(A) When SP, NSP, and unsorted control cells were grown for 4 weeks, there were 34.33 ± 2.84 colonies with 1,000 SP cells and 64 ± 4.93 colonies with 2,500 SP cells (n = 3 experiments), compared with absent colonies with control or NSP cells (n = 3 experiments). Arrowheads indicate colonies. *P < 0.001 compared with NSP. (B) Two-photon microscopy of the disordered cells on the organotypic culture (day 12) after seeding 10³ NSP cells isolated from GFP⁺ mice (green) with DAPI⁺ nuclei (blue); top view, z-stack, 53.8 μm in 0.78-μm increments. (C) Two-photon microscopy of the fully formed epithelium on day 12 after seeding 10³ SP cells isolated from GFP⁺ mice (green) with DAPI⁺ nuclei (blue); top view, z-stack, 60 μm in 1-μm increments. Arrowheads indicate basement membrane. (D and E) Unsorted GFP^– cells (3 × 10⁴) did not form a complete epithelium. (F) GFP^– immunohistochemistry and (G) DAPI⁺ nuclei. (H and I) Sorted 10³ NSP cells from GFP⁺ mice mixed with 3 × 10⁴ unsorted GFP^– cells did not form a complete epithelium. (J) Mosaic GFP⁺ pattern and (K) DAPI⁺ nuclei. (L and M) SP cells (10³) from GFP⁺ mice mixed with 3 × 10⁴ unsorted GFP^– cells. There was complete epithelial formation with keratinization. (N) GFP⁺ immunohistochemistry and (O) DAPI⁺ nuclei. Dotted line in B–O indicates basement membrane. Scale bars: 5 mm (A), 25 μm (B–O). Original magnification, ×20 (A); ×100 (D, H, and L); ×200 (B and C); ×400 (E–G, I–K, and M–O).