Fig. 3

R-spondin2 treatment during early mesoderm differentiation suffices to promote hPSC hematopoietic differentiation. a Representative immunofluorescence images of H1 cells with treatment of R-spondin2 (20 ng/mL) at different stages of differentiation showing the generation of CD43⁺ HPCs at day 7 (top) and CD45⁺ HPCs at day 10 (bottom) of mAGM-S3 co-culture differentiation. b Flow cytometry analysis of the percentage of CD43⁺ HPCs at day 7 (left) and CD45⁺ HPCs at day 10 (right) from H1 cells in mAGM-S3 co-culture differentiation with R-spondin2 treatment (20 ng/mL) at different stages of differentiation. Day0–3: R-spondin2 was added from day 0 to day 3 and withdrawn at other days. Day3–5: R-spondin2 was added from day 3 to day 5 and withdrawn at other days. Day5–7: R-spondin2 was added from day 5 to day 7 and withdrawn at other days. Day0–7: R-spondin2 was added from day 0 to day 7 throughout. Results are shown as means ± D (n = 3). NS, not significant, *P < 0.05, ***P < 0.001. c Representative immunofluorescence images of H1 cells with the treatment of R-spondin2 (20 ng/mL) at different temporal window of mesoderm induction showing the generation of CD43⁺ HPCs at day 7 (top) and CD45⁺ HPCs at day 10 (bottom) of mAGM-S3 co-culture differentiation. d Flow cytometry analysis of the percentage of CD43⁺ HPCs at day 7 (left) and CD45⁺ HPCs at day 10 (right) from H1 cells in mAGM-S3 co-culture differentiation with R-spondin2 treatment (20 ng/mL) at different temporal window of mesoderm induction. Day0: R-spondin2 was added at day 0 for 24 h and withdrawn at other days. Day1: R-spondin2 was added at day 1 for 24 h and withdrawn at other times. Day2: R-spondin2 was added at day 2 for 24 h and withdrawn at other times. Day0–3: R-spondin2 was added from day 0 to day 3 and withdrawn at other times. Results are shown as means ± SD (n = 3). NS, not significant, *P < 0.05, **P < 0.01