OBJECTIVETo establish a new inducing system for differentiation of human embryonic stem (ES) cells to dendritic cells (DC), and further explore how microRNA-223 (miR-223) regulates DC differentiation from ES cells.

METHODSHuman ES cells were cultured on plates coated by IV type collagen and differentiated into hematopoietic stem/progenitor cells, common myeloid progenitor cells and DC step by step via adding different hematopoietic growth factors. The differentiated cells were identified by morphology, flow cytometry and hematopoietic colony forming unit (CFU) assays. Human ES cells were transfected with lentiviral vectors to overexpress miR-223 or inhibit miR-223 expression, then initiated the differentiation to DC. The differentiated cells at the different miR-223 levels were compared by the numbers of hematopoietic CFU and the expressions of specific surface markers. Dual-luciferase reporter assay was performed to test whether miR-223 directly targets TGFBR3.

RESULTSHuman ES cells were successfully induced into DC as the percentage of CD83 was approximately 82%, and the expression of miR-223 was up-regulated during the whole process. Supplementing miR-223 level using synthetic miR-223 mimics improved the proportions of CD34CD45, CD34CD45and CD83in differentiated cells, which were significantly higher than those in synthetic miR-223 inhibitor group and negative control (P<0.05). The expressions of cell makers at each differentiated phase in miR-223 inhibitor group were significantly lower than those in negative control (P<0.05). The differentiated cells in miR-223 mimics group showed approximately 759 CFUs per 10cells, which was significantly higher than that in others (P<0.05). Compared with negative control, miR-223 substantially inhibited the luciferase activity of Tgfbr3 3'UTR construct (by 37%) (P<0.05). In addition, the luciferase activity of the mutant construct was significantly higher than that of the WT construct in the presence of miR-223 mimics (P<0.05). With DC mature, the protein level of TGFBR3 gradually decreased using miR-223 mimics, and increased in miR-223 inhibitor group due to the suppression of the endogenous miR-223.

CONCLUSIONMiR-223 promotes the differentiation of human ES cells to DC, probably through direet target to TGFBR3.