Wnt3a induces expression of matrix molecules, and one of them mediates most Wnt3a-regulated changes in murine hematopoiesis. (A) RT-PCR was performed using Wnt3a-transduced OP9 stromal cells. The results are expressed as the fold changes relative to the vector control OP9 cells and are representative of those obtained in 3 independent experiments. (B) Murine Lin⁻Sca-1⁺c-Kit^hiFlk-2⁻ HSCs were then cocultured with decorin plus OP9 or on OP9-Wnt3a in the presence of IL-7, SCF, and FL for 3 days. Levels of c-Kit are shown as typical histograms or mean fluorescence intensities (MFI). (C) Lin⁻Sca-1⁺c-Kit^hiRag1-GFP⁺ ELPs were held for 3 days in primary cultures before subculture in stromal cell–free, erythropoiesis-supporting conditions. After an additional 2 weeks of culture, the generation of Ter119⁺ erythroid cells was analyzed using flow cytometry. (D) Numbers of Ter119⁺ erythroid cells were determined by flow cytometry after Lin⁻Sca-1⁺c-Kit^hi cells were held for 2 weeks of culture under stromal cell–free, erythroid-supporting conditions. (E) Proliferation of Lin⁻Sca-1⁺c-Kit^hiFlk-2⁻ HSCs was unaffected by decorin, as reflected in Ki-67 staining. (F-G) The influence of recombinant decorin on B lymphopoiesis was tested with 1-week stromal cell cocultures (F) or stromal cell–free cultures (G) initiated with ELPs. Generation of B220⁺ lymphocytes in triplicate wells ± SD is shown. (H) The same transmembrane culture system shown in Figure 2D was used to analyze gene-expression patterns in OP9 cells exposed to diffusible Wnt3a produced by transduced cells in the lower wells. Similar results were obtained in 3 independent experiments. Statistical significance was determined by unpaired 2-tailed t test analysis: *P < .05; **P < .01.