FIG. 4.

Strategy for flow cytometric gating of human CB CD133⁺ subsets. (A) Representative flow cytometric gating strategy is shown for human CB CD133⁺ cells selected at day 0 of culture. Single, live (top density plots), and lineage-negative (Lin⁻) cells were discriminated into two subsets based on their expression of CD34 and CD38 or analyzed for CD34⁺CD133⁺ cell content. The CD34⁺CD38^−/low population was further distinguished based on CD90 and CD45RA staining to distinguish HSC, MMP, and CLP. In addition, CD90⁺CD45RA⁻ HSCs were characterized for CD49f expression. The CD34⁺CD38⁺ population was segregated into the progenitor subsets, GMP, CMP, and MEP, based on their expression of CD123 and CD45RA (Supplementary Data 2; MIFlowCyt configuration file). (B, C) Fold expansion of (B) total nucleated cells and (C) Lin⁻CD34⁺CD133⁺ cells after 8-day culture of 2,500 human CB CD133+ cells on 3D nanofiber scaffolds. Cytokines were added by medium change for the protocols shown (379, 747, 245, 241, and basal) at specific days of culture as described in Table 2. Individual biological replicates and average values are shown. Values are mean ± SEM for three to five donors (biological replicates). Statistical analysis was performed in GraphPad Prism using one-way ANOVA and Tukey's ad hoc test and P < 0.05 was considered as statistically significant.