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Fig. 2 | Cell & Bioscience

Fig. 2

From: Rapid and robust derivation of mesenchymal stem cells from human pluripotent stem cells via temporal induction of neuralized ectoderm

Fig. 2

Generation of MSCs from pluripotent stem cells by two-step induction via neuralized ectoderm as a differentiation intermediate. a Schematic illustration of two-step procedure for MSCs induction. Human pluripotent stem cells are treated with SMAD inhibitor and WNT activator for 5–7 days to differentiate to the neural ectoderm. Cells are then split before further incubated with medium supplemented with bFGF/EGF. Fibroblast-like cell morphology were observed around day 4–10. Cells were expanded for another 2–3 passages followed by FACS analysis of typical MSC surface markers. Lower panel shows the morphology of initial human iPSCs, intermediate neural ectoderm and fibroblast-like MSCs. Scar bar 100 μm. b Expression of neural ectoderm genes (SOX10, SOX2, SOX9, FOXD3, p75, PAX3, ZIC, and AP2a) and pluripotency related genes (OCT3/4, NANOG, and REX1) examined by qPCR. Gene expression was normalized to endogenous GAPDH. Relative mRNA levels were plotted against that in iPSCs. Data represent mean ± S.D. n = 3; *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant; one-way ANOVA coupled with Tukey’s post hoc test was used for statistical analysis. c Flow cytometry analyses of typical positive (CD73, CD90, CD105 and CD44) and negative (CD45, CD34, CD11b, CD19 and HDL-DR) MSCs surface markers. d MSCs are induced toward adipogenesis, chondrogenesis and osteogenesis in vitro for three weeks. Adipocyte, chondrocyte, and osteoblast are detected by staining of Oil red O, Alizarin red S and Alcian blue. Scar bar 100 μm. e Colony formation of MSCs. MSCs are stained with crystal violet. A representative colony in high magnification is shown on the right

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