Preparation of HSPCs from frozen human UCB units and their transplantability shown in xenotransplantation. (A) Comparison of procedures and cell-collection efficiencies between a manual method and that using CliniMACS Prodigy. Shown are the purity of CD45⁺CD34⁺ cells (top right) and %recovery (bottom right). n = 5 (manual); n = 8 (CliniMACS Prodigy). Percent recovery (CD34⁺ cells) was calculated by the ratio between the number of CD34⁺ cells estimated before freezing (provided by Japan Red Cross Society) and the number of CD34⁺ cells isolated. (B) Percentages of human CD45⁺ cells with further characterization by CD33, CD19, CD4, and CD8 (left) and by lineage markers, CD34, and CD38 (right) within BM of individual recipients at 12 wk after transplantation. n = 5 in each group. Each bar indicates the chimerism of individual recipients. †, death. (C) The experimental protocol to obtain CD34⁺ cells from multiple UCB units at one time and test their transplantability. One donor UCB was used as a model of principal unit, whereas multiple units were combined before purification of CD34⁺ cells. (D) Results of transplantation that used four unrelated UCB units. A dose of 3.2 × 10⁴ cells was defined as a single unit. Percentages of human CD45⁺ cells in BM of individual recipients at 21 d after transplantation. n = 6 in each group. One-way-ANOVA with Tukey’s multiple comparison test was used for statistical analysis. Mean values are indicated as bars. P < 0.05 was considered statistically significant. *, P < 0.05; **, P < 0.01. (E) Results of transplantation that used 10 unrelated UCB units. A dose of 1.0 × 10⁴ cells was defined as a single unit. (left) Percentages of human CD45⁺ cells in each indicated recipient BM at day 20. n = 5 or 6 in each group. One-way ANOVA with Tukey’s multiple comparison test was used for statistical analysis. Mean values are indicated as bars. P < 0.05 was considered statistically significant. **, P < 0.01; ***, P < 0.001. (right) Percentages of HLA-A2⁻ cells (representing combined multiple units) in human CD45⁺ cells among recipients. Mean values are indicated as bars. The setting shown in D was tested twice independently, whereas the 10-unit experiment in E was performed once.