Four aspects of human hematopoiesis are altered by Wnt3a. (A) Human Lin⁻CD34⁺CD38⁻ BM cells (15 000 cells/well) were cocultured with Wnt-related gene (ie, Wnt3a, Wnt5a, or Dkk-1)–transduced OP9 cells for 3 weeks under B-lineage supporting conditions (ie, IL-7, SCF, and FL). Flow cytometry was used to evaluate differentiation and representative gating for CD14⁻CD19⁺ lymphocytes is shown in the top panels. The fold changes of recovered CD19⁺ cells numbers to control are given in the bottom panel. (B) CB CD34⁺ cells were cocultured for 17 days with Wnt3a-producing OP9 cells in the presence of IL-7, SCF, FL, and IL-15. The numbers of B cells (CD14⁻ CD19⁺), natural killer cells (CD14⁻CD56⁺), and plasmacytoid dendritic cells (CD14⁻CD11c⁻CD19⁻CD56⁻CD123⁺) recovered were used to calculate yields per input cells. (C) Lin⁻CD34⁺CD38⁻ HSCs or Lin⁻CD34⁺CD38⁺ HPCs were sorted from BM and cocultured on Wnt (ie, Wnt3a, Wnt5a, or Dkk-1)–producing OP9 cells for 48-72 hours under cytokine-free conditions to evaluate changes in phenotypes. The 2 bottom panels illustrate retention of c-Kit as reflected in mean fluorescent intensity normalized to control values. (D) The same culture conditions were used to investigate proliferative status, and repre-sentative staining for Ki-67 is shown. Averages ± SD are given in the right panel. (E) A 2-step culture system was used to assess de-differentiation of CB ELPs (Lin⁻CD34⁺CD38⁻CD7⁺) or CLPs (Lin⁻CD34⁺CD38⁺CD10⁺). After coculture on OP9-Wnt3a with IL7, SCF, and FL for 3 days, their potential to generate glycophorin-positive erythroid cells during an additional 2 weeks under stromal cell–free, erythropoiesis-supporting conditions (ie, EPO, SCF, and FL) was determined. Similar results were obtained in 3 independent experiments using ELPs and CLPs isolated from either CB or BM. Statistical significance was determined by unpaired 2-tailed t test analysis. *P < .05; **P < .01.