Transplantation Of Mesenchymal Stem Cell To Treat Rat Acute Liver Failure

Background: By definition, stem cells are capable of both self-renewal and differentiation into at least one mature cell type. Stem cells, with their potential to differentiate into a wide variety of cell types in culture, would be invaluable for studies of some aspects of human embryogenesis and for transplantation therapies. Numerous previous reports demonstrated that bone marrow (BM) contains stem cells which differentiate into multitypical cell lineages. The most useful and important property of these cells is their ability to differentiate into ectodermal, endodermal, and mesodermal derivatives in vitro and in vivo, including hepatocyte, pancreatocyte, osteocyte, chondrocyte, adipocyte and endotheliocyte. The plasticity of marrow-derived stem cells is very important for the treatment of congenital diseases, age-related diseases and tissue defect.Stem cells have been identified in most organ tissues, including hematopoietic, neural,gastrointestinal, epidermal, hepatic, and mesenchymal stem cells (MSCs).Embryonic stem (ES) cells are pluripotent cells derived from blastocysts that can be propagated indefinitely Undifferentiated in vitro, can differentiate into all cell lineages in vivo, and can be induced to differentiate to most cell types in vitro. Although ES cells have been isolated from humans, their use in research as well as therapeutics is encumbered by ethical considerations. The ability to purify, culture, and manipulate MSCs from nonembryonic origin would provide investigators with an invaluable cell source to study cell and organ development: First, MSCs have the basic characteristic of stem cells. MSCs can not only differentiate into mesodermal tissues but also ectodermal and endodermal tissues. Second, the adult have lifelong MSCs. The adult stem cells are not limited by MHC when they are used to repair defected tissues,which makes the clinical use of adult stem cells more abstractive. Third, MSCs are easy to harvest and separate. At the same time they are relatively easy to expand in culture by capitalizing on their tendency to adhere and proliferate on tissue culture surfacesLiver failure is still associated with a high mortality rate despite various therapeutic attempts. Liver transplantation is the effective therapeutic modality that significantly improves the prognosis of patients with hepatic insufficiency. However, liver transplantation remains limited by some problems such as shortage of donor, major surgical trauma, rejection and expensive costs. It is important to develop an alternative procedure to treat liver failure. A possible alternative to liver transplantation is the hepatocyte transplantation. However, it is also limited by shortage of donor. The harvesting of hepatocytes is also associated with major trauma. Now the research of stem cell provides a new alternative method to treat liver failure. The MSCs were shown to be multipotential in that they differentiated in culture or after implantation in vivo and recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. The source of MSCs is sufficient and accessible. As a type of stem cell MSCs are relatively easy to propagate and amplify in vitro, which lead them to meet the requirements of cell transplantation. The rejection of the organ transplantation is a large problem which leads to the patients to require lifelong immunosuppressive therapy. While the autoallergic bone marrow cells transplantation overcomes this problem.Some reseachers found that stem cells, in the presence of cells from damaged liver tissue, developed into liver cells in as little as seven hours. Makowka L et al. reported that intraperitoneally transplanted syngeneic free BMCs were effective in D-galactosamine induced acute liver failure rats. Some reseachers transplanated bioencapsulated BMCs into the peritoneal cavity of 90% hepatectomized rats. They found bioencapsulated bone marrow cells could transdifferentiate into hepatocyte-like cells in the peritoneal cavity of 90% hepatectomized rats and increased the survival rates of these rats. These sduties show Bone marrow stem celss can be used to treat acute liver failure. However, However, No data is available about whether bone marrow cells transdifferentiation could occur in liver of surgically induced liver failure models. At the same time there is no report about whether mesenchymal stem cells can be used to treat acute liver failure.Objective: First, to study the method of separation and cultivation of MSCs from rat bone marrow. Establishing a stable MSCs' cell lineage of rat to provide a cell model for the further study of stem cells. Second, to establish the rat model of acute liver failure induced by hepatectomy. MSCs cultivated in vitro were transplanted into the acute liver failure rats to study the effect and possible mechanism of MSCs in treatment of acute liver failure