Fig. 1. Expression of pluripotency-related markers in APOSCs isolated from the human olfactory mucosa.

a–d Immunocytochemistry (green) showed expression of Nanog (a), Oct-4 (b), Sox-2 (c), and SSEA-4 (d) proteins in isolated human APOSCs. Cell nuclei were stained with DAPI (blue). e RT-PCR analyses of isolated human APOSCs. Human ES (hES) cells served as the positive control (twofold diluent cDNA). Templates without reverse transcription served as the negative control (-). f Flow cytometric analysis of isolated human APOSCs for Nanog, Oct-4, Sox-2, and SSEA-4 expression using specific antibodies (open area curve) or respective isotype control antibodies (filled area curve). Proportions of positive cells (%) are shown on upper right of each panel as compared to controls. g Expansion capacity of long-term cultivated human APOSCs. Shown are averages in duplicates of APOSCs. h Flow cytometric analysis of APOSCs distribution according to their CFSE fluorescence after 4-day cultivation. Peaks represent the number of cells corresponding to each of the subsequent daughter generations (number of cells analyzed: 10⁴). i–n Characterization of human APOSC-formed primary spheres. Morphology (i) of a sphere under a bright-field microscope. A BrdU-labeled (green, j) sphere. Cell nuclei were stained with PI (red). ALP staining (red, k) of an APOSCs sphere. Immunocytochemistry (green) and DAPI staining (blue) of APOSCs spheres for Nanog (l), Oct-4 (m), and SSEA-4 (n) expression. Scale bar, 20 μm