FIGURE 3. In vitro proliferation and colony formation of wild-type and triple mutant HSCs and multipotent progenitors.

a, Proliferation of wild-type and triple mutant HSCs and multipotent progenitors in expansion media. Wild-type and triple mutant HSCs are responsive to cytokines that promote their expansion and proliferate considerably better than wild-type and triple mutant multipotent progenitors in serum-free StemSpan media containing SCF, TPO, IGF-2, FGF-1 and Angptl3 (STIFA) media. Wild-type and triple mutant multipotent progenitors show a doubling in their proliferation rates with the addition of Flt3 ligand to the STIFA media. Five cells were double-sorted into U-bottom 96-well plates. Replicates of at least 21 wells for each experimental group were counted. b, Triple mutant multipotent progenitors show an increase in the number of secondary colonies that they form compared to wild-type multipotent progenitors in Methocult and Methocult plus media, demonstrating their proliferative advantage. Triple mutant multipotent progenitors are also very responsive to the additional cytokines IL-11, GM-CSF, Flt3 ligand and TPO added to the Methocult media and can form secondary colonies even better than wild-type HSCs. Single cells were double-sorted into U-bottom 96-well plates for the colony formation experiments. Replicates of at least 20 wells were scored in the methylcellulose colony formation experiments. For a and b: asterisk, P < 0.005; double asterisk, P < 0.0001 (Student's t-test). Error bars denote s.e.m. c, Representative images of wild-type and triple mutant colonies from single double-sorted HSCs and multipotent progenitors taken at 25 magnification.