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BACKGROUND: Bone marrow mesenchymal stem cells can be induced into myocardial tissue-like structure in vitro. Myocardial tissue lysates from different parts of the myocardium can be used to construct differential microenvironments. Few studies have investigated the targeted induction efficiency of bone marrow mesenchymal stem cells and the expression of related genes. OBJECTIVE: To investigate the effects of lysates from different parts of the myocardium on the differentiation of bone marrow mesenchymal stem cells into myocardial tissue-like structures and to assess the correlation between the expressions of related genes during induction and myocardial tissue engineering. METHODS: Passage 3 bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured. Whole heart tissue lysate, atrial muscle tissue lysate, and ventricular muscle tissue lysate were used to construct different microenvironments to induce bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells. The morphological changes and ultrastructure of cells were observed with an inverted phase contrast microscope and transmission electron microscopy. Expression of α-actin and cTnI was detected by immunofluorescence staining. qRT-PCR was used to detect the expression of upstream molecules ANP, HCN4 and downstream molecules MLC-2v in the cAMP/PKA signaling pathway after induction. RESULTS AND CONCLUSION: (1) Each induction cell group followed similar morphological changes: Cardiomyocyte-like cells were capable of autonomic beats, rhythmic contractions and relaxations; under the transmission electron microscope, there were a large number of arranged myofilaments; immunofluorescent staining showed positive expression of α-actin and cTnI. (2) The whole heart tissue lysate was able to induce the differentiation of bone marrow mesenchymal stem cells into rice grain-sized myocardial tissue-like structures with collagen fibers. (3) Atrial muscle tissue lysate could induce bone marrow mesenchymal stem cells to differentiate into atrial muscle-like cells. Autonomic beats appeared earlier, but the beat frequency and duration were shorter. ANP and HCN4 were highly expressed in atrial myocytes. (4) Ventricular muscle tissue lysate induced bone marrow mesenchymal stem cells to differentiate into ventricular muscle-like cells with no secretory granules, and MLC-2v was highly expressed in ventricular muscle-like cells. (5) To conclude, the whole heart tissue lysate can induce bone marrow mesenchymal stem cells to form myocardial tissue-like structures. Atrial muscle tissue lysate can induce bone marrow mesenchymal stem cells to differentiate into atrial muscle-like cells. Ventricular muscle tissue lysate can induce bone marrow mesenchymal stem cells to differentiate into ventricular muscle-like cells. By constructing a specific microenvironment, bone marrow mesenchymal stem cells can be differentiated to cardiomyocytes in different parts, providing laboratory data for the construction of myocardial tissue engineering.
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BACKGROUND: Elabela is a new type of endogenous receptor of APJ discovered in recent years. It is widely distributed in the adult cardiovascular system and has a certain influence on cardiovascular diseases. However, the effect of Elabela on the differentiation of stem cells into cardiomyocytes and the expression of APJ in cardiomyocyte differentiation has not been studied yet. OBJECTIVE: To investigate the effect of Elabela on the differentiation of Wharton’s jelly-derived mesenchymal stem cells into cardiomyocytes. METHODS: The frozen mesenchymal stem cells were resuscitated. 5-Azacytidine was used to induce Wharton’s jelly-derived mesenchymal stem cells to differentiate into cardiomyocytes when the cell confluence reached 80%-90%. After 24 hours, the medium was replaced by low-glucose medium containing Elabela and 10% fetal bovine serum in the experimental group, and by low-glucose medium containing 10% fetal bovine serum in the control group. At 7, 14, 21, and 28 days after induction, cell morphology was observed. The total RNA and total protein of each group were collected. The myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein expression levels were detected by real-time fluorescent quantitative PCR and western blot assay. The expression of APJ in the induced cardiomyocytes was detected by real-time fluorescent quantitative PCR and flow cytometry. RESULTS AND CONCLUSION: (1) The expression levels of myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein were higher in the experimental group than in the control group in all stages of differentiation, and the expression of APJ was also higher in the experimental group than in the control group. (2) In summary, Elabela plays a certain promoting role in the differentiation of Wharton’s jelly-derived mesenchymal stem cells into oriented cardiomyocytes. Elabela, as another agonist of APJ, can promote the expression of APJ during the induced cell differentiation.
