ABSTRACT
Objective: To prepare graphene oxide(GO)/polycaprolactone(PCL)composite matrix nanomaterials with different concentrations, and investigate effects of the nanomaterials on the myocardial differentiation of rat brown adipose stem cells (BASC)in vitro. Methods: The GO/PCL composite nanofiber materials were prepared by electrospinning technology. The biocompatibility of the nanomaterials was tested by the CCK-8 assay after cultivation of the BASC on the pure PCL and GO/PCL composite nanofibers, and the characterization of the nanofiber materials was performed by the scanning electron microscopy(SEM)and electrical conductivity measurement. The expression of cardiomyocyte-related proteins was examined by the cytological method and immunofluorescence staining. The GO/PCL composite nanofiber materials were systematically evaluated for their effect on the viability, proliferation and myocardial differentiation of BASC. Results: The GO/PCL composite nanofiber materials showed no obvious cytotoxicity at the 0.1 mg/ml GO concentration. The statistical results for the protein fluorescence intensities showed that the expression of the cardiomyocyte specific protein, α-actinin, and the intercalated disc-related protein, connexin-43(CX-43), was significantly increased in the GO/PCL group than in the PCL group(P<0.05). The cytoskeletal staining results showed that, compared with the PCL group, the cells in the GO/PCL group showed a long spindle-like stretch similar to the natural myocardial cell bundle, and the growth direction had a certain polarity. Conclusion: This study successfully prepared GO/PCL composite nanomaterials, which could promote the differentiation of BASCs into cardiomyocytes and the expression of intercalated disc-related proteins.
ABSTRACT
Objective To compare the characterization and myocardial differentiation capacity of amniotic fluid-derived mesenchymal stromal cells (AF MSCs) and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells (WJ MSCs). Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton's Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes (GATA-4, c-TnT, α-actin, and Cx43) were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities of WJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility (MHC I) antigens (HLA-ABC), and were negative, or mildly positive, for MHC Class II (HLA-DR) antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, α-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.
ABSTRACT
Objective: To study the role of Nkx2-5 gene in 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells. Methods: The experiment was divided into two groups: an experimental group and a control group. The cells in the experimental group were P19 cells stably expressing Nkx2-5 gene, and cells in the control group were P19 cells. Under the monolayer-culture condition, the cells of two groups were induced by 5-azacytidine (1 μmol/L). The growth of cells were observed by inverted microscope. On the 4th day, 8th day, 12th day and 16th day after induction, RT-PCR was used to detect the expression of GATA-4, α-MHC and ANP gene. Results: In control group, there was no ANP expression after induction; GATA-4 expression was seen on the 8 th day, 12th day, and 16th day after induction; and α-MHC expression was found on the 12th day and 16 th day. In experimental group, the expression of GATA-4 was detected on the 4th day, 8th day, 12th day and 16 th day after induction; Alpha-MHC and ANP expression was noticed on the 8th day, 12th day and 16th day after induction. RT-PCR results showed that the expression of GATA-4 and α-MHC in the experimental group was earlier than that in the control group. And at all time points of observation, the expression of GATA-4 and α-MHC in the experimental group was significantly increased compared with that in the control group (P<0. 05,P<0. 01), except for α-MHC expression on the 12 th day. Conclusion: Nkx2-5 gene can promote 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells.