Figure 4. Differentiation of HFKs Expanded with the EpiX Medium.

(A) Addition of 1 mM CaCl₂ to the EpiX medium induced the HFKs to differentiate in 24 hr.

(B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl₂ for 7 days. ZO-1, zonula occludens-1.

(C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl₂ (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl₂.

(E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl₂ for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C.

(F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium.

(G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis.

(H) Most cells in the basal layer were stained positive for the proliferation marker Ki67.

(I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).