Curative effects of transplantation of hepatocytes differentiated from embryonic stem cells on treatment of fulminant hepatic failure: experiment with mice

Abstract

To investigate the curative effects of transplantation of hepatocytes differentiated from embryonic stem cells (ESCs) on treatment of fulminant hepatic failure (FHF). Mouse ESCs transfected with green fluorescent protein were cultured. RT-PCR was used to detect the mRNA expression of alpha-fetoprotein (AFP), transthyretin (TTR), hepatic nuclear factor (HNF), albumin (ALB), glucose-6-phosphate (G6P), and tyrosine aminotransferase (TAT) at different time points. Immunocytochemistry (ICC) was used to detect the expression of AFP, ALB, cytokeratin 8 (CK8), and CK11 in the cells. The morphology and distribution of the cells were observed with microscope. Forty mice were randomly divided into 2 equal groups: ESC transplantation group, undergoing transplantation of ICC positive ESCs (hepatocytes) into the hepatic capsule, and control group, undergoing injection of normal saline in the hepatic capsule. Twenty-four hours later carbon tetrachloride was injected intra-peritoneally to induce FHF. The survival status of the mice was observed. Twenty-four hours later venous blood samples were collected to examine the levels of total bilirubin (TB), alanine aminotransferase (ALT), ALB, blood sugar (BS), and prothrombin time (PT). After the death of the mice, their livers were taken out to undergo microscopy and immunohistochemistry to observe the structure of liver tissue, growth of tumor, and expression of ALB. RT-PCR showed that the mRNA expression of AFP, TTR, HNF, ALB, G6P, and TAT emerged since days 3, 3, 5, 9, and 11 respectively and then gradually increased till day 19. ICC showed that the EB cells began to express AFP since day 7, to express CK8 and CK11 since day 9, and to express ALB since day 11. The ICC-positive cells were consistent with the mouse hepatocytes morphologically and were distributed only in the central and marginal areas of the EB cell community. Injected with carbon tetrachloride, the mice showed manifestations of FHF. However, the symptoms of central nervous system emerged later in the mice implanted with the hepatocytes in comparison with the control mice; 8 of the 20 mice in the transplantation group showed ascites in comparison with 14 in the control group. The mean survival time of the transplantation group was 62 hours, significantly longer than that of the control group (23 hours, P < 0.05). Compare with the normal mice, the FHF mice in both groups showed higher ALT and TB and lower ALB and BS (all P < 0.01), however, the levels of ALT and TB were lower, the level of BS was higher, and PT was shorter significantly in the transplantation group than in the control group (all P < 0.05). Pathology showed that no tumor formation was found in both groups and that the transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed ALB. Hepatocytes originating from ESCs exercise the functions of normal hepatocytes. Transplantation of hepatocytes differentiated from ESCs is able to improve the life quality and lengthen the survival time of FHF mice.