HyperD-ALL primary blasts show a delay in early mitosis associated with chromosome-alignment defects in prometaphase. (A) Schematic depicting the experimental design for ex vivo growth of primary B-ALL blasts onto Nestin-positive hBM-MSCs. (B) Left, representative images of primary non-HyperD and HyperD-B-ALL ex vivo cultures on Nestin-positive hBM-MSCs at the indicated time points. Right, absolute counts of B-ALL primary blasts at the indicated time points; n = 2. (C-D) Frequency of apoptotic (SubG₀/SubG₁) (C) and G₂/M (D) non-HyperD and HyperD-ALL primary cells from BM samples; n = 3 patients of each. (E) Schematic depicting the PDX model used to expand primary B-ALL blasts in vivo. (F) Representative DNA (blue)-Kinetochore (purple)-spindle (red-green) IF staining identifying the different mitotic phases in PDX-expanded B-ALLs. The SAC identifies the transition from early to late mitosis. (G) Mitosis progression of PDX-expanded B-ALL primary cells. Left, progression from early to late mitosis. Right, frequency of cells at the indicated mitotic phases; n = 3 non-HyperD and n = 5 HyperD PDX-expanded B-ALLs. (H) Left, representative images of mitotic cells with nonaligned and aligned chromosomes at the metaphase plate. Right, frequency of PDX-expanded B-ALL primary blasts showing chromosome alignment at prometaphase/metaphase; n = 4 non-HyperD and n = 4 HyperD PDX-expanded B-ALLs. (I) Schematic depicting the chromosome-biorientation assay. (J) Representative images of the DNA-Kinetochore-spindle staining in monastrol/MG132-treated cells with 0 (left), 1 (middle), and >2 (right) misaligned chromosomes. (K) Quantification of metaphase cells showing misaligned chromosomes; n = 3 non-HyperD and n = 3 HyperD PDX-expanded B-ALLs. Graphs represent the mean, and error bars represent the standard error of the mean. *P < .05, **P < .01; ***P < .0001 (2-way ANOVA). Scale bars, 10 µm.