| BackgroundLiver disease is one of the major diseases that threaten human health. Especially acute liver failure (ALF), carries a high morbidity and mortality, as high as70～80%, because of severe acute liver damage with hurt synthetic function in the absence of pre-existing liver disease. Orthotopic liver transplantation is the most effective therapy for ALF. But it also has several disadvantages, such as a shortage of donor organs, irreversible, highly intrusive, high expense, and the necessity of lifelong immunosuppressive treatments. Cell transplantation represents an attractive tool for the establishment of cell-based therapy for ALF.Stem cells can differentiate into hepatocyte cells in certain internal environment, which can secrete cytokines to promote the repair and regeneration of damaged liver. The preeminent candidate cell is mesenchymal stem cell (MSC), which obtained from bone marrow (BM) first of all. MSC is an important member of the stem cells family, because of their wide range of sources, with a high degree of self-replication and differentiation potential, and a unique low immunogenicity.Thus it become the appropriate cell transplantation source. Bone marrow-derived MSC is the most extensive study of seed cell, but it is restricted by limited sources and low MSC content. Now some researchers find MSC can also from umbilical cord blood (UCB), amniotic fluid (AF), placenta, tooth, dermis, scalp tissue, and adipose tissue (AT). Especially human placenta-derived mesenchymal stem cells (hPMSCs) have a very consistent biological characteristics with bone marrow MSC, and less immunogenic, stronger proliferation and differentiation ability. While the placenta is in clinical waste, drawn almost unlimited, and does not involve ethical issues; therefore, it will not only be able to become an ideal alternative to bone marrow MSC, but also has a wider application potential.In this study, we cultured the placenta-derived mesenchymal stem cells and detected their biological characterizations.ALF pathogenesis is very complex, so establishment a stable and reliable animal model is essential. Generally, models of mice, rats and other rodents are used to evaluate the potential of MSCs from the bone marrow and placenta in repairing liver damage. In this study, stable ALF models of Chinese miniature pigs were established by injected D-galactosamine (GalN) to study the feasibility, safety and efficacy of hPMSCs transplantation therapy.Liver plays a major role in metabolism with a wide range of functions such as protein synthesis, detoxification, and glycogen storage. Normal liver can maintain a relative balance of lipid metabolism. When mild liver damage occurred, liver’s compensatory role helps to maintain the balance.Serum concentration of many endogenous substances will be changed when in liver failure, such as aromatic amino acids, bilirubin, free fatty acids, serum ammonia, benzodiazepine class and pro-inflammatory cytokines. As the disease progresses, the levels of endogenous metabolites in vivo also changed accordingly. Metabonomics refers to the measurement the metabolism of tissues and biofluids that lead conventional biochemical and pathological changes. Metabonomics offers a rapid and simultaneous measurement of significant number of metabolites in biological samples. Today, ultra performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-Q/TOF-MS) has become one of the widely used techniques in metabonomics studies with higher resolutions, higher sensitivities, and more rapid separations.In the present study, a metabonomics method based on UPLC-Q/TOF-MS was employed to investigate the mechanism underlying the therapeutic effect of human placenta mesenchymal stem cells (hPMSCs) on GalN-induced liver injury in Chinese miniature pigs. The experimental goals were to explore the dynamic patterns of endogenous metabolites in the whole process of ALF and hPMSCs transplantation, and to characterize the serum metabolic profiles associated with acute liver injury as well as hPMSCs-mediated recovery.MethodshPMSCs were isolated by collagenase digestion and density gradient centrifugation. Then the biological characterization, including determination of cell surface markers by flow cytometry; osteogenic, adipogenic, and hepatic differentiation and their functional specificity were measured.To study the effectiveness and safety of hPMSCs for treatment of liver injury, ALF models of Chinese experimental miniature pigs were established and separated into four groups:no cell transplantation; hPMSC transplantation through the jugular vein; X-ray-treated hPMSC transplantation through the portal vein; and hPMSC transplantation through the portal vein. Mortality rates, serum biochemistry, pathological and molecular biological analysis of liver tissues were determined1-7days after cell transplantation and then every2weeks beginning at the second week until death.UPLC-Q/TOF-MS-based metabonomics approach was employed to analyze serum from GalN treated pigs with the goal of identifying potential biomarkers for acute liver injury. Both positive and negative modes were analyzed in this study. The time-of-flight analyzer was used in V mode and was tuned for maximum resolution (10,000resolving power at a mass-to-charge ratio556.2771m/z) in the positive mode or at m/z of554.2615in the negative mode). The raw data files generated from the UPLC-MS analysis were processed using MassLynx v4.1and MarkerLynx XS v4.1software. The preprocessed data obtained by MarkerLynx were exported and analyzed by principal components analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) using software SIMCA-P+12.0. Potential biomarkers of ALF were selected according to VIP values (VIP>1.0, and p<0.01in paired t-test) and the S-plot (a further loading plot, is a visual method that can be used for selection of biomarkers). The final selection of biomarkers was based on a comparison with corresponding standards.ResultsPlacenta derived mesenchymal stem cells can be cultured long-term in vitro and they express high levels of HLA-I, CD29, CD13, CD73, and CD90, but hardly express HLA-Ⅱ, CD34,CD45%, CD133, CD79b, and CD31. hPMSCs can also differentiate to functional osteoblasts,adipocytes and hepatocyte-like cells in vitro.Multivariate analysis showed that obvious metabolic disturbance occurred during acute liver failure (ALF), which can be ameliorated by the hPMSCs transplantation. After hPMSCs were transplanted via the portal vein, the liver function of ALF model pig was improved significantly.Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholinesterase (CHE), total bilirubin (TBIL) and total bile acid (TBA) concentration gradually returned to the normal range.The metabolic profiles of the hPMSCs transplantation group returned back to the original state5weeks after the hPMSCs transplantation. Serum levels of several metabolites including glycoursodeoxycholic acid, glycochenodeoxycholic acid, aromatic amino acids, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, were significantly modified during the process of ALF and cell treatment. In addition, the result obtained from metabolic trajectory analysis correlated well with those from biochemical analysis and histological examination. Pathology results showed significant improvement in hepatocytes degeneration and moderation in liver inflammation and necrosis after hPMSCs transplantation through the portal vein; while the other three groups of indicators obvious change.The results of immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) showed the transplanted hPMSCs colonization in recipient pig livers.ConclusionsMSCs from human placenta displayed characteristics similar to those of MSCs from bone marrow. hPMSCs can differentiate into functional hepatocyte-like cells in vivo and in vitro. They can also improve liver functions in vivo after transplantation into ALF model pigs and increase the survival of pigs with liver failure signally.This represents an attractive stem cell-based candidate therapy for liver disease because it is free of ethical concerns, non-invasive, uses abundant cells, and is strongly immunosuppressive.UPLC-Q/TOF-MS-based metabonomics has gained new insight into the molecular mechanism on how hPMSCs administration facilitates the recovery of ALF, and provided strong support for treating liver diseases with stem cell-based therapies. |
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