Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation

Abstract

Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated β-galactosidase (SA-βgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. 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. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=−0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=−0.94, p=0.0001) 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.

Residual γH2AX foci induced by low dose x-ray radiation in bone marrow mesenchymal stem cells do not cause accelerated senescence in the progeny of irradiated cells

“…6A ) to evaluate a possible influence of the IR exposure on senescence in the progeny of irradiated cells. This enzyme is commonly used as a marker of cellular senescence since its expression is substantially elevated in senescent cells [ 45 , 46 ].…”

“…The cells were stained according to supplemented manufacturer protocol with the following modification: at the final РВS washing step, the cell nuclei were stained with 1 μg/ml Hoechst 33342 (Molecular Probes, USA). Such modification significantly improves the quality of counting of β-galactosidase negative cells [ 46 ]. The stained cells were visualized using Excitation/Emission Interference Filters (СKX-U: 340-380nm/435-485 nm) on the inverted fluorescent microscope Оlympus СКХ 41 SF (Оlympus, Japan) equipped with Infinity 3-1 (Lumenera Copr., Canada) CCD camera and 20X objective.…”

MeCP2-mediated epigenetic regulation in senescent endothelial progenitor cells

“…Gradual loss of cellular functions including the ability to proliferate with an increase in the number of cell divisions during cell population development was called replicative aging or cellular senescence [ 21 ]. During aging, the cell undergoes substantial changes in functional activity, morphology, and proliferative potential [ 22 , 23 ]. The number of EPCs decreases with aging, along with their ability for proliferation, migration, and angiogenesis [ 24 , 25 ], as we found formerly in senescent EPCs.…”

Identification of senescent cells in multipotent mesenchymal stromal cell cultures: Current methods and future directions

“…Despite this assay being useful for the identification of senescent MSC passages (those containing no colony forming cells), it notably measures highly-proliferative, as opposed to senescent cells, and is time consuming and highly subjective. In a recent study by Zorin et al, the group confirmed that cells that tested positive for senescence were the highest in small diffuse colonies of human skin fibroblast cultures, and they originated from the cells with the least proliferative potential [54]. However, counting such small colonies as representative of pre-senescent cells remains inaccurate and very subjective.…”