Derivation of macrophages from hES cell derived and human fetal CD34 cells
CD34 cells were cultured initially in semisolid media to derive myelomonocytic colonies followed by liquid culture in cytokine supplemented media as described below. Purified CD34+ progenitor cells (~2.5 × 105 to 4.0 × 105) were placed directly into Methocult semisolid medium (Stem Cell Technologies, Vancouver, BC), mixed, and cultured in 35 mm plates. Myeloid colonies were allowed to develop for 12–15 days. Upon differentiation and proliferation, myelomonocytic colonies were harvested by the addition of 5 ml DMEM containing 10% FBS, 10 ng/ml each GM-CSF and M-CSF. Cells (~106) were placed in a 35 mm well and allowed to adhere for 48 hours. At two and four days post-harvest, medium was replaced with fresh complete DMEM supplemented with 10 ng/ml GM-CSF and M-CSF. By 4–5 days, cells developed into mature macrophages which were used for subsequent phenotypic and functional characterization.