Figure 5.
Cell-cycle entrance is delayed in HSCs with lower mitochondrial activity. (A) Representative plot (left) of the percentage of cumulative first division of total initial cells (fitted into sigmoidal curve) in MMP-low or MMP-high CD90+ PB HSC single-cell culture in cytokine-supplied STEM SPAN. R2 > 0.997. Dotted arrows represent Khalf: hours for cumulative 50% cells to finish first division (deducted from the curve). Mean best fit value (right) of Khalf of repeated experiments (P = .06, n = 2). ΔKhalf: difference of Khalf between MMP-low and MMP-high CD90+ HSCs. (B) Representative plot of the percentage of newly finished first division of total initial cells (fitted into spline curve). (C) The mean time (recorded) to complete the first division. (D) The mean interval time (recorded) between the first and second divisions. In total, 75 to 84 single live cells were scored (n = 2). (E) Total number of live cells from a 9-day expansion of 600 seeded cells in cytokine-supplied STEM SPAN (top; n = 2). Representative bright field light microscopy images of cell clusters (bottom; scale bar, 0.15 mm). Arrows indicate the diameter of the clusters. (F-H) Representative immunofluorescent confocal images of CDK6 (F; scale bar, 5 μm), quantification of total CDK6 fluorescent intensity (G), and the distribution of nuclear sizes (H) of MMP-low and MMP-high CD90+ PB HSCs after 34 hours in culture. Data are presented as mean ± standard deviation; Student t test, *P < .05, **P < .01.