*Mann-Whitney test p≤ 0.05
Figure 4: Influence of ASCs on senescence in vitro.
A: Morphological analysis using HPS staining, Ki67 and p16 expression and B-Galactosidase assay. Comparison with the young control on D42 obtained with dermal equivalent prepared with fibroblasts which is similar to the young control on D42 obtained with each condition. β-Galactosidase assay gave rare patches of low intensity blue dying for all SEs from D42 and in SE of D100 prepared with fibroblasts alone, patches in the dermis increased in number and in intensity. However, in SE prepared with 25% of ASCs, the blue staining appears close to that observed in short time SEs at D42.
B: number of proliferating cells expressing Ki67 on D100.
C: Epidermal thickness on D100 (µm). At D100, with fibroblasts alone, the epithelium was fully differentiated into a stratum corneum devoid of nuclei and therefore presenting only 0.1 cells per field expressing proliferation markers such as Ki67. After this period of culture, only the addition of 25% ASCs maintained a living epidermis and the level of proliferating cells expressing Ki67. D: Percentage area labeled with p16 in relation to total area of dermis on D42 (42) and D100 (100). On D100, the presence of 25% ASCs significantly reduced its expression (p = 0.05). Indeed, the percentage of area labeled with P16 in the dermis as compared to the total surface area of the dermis was 3.37% without ASCs and 2.33% with 25%, resulting in percentage drop to a level near to and statistically similar to that of young skin, which is around 2% (D42). Means calculated for 3 fields in 3 samples for each condition.