Fig. 6.

Non-canonical Wnt signaling promotes vascular specification of hPSC-derived CPCs. (A,B) The CP/CM population was isolated at day 6 and cultured in the presence of CHIR99021, Endo-IWR1 or recombinant WNT5A or WNT11; the total number of NKX2.5^GFP+ CD31^neg and CD31⁺ cells was quantified after 3 (A) and 6 (B) days. (C) The CP/CM population was isolated at day 6 of differentiation and transduced with lentiviral particles containing a luciferase reporter driven by a TCF/Lef-based promoter; the activity of the reporter within NKX2.5^GFP+ CD31^neg derivatives was measured after 4 days. (D-F) CP/CMs were isolated at day 6 of differentiation and incubated with CellTracker reagent to monitor cell proliferation; after 6 days in various growth conditions, the mean fluorescent intensity of the CellTracker reagent was measured among NKX2.5^GFP+ CD31^neg and CD31⁺ derivatives. (E,F) Following 3 or 6 days of CP/CM culture in various conditions, the mean fluorescent intensity of FLK1 was measured on NKX2.5^GFP+ CD31^neg (E) and CD31⁺ (F) derivatives. (G-I) Endogenous WNT5A transcript level was reduced in hPSCs using lentiviral shRNA (G), resulting in a reduced percentage of ECs among hPSC derivatives (H) and a reduced proportion of ECs within the NKX2.5^GFP+ population (I). Error bars represent s.d. between six (A-F) or five (G-I) biological replicates. *P<0.05, **P<0.01.