Fig. 5. Self-renewal and antisenescence potential of APOSCs requires CBX7 expression.

a The olfactory mucosa from CBX7^−/− showed no CBX7 expression (green) and decreased numbers of proliferating cells (Ki67, red) compared to the CBX7^+/+ mice (Ki67, red, white arrow). b, c Uninjurbed (b) or methimazole-injured (c) olfactory mucosa tissues were examined for spontaneous or stimulated proliferation of APOSCs, respectively. Double-labeled immunohistochemistry for Ki67 (green) and K14 (red) revealed significantly reduced numbers of proliferating APOSCs in CBX7^−/− olfactory mucosa (right column), as compared with CBX7^+/+ olfactory mucosa (left column). d, e Defective olfactory neuron differentiation in CBX7^−/− mice was indicated by reduced Tuj-1⁺ (d, green) and NeuroD1⁺ (e, green) cells. Cell nuclei were stained with DAPI (blue). Scale bar, 20 μm. f Hematoxylin/eosin (HE)-stained olfactory mucosa tissues. g Adult (8-weeks) olfactory mucosa tissue of CBX7^−/− mice showed SA-β-Gal activity (blue staining) in OE. Basal membrane is indicated by the dotted line. h–i Isolated- CBX7^−/− APOSCs exhibited flattened/enlarged morphology (h, right panel, on day 21) and SA-β-Gal activity (i, right panel, on day 32), as compared with the CBX7^+/+ APOSCs (h or i, left panels, on day 21 or 32, respectively). j The percentages of Ki67-, NeuroD1-, Tuj-1-, and K14-expressing cells were quantitated in uninjured CBX7^+/+ and CBX7^−/−olfactory mucosa. k Quantitative RT-PCR analysis of p16^Ink4a in CBX7^+/+ and CBX7^−/−olfactory mucosa. l Proliferation arrest of CBX7^−/− mouse-derived APOSCs in culture. Cumulative cell numbers of mouse APOSCs derived from the indicated genotypes are shown. m ShRNA-mediated downregulation of CBX7 attenuated long-term expansion capacity of human APOSCs. OE: olfactory epithelium, SA-β-Gal: senescence-associated β-galactosidase. Data are presented as mean ± SD from two independent experiments of triplicate measurements; *P < 0.05, and **P < 0.01, Scale bar, 20 μm