| ObjectiveHepatic injury is the common pathological basis of various types of liver disease. Severe liver damage can develop into liver failure or death within several days. At present, stem cell therapy which is able to dramatically improve the treatment of hepatic injury has become a research hot spot, although it is limited because of the difficulty in the in vitro enrichment and purification of hepatic stem cell and the poor knowledge about mechanism of stem cell differentiation. In this study we investigate the therapeutic effect of fetal hepatocytes which is rich in stem cells with directed differentiation potential on severe liver injury, the advantages of 3D Soft-Agar-Culture-System (SACS) for the culture of mouse embryonic hepatic stem cells was also demonstrated. We showed a satisfactory ability of SACS to maintain the undifferentiated state and pluripotency of the suspended hepatic stem cells for a long time. Then suspension of embryonic hepatic stem cells was transplanted into injured livers of rats via portal vein. The therapeutic effect of hepatic stem cells on CCl4-induced hepatic injury was evaluated. Moreover, we determined the protein expression of HNF4αin rat livers at different time points in order to underline its promotive role on the recovery of acute hepatic injury. This study was divided into two parts: 1. Evaluation of the differentiation-inductive effect of suspension culture in the SACS on embryonic hepatic stem cells. 2. Study on the promotive role of HNF4αon the recovery of acute hepatic injury. Current studies provided a better understanding of the molecular mechanisms of the embryonic hepatic stem cells-based cell therapy which warranted the clinical implication of this novel modality for liver disease in the future.Methods1. Suspension of ED 14 Fischer (F) 344 rat embryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly, Group 1 were seeded into type I collagen-coated plates for adherent culture while Group 2 were seeded into soft agar medium for suspension culture. Suspension medium was composed of two parts, the basal layer contained 20% fetal bovine serum, 0.6% agar, and EGF with 20μg/ml final concentration. The upper layer was composed of 0.3% agar into which the hepatic stem cells were seeded. After 2-week culture, the morphology and ultrastructure of cells in both two groups were observed by inverted microscope and transmission electron microscope (TEM). Expressions of CD90.1 and CD49F, two specificstem cell surface markers, were tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase staining was performed to detect stem cell differentiation by immunohistochemistry, and the expressions of AFP and ALB were tested by RT-PCR.2. In the following experiments, SD rats were randomly divided into transplantation group (n = 20) and control group (n = 20) after CCl4- induced hepatic injury. Hepatic stem cells were transplanted into injured livers of rats in transplantation group via portal vein. Equal volume of saline was transplanted in control group. The expression levels of ALT, AST in the serum and histopathological injury of hepatic tissues were analyzed 6 h before, 1w, 3w and 5w after transplantation. Evaluation of HNF4αprotein expression in rat livers both by immunohistochemistry and western blot was simultaneouly performed.Results1. A minor part of cells from suspension culture could not proliferate and form clone ball, and then they experienced apoptosis and disintegrated. However, majority of cells developed into colonies containing more than 3-5 cells after 2days. Shapes of the newly formed colonies were irregular and mulberry-like. Newborn cells budding in these clumps could also be observed. Most of the colonies contained more than 20-25 cells which are well refractive and contacted with each other closely. The morphological study of the colonies in SACS showed that all cells were stem cell-like. Primary cells culturing in adherent cultures condition adhered to bottom wall within 24 h after inoculation and began to divide and proliferate within 48 h. The adhered cells developed into colony-forming units finally. Cells passages significantly accelerated the growth rate of adhered cells and their differentiation into mature hepatocytes.2. TEM revealed that hepatic stem cells in SACS can maintain an undifferentiated state for a long time interval with a mean diameter of 7μm~13μm and rare microvilli in cell surface. Most of the cells had a relatively large nucleus, higher nucleus-cytoplasm ratio, and less cell organelles such as ribosomes, mitochondria, endoplasmic reticulum. The diameter of adhesive culture cells was significantly greater than suspension culture cells (20~40μm). In addition, ultrastructural feature of these cells showed a mature hepatocyte-like phenotype. Flow cytometry demonstrated that soft agar culture resulted in a larger population of cells expressing the undifferentiated stem cell markers CD49F, CD90.1, compared with a smaller population of the cells which were under adhesive culture conditions. The undifferentiated hepatic stem cells in SACS showed strong ALP staining, while cells in adhesive culture conditions stained weakly for ALP. RT-PCR showed a significantly decreased AFP expression along with a incresed ALB expression in the cells under adhesive culture(P<0.01). There was no significant change before and after culturing in the SACS.3. Rats in each group suffered different degrees of injury after CCl4-induced hepatic injury. In treatment group, the injured hepatic tissues were markedly repaired after stem cell transplantation and the liver function gradually returned to normal physiological level. By contrast, the liver function of control group was slightly repaired and experienced slow recovery, as evidenced by significant increased ALT and AST in serum. Survival rate of the rats in transplantation group was significantly higher than control group at each time point. Western blot and immunohistochemistry demonstrated that expression of HNF4αnotably increased when liver injured, and then slowly decreased in accordance with the gradual restoration of liver tissue.ConclusionsEmbryonic hepatic stem cells cultured in SACS experienced less differentiation than those were adherently cultured in serum-added culture medium, and could proliferate and form clone ball with a specific stem cell feature. HNF4αplays a protective effect in the early stage of hepatic injury. It was carried out probably through promoting the restoration of acute hepatic injury by promoting the migration to damaged hepatic lobule and facilitating cell differentiation, proliferation of embryonic hepatic stem cell.
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