding to induce angiocrine signals in KDR- and KDR+ cells during the liver bud stage. We hypothesized that mechanical stimuli of cyclic stretching/pushing by the fetal heart adjacent to the liver bud could be the main contributor to promoting angiocrine signals in KDR- and KDR+ cells during the liver bud stage. In this study, we show that an organ-on-a-chip platform allows the emulation of an in vivo-like mechanical environment for the liver bud stage in vitro and investigate the role of cyclic mechanical stretching (cMS) to angiocrine signals in KDR- and KDR+ cells derived from hPSCs. RNA sequencing revealed that the expression of genes associated with epithelial-to-mesenchymal transition, including angiocrine signals, such as hepatocyte growth factor (HGF) and matrix metallopeptidase 9 (MMP9), were increased by cMS in cocultured KDR- and KDR+ cells. The expression and secretions of HGF and MMP9 were increased by 1.98- and 1.69-fold and 3.23- and 3.72-fold with cMS in the cocultured KDR- and KDR+ cells but were not increased by cMS in the monocultured KDR- and KDR+ cells, respectively. Finally, cMS during the liver bud stage did not lead to the dedifferentiation of hepatocytes, as the cells with cMS showed hepatic maker expression (CYP3A4, CYP3A7, ALB, and AAT) and 1.71-fold higher CYP3A activity than the cells without cMS, during 12 day-hepatocyte maturation after halting cMS. Our findings provide new insights into the mechanical factors during the liver bud stage and directions for future improvements in the engineered liver tissue. Impact statement This study demonstrates that cyclic mechanical stretching using an organ-on-a-chip platform promotes angiocrine signals including hepatocyte growth factor and matrix metallopeptidase 9 in human pluripotent stem cell-derived cells negative and positive for KDR (VEGFR-2; KDR- cells and KDR+ cells) through intercellular communication. Our approach emphasizes the importance of considering mechanical influences during the liver bud stage and provides direction for future improvements in the engineered vascularized liver tissue.