ABSTRACT
AIM:To direct embryonic stem cells(ESCs)into hematopoietic stem cells(HSCs)in vitro by simulating the hematogenic microenvironment in human early embryonic aorta-gonad-mesonephero(AGM)region.METHODS:Murine E14 embronic stem cell line was used for two-step differentiation.In the first step of primary differentiation,E14 ESCs were seeded into semisolid methylcellulose-based medium containing bone morphogenesis protein 4(BMP4)and vascular endothelial growth factor(VEGF)for embryoid body(EB)formation.On days 3,6,9,12 and 15,single EB cells were analyzed for Flk-1+ cells amount through flow cytometry.In the second step,single cell from EB containing most Flk-1+ cells was further co-cultured with human AGM stromal cells in non-contact system.On co-culture days of 3,6,9 and 12 days,cells were collected for cell count,flow cytometry for Sca-1+c-kit+ cells analysis,and colony forming cell assay.RESULTS:During the EB formation,BMP4+VEGF promoted Flk-1+ cell genesis on day 9 at peak pencentage value of 27.53%?2.84%,which was statistically higher than that in control group as 8.77?1.10(P