Wnt5a Inhibits Wnt3a-Mediated HSC Differentiation.

Activation of the canonical Wnt signaling pathway by Wnt3a has been implicated in hematopoietic stem cell (HSC) self-renewal (Reya et al., Nature, 2003). Wnt5a has been observed to inhibit Wnt3a signaling (Topol et al., J Cell Biol, 2004). We hypothesized that Wnt3a and 5a act as antagonists on HSC function. 1 x 10⁶ lineage negative cells (lin⁻) were cultured for 4 days in the presence of 50 ng/ml SCF and Flt3L (control) plus 100 ng/ml rmWnt3a and/or 500 ng/ml rmWnt5a (all factors added on day 0 and day 2). Control lin⁻ cell numbers expanded more than lin⁻ cells cultured with Wnt3a, 5a, or both (control 8.3 ± 0.3-fold; Wnt3a 6.9 ± 0.2-fold (p < .01); Wnt5a 4.8 ± 0.2-fold (p < .001); Wnt3a and 5a 2.6 ± 0.6-fold (p < .001); n = 3). After 4 days, cells were analyzed for myeloid colony formation. Control cells and cells cultured in Wnt3a had similar numbers of CFU-GM/5000 lin⁻ cells (control 13.1 ± 11.1; Wnt3a 21.8 ± 15.3; p = .21; n = 8), while cells cultured in Wnt5a and Wnt3a and 5a had 2-fold and 5.9-fold more CFU-GM/5000 lin⁻ cells than control (Wnt 5a 26.8 ± 13.3 (p = .04); Wnt3a and 5a 77.9 ± 48.3 (p < .01); n = 8). To analyze repopulating ability, 4 x 10⁵ lin⁻ Ly5.1 cells, cultured under the same conditions, were transplanted with 2 x 10⁶ Ly5.2 bone marrow cells into lethally-irradiated Ly5.2 recipients. 16 weeks after transplant, repopulation by control lin⁻ cells increased 2-fold compared to lin⁻ cells cultured in Wnt3a or Wnt5a (control 7.3 ± 3.8%; Wnt3a 3.37 ± 1.2% (p < .01); Wnt5a 3.6 ± 1.1% (p < .01); n = 9-10). However, lin⁻ cells cultured in Wnt3a and 5a showed normal repopulating activity (n = 10; 8.7 ± 5.3%; p = .52). 1 x 10⁴ HSCs (lin⁻, c-kit^HI, Sca-1^HI, IL-7Rα ⁻) were cultured for 6 days with SCF, Flt3L, Wnt3a and 5a (factors added on day 0 and day 3) as described above. Control HSC numbers expanded more than HSCs cultured with Wnt3a, Wnt 5a, or both (control 20.7 ± 10.4-fold; Wnt 3a 7.0 ± 4.1-fold (p = .05); Wnt5a 1.7 ± 1.7-fold (p = .01); Wnt3a and 5a 1.2 ± 1.0-fold (p < .01); n = 4). Similar numbers of control HSCs and HSCs cultured with Wnt3a or 5a were lin⁺ (control 21.7 ± 0.2%; Wnt 3a 15.4 ± 5.3% (p = .10); Wnt5a 14.4 ± 5.2% (p = .07); n = 3). However, culturing HSCs with Wnt3a and 5a resulted in a 50% decrease in the number of lin⁺ cells compared to control (12.3 ± 2.0% (p = .001)). Cultured Ly5.1 HSCs were transplanted with Ly5.2 bone marrow cells at a 1:100 ratio. There was no difference in repopulation between control HSCs and HSCs cultured with Wnt3a (control 5.8 ± 6.1%; Wnt3a 3.6 ± 0.4%; p = .43; n = 5). To examine the effects of enforced expression of Wnt ligands in HSCs, 5-FU treated bone marrow was transduced with Wnt3a-IRES-GFP, Wnt5a-IRES-dsRED, or IRES-GFP retroviral vectors. Sorted IRES-GFP⁺, Wnt3a-GFP⁺ and Wnt5a-dsRed⁺ cells (Ly5.1) were transplanted with equal numbers of mock-transduced cells and 3 x 10⁵ Sca-1⁻ bone marrow cells (Ly5.2) into lethally-irradiated Ly5.2 mice. 16 weeks later, recipients of IRES-GFP⁺ and Wnt5a-dsRed⁺ cells contained a similar number of engrafted cells expressing the vector (3.4 ± 1.8% GFP⁺ Ly5.1 and 3.5 ± 0.4% dsRed⁺ Ly5.1 respectively; n = 8). In contrast, no GFP⁺ Ly5.1 cells were detected in Wnt3a-GFP⁺ recipients (n = 8). 33.4 ± 3.7% of bone marrow cells were Ly5.1⁺ indicating successful engraftment and retroviral DNA was detected by PCR, suggesting that transduction had occurred but that only cells in which the vector was silenced survived. We conclude that activation of the canonical Wnt pathway in HSCs promotes differentiation of primitive hematopoietic cells and that other signals, such as Wnt5a, are required to maintain the balance between HSC differentiation and self-renewal.