| Background:Embryonic stem cells (ES cells) are capable of self-replication and renewal. Under the certain condition, they will differentiate into multiple lineages cells, including osteoblast, chondrocyte, adipocyte, neurocyte, muscle cells and so on. A big number of evidence have indicated that ES cells transplanted into the ischemic heart will regenerate the necrotic cardiomyocytes and vascular endothelial cells, can also secrete various growth factors, promote the establishment of a micro-circulation, increase perfusion and improve heart function after infarction. The clinical therapy based on the utilizing of pluripotential ES cells has become the new hope for numerous chronic heart failure patients after myocardial infarction.ES cells can be induced to differentiate into cardiomyocyte-like cells in vitro, these cardiomyocytes express part of the phenotype of myocardial cells, cardiac-specific markers, or even show spontaneous beating. Cardiomyocytes derived from the stem cells are promising candidate cell sources used for regenerative medicine in the treatment of heart failure disease. But the in vitro spontaneously cardiomyocytes differentiation from ES cells is low efficiency. Researchers have studied many inducible factors to induce cardiac differentiation from ES cells in vitro. Although the effects of these chemicals on cardiac differentiation are impressive, it still does not meet the request of quantity and purity of cells used in clinical transplantation. How to further enhance the efficiency of cardiac differentiation of ES cells is the key problem to be solved. Looking for a new and more effective, safer, more economical inducer is still a hot research topic. Baicalin is one of the active ingredients of traditional Chinese medicine, it possesses anti-oxidant and anti-inflammatory activities, and thus can protect cardiomyocytes exposed to ischemia/reperfusion. Baicalin has been widely used in clinical treatment of various cardiovascular diseases. The purpose of this project is to study the effect of baicalin on cardiac differentiation of mouse embryonic stem cells (mES cell) and its underlying mechanism.The differentiation of ES cells draws assistance from the regulation of stimulus signal coming from outside accompany with intercellular specific transcription course. It has been approved that ES cells differentiation into cardiomyocytes-like cells is partly similar to embryonic development. Some regulation factors can be reactivated during the cell differentiation. However, the expression pattern has not yet been elucidated. Based on the existing status of traditional Chinese medicine-induced cardiac differentiation of ES cells, we try to use baicalin as an inducer to study the cardiac differentiation of ES cells to make discoveries in the following areas:(1) Whether baicalin can induce cardiac differentiation from mouse embryonic stem cells? (2) Whether the expression of myocardial cell marker after induction is different at every differentiation time points? (3) Which underlying signaling pathways is affected by baicalin during cardiac differentiation from ES cells? Clarifying the above issues can provide a theoretical basis to promote the efficiency of myocardial cell differentiation and speed up the process of clinical use of ES cells derived cardiomyocytes.Aims:(1) To observe the effect of baicalin on the proliferation of mouse embryonic stem cells;(2) To observe the size of embryoid body (EB) after induction by baicalin at different development time points;(3) To detect the expression of cardiac-specific markers at the mRNA and protein levels; (4) To study the expression of genes related to cardiac differentiation during different differentiation stages after baicalin treatment, revealing the possible mechanism of baicalin affecting myocardial differentiation.Methods:1.MTT (3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide the tetramethyl azo oxacillin salt) assay was used to detect the effect of different concentrations of baicalin on the proliferation capacity of mES cells, screening the best concentration used to induce cardiac differentiation of embryonic stem cells.2. Differentiation of mouse embryonic stem cells:The classic "hanging drop-suspension culture-adherent culture" three-steps method was used to initiate cardiac differentiation. A final concentration of 10μmol/L or 50μmol/L baicalin was maintained in the differentiation medium during all the stages of differentiation. The medium was changed every 2 days.3. Morphological observation:Observe the morphology of embryonic stem cells before differentiation. After differentiation, We use an inverted microscope to observe the size of the embryo (embryoid body, EB) and the percentage of beating EBs (EBs with beating area).4. Flow cytometry analysis:We evaluated the percentage of cardiomyocytes at 10,16,20 days of EB by PE labeledα- sarcomeric actinin positive cells.5. Immunofluorescence analysis:At day 16 of differentiation, the expression of cardiac-specific markerα-sarcomeric actinin and ventricular muscle-specific myosin light chain 2 (myosin light chain-2, ventricular, MLC-2v) was detected using immunofluorescence staining.6. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative polymerase chain reaction (real-time PCR): The total cellular RNA was extracted from the EBs of days 2,4,6,8,10,12,14,16,20, cDNA was obtained by reverse transcriptase reaction (RT), housekeeping gene 3-phosphate dehydrogenase (GAPDH) was used as internal control, the mRNA levels of three germ layer marker and cardiac-specific genesα-cardiac myosin heavy chain (α-MHC), the MLC-2v, atrial natriuretic peptide(ANP), hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and myocardial developmentally regulated signaling pathway genes such as TGF-β1, TGF-β2, TGF-β3, BMP2, BMP4, Wnt11, Wnt3a and cardiac transcription factor such as Nkx2.5, MEF2c, TBX5, of GATA4 were detected by RT-PCR and real-time PCR.7. Western blot:The total protein was extracted from EBs at day10,16,20. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane. After western blotting, ECL color development, exposure, the a-actinin and MLC-2v was detected.8. Patch clamp analysis:Electrophysiological characteristics and hormonal regulation of the cardiomyocytes derived from ES cells were detected using patch clamp technique.Results:Baicalin inhibited the proliferation of mES cells in a concentration dependent manner. During all the differentiation stages, the size of EBs was significantly decreased in baicalin treatment groups than controls.50μmol/L baicalin successfully promoted the late stage cardiac differentiation in vitro, as demonstrated by increases in the percentage of beating EBs,α-actinin+ cells, and expression of cardiac specific markers. Typical pacemaker-like, atrial-like and ventricular-like action potentials (APs) were recorded by patch-clamp. Baicalin enhanced the percentage of working cardiomyocytes while decreased the percentage of pacemaker-like cells.β-adrenergic and muscarinic signaling cascades are present and functional in baicalin-induced ES-CMs. Further analysis revealed that baicalin strikingly up-regulated Wnt3a which is an activator of Wnt/β-catenin signaling pathway at the intermediate stage while dramatically down-regulated it at the late stage, but the expressions of TGF-β1/β2/β3, BMP2/4 and Wnt11 were not affected by baicalin. Downstream cardiac transcription factor Nkx2.5 was elevated by baicalin at the late stage, while Mef2c, Tbx5 and Gata4 were not significantly affected by baicalin.Conclusion:1. Baicalin inhibited the proliferation of ES cells in a concentration dependent manner.2.50μmol/L of baicalin successfully promoted cardiac differentiation of mES cells in vitro at the late stage.3. Baicalin enhanced the percentage of working ceardiomyocytes. The cardiomyocytes derived from ES cells had intact hormonal regulation.4. Canonical Wnt/β-catenin signaling pathway might play an important role in this effect of baicalin.
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