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. 2020 May 30;23(6):101222. doi: 10.1016/j.isci.2020.101222

Figure 5.

Figure 5

The Wnt Signaling Pathway Facilitator R-Spondin 2 Acts in Concert with the Stem Cell Factor (SCF) to Amplify Ex Vivo Hematopoietic Precursors

(A) Expression of R-spondin 2 in peri-sinusoidal cells. R-spondin 2-positive/Leptin receptor-positive (LEPR) peri-sinusoidal cells: continuous arrows; R-spondin 2-negative/LEPR-positive peri-sinusoidal cells: discontinuous arrows; S, sinusoids; IsoB4, isolectin B4 labeling of endothelial cells and macrophages. Scale bar, 10 μm.

(B) Correlation of Rspo2 to Kitl expression values in all studied samples.

(C) Effect of R-spondin 2 on Linneg/Sca-1pos/Kitlpos(LSK) cell number. SCF (10 ng/mL) was present in all tested conditions, whereas Wnt-3a (100 ng/mL) and R-spondin 2 (20 ng/mL) were as indicated. N = 60 per condition (12 experiments with 5 wells per condition). Data are presented as mean ± SEM. Paired t test ∗∗∗p < 0.0001, ∗∗p < 0.001 in paired comparisons.

(D) Effect of R-spondin 2 on LSK cell phenotype (one representative experiment out of 8). Flow cytometry analysis was carried out on cells collected at day 12 and cultured in the presence of 10 ng/mL SCF alone (upper panels), 100 ng/mL SCF + Wnt-3a (middle), or 20 ng/mL SCF + R-spondin 2 (lower). To obtain a more precise evaluation of the Sca-1pos/Kitpos cells we first selected the Ter119neg/Gr-1neg cells before evaluating in this gate the % of Sca-1pos/Kitpos cells.

(E) Effect of R-spondin 2 and Wnt-3a on Wnt downstream targets Tcf7, Bmp4, and Axin2 and on the R-spondin receptor Lgr6. Expression level as 2-ΔCq, ΔCq = Cq(gene of interest) - Cq(housekeeping gene), housekeeping gene being Gapdh. N = 5 per condition. Box limits are the 25th and 75th percentages, center lines are mean, and the whiskers are the mean SD values. Data comparison by paired t test. ∗∗p < 0.01, ∗p < 0.05 in paired comparisons.

(F) Effect of the ß-catenin signaling inhibitor increasing concentrations of PKF118-310 (ng/mL) in the presence (left) or absence (right) of R-spondin 2 on LSK cell number. N = 14 per condition. Legend as in (E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 in paired comparisons.