Experimental Study On The Hepatocyte-like Cell Differentiated From The Human Amniotic Epithelial Cells And Its Intrasplenic Transplantation

In the process of embryonic development, the development of the liver is involved in a multi-gene, multi-link, multi-channel network and the hepatic stem cells play a very important role in this process. This kind of stem cells was developed from the foregut. it is similar to bile duct epithelial cell in morphology and to embryonic stem cells in biochemical.In the end of the 20th century, numerous studies show that trandifferentiation could happen when adult stem cells were cultured in special condition. In recent years, American scientists announced that they found some stem cells in the amniotic fluid as effective as embryonic stem cells, and they found that those stem cells can differentiated into muscle, bone, fat, blood vessels, nerves and liver cells in the laboratory. Compared to embryonic stem cells, they could differentiate into various organizations more easily, and could not formate teratomas. Human amniotic epithelial cells, extracted from the abandoned placenta is such a great research potentiality cells. Current domestic and international research in this area is still in its infancy. The aim of theasis was to discuss the method of isolation and identification of the human amniotic epithelial cells, to invstigate the posibility of its transdifferentiation into hepatocyte-like cells and the Intrasplenic Transplantation. This study is divided into three parts, the main research methods and results are as follows: Objectives: To isolate hAECs from amnion and establish a culture system for hAECs in vitro. To identify the stem cell related characteristics of these isolated hAECs . To test the AAV transfection efficiency of hAECs.Methods: Trypsin-EDTA was used to digest the amnions we collected. The cell sample of P1 passage was collected and then semi-quantity RT-PCR was performed to examine the pluripotency-related genes expression, such as TERF,THY1,LEFTYA,Rex-1,Sox-2,Oct-4 and nanog. Immunocytofluorescental analysis was taken to test the expression of stem cell related markers, such as SSEA-K SSEA-3,SSEA-4 and TRA-1-60,TRA-1-81. The AAV transfection efficiency to hAECs was analyzed with AAV-GFP in vitro.Results: RT-PCR showed that TERF,THY1,LEFTYA,Rex-1,Sox-2,Oct-4 and nanog were expressed in P1 hAECs. Immunocytofluorescent analysis showed that most of hAECs were SSEA-3⁺,SSEA-4⁺,TRA-1-60⁺ and TRA-1-81⁺. The transfection efficiency of hAECs estimated by EGFP staining was about 58.2%±1.3 % with AAV concentration of 10^-3 pfu/ml. Conclusions: We isolated hAECs from amnions successfully and stablished a culture system for hAECs in vitro. hAECs was a kind of stem cells. hAECs cultured in vitro can express GFP and its expression efficiency is more than 50 percent. Objective: To establish a optimizing culture system of the inducing process and study the contribution of the three factors in the system: Dex, HGF, IGF. To discuss detecting index of the hepatic transdiffernetion of HAECs.Methods: The induction cycle lasted for two weeks, in the end of induction functional genes and transcriptional factors were selected to detect the hepatic fate commitment. For example, the expression of albumin, CYP1A1, CYP1A2, IGFR, c-met, and HNF3, HNF4 and C/EBP were examined by the RT-PCR. Then flow cytometric analysis were performed to test the cell surface markers such as ALB, AFP and CK18 to explore the hepatocyte-like cell maturation; In order to optimize the induction system, detected the dose-dependent of the Dex, HGF, IGF cytokines; Albumin gene expression of different generation's amniotic epithelial cells were examed in the processes of the amniotic epithelial cells differentiated into liver-like cell.Results: Two weeks after the induction, the expression of albumin, CYP1A1, CYP1A2, IGFR, c-met and other liver cell-related key functional gene's can be detected in the human amniotic epithelial cells. Further study found that these gene's expression level showed the trend of gradual increasement which suggested that hAECs may have successfully be induced into hepatocyte-like cells. After inducement, HAECs expressed HNF3, HNF4 and C/ EBPa which were not detected before the induction; and the expression level of HNF1 is significantly higher than that not been induced. The detection of cell surface markers found that: 6 days after be induced, hAECs mainly were stained AFP+, the positive rate was about 15.1±2.1% ; with the time extension, 10 days after be induced, part of the hAECs showed AFP+/ALB+, which was about 6.5±1.4%; and on 14 days, hAECs basically only showed ALB+, the rate was about 13.9±2.3%; the positive rate of the ALB+ cells increasing indicated a grandual founctional maturation from the hAECs to hepatocyte-like cells. Similaritly, the CK18+ cells in the whole population were also increased in the number, for example on the 10 day, the rate was 16.1±1.2%; on the 14^th day, 21.3±4.6%, which proved the above hypothesis of the trandifferentiation. Experimental results of the dose-dependent study show that: IGF, HGF were dose dependent,while Dex's dose-dependent experimental results was not statistically significant, suggested that dose-dependent manner does not exist. Three passages of HAECs were compared in the gene expression of albumin during the induction. It showed that all the third generation cells which were induced, could all expressed the albumin gene and the expression had no obvious distinction.Conclusion: hAECs could indeed been induced to differentiate into hepatocyte-like cells in vitro, and could be successfully induced to form special functional hepatocyte-like cells; In the induction process, had a dose-dependent on HGF and IGF, whereas this dose-dependent is not obvious on Dex; In the passage of three generations, the expression of albumin,which was the hepatocyte -like-cells-specific gene, had not significantly difference. Objective To observe whether human Amniotic Epithelial Cells (hAECs) have the potential to possess hepatocyte-like function after intrasplenic transplantation in nude mice with liver injury as well as the therapeutic feasibility to repair damaged liver.Methods 40 nude mice were randomly divided into three groups: Group A ( n=20) : 5×10⁶hAECs labeled with green fluorescent protein were transplanted into the spleen of nude mice undergone 2/3 hepatectomized; Group B: treated with same cells as in A,but without hepatectomized; Group C: 0.2 saline was injected into the spleen of nude mice undergone 2/3 hepatectomized. The hAECs were detected with immunofluorescent microscope and their albumin expression were studied. Two and four weeks after the cell transplantation, Liver function and quantitative human albumin were measured respectively in all groups.Results The hAECs labeled green fluorescent protein were observed in liver and spleen tissue in group A and mostly expressed albumin.While in group B,C ,there is no such findings. The human albumin was detectable in serum of group A .The level of serum human albumin in group A in 4 weeks after transplantation was higher than that in 2 weeks after transplantation (3.9±1.34vs0.37±0.14, P<0.01). Four weeks after transplantation, the liver functions in Group A were restored significantly compared with those of Group C,The liver functions in Group A in 4 weeks after transplantation were improved significantly compared with those in 2 weeks after transplantation while the level of ALT was also different between two groups(121.2±20.8 vs. 191.8±28.9, P＜0.05).Conclusion The stimulus of liver damage might enhances the migration of hAECs to the liver ,in which a majority of the hAECs have the potential to possess hepatocyte-like function in nude mice . Transplantation of hAECs might amend the damaged liver function to a certain extent.