Studies On Isolation And Culturation In Vitro And Transplantation In Vivo Of Murine Fetal Liver Stem Cells

Subject Resource:This work was supported by grants from the Major Project of Chinese National Programs for Fundamental Research and Development (973Program)(2010CB945502).BackgroundStem cells, a kind of cells with self-renewal ability and multiple differentiation potentials, exist in many organizations; According to the different development stages, stem cells are divided into embryonic stem cells (ESC) and adult stem cells. Hepatic stem cell (HSC) is a kind of adult stem cells, which can differentiate into mature hepatocytes, biliary epithelial cells (BEC) and pancreatic epithelial cells. According to different origins, hepatic stem cells can be divided into hepatogenous hepatic stem cells and non-hepatogenous hepatic stem cells, the former mainly include the hepatic oval cells and small hepatocytes, while the latter are originated in the bone marrow stem cells and embryonic stem cells. Recent study on hepatic stem cells has become a worldwide hotspot due to their extensive sources, high proliferation, small transplantation size and other advantages that traditional hepatocytes cannot compare, therefore, it can be predicted that it will become an important source of cells which will replace the liver transplantation and hepatocyte transplantation in the treatment of organ failure. Because of the incomparable advantage of hepatic stem cells to treat liver disease due to the varieties of causes, there is an important practical significance in discussing how to obtain high purity of hepatic stem cells and their specific markers.Under normal conditions, hepatic stem cells in vivo are very few and mostly in the resting period. Drugs or surgery are often used to cause liver injury during separation and the hepatic stem cells are then obtained by liver perfusion or separation combined with cellular extract liquid, which is complex and costly. Compared with traditional cultivation and separation of hepatic stem cells, it is the first time to separate hepatic stem cells through mechanical cutting and enzyme digestion methods in this research. Simple centrifugation is combined with trypsin selective digestion methods to separate hepatic stem cells from the rats with15days’ pregnancy successfully. LSCs with uniform morphology, fast proliferation and stable characters can be obtained in the original generation by this method, which has the advantages of simple operation, quick, practical and small damage to cells, thus providing experimental basis for the seed cells of the tissue engineering.ObjectiveTo improve the isolation and purification methods of rat fetal Hepatic stem cells(HSCs), and therefore to further improve the purity and vitality of the isolated HSCs.Methods1. Inbred SD rats with15day’s pregnancy (clean grade) were purchased. With mechanical cutting and enzyme digestion methods to separate the hepatic stem cells, and simple centrifugation and trypsin selective digestion method for further purification, relatively purified hepatic stem cells were achieved. The cells were added into the DMEM medium containing10%fetal bovine serum, and their adherence as well as morphology changes were observed under microscope.2. Oval cells were inoculated on the cover glass processed with0.2%gelatin in advance on24-hole culture plate, and cultured for24hours in the incubator at37℃and5%CO2. Immunofluorescence chemical detections were raised when the cells grown into monolayer. The cells had digestive transfer culture, and cells of P10generation were detected with immunohistochemical detection. The expression of the markers CD34, C-Kit, OV6and CK19, which were on the surface of the hepatic stem cells of P1and P10generations, was detected respectively.ResultsThe isolated rat LSCs in inoculation culture bottle initially attached after24hours, the cells increased in size after5days, with a spindle shape or polygon. By1-2times difference digestion transfer of culture, the cells grow in colony sample, with clear margin and strong refractive index. With the Immunohistochemistry method,we can draw this conclusion:P1generation of rat fetal LSCs C-Kit, alpha-fetoprotein (AFP) antigen, OV6, cytokeratin (CK)19take on positive attributes, while ALB doesn’t.p10generation of LSCs CD34, C-Kit,OV6,CK19take on positive attributesConclusionsThe methods of isolating, purifying, cultivating, amplifying and appraising rat fetal LSCs are simple and practicable; they give a material basis for further research on LSCs transplantation and biological characteristics in the experiment. Influences of mature liver cells and oval cells on Liver Regeneration After Liver InjuryBackgroundFor a long time, that how mature hepatocytes and oval cells play a role in the regeneration of injured liver has been the key issue in the research of the hepatic stem cell and their effects in the regeneration of injured liver are of different viewpoints. On the one hand, some reports showed that after partial hepatectomy (PH), rat livers could be regenerated and recovered to the original size in two weeks, and it was found that after12consecutive PH the liver regeneration still could be achieved, which was regarded as separate proliferations of mature hepatocytes. On the other hand, oval cells (OVC), which are originated from the small biliary duct and Hering’s duct in portal area and play a role in the regeneration of the injured liver, are regarded as the hepatic-endogenous adult hepatic stem cells with liver-and-gall two-way differentiation potential. Currently, in order to study the proliferation and differentiation of hepatic stem cells in adult liver, the researchers used a variety of different ways to inhibit hepatic cell proliferation and used drugs (such as CC14) or partial hepatectomy etc. to induce acute liver injury, thus activating the hepatic stem cells. Therefore, the relationships