Fig. 4

616452 on selectively isolated CD41⁺ cells. CB CD34⁺ cells were cultured with TPO, SCF, and IL-3 for 7 days prior for CD41 selection by flow cytometry (a). Control- (DMSO induced) and chemical-induced cells were grown (n = 2) in StemSpan containing only TPO for 8 days prior to cell cycle analysis (b). In control cells, we observed only 6% of the cells increased their ploidy numbers with no cell reaching a ploidy of 16 N or greater, while 54% of the chemically induced cells had a nuclei of 4 N or greater with a 4.5-fold increase in the number of 4 N, 30-fold increase in 8 N, and the appearance of cells with even greater ploidy reaching as high as 64 N within 8 days of culture (c, d). CB CD41⁺ cells were isolated using flow cytometry and cultured in the absence (e) or presence (f) of 616452(10 uM). Cells appeared mostly as single cell on prior to day 6 (e, f) but began to form clusters thereon, while the control had little to none (g). By 8 days of induction, the clusters were large (h) and covered the entire plate (i, j). A CD41 staining of these clusters illustrates the composition of these clusters as mainly large MK-like cells (k, l)