Researchers here set forth the basis for a novel approach to assessing the level of cellular senescence present in a patient, using a skin sample as a starting point. The current situation for assays of cellular senescence is very biased towards laboratory research needs, with little innovation over the past twenty years. The present standard assays are unfortunately not a suitable basis for the efficient, discriminating, and above all easy and low-cost clinical tests that will be needed in the years ahread. Senolytic therapies capable of clearing senescent cells as a form of rejuvenation treatment will become available in the next few years, and adventurous souls can already self-experiment with some of the drug candidates. Tests capable of clearly establishing the results of such experimentation are much needed.
Fibroblasts form one the most important cellular components of the skin derma. During aging, skin fibroblasts undergo substantial changes in their functional activity, morphology and proliferative potential. The number of dermal fibroblasts decreases with aging, along with their ability to synthesize active soluble factors and to maintain proteostasis of components of the intercellular dermal matrix. The skin thinning, the loss of skin flexibility and elasticity, and wrinkle formation are natural consequences of such a decline. Therefore, we suggested that evaluating the proliferative potential of dermal fibroblasts is of great significance.
Measuring the ability to form colonies in vitro represents one of the "gold standard" methods for the assessment of the clonogenic survival of cells. The method was initially developed to evaluate the loss of reproductive capacity (reproductive death) of cells after exposure to damaging agents, particularly ionizing radiation. Later it was shown that cells isolated from biopsy material from different patients had varying ability for colony formation. This allows for comparative assessment of different patient's cell capacity to proliferate and may represent a promising avenue for personalized medicine.
Beside a colony-forming efficiency of fibroblasts, defined as percentage of plated cells that are able to form colonies, the evaluation of colony size/type distribution provides additional important information especially for heterogenic cell populations such as primary fibroblasts. In this case, the size of the colony depends directly on the proliferative capacity of cell-precursors: cells can form morphologically distinct colonies that can be broken down into the following three types: dense (or compact), diffuse and mixed colonies. If the fractions of each of these colony phenotypes are known, one can evaluate the proliferative potential of the entire fibroblasts culture. Cellular aging, traditionally assessed by the fraction of senescence associated β-galactosidase (SA-βgal) positive cells, along with the degree of differentiation are closely associated with the proliferative capacity of cells. With aging, intracellular β-galactosidase accumulates in lysosomes and a sharp increase in the β-galactosidase activity in older cells is traditionally considered to be a classic marker of cellular aging. Therefore, it could be anticipated that the fraction of aging cells in colonies of the diffuse phenotype would be larger than that in the colonies of the dense phenotype.
The aim of this work was to verify the assumptions regarding the relationship of cellular aging with the formation of fibroblast colonies of different phenotypes, and to examine whether such enriched analysis of colony formation may be used for evaluating the degree of cellular senescence. To this end, we measured the fraction of SA-βgal positive cells (SA-βgal+) in the three types of colonies (dense, mixed and diffuse) of human skin fibroblasts from donors of various ages. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Moreover, a significant inverse correlation between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.