[Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes I and IV types].

The concept that stem cells form an independent subpopulations of somatic cells assumes the heterogeneity of cellular populations in adult tissues. As skin keratinocytes have natural affinity to extracellular matrix proteins, we made an attempt to reveal subpopulations of these cells depending on the time of their adhesion to substrates of collagen I and IV types and fibronectin. After selection for 10, 20 and 30 min the keratinocytes were cultivated for 24 h. The area of cell projection on a substrate and the spreading coefficient were measured (Kuzminykh, Petrov, 2004; Petrov et al., 2007). In 24 h statistically reliable morphological differences between the cells depending on the substratum were found. The size of the cells growing on collagen I type was twice as large as that of the cells cultivated on collagen IV type or in fibronectin. Irrespective of the substratum, up to 60-65% of the cells had a rounded form. The cultivation on collagens revealed the heterogeneity of keratinocytes both in the control cultures and under selection by adhesion time, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interaction with extracellular matrix proteins in the organism.

[Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and the keratin 19 to actin ratio].

Keratin 19 is reported to be a marker for skin stem cells and that assumes an independent subpopulation of these cells in keratinocyte population. In addition, keratinocytes have natural affinity to extracellular matrix proteins. The aim of this work was to reveal subpopulations ofkeratinocytes cultivated on the substrates of collagen I type, laminin-2/4 and fibronectin, and distinguished by keratin 19/actin ratio (G/R). The area of cell projection on a substrate, perimeter, the spreading coefficient and G/R were measured. Both on fibronectin and laminin-2/4, keratinocyte populations were morphologically homogeneous with large cells comprising 12 and 20 percents of the population, respectively. On fibronectin, correlation between morphological parameters of the cells and the keratin 19/actin ratio was not found. The cells growth on collagen behaved as a heterogeneous population, with the large cells compressing more than 50 percents of the population. An average, the size of the cells growing on collagen was twice as large as than of the cells cultivated on fibronectin. On the other hand, correlation between the cell size and G/R was revealed in the cells growth both on collagen and laminin-2/4. The cells with lower G/R value display a larger size and extent of spreading. We assume that this correlation may be determined by alphal and alpha2, integrins characteristic of these cells. Our results cast doubts on whether stem cells are present in the culture of human skin keratinocytes.

[Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins].

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.