Hepatocyte Differentiation from iPSCs or MSCs in Decellularized Liver Scaffold: Cell-ECM Adhesion, Spatial...

Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.¹⁵ This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin-eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell-ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.