Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

Abstract

OBJECTIVE:  Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell-to-cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS:  Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL2. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT-PCR, and alpha-fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP-positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP-positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS:  After coculture with BNL.CL2 cells, the differentiated ES cells had the same morphology as the BNL.CL2 cells, and albumin, transthyretin, glucose 6 phosphates and SEK-1 mRNA were found by RT-PCR, and alpha-fetoprotein was detected immuno­histochemically. The transplanted GFP-positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP-positive ES cells retained an albumin-producing ability. CONCLUSIONS:  Cell-to-cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.