Figure 4.

cKit⁺ progenitors exhibit age-dependent differentiation defects. (A) A representative contour FACS plot from strain 353P, showing the gating logic used to enrich cKit⁺ from 4^th passage HMEC strains. (B) Histograms representing average lineage distributions from five individuals <30y (strains 240L, 407P, 168R, 123, and 124) or five >55y (strains 122L, 881P, 353P, 464P, and 451P) in unsorted HMEC (left column), and cKit⁺ progenitors (right column) after 48h of culture on tissue culture plastic. Histograms represent log₂ transformed ratios of K14 to K19 protein expression in single cells, histograms are heat mapped to indicate cells with the phenotypes of K14⁻/K19⁺ LEPs (green) K14⁺/K19⁺ progenitors (yellow), and K14⁺/K19⁻ MEPs (red), error bars represent SD (n=2500 cells/histogram). (C) Scatter plot representing EdU incorporation in the different lineages, as defined by K14 and K19 expression, in (top) unsorted 4^th passage HMEC strains and (bottom) cKit⁺-derived cells after 48h culture. Lines indicate average and error bars represent SEM (n=2500 cells per age group). (D) Representative images of unsorted, CD227- and cKit-enriched HMEC from two individuals, after 48h of culture; Protein expression of K14 (red), K19 (green) are shown, and nuclei are stained with DAPI (blue). Scale bar represents 20μm.