Fig. 5

EZH2 promotes the proliferation of human neural progenitor cells (NPCs) in vitro. a Schematic of the default neural differentiation and random differentiation strategy for human embryonic stem cells (hESCs) with over-expression of EZH2. b Western blot for EZH2-FLAG proteins in the EZH2-overexpressed hES cells. c Morphology of the wild-type (WT) H1 and EZH2 over-expressed hESCs under NPC differentiation conditions at day 0 and day 16. Scale bar, 200 μm. d Immunostaining for the pluripotent marker OCT4 and the NPC markers SOX2/NESTIN in wild-type (WT) and EZH2 overexpressed rosette-like NPCs. Scale bar, 50 μm. e qRT-PCR analysis for the expression of EZH2, the NPC genes SOX2/SOX1/PAX6, and meso-endoderm genes ZIC1/KRT18/VEGFA /DLX5 at day0, day8, day 16 of neural differentiation. Significance was determined using unpaired two-tailed Student’s t-tests. The data represent the mean ± SD (standard deviation) from three independent replicates (n = 3). **, P < 0.01. f Left panel, morphology of indicated NPCs maintained as neural spheres at passage 2 (P2) or passage 4 (P4). Scale bar, 200 μm. Right panel, proliferation curve of the indicated NPCs at different passages. Significance was determined using unpaired two-tailed Student’s t-tests. *, P < 0.05. **, P < 0.01. The data represent the mean ± SD (standard deviation) from three independent replicates (n = 3). g Apoptosis assay in the indicated cells at passage 2 (P2) or passage 4 (P4). PI- and/or annexin V-positive cells were analyzed by FACS. The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments (n = 3). h qRT-PCR analysis for the expression of EZH2, the NPC genes SOX2/SOX1/PAX6, and meso-endoderm genes ZIC1/KDR/DLX5/TFAP2A /PAX3/VEGFA in the indicated NPCs at passage 4 (P4). The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments (n = 3). i Morphology of NPCs and their subtype neuron/glia cells with over-expression of EZH2. Scale bar, 50 μm. j Immuno-staining analysis for SOX2/NES, MAP2/GFAP in NPCs with forced EZH2 expression during random differentiation. The numbers of MAP2⁺ or GFAP⁺ cells were analyzed. The significance level was determined using unpaired two-tailed Student’s t-tests. **, P < 0.01. Error bars represent the mean ± SD from three independent experiments (n = 3)