between mature liver cells, oval cells and liver regeneration after liver injury were studied.Considering RS has been recognized as a chemical toxicant with the long-term inhibition to mature hepatocytes proliferation and PH can provide strong demand of liver regeneration, rat models with acute liver injury were set up in this research because of the combined application of the PH and RS. According to comparative pathology research of mature hepatocytes and OVC in the liver regeneration after PH/PH and1/3PH, and a series of observation during the study, the relationship between mature hepatocytes, OVC and liver regeneration after liver injury was discussed.ObjectiveCombine retrorsine (RS) and a third liver resection (1/3PH) to establish a rat liver injury model. Observe the proliferation of liver cells and oval cells after liver injury in rats, and discuss the relationship between liver regeneration and mature liver cells and oval cells after liver injury.Methods1.30rats had been randomly divided into2groups with15in each. Group of retrorsine/1/3partial hepatectomy (RS/PH group):SD rats of around150g were intraperitoneally injected with retrorsine solution of30mg/kg twice with the internal of2weeks.1/3partial hepatectomy (1/3PH group):Normal saline solution instead of retrorsine was intraperitoneally injected with30mg/kg twice with the internal of2weeks,4weeks after the second intraperitoneally injection,1/3partial hepatectomy was improved in two groups of rats.2. Rats were executed respectively on the postoperative3,7,14,20th days. Cells were fixed with10%formaldehyde, embedded in paraffin, sectioned in routine method and stained with hematoxylin and eosin. BrdU injection, BrdU immune fluorescent chemical detection, CK19as well as C-Kit immunohistochemical detections were used. Liver pathological morphological changes, cell proliferation and immunohistochemical detection of CK19, C-Kit at different postoperative time points between the two groups of rats were observed.Results20days after operation, the body quality of RS/PH group rats is still lower than that of preoperative, liver increase is obviously less than the a1/3PH group, cell body swelling with HE staining, loose cytoplasm, extensive vacuoles degeneration of liver cells, cell proliferation can be seen clustered or scattered around the portal area of small bile duct and lobular;20days after operation, the body quality of1/3PH group is close to that of preoperative, liver damage is lighter than that of RS/PH group, a large number of mature liver cells proliferating under Brdu immunofluorescence,14days after operation, liver OVC proliferates, appearing in the liver cell line, morphology and immunohistochemical markers consistent with OVC of the RS/PH group.ConclusionsAn acute rats liver injury model combining retrorsine alkali (RS) and a third liver resection (1/3PH) is successfully built, the phenomenon of liver toxicity and liver stem cells and mature liver cells’ proliferation after poisoning can be seen in the experiment, which firmly confirm that liver cells’ renewal and regeneration after injury is the outcome of combined action of liver stem cells in liver basin and mature liver cells. Studies on Isolation and culturation in vitro and transplantation in vivo of Embryonic hepatic stem cells of GFP transgenic miceBackgroundMorbidity and mortality of acute and chronic liver failure due to a variety of causes are very high, which has become the main threat to lives of patients with liver disease. At present, orthotopic liver transplantation (OLT) has been proven to be the most effective way to treat varieties of hepatic dysfunction and liver failure, but due to the deficiency of donor liver, costly outlay and many complications after transplantation, the widely applications of OLT have been limited. In recent years, with the constant appearance of new interventional treatments, hepatocyte transplantation has been used in some animal models and human clinical trials, but due to the insufficient sources, an abundant and safer source of cells is still in demand. For this demand, a new way is opened up by means of transplantation of hepatic stem cells, which is a kind of pluripotent stem cells with the abilities of self-renewal, high proliferation and differentiation to hepatocytes and cholangiocytes. The hepatic stems may differentiate into hepatocytes and cholangiocytes in vivo and participate in the physiological and pathological process such as the development and progression of the liver and its repair process after injury. If multiplied vigorously and then transplanted, thus the problem of cell source shortage can be solved.Hepatic stem cell transplantation has greater advantages when compared with the hepatocytes:(1) Higher proliferation makes the hepatic stem cells proliferate greatly after implanted into the receptor, so less transplanted cells can achieve a good proliferation effect;(2) After transplantation into the liver, the HSCs can differentiate into hepatocytes and biliary epithelial cells, which is more beneficial for the reconstruction and functional recovery of liver tissue, and more importantly, the receptor liver needs selective pressure during hepatocytes proliferation, while the hepatic stem cells are multiplied in normal liver which provide opportunities for the regeneration of the receptor liver and liver function recovery;(3) Hepatic stem cells are not mature with less immunogenicity, Kubota etc. had studied that classical MHC-1were not expressed in hepatic progenitor cells, and immune escape occurred upon hepatic stem cells allograft. Transplant failure was relatively less than that of hepatocyte transplantation, and the effects on receptor were small;(4) The diameter of liver stem cells is smaller than (10-12μm) that of hepatocytes (20-35μm), so the liver stem cells are more easily going through the hepatic sinusoid to the liver lobes, thus the efficiencies of