Dose-dependent effect of Y-27632 on HFSC enrichment. HFSCs were cultured in basal, Y-27632, or EGF medium. After P3, fibroblasts grew in basal medium from primary cultures (A1-A3) without the existence of HFSCs. Cells cultured in Y-27632 medium maintained clustered HFSC colonies labeled by anti-K15 until P15 (B1-B3). HFSCs cultured in EGF medium (C1-C3) differentiated into keratinocyte-like cells without K15 expression at P6 (D1-D3). (E) P5 HFSCs were maintained with increasing concentrations of Y-27632 (0, 0.5, 1, 2.5, 5, 10, 20, or 50 μM). After 3 d in culture, HFSCs were labeled by anti-K15. HFSC ratio was calculated as the number of HFSCs divided by the total cell number (**P < 0.01). When Y-27632 was removed from the culture medium, P10 HFSCs differentiated into cells that lacked K15 expression after 3 d (F1-F3). Scale bar = 10 µm.