Figure 4
Downregulation of miR-302 suppresses self-renewal and promotes differentiation. (a) Immunofluorescence staining of SSEA4 in hESCs and hNT-2 cells when endogenous miR-302 was inhibited by miR-302s antagomirs. The negative control oligonucleotide antagomir was transfected as a control. Scale bars, 200 μm. (b) miR-302s-downregulated hESCs were cultured in non-adherent conditions to form EBs. EBs were then dissociated and plated back in ESC culture conditions at day 15. ESC-like colonies were analyzed by AP staining after 6 days under EB to ESC conditions. (c) The numbers of AP-positive ESC-like colonies were calculated under EB to ESC conditions in miR-302s-downregulated hESCs. Negative control oligonucleotide antagomir was transfected as control. Data are presented as mean±S.D. (n=5) and are representative of three independent experiments. (d) hNT-2 cells were treated with vehicle or 1 mM retinoic acid (RA) for 4 days and the expression levels of lineage-specific markers were determined by qRT-PCR; GAPDH was used as an internal normalization control. Data are presented as mean±S.D. (n=3) and are representative of three independent experiments. (e) Morphological changes of hNT-2 cells were investigated after treatment with vehicle or 1 mM retinoic acid (RA) for 4 days in miR-302s antagomir-transfected hNT-2 cells and negative control cells. Scale bars, 100 μm. P-values were obtained using Student's t-test
