Human CD34+ cells, isolated from peripheral blood of granulocyte colony-stimulating factor-mobilized healthy volunteers, were purchased from the Fred Hutchinson Cancer Research Center. The cells were maintained and differentiated as previously described28 (link),73 . Briefly the cells were expanded in StemSpan medium (Stem Cell Technologies Inc.) supplemented with StemSpan CC100 cytokine mix (Stem Cell Technologies Inc.) and 2% P/S for a total of 6 days. After six days of expansion the cells were stimulated for 2 h with rhBMP4 (R&D) at a final concentration of 25 ng/ml and harvested for performing all the experiments corresponding to D0 time point. For studying differentiated cells after day 6 of expansion, cells were reseeded in differentiation medium (StemSpan SFEM Medium with 2% P/S, 20 ng/ml SCF, 1 U/ml Epo, 5 ng/ml IL-3, 2 mM dexamethasone, and 1 mM b-estradiol), at a density of 0.5–13 106 cells/ml. Prior to harvesting at H2, H6, D1-D8 the cells were treated with 25ng/ml hrBMP4 for 2hrs.
For testing the effects of BMP4 and dorsomorphin, cells at the beginning of the third day of differentiation were treated with either 25 ng/ml hrBMP4 or 20 μM dorsomorphin until the beginning of the fifth day of differentiation. At D5, cells were isolated for flow cytometry and qPCR analysis. Cells treated with DMSO were used for control experiments.
For testing the effects of BMP4 and dorsomorphin, cells at the beginning of the third day of differentiation were treated with either 25 ng/ml hrBMP4 or 20 μM dorsomorphin until the beginning of the fifth day of differentiation. At D5, cells were isolated for flow cytometry and qPCR analysis. Cells treated with DMSO were used for control experiments.
