Figure 4. Pseudotime analysis and metacluster identification in young versus middle-aged NMR keratinocytes. (A) Colored pseudotemporal ordering of keratinocytes for MA (middle-aged) and Y (young) NMR separately. No significant differences were observed between the 2 groups. (B) Relative expression of Krt14, Krt10 and Lor markers plotted along the pseudotime axis for each condition. The same relative expression profile was found between MA and Y NMR, validating the 3 cellular states for NMR keratinocytes and the no age-related impact of aging in the differentiation process. (C) Pseudotime estimation showing epidermal progression along the differentiation process with individualized cluster visualization, for each condition. Five metaclusters were defined according to their differentiation engagement: basal 1, basal 2, intermediate 1, intermediate 2 and corneum. (D) Unsupervised differentiation trajectories for MA and Y animals. The same repartition of metacluster among the trajectory was observed. BSC = basal and stem cells; BPSC = basal proliferating and stem cells; Sp = Spinous layer cells; Gr = granular layer cells; Cor = corneous layer cells.