Fig. 3

WNT5A is highly expressed by the liver endothelial niche and promotes CD45⁺CD34⁺ cell generation in vitro. A RT-qPCR of WNT5A expression (n = 3 donor sets, mean ±SEM, **p ≤ 0.01). B Quantitative measurement of WNT5A protein via ELISA assays (n = 3 donor sets, mean ± SEM, **p ≤ 0.01). C Knockdown of WNT5A in human fetal liver ECs decreases the number of CD45⁺CD34⁺ cells and colony-forming progenitors generated following co-culture with human fetal liver CD45⁻CD43⁺ HPCs. (i, ii) Flow cytometric analysis of CD45⁺CD34⁺ cells generated from HPCs following 10 days of co-culture on E4LEC (i) or KD-WNT5A E4LEC (ii). (iii) Quantification of CD45⁺CD34⁺ cells produced following co-culture, normalized to those produced from co-culture with E4LECs (n = 3 donors, mean ± SEM, ***p ≤ 0.0001). (iv) Quantification of colony-forming progenitors following co-culture on E4LEC, KD-WNT5A E4LEC, or scrambled control (sr) E4LEC (n = 3 biological replicates, mean ± SEM, **p ≤ 0.01, ***p ≤ 0.001). D Overexpression of WNT5A in E4ECs increased the number of CD45⁺CD34⁺ cells and colony-forming progenitors generated following co-culture with human fetal liver CD45⁻CD43⁺ HPCs. (i, ii) Flow cytometric analysis of CD45⁺CD34⁺ cells generated from HPC following 10 days co-culture on E4ECs (i) or OE-WNT5A E4EC (ii). (iii) Quantification of CD45⁺CD34⁺ cell numbers (n = 3 donors, mean ± SEM, **p ≤ 0.01) and (iv) colony-forming progenitors (n = 3 donors, mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01)