Replicative Senescence and Differentiation Properties of Reconstituted Skin and Monolayer Human Keratinocytes in Vitro

Doh, Yeung Ho; Liu, Yin; Sung, Hoon Ki; Jung, Tae Eun; Park, Jeong Hyun; Kim, Joo Young; Song, In Hwan; Lee, Yung Chang; Sung, Eon Gi

Abstract

This experiment tried to elucidate the characteristics of senescence and differentiation in the reconstituted skin and the monolayer cultured human keratinocytes in vitro, respectively. While the keratinocytes were cultivated from undifferentiated state to completely senescent and differentiated, the monolayer cultured cells of every passage were doubly stained with SA-beta-gal initially, then keratins or involucrin. We also performed the SA-beta-gal enzyme staining and the immuno-reaction such as keratins or involucrin in the reconstituted skin. The results were as follows: Lack of reactivity against SA-beta-gal in the reconstituted skin indicated that there was no senescence occurred. The reconstituted skin showed decreased expression of K10 and preceded expression of involucrin compare to in vivo skin. Nevertheless, the reconstituted skin which did not express the K10 or involucrin in the basal cell maintained the differentiation system similar to that of in vivo skin. On the other hand, the monolayer cultured keratinocytes showed a thoroughly different pattern in the senescent and differentiating process. SA-beta-gal was colocalized with K10 or involucrin in the cells of high percentage ratio by the double staining method, and this indicated that the senescence and differentiation in the kratinocytes were simultaneously progressed. Reaching the nearer stage leading to the cell death, the cells choosed the one of senescence or differentiation pathway. It was supported by the fact that the percentage index of double staining together with SA-beta-gal and involucrin was lower at passage 5 than passage 1~4. The SA-beta-gal's reactivity was maximally reached at passage 4 and the involucrin maximally reached at passage 5. These trends suggested that the senescence was preceded by the differentiation. In conclusion, the reconstituted skin maintained only the differentiation system without the cell senescent process similar to the in vivo while the senescent and differentiating events were simultaneously processed in the monolayer cultured keratinocytes.