Expression And Metabolic Function Of The Microsomal Phase â…¡ Enzymes In The ES Derived Hepatocytes

Embryonic stem (ES) cells are derived from the inner cell mass of the pre-implantation blastocyst or the morula cells, which can be cultured and maintained in undifferentiated state in vitro. Unlimited differentiation potential is one of the marked characters of ES cells, in other words, ES cells can differentiate into cell types of all three primary germ layers if cultured under the correct conditions. Accumulated evidence suggested that hepatic differentiation of ES cells by interactions between three primary germ layers in EBs in vitro faithfully recapitulated hepatic differentiation in vivo and the hepatocytes derived from ES cells display properties similar to those observed in vivo or in primary cultures cultures: 1) Embryoid bodies (EBs) were developed before hepatic differentiation, which was characterized with three primary germ layers; 2) Hepatic specific transcriptors or genes are expressed in a developmentally controlled manner as differetiation; 3) The ES cells derived hepatocytes ,which have a typical binuclear structure, can synthetize and secrete the albumin; 4) The derived hepatocytes have metabolic function of phase I enzymes; 5) The derived hepatocytes have been successfully applied in the toxicity research, which suggested that the hepatocytes can be applied for toxicant preliminary screening. For these reasons, the application of the differentiation in the investigation perspective, such as hepatic developmental biology, drug metabolism, pharmacodynamic action ,toxicity and hepatic disease therapy, is gradually payed close attention to.It must be taken on wide and deep research on the function of ES cells derived hepatocytes as above application. The metabolism, by which the endogenous and exdogenous chemicals are evacuated and maintained the homeostatic in vivo, is the most important characteristic function of the liver. Till today the investigations on the metabolic enzymes of the ES cells derived hepatocytes are few and none had been reported about the expression and the metabolic function of the microsomal phase II enzymes.What'more, the ES cells derived hepatocyts have unique superiorities in the drug research compared with other cells systems, such as primary hepatocytes and HepG2 ,an immortal cell lines. ES cells derived hepatocytes have complete function which can sustain for a long period. Therefore the purpose of our present work is to construct the ES cells derived hepatocytes and confirm the phase II metabolic function in the cells for supporting an alternative model in drug screening and other purposes.Part I Morphological and functional changes as the differentiation of the murine embryonic stem cell derived into hepatocytesAim: to construct a simple and effective hepatic differentiation model from ES cells in vitro for the further study on the metabolic function in it.Methods : Cultures of differentiating ES cells were established by the formation of EBs in hanging drop cultures without additional induction factors. Differentiation cultures were monitored every other day with light microscopy to record the morphology. Hepatic development-dependent gene expression were detected by RT-PCR. Immunofluorescence was adapted to identify hepatocytes derived from ES cells. Albumin production was measured in the culture medium by ELISA.Results: The development-dependent feature was detected as the differentiation went on. The level of OCT3/4 gene expression decreased dramatically during the differentiation course and was not detected on d 18. There is a first increase and then a decrease in the AFP gene expression which slightly detected on d 18.Gene expression of ALB was detected on the d 8 and the level increased since then. Cyp7a1, a marker of mature hepatocyte, was detected on d 18. From these, the ES cells had differentiated into mature hepatocytes on d 18 in the profile of gene expression. The derived hepatocytes were positive for ALB, which suggested that the mature hepatocytes had been derived from ES cells. Some hepatocytes were binuclear, which indicated that the derived hepatocytes were mature.Albumin production was measured on d 8 and the level increased in the following differentiation and went into a platform on d18. This revealed the ES derived hepatocytes had the function of albumin production.Conclusions: By hanging drop method, the ES cells derived hepatocytes on d 18 presented the typical binuclear structure of hepatocytes and positive staining for albumin. The derived hepatocytes shown an increase tendency in albumin production first and then a platform, which suggested the ES cells derived hepatocytes became mature eventually.Part II Expression of microsomal phase II enzymes in the ES cell derived hepatocytesAim: to detect the expression of some important kinds of phase II enzymes in the ES cell derived hepatocytes, thus set up the foundation for metabolic function study.Methods: RT-PCR was adapted to detected the expression of UGT1a1, UGT1a6, UGT1a9, UGT2b5 and mGST1 in the derived hepatocytes during the differentiation. Western-blot was applied to measure the protein expression of UGTa1, UGT1a6, mGST1 in the derived cells.Results and results: UGT1a1 and UGT1a9 mRNA steadily expressed in the ES derived hepatocytes during the differentiation culture. UGT2b5 was not detected in the process. The level of gene expression UGT1a6 and mGST1 increased as differentiation. Protein expression of UGT1a1 show an increase tendency in the culture. Protein of UGT1a6 and mGST1 were not detected in earlier and intermediate stage but obviously expressed on d 18. These indicated that the ES derived hepatocytes on d 18 may have the metabolic function of phase II enzymes from the profile of protein expression.Part III Metabolic function of the microsomal phase II enzymes in the derived hepatocytesAim: to study the metabolic function of the phase II enzymes in the derived mature hepatocytes for providing an alternative model for drug metabolizing study and an experimental evidence for ES cells in hepatic disease therapy. Methods: The derived cells were broken into pieces by ultrasound and then added with the UDPGA and 7-HFC. After incubation in vitro, the formation of 7-HFC glucuronide was detected by HPLC to confirm the activity of the UGTs.The microsomes of the derived mature hepatocytes were added with CDNB and GSH. The formation of the conjunction of CDNB and GSH was measured by spectrometry to determine the mGST1 activity.Results and conclusions: The metabolic activity of UGTs in ES cells was 0.069 nmol/min/mg. The metabolic activity of UGTs in the derived hepatocytes on d 18 was 0.445 nmol/min/mg, which is more than six times as the ES cells. The metabolic activity of mGST1 in the derived hepatocytes on d 18 was 7.65 nmol/min/mg.All these suggested that the derived hepatocytes on d 18 have the metabolic function of UGTs and mGST1.Summary1. The mature hepatocytes in phenotype ,which can synthesize and secrete albumin, can be derived from ES cells by the formation of EBs in hanging drop cultures.2. The expression of microsomal UGTs and mGST1 in the ES cells derived are development-dependent during differention of mature hepatocytes from ES cells.3. The ES cells derived mature hepatocytes have the metabolic function of the UGTs and mGST1.