Cyclic mechanical stretching stimuli promotes angiocrine signals during in vitro liver bud formation from human pluripotent stem cells

Liver organoids derived from human pluripotent stem cells (hPSCs) allow elucidation of liver development and have great potential for drug discovery. However, current methods for generating liver organoids using biochemical substances do not realize the vascular network of the liver lobule, due to the lack of knowledge of the role of in vivo mechanical environments during liver development. Here, we investigate the role of cyclic mechanical stretch (cMS) to angiocrine signals of hepatoblasts (HBs) and endothelial progenitor cells (EPCs) using an organ-on-a-chip platform to emulate in vivo -like mechanical environments and hPSCs to recapitulate hepatic differentiation. RNA sequencing revealed that the expression of angiocrine signal genes, such as HGF and matrix metallopeptidase 9 ( MMP9 ), was increased by cMS in co-cultured HBs and EPCs. The secretion of HGF and MMP9 increased by 3.23-folds and 3.72-folds with cMS in the co-cultured HBs and EPCs but was not increased by cMS in the mono-cultured HBs and EPCs. Immunofluorescence micrographs with anti-KRT19, HGF, and MMP9 antibodies also revealed that cMS increased HGF and MMP9 expression when HBs and EPCs were co-cultured. cMS increased HGF and MMP9 expression and secretion when HBs and EPCs were co-cultured. Our findings provide new insights into the mechanical factors involved in the vascular network of human liver bud formation and liver organoid generation. ### Competing Interest Statement The authors have declared no competing interest. * cMS : cyclic mechanical stretching CYP3A : cytochrome P450 3A D9 : day 9 D12 : day 12 DEG : differentially expressed gene EC : endothelial cell EMT : epithelial-to-mesenchymal transition EPC : endothelial progenitor cells FACS : fluorescence-activated cell sorter FOXA2 : forkhead box A2 HB : hepatoblast HE : hepatic endoderm hESCs : human embryonic pluripotent stem cells HHEX : hematopoietically-expressed homeobox protein hiPSCs : human induced pluripotent stem cells HNF4α : hepatocyte nuclear factor 4α hPSCs : human pluripotent stem cells KRT19 : keratin 19 MMP9 : matrix metallopeptidase 9 NEAA : MEM Non-essential Amino Acid Solution without L-glutamine, liquid, sterile-filtered Bioreagent suitable for cell-culture OoC : organ-on-a-chip PDMS : polydimethylsiloxane RNA-seq : RNA sequencing RPMI : Roswell Park Memorial Institute SD : standard deviation SOX17 : SRY-box transcription factor 17 TPM : transcripts per kilobase million TRPV : transient receptor potential vanilloid