ИСТИНА

The epidermis of human skin is stratified and consists of several layers - stratum basale, stratum spinosum, stratum granulosum and stratum corneum. During the transition from the basal to the overlying layers, skin keratinocytes undergo the specialized type of terminal differentiation - keratinization. However, immortalized monolayer cell lines of epidermal origin usually do not reach terminal differentiation under standard cultivation conditions. Nevertheless, in the presence of calcium contained in standard culture media, a subset of cells from the population can express markers of early differentiation. We investigated the presence of some markers - EdU replicative labeling, immunocytochemical staining for Ki67 (proliferating cells) and specific differentiation markers - keratin 14 (K14) (undifferentiated keratinocytes), keratin 10 (K10) and involucrin (Inv) (differentiated keratinocytes) in immortalized human keratinocytes HaCaT, cultured under standard conditions (DMEM+10% FBS) for 72 hours. EdU labeling showed that 37% of cells were in the S/G2 phase of the cell cycle, and 96.3% of cells were Ki67-positive. K14 was detected in 75% of keratinocytes, while K10 - only in 1.1% of cells. Double labelling for K10 and K14 revealed four types of cellular subpopulations: K10-K14- and K10-K14+ (undifferentiated phenotype), K10+K14+ (intermediate phenotype), K10+K14- (differentiated phenotype). The cells from K10-K14- subpopulation were usually clustered, and K10+K14- keratinocytes often grew above the other cells, that may indicate the initial stages of stratification. In K10+K14+ keratinocytes, both markers were usually colocalized, and in some bundles we observed the alternating pattern of keratins distribution along the filament length. The number of Inv+ cells was small (2%) and only 1.37% of them were Ki67-positive. At the same time, rare non-proliferating Inv+Ki67- cells (0.63%) were distinguishable from Inv+Ki67+ cells due to large size and spreading. Thus, immortalized human keratinocytes HaCaT, cultured under standard conditions, show heterogeneity in appearance of early differentiation markers and allow to trace the dynamics of certain parameters of early differentiation.