Human FOXA3 mRNA

To generate hepatocyte-like cells, the researchers transfected mouse embryonic fibroblasts with mRNA encoding the hepatic transcription factors Foxa3 and HNF4α. After 10-12 days, the fibroblasts transformed into an epithelial morphology and formed hepatocyte-like cell colonies (R-iHeps). The generated R-iHeps expressed hepatocyte-specific marker genes and proteins, including albumin, alpha-fetoprotein, HNF4α, CK18, and CYP1A2. To assess liver function, the researchers evaluated indocyanine green uptake, periodic acid-Schiff staining, and albumin secretion. In addition, they implanted mCherry-positive R-iHeps into the livers of Alb-TRECK/SCID mice and confirmed the expression of the FAH enzyme in the Fah1RTyrc/RJ model. Together, these data suggest that the non-integrating approach using mRNA has the potential for cell therapy.

Here, the researchers transfected Foxa3 mRNA and HNF4α mRNA into mouse embryonic fibroblasts (MEFs) at day 0 and day 3, respectively, and cultured them at 37°C for 4 hours (Figure 1). Two days after transfection, the medium was changed to direct transformation medium to allow efficient transformation into the hepatocyte lineage. On day 6, MEFs began to move and steadily transformed their morphology (Figure 2). Finally, 12 days after transfection, the researchers found epithelial colonies similar to hepatocytes, which had abundant cytosol, small nuclei, and formed bile ducts. These results indicate that directly transformed R-iHeps can effectively generate hepatocyte-like cells from MEFs using mRNA.

Figure 1. Scheme of generation of R-iHeps. (Yoon S, et al., 2018)

Figure 2. The morphology of directly converted R-iHeps by mRNA. (Yoon S, et al., 2018)