Figure 4
The maintenance of self-renewing and terminal differentiation abilities in keratinocyte stem/progenitor cells expressing EGFP. (A) Images of phase-contrast (left column) and enhanced green fluorescent protein (EGFP) expression (middle column) in a progressively growing colony. The merged image (right column) shows clonal expansion of EGFP-expressing single keratinocytes. Keratinocytes (2 × 103 cells) were seeded on mitomycin C-treated 3 T3 cells, and transduced with the EGFP-expressing lentiviral vector at multiplicity of infection (MOI) 1. After six days of cultivation, the expression of EGFP and keratinocyte marker proteins were analyzed by immunofluorescence microscopy. (B-D) The left column shows the expression of EGFP in growing colonies. The middle column shows the expression of ITGA6 (B), p63 (C), or involucrin (INV) (D). DNA was also visualized with Hoechst 33258, and merged images (right column) show clonal expansion of EGFP-expressing single keratinocytes. (E) The phase-contrast image and the expression of ITGA6, p63 and INV in progressively growing colonies derived from untransduced keratinocyte stem/progenitor cells. Bars, 100 μm. (F) Long-term maintenance of EGFP expression in keratinocyte stem/progenitor cells. The EGFP-expressing colony was isolated, trypsinized and expanded in a new culture dish. EGFP expression was maintained for at least 18 days after the lentivirus was transduced. Bars, 100 μm.