To evaluate the maturation and differentiation state of cultured keratinocytes, the author investigated expression of differentiation markers in cultured keratinocytes. The specimens were divided into three experimental groups, 3rd passage keratinocytes cultured in serum free media (3rd SFM group), 6th passage keratinocytes cultured in serum free media (6th SFM group) and 3rd passage keratinocytes cultured in DMEM (DMEM group). CK14, marker of basal layer, expressed in all groups. The expression was localized and condensed in the SFM groups but spreade in the DMEM group. Most of the cells in both SFM groups were positive but a few cells in DMEM group were also positive. CK10, marker of initiation of differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. Involucrin, marker of terminal differentiation, expressed weakly in DMEM group but there was no expression in both SFM groups. CK16 and 17, markers of fast turnover of keratinocytes, were not expressed in SFM groups. Weak positive reactions were observed in DMEM group. With these results the authors concluded that the keratinocytes from 3rd passage to 6th passage, cultured in serum free media with calcium less than 0.1 mM, had highly homogeneous basal cell characteristics.
Antigens, Differentiation ; Calcium ; Culture Media, Serum-Free ; Humans* ; Keratinocytes* ; Keratins*

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.
Aging ; Cell Aging* ; Cell Death ; Cells, Cultured ; Hand ; Humans* ; Keratinocytes* ; Skin*