ISOLATION OF HEPATOCYTE-LIKE CELLS FROM MOUSE EMBRYOID BODY CELLS

Abstract

P1103 Aims: We previously reported that embryoid body cells (EBs) derived from embryonic stem (ES) cells differentiated into functional hepatocytes both in vitro and in vivo. Transplantation of these cells in vivo resulted in a low incidence of teratoma formation. Thus, purification of hepatocytes will be required for prevention of teratoma formation. The aim of this study is to purify hepatocytes from cultured EBs and to examine the effect of cell transplantation using the purified hepatocytes on injured liver induced by CCl4 administration. Methods: For the isolation of hepatocytes from EBs, EBs cultured for 15 days were treated with 0.25% trypsine-EDTA, and disaggregated cells were plated on a 0.1% gelatin-coated dishes. Expressions of the mRNAs of hepatocyte specific markers such as albumin, methionine adenosyltransferase 1A, tyrosine aminotransferase and cytochrome P450 7A1 were examined by RT-PCR using the monolayered cells. These cells were then purified by Percoll gradient centrifugation and magnetic cell sorting (MACS). The purified monolayered cells were transplanted into mice to observe whether the frequency of teratoma formation reduced and liver function was improved. Results: RT-PCR analysis revealed the monolayered cells to express mRNAs of hepatocyte specific markers. Liver function was improved by the transplantation of purified monolayered cells. Teratoma formation was not observed when the purified monolayered cells were transplanted. Conclusions: Teratoma formation could be prevented by the purification of the hepatocytes derived from mouse ES cells using Percoll gradient centrifugation, and cell transplantation therapy was effective for liver failure.