Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and ␤-nerve growth factor (␤-NGF). RA, HGF, and ␤-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of ␣ 1 -antitrypsin (␣ 1 -AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and ␤-NGF to the cell culture, many epithelioid cells were noticed. ␣ 1 -AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and ␤-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited. E mbryonic stem (ES) cells are continuously growing cell lines derived from the inner cell mass of 3.5-day blastocysts. Under proper experimental conditions in vitro, ES cells have full developmental potential to differentiate into cell types of all three germ layers. 1,2 In addition, when allowed to form three-dimensional structures known as embryoid bodies (EBs), ES cells initiate the progenitor from a continuously growing stem-cell population in vitro, providing a unique system for the analysis of both cellular and molecular events involved in the first stage of lineage determination. ES cells also have been used to study the differentiation of various cell types and tissues in vitro, such as neural cells, 3 hematopoietic lineages, and cardiomyocytes. 4 ES-derived cells have been transplanted successfully into the neuronal system and liver of fetal and adult mice, where they show integration of morphological characteristics and function. 5 Because of the shortage of liver donors, hepatocyte transplantation currently is being evaluated as a potential method of providing metabolic support during acute and chronic liver failure and can be used to compensate specific liver functions in patients with inherited metabolic liver diseases. 6 Hepatocytes currently have not been considered an ideal source for cell transplantation. We wanted to know whether ES cells could be a new cell source for cell transplantation to treat liver diseases.The aim of our study is to investigate the effects of retinoic acid (RA), hepatocyte growth factor (HGF), and ␤-ner...

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.