Fig. 1: MSC generation from hPSCs via definitive endoderm progenitors.

a Stage-wise differentiation strategy to induce DE-MSCs with definitive endoderm (DE) origin from hESCs. DE-MSCs were obtained on day 24. b Cell morphology (upper panel) and expression of SOX17-GFP (lower panel) from day 0 to day 24. Spindle-like cells were observed on day 24 and they were SOX17-GFP negative on day 24. Scale bar = 100 μm. c RT-qPCR analysis for mRNA level of SOX17, CD44, NT5E (CD73), and ENG (CD105) on day 3, 5, 10, 15 (Passage 0, P0), 18 (Passage 1, P1), 21 (Passage 2, P2), 24 (Passage 3, P3) (n > 3), *p < 0.05, **p < 0.01. d Flow cytometry analysis of percentage of CD44⁺, CD73⁺, CD105⁺, CD45⁺ cells. The blue line represented IgG isotype control for the gating strategy, the red line represented the percentage of CD44⁺, CD73⁺, CD105⁺, and CD45⁺ cells. e Flow cytometry analysis of the percentage of SOX17⁺ cells on day 0, 3, 5, 10, 15, 18 (P1), 21 (P2), 24 (P3) (left panel), and flow cytometry analysis of the percentage of CD44⁺, CD73⁺, and CD105⁺ cells on day 18 (P1), 21 (P2), 24 (P3) (right panel) (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001. f Total cell number of DE-MSCs from passage 3 (P3) to passage 6 (P6) (n = 3). g RT-qPCR analysis was used to detect adipogenic (C/EBPβ), chondrogenic (COL2A1) and osteogenic (RUNX2) marker genes (n = 3), **p < 0.01, ***p < 0.001. h DE-MSCs were induced into adipocytes (stained with Oil Red O), chondrocytes (stained with Alcian Blue), and osteocytes (stained with Alizarin Red). Scale bar = 150 μm for an upper panel, scale bar = 75 μm for lower panel.