Wnt3a regulates human hematopoiesis via stromal cells. (A) Human MSCs were cocultured with Wnt (ie, Wnt3a, Wnt5a, or Dkk-1)–transduced OP9 cells for 3 days, with recombinant human TNFα present for the first 24 hours to enhance VCAM-1 expression. (B) MSCs were similarly cultured along with recombinant human Wnt3a. Flow cytometry histograms of VCAM-1 staining are shown. (C) BM CD34⁺ cells were cultured without stromal cells for 3 weeks under B-lineage conditions (G-CSF and SCF). The numbers of recovered CD19⁺ cells are given in the panel below. (D) An experimental design was used to determine whether direct contact between hematopoietic cells and stromal cells was required. The top figure in each row depicts the experimental design, whereas the next shows results from a 4-week culture of CD34⁺ cells under lymphoid conditions. The bottom 2 rows show results of 2-day cytokine-free cultures initiated with CD34⁺ cells. Suppressed B lymphopoiesis (expression of CD19), retention of c-Kit, and diminished replication (Ki-67 staining) all required that stromal cells were present in the top chambers. Results from controls are given as gray lines, and those for OP9-Wnt3a are shown as black lines. Similar results were obtained in 3 independent experiments using either CB or BM together with MS5 or OP9 stromal cells in the top chambers. Statistical significance was determined by unpaired 2-tailed t test analysis: **P < .01.