Postnatal mouse adrenal glands contain sphere-forming cells expressing markers of NCSCs and SAPs.

<p>Mouse adrenals were dissociated to single cells and cultured using differential plating in low-attachment plastic dishes with serum-free medium containing bFGF and EGF. (<b>A</b>) <b>Dissociated postnatal mouse adrenal glands form spheres in non-adherent serum-free conditions.</b> Phase contrast image of spheres derived from the adrenal glands of a 2 d old mouse growing in suspension culture. Scale bar equals 200 µm. (<b>B</b>) <b>Early spheres contain proliferating cells.</b> BrdU was added to the culture medium of spheres at different time of culture. 24 h later BrdU was detected by immunofluorescence microscopy. Micrograph of a sphere cultured for 7 d stained with anti-BrdU and DAPI is shown in the left panel, scale bar equals 50 µm. Quantification of BrdU-incorporating sphere cells is depicted in the right panel. The means of two independent experiments are shown (15 wells/time-point). Statistical analysis was performed using the t-test. ***, p<0.001. (<b>C</b>) <b>Frequency of sphere forming cells decreases with age of mice.</b> 1.2×10⁵ cells/ml derived from adrenal glands of mice of increasing age were seeded in non-adherent serum-free condition. Phase contrast images of spheres are shown, scale bar equals 200 µm. Sphere-forming frequency was calculated as frequency of spheres formed per number of cells seeded (histogram). The means of three independent experiments are shown. Statistical analysis was performed using the t-test. ***, p<0.001. (<b>D</b>) <b>Sphere cells express markers of SAPs and NCSCs.</b> RT-PCR analysis was performed on RNA isolated from adrenal-derived spheres.