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BACKGROUND: Studies have shown that miRNA-148a can promote human bone marrow mesenchymal stem cells to differentiate into mature cardiomyocyte-like cells, but the effect of miRNA-148a on the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells has not been reported. OBJECTIVE: To investigate the effect of miRNA-148a on the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells. METHODS: Human induced pluripotent stem cells differentiating into cardiomyocyte-like cells were divided into three groups. Cells in the control group were not treated. Cells in the low expression group were treated with miRNA-148a for 28 days, and those in the high expression group were treated with mimics of miRNA-148a for 28 days. In addition, human induced pluripotent stem cells cultured for 28 days were taken as the blank control group. CCK-8 was used to detect cell proliferation activity. qRT-PCR was used to detect the expression of miRNA-148a. Immunofluorescence staining and western blot analysis were performed to detect the expression of MHC and cTnT protein. RESULTS AND CONCLUSION: The expression of intracellular miR-148a mRNA and cell proliferation activity in the low expression group were lower than those in the blank control and control groups, while those in the high expression group were significantly higher than those in the other three groups (P < 0.01). There were no positive expression of MHC and cTnT in the blank control group. There were positive expressions of MHC and cTnT in the control, low expression and high expression groups. The expression of MHC and cTnT protein in the low expression group was significantly lower than that in the control group, and that in the high expression group was significantly higher than that in the other three groups (P < 0.01). These results suggest that miRNA-148a can promote the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells.
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Objective To identify the miR-122 which regulateing GATA4 expression during the induction of rat bone marrow mesenchymal stem cells (bMSCs) differentiating into cardiomyocyte-like cells. Methods BMSCs were isolat-ed from bone marrow and induced to differentiate into cardiomyocyte-like cells using 5-azacytidine. The miR-122 which may regulate expression of GATA4 were predicted using miRanda and TargetScan softwares and identified by dual luciferase report system. The expressions of miR-122 and GATA4 were determined using q-PCR during the differentiation of bMSCs into cardiomyocyte-like cells. Results The induced cells were completely in contacted with adjoining cells and uniform in shape and aligned parallelly. Cardiac troponin I (cTnI) expression was detected by immunofluorescence cytochemistry. Using dual luciferase reporter system in vitro, miR-122 were proved to be able to effectively inhibit GATA4 expression by binding the 3′UTR of GATA4 mRNA. q-PCR results showed that the expression of miR-122 is negatively correlated with that of GATA4 mRNA transcription. Conclusion These results indicated that miR-122 regulate the expression of GATA4 during the induction of cardiomyocyte-like cells.
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@#Objective To detect whether the cardiomyocyte-like cells differentiated from human marrow mesenchymal stem cells (hMSCs) could produce action potential (AP). Methods Isolated and cultured hMSCs were induced into cardiomyocyte-like cells with 5-Azacytine in vitro. They were measured for their AP by patch clamp technique, and compared with those of hMSCs of the same generation and beating cardiomyocytes (CMs) derived from 2 day-old SD rats. Results 6/30 cardiomyocyte-like cells produced AP. The CMs produced significant AP, hMSCs appeared no AP, and cardiomyocyte-like cells appeared weak AP. Conclusion The hMSCs manifested the potential to differentiate into CMs in the electrophysiology characteristics following 5-Azacytine induction.
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0.05) respectively in groupC1-C4,while in groups T1-T5,the rates were 21%,28%,36%,40%,37.5% ( P 0.05).However,the differentiation rate of MSCs increased probably with the incubation time prolonged.During the procedure of MSCs differentiation,the expression pattern of some genes such as GATA4 and cTNT may make some difference.
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Objective To establish a new cell resource of stem cell transplantation for heart failure therapy,by using cardiomyocytes from rat marrow mesenchymal stem cells(MSCs). Methods MSCs were isolated and cultured from rat bone marrow,then induced by 5aza(5,10,15,20 ?mol/L) for 24 hours.The cultured cells were observed with phase-contrast microscopy.The immunohistochemical technique and laser scanning confocal microscopy(LSCM) were applied for revealing the expressions of desmin,?-sarcomeric actin and C-TnT.The induced cells were evaluated by a transmission electron microscope.GATA4 and(?-MHC) expressions were detected by relative quantitative RT-PCR after 7,21,28 days of inducement respectively. Results MSCs induced in vitro by 5-aza could be identified by the positive staining for desmin,?-sarcomeric actin and C-TnT.The number of cells stained positively in the 5 ?mol/L was greater than that of other groups.Transmission electron microscope showed that paralleled myofilaments were formed.RT-PCR assessment showed that the differentiated cells began to express GATA4 from day 7 to day 28 of differentiation and began to express ?MHC from day 21 to day 28 of differentiation.Conclusion Cardiomyocyte-like cells can be generated from MSCs in vitro and the marrow mesenchymal stem cells are one of the best resources of donor cardiomyocytes.