Enforced expression of Wnt ligands in bone marrow. (A) Diagram of the control MSCV MGirL22Y IRES-GFP vector (Top), the Wnt3a-IRES-GFP vector (Middle), and the Wnt5a-IRES-DsRed vector (Bottom). (B) Average repopulation of whole bone marrow by CD45.1 bone marrow transduced with either the control vector (n = 9), Wnt3a-IRES-GFP (n = 7), or Wnt5a-IRES-DsRed (n = 8). FACS analysis of whole recipient whole bone marrow was performed 16 weeks after transplant. Data represent the pooled results of two independent experiments. Error bars in B–D represent standard deviation, and P values were generated by Student's t test. (C) Average percentage of CD45.1⁺ bone marrow cells positive for GFP (control and Wnt3a vectors) or DsRed (Wnt5a vector). (D) Average percentage of CD45.1⁺ lin⁻ bone marrow cells positive for GFP (control and Wnt3a vectors) or DsRed (Wnt5a vector). (E) PCR was used to amplify a fragment from the GFP cDNA in recipients of bone marrow transduced by Wnt3a-IRES-GFP. PCR plasmid was used as a positive control (+), and DNA isolated from wild-type bone marrow was used as a negative control (−).

Inhibition of canonical Wnt signaling in HSCs by Wnt5a. (A) Semiquantitative duplex RT-PCR analysis of lacZ mRNA in TOPGAL LKSI cells after a 48-h in vitro culture as described in Materials and Methods. LacZ mRNA levels were quantified within the linear range of amplification by densitometry and normalized to β2m expression. (B) Average lacZ mRNA levels relative to β2m. The amount of lacZ mRNA in TOPGAL LKSI cells was normalized to β2m mRNA (β2) by the formula (densitometry value: lacZ/densitometry value: β2m) (n = 3 for all groups). The normalized TOPGAL lacZ mRNA level in control LKSI cells was set to 1. The data represent the average results of three independent experiments. Error bars represent standard deviation. P values were generated by Student's t test. (C) Representative Western blot analysis of β-catenin levels in lin⁻ cells after a 48-h in vitro culture. The experiment was performed three times with independent cultures.

Analysis of Wnt-mediated HSC expansion and apoptosis. (A) Average fold expansion of wild-type LKSI cells after 4 days in culture (n = 3 for all conditions). Error bars represent standard deviation. P values were generated by Student's t test (*, P < 0.05; **, P < 0.01). (B) Representative FACS analyses of apoptotic wild-type LKSI cells cultured for 4 days under control conditions (Top), with recombinant Wnt3a (Middle), and recombinant Wnt5a (Bottom). Actively apoptotic cells actively were defined as Annexin V⁺ and PI⁻. (C) Average percentage of apoptotic wild-type LKSI cells after 4 days in culture (n = 4 for all conditions). Error bars represent standard deviation. P values were determined by Student's t test. (D) Average fold expansion of H2K-BCL-2 LKSI cells after 4 days in culture (n = 4 for all conditions). Error bars represent standard deviation. P values were generated by Student's t test

Analysis of Wnt-mediated effects on hematopoietic repopulation. (A) Raw data of short-term (6 weeks) engraftment of cultured LKSI cells. Recipients were defined as positive for engraftment if they contained >1% hematopoietic chimerism. (B) Average short-term repopulation of cultured LKSI cells. Data represent the pooled results of two independent experiments. Only recipients with >1% chimerism were included in this analysis. Error bars represent standard deviation. P values were generated by Student's t test. (C) Raw data of long-term (16 weeks) engraftment of cultured LKSI cells. Recipients were defined as positive for engraftment if they contained >1% hematopoietic chimerism. (D) Average long-term repopulation of cultured LKSI cells. Data represent the pooled results of three independent experiments. Only recipients with >1% chimerism were included in this analysis. Error bars represent standard deviation. P values were generated by Mann–Whitney nonparametric analysis.

Analysis of Wnt5a-mediated effects on gene expression. (A) Representative duplex RT-PCR analysis of Hes-1 mRNA in LKSI cells. (B) Average Myc mRNA levels relative to β2m (n = 3 for all groups). The data represent the average results of three independent experiments. Error bars represent standard deviation. P values were generated by Student's t test. (C) Average Cdkn1b mRNA levels relative to β2m (n = 3 for all groups).

Analysis of Wnt-mediated effects on HSC cell cycle status. (A Left) Representative cell cycle histograms obtained through propidium iodide (PI) staining of wild-type LKSI cells cultured for 4 days under control conditions (Left) or recombinant Wnt5a (Right). Histograms were analyzed as described in Materials and Methods. (Right) Average percentage of wild-type LKSI cells in S/G₂/M phases of the cell cycle after 4 days in culture. Data represent the average of three independent experiments for each condition. Error bars represent standard deviation. P values were generated by Student's t test. (B Left) Representative FACS analyses of G₀/G₁ LKSI cells after 4 days in culture under control conditions (Left) or recombinant Wnt5a (Right). LKSI cells in the G₀/G₁ phase were defined as cells with 2n DNA content as detected by 7-AAD staining (data not shown). G₀/G₁ cells were gated and analyzed for the percentage of cells that were in the G₀ phase, defined as Ki-67⁻. Regions were drawn based on isotype controls. (Right) Average percentage of wild-type G₀/G₁ LKSI cells in the G₀ phase after 4 days in culture. Data represent the average of three independent experiments for each condition. Error bars represent standard deviation. P values were generated by Student's t test. The approximate percentage of total cells in G₀ under control conditions, with Wnt3a, or with Wnt5a were 10%, 25%, and 35%, respectively (determined by multiplying the percentage of total cells in G₀/G₁ (A) by the percentage of G₀/G₁ cells in G₀ (B).