Embryonic Stem Cell-Derived Embryoid Bodies in Three-Dimensional Culture System Form Hepatocyte-Like Cells in Vitro and in Vivo

Pluripotent embryonic stem (ES) cells can be a source of hepatocytes for bioartificial livers or transplantation. In this study, embryoid bodies (EBs) were formed from ES cells cultured in polypropylene conical tubes. The EBs were then inserted into a collagen scaffold three-dimensional culture system and stimulated with exogenous growth factors and hormones to induce hepatic histogenesis. The EB-derived cells expressed liver-specific genes, and albumin-positive cells formed cordlike structures that were not present in two-dimensional monolayer culture systems. However, these albumin- positive cells were not cytokeratin 18 positive. Electron microscopy showed immature hepatocyte- like cells having tight junctions, rough endoplasmic reticulum, and intercellular canaliculi. The scaffold including EB-derived hepatocyte-like cells was transplanted into the median lobes of partially hepatectomized nude mice. After 7 and 14 days, cells positive for both albumin and cytokeratin 18 appeared in the transplant and formed clustered aggregates. Thus the collagen scaffold threedimensional culture system and the liver regeneration environment induced hepatocyte-like cells and hepatic lobule-like aggregates from EBs. Therefore, differentiating EBs in the scaffold culture system may be useful in developing bioartificial livers, secondary livers, and as pharmaceutical models.