Differentiation of ES-D3 embryonic stem cells into dendritic cells in vitro

In order to differentiate directly the ES-D3 embryonic stem cells(ESC)into dendritic cells,single ESC suspensions were plated onto bacteriological plastic to encourage the formation of EBs,and after 14 days in culture,EBs were plated onto tissue culture grade plastic in complete medium supplemented with 25 ng/ml of murine GM-CSF and 10 ng/ml of IL-3 and cultured for 10 days.The expressions of MHCⅠandⅡmolecules,CD11c,CD80,CD86 and CD83 on the surface of the differentiated cells were evaluated by flow cytometry(FCM).Meanwhile,LPS was used to induce maturation of these cells,and then the morphological changes and expressions of MHCⅡmolecule and CD80 of these cells were investigated.Also,the mixed leukocyte reactions(MLR)was used to study the allogeneic immunostimulatory function of matured or immature DCs derived from embryonic stem cells(esDCs).By cultivation with IL-3 and GM-CSF,the early esDCs first appeared around days 4～5 of culture and rapidly expanded over the ensuing days.This population consistently regenerated following routine harvesting. Moderate expression of MHCⅠmolecule and high expression of CD11c could be demonstrated on these cells;but there was no expression of CD80,CD86,CD83 or MHCⅡmolecules.indicating that they were all immature DCs.However,when stimulated with LPS,these cells presented the morphology of matured DCs and could up-regulate the expressions of MHCⅡand CD80 molecules on cell surface.Result of allogeneic MLR suggested that the mature esDCs showed a significant immunostimulator activity(P0.05),but the immature esDCs did not show this activity(P0.05).It is evident that the dendritic cells derived from the embryonic stem cells was successfully acquired though the direct differentiation ES-D3 embryonic stem cells into DCs by use of cytokine GM-CSF and IL-3.