engraftment and amplification are increased. While at high transplantation concentration of hepatocyte, some cells cannot pass through the portal area to small blood vessels. So the liver stem cells can be used as the most ideal cell source for biological artificial liver and hepatocyte transplantation, thus playing an important role in the gene therapy for liver disease and being expected to replace the replacement therapy of liver transplantation of patients with severe liver failure, which has a great research prospect.Embryonic hepatic stem cells of GFP transgenic mice were transplanted to rats treated by L-CL2MDP/RS/PH in advance, the results showed that hepatic stem cells stains were implanted into livers of L-CL2MDP/RS/PH rats successfully in this laboratory. The implanted cells were survived in the receptor rat livers, and different degrees of proliferation were appeared. While GFP+liver stem cells were not seen in normal rats in the absence of pretreatment. We could not clearly know the proliferation degrees of transplanted embryonic hepatic stem cells, but from the comparison of the ratio of the GFP positive cells in liver, L-CL2MDP/RS/PH group had obvious advantages than the control group. So it was declared that the L-CL2MDP/RS/PH models might provide a relatively better environment for transplantation of the hepatic stem cells. Some valuable information was provided for the research on the experimental animal models which were suitable for cell transplantation. In the future, the achievements from this research will be extended through combination with clinical practice, and the problems of immunological rejection mechanisms in transplantation which are remained to be settled will be answered.Objective 1、 Fetal liver liver stem cells of fluorescent transgenic mouse stably expressing GFP protein2、Lower success rate of hepatic stem cell transplantation was still a problem to be solved in the research at home and abroad. In order to further improve survival of hepatic stem cell in liver,Methods1. Selected transgenic mice with green fluorescent protein (GFP),15days’ pregnancy, weight of40-60g; and30SPF male SD rats of around150g; Separation, cultivation and generation in vitro and immunohistochemical evaluation of the embryonic hepatic stem cells of the GFP transgenic mice were developed.2. L-CL2MDP/RS/PH animal models were established, the separated embryonic hepatic stem cells were transplanted into rat livers through spleen injection, expression of GFP protein were detected after cell transplantation, and immunohistochemical detection of C-Kit and AFP ensured whether there was proliferation of the transplanted hepatic stem cells within the liver.ConclusionsAn acute rats liver injury model combining retrorsine alkali (RS) and a third liver resection (1/3PH) is successfully built, the phenomenon of liver toxicity and liver stem cells and mature liver cells’proliferation after poisoning can be seen in the experiment, which firmly confirm that liver cells’ renewal and regeneration after injury is the outcome of combined action of liver stem cells in liver basin and mature liver cells. Conclusions1. The separation and purification methods of rat embryonic hepatic stem cells (EHSCs) were improved, and the purity and vitality in the hepatic stem cells separation were increased. With rat embryonic liver as source of the cells, through mechanical cutting and enzyme digestion methods, hepatic stem cells were separated and purified by simple centrifugation and further selective digestion of trypsin in order, thus pure rat EHSCs were obtained. Markers on the hepatic stem cells surface of the P1and P10generation were detected respectively with immunocytochemistry method, which demonstrated that the separated and cultured cells in this study had the properties of stem cells, namely hepatic stem cells.2. Influence of retrorsine combined with partial hepatectomy (RS/PH) on the regeneration and repair after liver injury was observed. Rat models of acute liver injury due to RS/PH were constructed successfully in this experiment. Liver intoxication and the later hepatic stem cell proliferation were observed in the experiment. Also the proliferation of oval cells were observed clustered or scattered around the biliary duct and in the hepatic lobule in the portal area of the RS/PH group after postoperative H-E staining. CK19and c-kit positive cells were appeared in the portal area, especially around the biliary duct with the shape similar as the cholangiocytes around. While the appearance mentioned above were not seen in the PH group. It was confirmed that this model could inhibit mature liver cells proliferation, at the same time realized the repair through stimulation of hepatic oval stem cells proliferation. This provided some valuable information to the experimental animal models which were suitable for hepatic stem cell transplantation.3.Lower success rate of hepatic stem cell transplantation was still a problem to be solved in the research at home and abroad. In order to further improve survival of hepatic stem cell in liver, in this laboratory liposome with dichloromethylene diphosphonate (L-CL2MDP) wrapped was prepared, which was used through intraperitoneal injection to specific eliminate macrophages in liver and spleen, and further a new model of hepatic stem cell transplantation was established by combination of the built rats liver injury model with retrorsine/partial hepatectomy. At present, we separated hepatic stem cell from the transgenic mice with fluorescent gene in our laboratory to obtain stable cell strains expressed GFP. The GFP+-LSC cells were transplanted through injection into spleen of rats treated by L-CL2MDP/RS/PH in advance. Proliferation in the liver was observed, tested and compared of the control group and treatment group, to determine whether this model can improve the efficiency of hepatic stem cell transplantation.