FIG. 6.

Development of a completely defined, standardized epithelial differentiation system. (A) Method for generating epithelial and epidermal progenitor cells from hPSCs using a completely defined system (i) with or (ii) without a subculture step. (B) Representative flow cytometry dot plots showing K14 expression in H9 hESC-derived cell populations at day 28 of differentiation generated using conventional substrates or defined substrates (top panels) with a subculture step or (bottom panels) without a subculture step compared to an isotype control. (C) Quantification of flow cytometry data measuring purity of K14+ cells at day 28 of differentiation from each condition shown in (B). N=3 independent replicates for each condition. (D) Yield of K14+ cells at day 28 of differentiation of conditions incorporating a subculture step. Yield measured by absolute number of K14+ cells at day 28 relative to the starting number of Nanog+ cells at day 0 of differentiation. Proliferative fraction of K14+ cells shown as measured by Ki67 coexpression as well as the nonproliferative (Ki67-) fraction of K14+ cells. Error bars indicate standard deviation of three independent replicates for each condition (* indicates p<0.05 compared to Matrigel/gelatin condition). (E) Immunofluorescence images of K14 expression in day 28 differentiated cell populations generated on Matrigel/gelatin or defined substrates. Scale bar in all panels is 100 μm. Color images available online at www.liebertpub.com/tec