rats.Methods: First, for the isolation of MSCs, F344 rats (6-weeks-old) were killed by cervical dislocation, and their limbs were removed. BMCs were flushed from the medullary cavities of the tibias and femurs using a 22-gauge needle. The cell suspension was filtered through a nylon sieve (75um). MSCs were separated from bone marrow cells (BMCs) by using Percoll. The cells were cultured with DMEM/F-12 containing 15% fetal calf serum, 50u/ml penicillin G and 50u/ml streptomycin. The cells were cultured at 37℃ in a humidified atmosphere containing 5% CO2. Growth curve was drawed and doubling time of MSCs during logarithmic growth phase was calculated by using Patterson formula. The activity of MSCs' teiomerase was measured by TRAP- argentation. The phenotype of MSCs was identified by flow cytometry. Second, Acute liver failure was surgically induced by 90% hepatectomy as described previously. 40 rats were divided into two groups : Group A (MSCs transplantation group):labeled bone marrow stem cells were suspended in 0.5 ml saline at a concentration of 1 ×10~6 cells and transplanted by portal vein injection after 90% hepatectomy (n=20). Group B (control group): 0.5 ml saline were injected, into liver via portal vein after 90% hepatectomy (n=20). Blood samples were taken from the rats on 1d, 3d, 5d and 7d after hepatectomy. Blood levels of albumin (ALB), amino alanine transferase(ALT), and aspartate amino transferase(AST) were determined by a biochemical kit according to the manufacturer's instructions. The rat survival time was defined as the period from partial hepatectomy to the time of death. Survival rate was defined as the percentage of surviving animals at any specific time point. The Liver tissues of Group 1 rats were sectioned into 3 μm slices using a cryostat. immunohistochemistry analysis and double Immunofluorescence was used to detect the BMSCs in the liver parenchyma and their differentiating state.Results: The primary passage cells were characterized with regular oat-like morphology. During logarithmic growth phase MSCs showed spindle-like or polygon morphology. They arranged regularly with clear bouncary. The doubling time of MSCs during logarithmic growth phase was 18hr. The teiomerase of MSCs showed positive when measured by TRAP- argentation, while telomerase of karyote in peripheral blood showed negative. The cell cycle and the content of DNA were measured by flow cytometry. The results were compared to those of tumor cell SKOV3. We found that all MSCs were diplont. While only 41.97% tumor cells were diplont. The amount of tumor cells in S phase was much less than that of MSCs (12.1% /3 4.7%). MScs express CD44 CD90, while CD34 CD45 MHC I and MHCII were negative when they were identified by flow cytometry.More than 90% of the cells were labeled by BrdU. More than 95% of the cells survived after labeling by 5-bromodeoxyuridine (BrdU). Liver failure was observed in the rats after 90% hepatectomy. The liver failure was characterized with decreaed motoe activity, downcast, hypnody, no response to stimulus and urinary incontinence. The survival rate was 20%. Laboratory measures of liver function showed that the serumal levels of ALT and AST increased, while the serumal level of ALB decreased. The changes of the serum levels of ALT, AST and ALB on 1d, 2d, 3d and 5d postoperative were significant when compared to those before surgery (P< 0.05). The survival rate of the group A was 70%, while that of the group B was 20%. The difference in survival rats after a 90% hepatectomy was significant in group A compared with group B (P< 0.05). The plasma albumin levels in two groups decreased from day 1 post-surgery and started to increase from day 3 post-surgery. Blood levels of ALT and AST increased and reached peak levels at day 1 post-surgery, then they decreased. There were significantly less in the ALT, AST levels on 3d and 5d in group A when compared to those in the group B. The results of immunohistochemistry showed that some transplanted stem cells have migrated into parenchyma, exhibiting the morphology of hepatocyte. Most of these cells were distributed at portal area. Double Immunofluorescence showed Co-expression of Brdu (green) and albumin (red); the albumin in co-expressing cells appeared as fine yellow particles under Confocal laser-scanning microscope. Conclusions: (1)The isolation and culturation of MSCs from bone marrow are feasible. The characteristics of cultured MSCs lineage is stable. The phenotype of the cell lineage is uniform. This cell lineage can be used for further research. (2)MSCs transplantation can increase the survival rate of 90% hepatectomy induced liver failure rats and promoted the recovery of liver functions. Transplanted stem cells migrated into parenchyma, exhibiting the morphology of hepatocyte and secreted ALB. These observations imply a possible alternative to liver transplantation for the treatment of liver failure.