Characterization Of Wnt/β-catenin Signaling During The Hepatocyte-like Differentiation Of BMSCs Induced By Galectin-3

Background:Acute hepatic failure(often abbreviated AHF) is refers to infection by hepatitis B virus, drug poisoning and other various reasons resulting in a large area of necrosis of liver cells in a short period of time, and thus the synthesis, detoxification, and excretion of biotransformation function obstacles or decompensation, appears in the blood coagulation mechanism of abnormal, jaundice, ascites of liver function obstacle and cause clinical syndrome. According to statistics, China’s annual such patients up to 20～300000, AHF of acute onset, rapidly progressive, mortality can be as high as 80%, so the domestic and foreign scholars are actively searching for effective treatment method. It is known that liver transplantation is the most effective method for the treatment, but due to the shortage of liver donors, most of patients, actually, are late to receive prompt treatment for help, and even until someone obtains the suitable liver source, he must face a series of transplant-related serious problems such as immunological rejection, surgical complications, expensive cost, and so on. The treatment based on liver transplantation can not meet the clinical demand far less. New treatments, such as cell transplantation therapies due to their minimally Invasive, fewer complications and other advantages, attract scholars attention widely all over the world, of which liver cell transplantation therapies once considered the most promising, and however, the problems due to the loss of function of liver cells in vitro, freezing and thawing low vitality and immune rejection greatly limit its clinical application. In recent years, the rise of stem cell biology, especially the research of bone mesenchymal stem cells transplantation treatment has brought new hope to patients with acute hepatic failure.Bone marrow-derived mesenchymal stem cells (often abbreviated BMSCs) is another type of adult stem cells derived from the bone marrow except hematopoietic stem cells with potential of self-renewal and differentiation, which has a strong plasticity and is able to across the lateral mesoderm for differentiating into many kinds of cells under certain stimulation conditions, including osteoblasts, fat-like cells, neuron-like cells, cardiomyocytes-like cells and hepatocyte-like cells. Although BMSCs’level is low in the bone marrow of the body, and the number of cells will gradually reduce with age and debilitating body, it may be amplified into a large number in vitro, and at the same time can be induced directed differentiation into hepatocyte-like cells on certain conditions and thus participate in the liver pathophysiological processes including the tissue repairing, cell renewal, functional reconstruction,etc. In addition, BMSCs have enormous potential applications in the field of liver disease treatment and liver tissue engineering because of the advantage of donor sources and immune tolerance, also a very broad prospects to treat end-stage liver disease taking advantage of its multi-directional differentiation ability. And the problem how to induce BMSCs to differentiate into hepatocyte-like cells efficiently becomes the scholars’research hotspot, solving which crucial for stem cell transplantation to use in clinical treatment of liver diseases. But recent studies on the differentiation mechanisms are still in a preliminary stage because of more concentrating on their differentiation characteristics researching on BMSCs’ potential of directional differentiation into hepatocyte-like cells, and research on the in vitro inducer has little progress, resulting in poor effect on clinical application of stem cell transplantation. Therefore, it is significant to explore the molecular mechanism of BMSCs into hepatocyte-like cells and find a new method for inducing efficient differentiation.Our previous research on the microenvironment of the process that BMSCs differentiate into hepatocyte-like cells using isobaric tags for relative and absolute quantitation (often abbreviated iTRAQ) proteomics technology successfully screened out the different proteins in the liver tissue microenvironment between the liver injury rats model and normal rats, and then screening out a "critical" protein named galectin-3 which may play an important role in the BMSCs’induced differentiation process by using proteomics related software and bioinformatics analysis. Further induction experiments demonstrated Gal-3 can induce rat BMSCs to differentiate into hepatocyte-like cells, indicating that the Gal-3 is a very potential and powerful factor to promote BMSCs differentiation into hepatocyte-like cells. However, the related signaling pathways molecular mechanisms involved in the induction process has not yet been clear, and further exploring of this issue will contribute to excavating the ability that Gal-3 induces the rat BMSCs to induce into hepatocyte-like cells.Galectin-3 is a special member of galectin family, widely expressing in normal tissue and tumor tissue, mainly situating in the cytoplasm and a part of situating in the cell membrane and extracellular matrix, which participating a variety of physiological and pathological processes including cell growth and apoptosis, cell adhesion, immune response modulatory, angiogenesis, tumor invasion and metastasis, etc., through a special amino-terminal structure and carboxy-terminal structure with the carbohydrate recognition domain. A large number of studies show that Gal-3 play an important role in the regeneration of various cells and the process of tissue repair, in particular closely related to the regeneration of hepatocytes. However, the exact mechanism of action has not yet reported, which is presumably related to the proliferation and differentiation of stem cells combining with our previous results.The process that BMSCs differentiate into hepatocyte-like cells is extremely complex and involves interactions among a variety of cytokines, proteins inside and outside the cells, extracellular matrix, etc., and crosstalk regulation of multiple signaling pathways, which are recently widely recognized that Wnt/β-catenin signaling pathway, Notch signaling pathway, Hh signaling pathway, PI3K/AKT signaling pathway and TGF-β signaling pathways participate in the regulation during the process of BMSCs differentiation into hepatocyte-like cells, and furthermore play an important role. Among them the Wnt/β-catenin signaling pathway is closely related to the proliferation and differentiation of mesenchymal stem cell, and regulating the expression of WNT signaling gene on varying degrees can prompt mesenchymal stem cells differentiation into osteoblasts, fat cells, skeletal muscle cells, neuron-like