| Background and Objective:Currently, reperfusion therapy has become an important treatment method for acutemyocardial infarction, however, a large number of clinical records showed that recoveryblood supply for ischemic myocardial always increased the myocardial injury, how to letmyocardial necrosis repaired become to one of the development trend of cardiovascularresearch in the future. In recent years, the development of molecular biological techniquesand stem cell research brought new hope for the regeneration of necrotic myocardium.Using specific differentiation protocols, mesenchymal stem cells (MSC) can beinduced to differentiate into a variety of mature target cells, and MSC are one of the bestoptions for the transplantation of adult stem cell (ASC). MSC have been used in numerousstudies involving regeneration of cardiac cells post myocardial infarction (MI), and theyare capable of enhancing myocardial perfusion and cardiac function in ischemic hearts ofpatients with acute or chronic heart diseases. However, MSC isolation from the bonemarrow of embryonic or adult tissue has many shortcomings, thus the focus of presentstudies is to identify other types of suitable stem cells.The study of directional amniotic fluid-derived mesenchymal stem cells (AFMSC)differentiation remains in its infancy, with few reports so far. The focus of cardiovascularregenerative medicine is improve the efficiency of directional differentiation of AFMSC.5Aza could promote the generation of myocardial sarcomeres and the product of myosinand muscle protein. Transforming growth factor β1is a multifunctional cytokine that regulates cell growth, differentiation, and death, and has emerged as a key player in theself-renewal and maintenance of stem cells. In this study, the traditional method wasreplaced by a combined induction method to improve the efficiency of directionaldifferentiation.Methods:In our preliminary work, high-quality AFMSC were successful isolated, we obtainedAFMSC from rabbit amniotic fluid at1618days gestation, western blot analysis was usedto analyze the expression of octamer-binding transcription factor4(OCT4), andtumorigenicity experiments were performed, BALB/C nude mice were divided into2groups randomly, control group and experimental group were observed in8weeks. Then,the AFMSC were divided into4groups, including group A: the control group, group B:transforming growth factor β1(TGFβ1)-induced group, group C:5Azacytidine(5Aza)-induced group, group D: combined TGFβ1-and5Aza-induced group, TGFβ1or/and5Aza were added to the media of the experimental groups as follows: vehicle ingroup A,5ng/mL TGFβ1for24h in group B,10μmol/L5Aza for24h in group C,5ng/mL TGFβ1combine with10μmol/L5Aza for24h in group D. Real-time PCR was usedto analyze the expression of cardiac specific transcription factor4(GATA4),immunofluorescence was used to analyze the expression of cardiac troponin T (cTnT),western blot was used to analyze the expression of connexin43(Cx43), and transmissionelectron microscopy was used to observe the ultrastructure of differentiated cells.Results:1. Positive OCT4expression was showed in AFMSC.2. The tumorigenicity experiment demonstrated that nude mice remained living anddid not develop tumors in both the experimental and control groups.3. After induction of AFMSC differentiation, no significant changes were observed inthe group A cells, which maintained their morphology after passage6. however, the groupB cells proliferated rapidly and became longer than group A cells after TGFβ1treatment.Cytomixis was observed in the group C cells, which elongated after5Aza treatment. Aftercombined TGFβ1and5Aza treatment, cytomixis was observed, and cells elongated andbecame more bulky 4. Real-time PCR analysis of GATA4expression:No increase of GATA4expression was observed in group A at4weeks after induction.however, GATA4expression was increased in the group B cells at4weeks after induction,in cells of the group C at3weeks after induction, and of the group D at2weeks afterinduction. GATA4expression was highest in group D, and this difference was statisticallysignificant compared to the other groups.5. Immunofluorescence analysis of cTnT expression:Immunofluorescence was used to analyze the expression of cTnT. After2weeks, verylow cTnT expression was observed in the group A cells and a slight increase was observedin groups B and C. However, the cTnT expression in group D was significantly higher thanthat of groups A, B, and C, Cx43in the union induction group were markedly higher thanthose of other groups.6. Western blot analysis of Cx43expression:Western blot analysis of Cx43revealed very low expression in group A and a slightincrease in groups B and C. However, Cx43expression was significantly higher in groupD than in groups A, B, and C.7. Transmission electron microscopy observe the ultrastructure:After AFMSC differentiation, the cells were observed under a transmission electronmicroscope. A myofilament-like structure, the specific structure of cardiomyocytes, wasfound under transmission electron microscopy in the group D cells.Conclusion:1. In the present study, AFMSC were successfully isolated and cultured from amnioticfluid using our direct adherence method. Western blot analysis demonstrated theexpression of OCT4, thus confirming that AFMSC are capable of multipotentdifferentiation. Moreover, the tumorigenicity experiment demonstrated that AFMSC arenot tumorigenic.2. After combined treatment with5Aza and TGFβ1, AFMSC showed positiveexpression of GATA4, cTnT, and Cx43, and the myofilament-like structure was observedunder transmission electron microscopy. These results showed AFMSC exhibitcardiomyocyte-like characteristics after differentiation, indicating that they have transformed into cardiomyocyte-like cells. These results indicated that combined inductioncould improve the directional differentiation efficiency of AFMSC.3. Our study provides an efficient and practical method for the directionaldifferentiation of AFMSC, increases the effectiveness of the transformation ofcardiomyocyte-like cells in vitro, and is promising for the regeneration ofcardiomyocyte-like cells. |
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