Ghrelin promotes cardiomyocyte differentiation of adipose tissue­derived mesenchymal stem cells by DDX17­mediated regulation of the SFRP4/Wnt/ß­catenin axis.

Adipose tissue­derived mesenchymal stem cells (ADMSCs) differentiate into cardiomyocytes and may be an ideal cell source for myocardial regenerative medicine. Ghrelin is a gastric­secreted peptide hormone involved in the multilineage differentiation of MSCs. To the best of our knowledge, however, the role and potential downstream regulatory mechanism of ghrelin in cardiomyocyte differentiation of ADMSCs is still unknown. The mRNA and protein levels were measured by reverse transcription­quantitative PCR and western blotting. Immunofluorescence staining was used to show the expression and cellular localization of cardiomyocyte markers and ß­catenin. RNA sequencing was used to explore the differentially expressed genes (DEGs) that regulated by ghrelin. The present study found that ghrelin promoted cardiomyocyte differentiation of ADMSCs in a concentration­dependent manner, as shown by increased levels of cardiomyocyte markers GATA binding protein 4, α­myosin heavy chain (α­MHC), ISL LIM homeobox 1, NK2 homeobox 5 and troponin T2, cardiac type. Ghrelin increased ß­catenin accumulation in nucleus and decreased the protein expression of secreted frizzled­related protein 4 (SFRP4), an inhibitor of Wnt signaling. RNA sequencing was used to determine the DEGs regulated by ghrelin. Functional enrichment showed that DEGs were more enriched in cardiomyocyte differentiation­associated terms and Wnt pathways. Dead­box helicase 17 (DDX17), an upregulated DEG, showed enhanced mRNA and protein expression levels following ghrelin addition. Overexpression of DDX17 promoted protein expression of cardiac­specific markers and ß­catenin and enhanced the fluorescence intensity of α­MHC and ß­catenin. DDX17 upregulation inhibited protein expression of SFRP4. Rescue assay confirmed that the addition of SFRP4 partially reversed ghrelin­enhanced protein levels of cardiac­specific markers and the fluorescence intensity of α­MHC. In conclusion, ghrelin promoted cardiomyocyte differentiation of ADMSCs by DDX17­mediated regulation of the SFRP4/Wnt/ß­catenin axis.

Composition and characteristics of distinct macrophage subpopulations in the mouse thymus.

The aim of the present study was to investigate the composition, morphology, characteristics, distribution and function of distinct macrophage subpopulations in the mouse thymus. Apoptosis of mouse thymocytes was induced by glucocorticoids and three monoclonal antibodies against Mac-2, F4/80 and ED1 were used for immunofluorescence staining and immunohistochemical analysis. The morphology of thymic macrophages was examined by transmission electron microscopy. Four subpopulations of mouse thymic macrophages were identified. Dendritic macrophages were identified using anti-Mac-2 and anti-F4/80 antibodies, and were demonstrated to be distributed in the entire thymus. Phagocytes were also observed. In addition, plate-shaped macrophages, identified using the anti-F4/80 antibody, were distributed under the thymic cortex capsule. Small oval macrophages, identified using the anti-Mac-2 antibody, were distributed in the thymic medulla and corticomedullary region (CMR), while phagocytes were not observed in these types of cell. ED1+ thymic macrophages with irregular forms were distributed in the CMR. All of the four subpopulations of mouse thymic macrophages described above exhibited acid phosphate activity. This study indicated the existence of macrophage subpopulations with different shapes, distribution and functions in the mouse thymus.