Abstract:

BACKGROUND: GATA-4 is a zinc finger transcription factor that is specifically associated with cardiac development. Previous experiments have found that exosomes secreted from GATA-4-overexpressed bone marrow mesenchymal stem cells (BMSCs) co-cultured with BMSCs can make BMSCs express more myocardial-specific antigens. When co-cultured with myocardial cells in a hypoxic environment, these exosomes can inhibit apoptosis in myocardial cells.
OBJECTIVE: To identify from a molecular level the basic and essential exosomes secreted from GATA-4-overexpressed mouse BMSCs that has a significant role in cardiac repair after myocardial infarction.
METHODS: (1) GATA-4-overexpressed mouse BMSCs were constructed by transfecting mouse BMSCs with GV308 (a lentiviral vector)-carrying GATA-4 before adding doxycycline for gene induction. ExoQuick-TC (SBI Inc.) was then used to extract the secreted exosomes. (2) The BMSCs were co-cultured with GATA-4-BMSCs-Exosome, free-vector-BMSCs-Exosomes, and BMSCs-Exosome. Another BMSCs and mouse myocardial cells were cultured alone. The expression levels of myocardial specific antigens, cTnT, α-actin, connexin 43, and Desmin, were assessed via qPCR quantification at 24 hours of culture. (3) The mouse myocardial cells were then co-cultured with GATA-4-BMSCs-Exosome, free-vector-BMSCs-Exosomes, and BMSCs-Exosome, and were subsequently mono-cultured in hypoxia and in serum-free cultures to construct the apoptosis-positive control group. Normally cultured myocardial cells were as negative controls. Cell apoptosis rates in different groups were determined by flow cytometry. (4) The microRNAs, which were associated with cellular differentiation or anti-apoptosis, in the exosomes secreted from GATA-4-overexpressed mouse BMSCs were detected by Agilent microRNA microarray.
RESULTS AND CONCLUSION: Results from Q-PCR and flow cytometry suggested that the exosomes secreted from GATA-4-overexpressed mouse MBSCs effectively facilitated the differentiation of BMSCs to form myocardial cells and reduce apoptosis. The Agilent microRNA microarray test results showed that the key microRNAs associated with differentiation included mmu-miR-199a-3p (up-regulated) and mmu-miR-1894-5p (down-regulated), while the key microRNAs associated with the anti-apoptotic function included mmu-miR-199a-3p, mmu-miR-20a-5p, and mmu-miR-330-3p, all of which were up-regulated. Among these microRNAs, mmu-miR-199a-3p was the only one associated with both cellular differentiation and anti-apoptosis, and it was therefore considered as the most primary microRNA to be validated. 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, GATA4 Transcription Factor, Exosomes, MicroRNAs, Tissue Engineering