cells, hepatocyte-like cells on certain conditions.Wnt signaling pathway is widely present in both invertebrates and vertebrates, highly conserved during the evolution of species, the most common of which is the Wnt/β-catenin signaling pathway. The Wnt pathway is mainly composed of extracellular signaling molecules, receptors in the cell membrane, a dynamic degradation complex named APC-Axin-GSK-3β complex in the cytoplasm, the key protein named p-catenin, downstream transcription factor Tcf/Lef, etc.. When Wnt signal is missing, the P-catenin in the cytoplasmic is mainly phosphorylated by the GSK-3β complex and then ubiquitinated into degradation in order to maintain a low P-catenin level in the cytoplasmic, wherefore the passage is closed; when the Wnt signaling molecules is specifically binding to the membrane receptor and then formed Wnt-Frizzled complex structure, resulting in the inhibition of GSK-3P phosphorylation activity, the P-catenin accumulates in the cytoplasmic and transfers into nucleus to activate transcription factors, playing a role in gene expression and regulation. It has shown that Gal-3 and Wnt/β-catenin signaling pathway have a very close relationship with extremely similar structures and functions to the important molecules on the pathway, such as Axin, GSK-3β,β-catenin, and even some scholars have pointed out that Gal-3 is very probably the important regulatory factor on the Wnt/β-catenin signaling pathway with functions similar to the P-catenin. Thus, we speculate that the canonical Wnt pathway may also occupy a vital role during the process of which Gal-3 induced rat BMSCs to differentiate directionally into hepatocyte-like cells, and studying on the expression characterization of the process can verify this hypothesis, and thereby providing experimental basis for the basic research and clinical application of Gal-3 and MSCs in aspects of liver disease.Objective:To explore the associated molecular mechanisms of WNT signaling pathway during the hepatocyte-like differentiation process of BMSCs induced by Gal-3.Methods:1. Isolated culture and identification of the rat bone marrow mesenchymal stem cells:SD rats were executed by using cervical dislocation to collect the bone marrow fluid from femurs and tibias, and then we jointed density gradient centrifugation and adherent culture to isolate the bone marrow mesenchymal stem cells for subculture. The fourth generation of the adherent cells were preparated into a single cell suspension, of which surface marker molecule were detected and identified using Flow Cytometry, demonstrating that bone marrow mesenchymal stem cells had been obtained successfully.2. Detection of the expression characteristics of important molecular on WNT signaling pathway during the hepatocyte-like differentiation of BMSCs induced by Gal-3:The fourth generation of BMSCs were culture induced by prepared complete medium containing 0.5μg/ml Gal-3, and the medium were changed once every two days. According to the different induced stages, the experiment were divided into five groups, including Od group,7d group,14d group,21 d group,28d group. Of each group total RNA and total protein were extracted to detect the mRNA expression of Wntl, Wnt3a, GSK-3β, β-catenin using Q-PCR technology, respectively, and to detect the protein expression levels of β-catenin by western blot, among which the Od group was as the reference group.3. Effect measurement of the agonist and antagonist of Wnt signaling pathway: The fourth generation of BMSCs was cultured in the medium containing Gal-3+ DKK-1, Gal-3+SB216763, respectively. Morphological changes were observed under inverted microscope and photographed on Od,7d,14d,21d,28d, respectively. Then we extracted total RNA and total protein to detect the mRNA expression of AFP and ALB by using Q-PCR, of which Od time as reference group, and the expression of P-catenin, and ALB by using western blot.Results:After the SD rat BMSCs Isolated were cultured for 24h, most of the cells had adhered and were in colony growth, and with the extension of culture time, cells in each colony continued to proliferate and grew in whorls, which like the appearance of fibroblast-like cells, fusiform, pseudopodia, arrayed with clear direction, and then the various colonies were of gradual integration. The result of Flow cytometry used to detect the cell surface marker molecules showed that CD29, CD44 and CD90 expressed positively, while the expression of hematopoietic stem cell marker molecules CD34 and CD45 were not obvious, which was consistent with the properties of bone marrow mesenchymal stem cells. Through detecting four genes’ expression at the different period of Gal-3 induced rat BMSCs differentiation into hepatocyte-like cells, we find that the mRNA expression of Wntl, Wnt3a, GSK-3β and β-catenin at different time group was in the presence of significant differences (P <0.01); At the same time, in addition to the 21 d group and 14d group (P= 0.07) of Wntl,and the 7d group Od group (P= 0.616), the 28d group and 21d group (P= 0.616) of GSK-3β,the remaining mRNA expression of each gene were significantly different with that at the previous time (P<0.05). Generally speaking, the expression of Wnt1, Wnt3a β-catenin showed a gradually trend, while GSK-3β showed a downward trend, and the protein expression of β-catenin by Western blot detection were consistent with the mRNA expression levels. After DKK-1 and SB216763 were added, respectively, the protein expression levels of β-catenin in both groups were gradually up-regulated with the extension of induction time, and the difference was significant comparing SB216763 group with DKK-1 group at the same time point (P <0.05), and the former increasing larger than the latter. The protein expression levels of ALB were likely similar with the β-catenin’situation. Detecting mRNA expression of AFP and ALB found that in addition to the expression of ALB and AFP of DKK-1 group at 14d slightly down, the two sets of AFP and ALB mRNA expression levels generally exhibited increasing trend with the increase of induction time, and moreover the expressions of AFP and ALB of SB216762 group were higher than that of DKK-1 group at the same time point significantly (P<0.05).Conclusion:Combined application of density gradient centrifugation and adherent culture could isolated and purified SD rat BMSCs successfully, and the expression of CD29, CD44 and CD90 on the cells’surface was positive, while CD34 and CD45 negative. The process of Gal-3 inducing SD rats BMSCs to differentiate into hepatocyte-like cells was closely related to the activation of Wnt/β-catenin signaling pathway. SB216763 and Gal-3 activated the pathway and promoted differentiation, while DKK-1 resisted the pathway activation at some extent and influenced differentiation efficiency.