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

Keratin 19 is considered to be a marker for skin stem cells that assume a particular subpopulation of these cells in the keratinocyte population. It is also known that keratinocytes have a natural affinity for extracellular matrix proteins. The aim of this work was to identify subpopulations of keratinocytes by cultivation on various substrates (type-I collagen, laminin 2/4 and fibronectin) and by measuring keratin 19/actin ratio (G/R). The areas of cell projection on a substrate, perimeter, the spreading coefficient, and G/R have been assayed. Keratinocyte populations were morphologically homogeneous both on fibronectin and laminin 2/4. Large cells comprised 12 and 20% of the populations, respectively. No correlation between morphological parameters of cells and keratin 19/actin ratio was found in keratinocytes cultivated in fibronectin. The cells grown on collagen behaved as a heterogenous population with large cells comprising more than 50% of the population. On average, the size of the cells grown on collagen was twice as large as the cells cultivated on fibronectin. On the other hand, the correlation between cell size and G/R was revealed in cells grown on both collagen and laminin 2/4. The cells with lower G/R values display a larger size and higher spreading. We assume that this correlation is determined by the presence of α1 and α2 integrin subunits in these cells. Keratin 19 assay did not reveal keratinocyte subpopulations. Our results raised doubts regarding the presence of stem cells in cultures of humanskin keratinocytes.