Characterization Of Notch Signaling During The Differentiation Of BMSCs Into Hepatocyte

Background:Cell differentiation is a course that one cell type is tansformed to another cell type with different morphology, frame and function during cell division. Bone Mesenchymal Stem Cells (BMSCs) is one kind of early stage cell type in mesoderm, it can differentiate into mature cell type such as osteoblast, lipocyte, muscle cell even nerve cell. The ability of Multilineage differentiation of bone mesenchymal stem cell is well investigated during many fields, It has also been demonstrated that human umbilical cord blood-derived MSCs can differentiate into functional hepatocyte-like cells in vitro. Furthermore, oval cells may also be derived from bone marrow stem cells and differentiate into hepatocytic cells, Our previous work had established a special experiment system in which BMSCs could differentiate into hepatocytes successfully Though BMSCs can differentiate into hepatocytes, the precise mechanisms controlling this process remain unclear yet. The ability of Multilineage differentiation of bone mesenchymal stem cell be utilized in therapy of some diseases. Notch signalling is an evolutionary conserved intercellular signalling mechanism, which is crucial for proper development of most vertebrate organs. The balance whether stem cells maintain their sternness or commit to transiently proliferate and differentiate along a particular pathway is complex and involves many signal transduction pathways. In mammals, Notch families have expanded to four Notch genes (Notch1-4) and five ligands, two Serrate-like(Jagged1-2) and three Delta-like (Dill,3and4). Upon ligand-binding, the cytoplasmic domain of Notch is released from the cell-surface by a Presenilin(PS)/y-secretase-dependent proteolytic cleavage. The intracellular Notch protein then translocates to the nucleus, interacts with the CSL (CBF1, Su(H), Lag-2) transcriptional repressor, and converts it to transcriptional activator. Notch signaling is initiated by ligand-receptor interaction between two neighboring cells resulting in two successive proteolytic cleavages. The ability of the Notch pathway to inhibit differentiation has been reported. The classical example of Notch signaling regulating cell fate decisions at developmental branch points is the development of the peripheral nervous system. However, it has been reported that Notch signal could stimulate the differentiation of BMSCs to osteoblast. The role of Notch signal in differentiation of BMSCs to hepatocyte is still unclear. In this study, we isolated the BMSCs from rat according to recent reports, and induced the differentiation of BMSCs to hepatocyte by the material prepared from the tissue of rat liver. During the process of differentiation of BMSCs to hepatocyte, we detected the changes of some elements of Notch signal by Reverse Dot Blot. The differentiation of BMSCs to hepatocyte in vitro is new pathway to obtain plenty of hepatocytes for transplantation.Objective:To explore the effect of Notch signaling in bone marrow-derived mesenchymal stem cells during the process of differentiation into hepatocytes.Methods:1. Construction of the animal model:the rat was given gastric lavage with2-AAF for7days, and then was given peritoneal injection with Ccl4. The liver of the rat was impaired, this animal model would be utilized in the subsequent experiment.2. Preparation of material used for BMSCs differentiation:Extraction of rat liver was obtained from the rat liver tissue, this kind of extraction would be used in the subsequent experiment.3. Identification of obtained BMSCs from rat marrow:The SD rats were sacrificed by means of ether asphyxia. Bone marrow was collected from tibiae and BM cells were suspended in the medium (DMEM), passage was performed for four times, the cells were presented to flow cytometry assay to detect potential markers of BMSCs.4. Identification of the cells differentiated from BMSCs:The BMSCs was treated with the aboved metioned extraction for21days, then we detected some markers of the hepatocyte by RT-PCR assay, we found that M2-PK, GST-P, Albumin and Bst-1were expressed in the cells differentiated from BMSCs. It suggested that we successfully obtained the hepatocyte from BMSCs5. We cultured BMSCs together with Jaggedl, a soluble protein, at the concentration of10ng/ml in DMEM with tissue extract of damaged liver.6. The expression of some Notch signal regulated genes increased during cocultured BMSCs with Jagged1,such as Hesland Hey1.In contrast,the expression of two genes decreased when cocultured BMSCs with DAPT. BMSCs coculture with Jaggedl inhibit its differentiation into hepatocyte. so it indicated that the Notch signal might play an inhibitory role during the differentiation of BMSCs into hepatocyte.. using DAPT or combined with Jagged didn’t show any influence to the differentiation of BMSCs into hepatocyte.Results:We successfully constructed the animal model with impaired liver, and we obtained the material used for inducing BMSCs to differentiation. After4passages of primary culture, bottom-adhered BMSCs were isolated from floating blood cells and hematopoietic stem cells. The pureness of BMSCs was examined by Hoechst33342staining. It showed that all bottom-adhered cells were positive stained for Hoechst33342.At the same time4th passage BMSCs had high expressed by CD29and CD44, on the contrary CD34and CD45was negative.The cells which we isolated were bone marrow mesenchymal stem cells, whose surface feature was stable.The cells were monitored by a phase contrast microscope. Some adherent cells died with the induction extension, whereas the surviving cells began to proliferate and differentiate. On the7th and11th days after induction, cell morphology did not show great change, though the fibroblastic morphology was lost and cells developed a broadenedly flattened shape. On the21th day after induction, many round or cuboidal cells developed, whose morphology is extremely similar to hepatocytes. Simultaneously At the beginning, BMSCs only expressed the Bst-1mRNA. After treatment with tissue extract of damaged liver for11days, RT-PCR analysis revealed that both the M2-PK and GST-p mRNAs were found in BMSCs; and for20days, expression of Albumin mRNA became detectable in BMSCs. On the contrary, the expression of Bst-1mRNA in BMSCs disappeared gradually following treatment with tissue extract of damaged liver. In the control group, BMSCs only expressed Bst-1during the whole differentiation process.Concusion:The BMSCs was successfully induced to differentiate into hepatocyte proved by flow cytometry assay. At the same time We investigated the effects of Jagged1on the expression of Hesl and Heyl, the target genes of Notch signaling, in BMSCs. Transduction with Jagged1significantly increased the mRNA expression of Hesl and Heyl as revealed by real-time RT-PCR analysis.The changes of Notch signal suggested that this pathway might play a inhibitory role during BMSCs differentiating to hepatocyte. This investigation may be significant for the hepatocyte transplantation for some liver diseases.