Rapamycin Conditioning of Dendritic Cells Differentiated from Human ES Cells Promotes a Tolerogenic Phenotype

Figure 2

Time course of DC differentiation from H1 hESC. Cells were harvested from cultures at various time points and analysed by flow cytometry for the onset of hematopoiesis and the appearance of DC. (a) Cells harvested at day 20 of culture showing expressing of CD45 but lack of myeloid commitment, as evidenced by staining for CD13, CD14, and CD11c. Open histograms show levels of background staining using isotype-matched control antibodies. (b) Appearance of CD45^int cells at day 27 of culture, accompanied by the upregulation of myeloid-specific markers. (c) Photomicrograph, taken at day 28 of culture, showing the morphology of DC, including veils of cytoplasm and long dendrites (inset) (×40 magnification). (d) Cells harvested at day 33 of culture, showing the appearance of a CD45^hi population containing predominantly DC progenitors expressing CD14, CD11c, CD86 and MHC class I. (e) Phenotype of immature and mature H1-DCs compared with human moDC. DCs were cultured either in medium alone or medium supplemented with the maturation cocktail and stained for MHC class II, the maturation marker CD83 and classical costimulatory molecules. Dead cells were excluded from the analysis using 7-AAD. Dashed histograms show the phenotype of immature DCs while the filled histograms represent mature DCs. Open histograms depict background staining using isotype-matched controls.