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

Clonogenic analysis

The primary cultures of human fibroblasts were isolated from skin biopsies taken from 10 female patients of 33 to 54 years of age, according to the standard procedures [17], and the fibroblast colonies from these cultures were phenotyped in terms of the dense, mixed and diffuse plaques (Figure 1). Although the age span of the patients was not great and is sometimes used as a single age category in aging studies [18,19], a remarkable inter-individual variability in the clonogenic capacity of their skin fibroblasts was found as evident from the results of their clonogenic analysis (Table 1). Indeed, the variability was demonstrated both in ECO-f (from 9.7 up to 61.0%) and in the fractions of each colony phenotype. A greater range (from 8.6 up to 89.6%) was seen for the fraction of diffuse colonies that are formed by descendants of cells with a low proliferative potential. Regression analysis revealed a strong inverse correlation between ECO-f and the fraction of diffuse colonies (Table 2), indicating that colony phenotype is related to the colony formation capacity. Consequently, diffuse colonies were predominantly formed in cell cultures that have low colony formation capacity. No correlation between any of the four clonogenic parameters and the age was seen (Table 2). It follows that the clonogenic parameters studied may reflect the proliferative capacity of a fibroblast population of a particular individual and are not related to the patient’s chronological age.

Analysis of SA-βgal positive cells in different types of colonies

Cells from different types of colonies had different morphological appearance, with those in diffuse colonies reminiscent of senescent cells. We therefore determined the fraction of SA-βgal+ cells in all three types of colonies of human skin fibroblasts. Indeed, the highest fraction of SA-βgal+ cells (40.6±4.9%) was found in the diffuse colony phenotype, whereas the dense colonies contained the lowest fraction of such cells (9.6±1.2%) (Figure 2). Both mixed and diffuse colonies contained significantly (p=0.002 and p=0.0001, respectively) higher fraction of SA-βgal+ cells. This observation is consistent with the proposed subdivision of human skin fibroblasts into three types [10]. Therefore, the fraction of diffuse colonies may be representative of the fraction of cells that are close to exhausting their proliferative capacity.

Relationship of diffuse colonies to proliferation and degree of senescence

To examine the relationship of the fraction of diffuse colonies to proliferation in a fibroblast culture, we measured the fraction of proliferating cells in mass cultures using the Ki67 marker, which is in our opinion an excellent marker for quantification of the growth fraction of a given cell population (Figure 3A). The fractions of Ki67+ cells were quantified and are presented in Table 3. In parallel, to assess the level of cellular aging in cultures of human fibroblasts, we measured the fraction of SA-βgal+ cells in mass cultures (Table 3). The fractions of both of SA-βgal+ and Ki67+ cells failed to correlate (r=-0.17; p=0.32 and r=0.18; p=0.31, respectively) with the chronological age of the human donors. However, since the donors participated in this study may be grouped into a single age category, the range of the ages may not be great enough to detect a possible correlation of SA-βgal+ cells with chronological age. A correlation between SA-βgal+ and Ki67+ cells was revealed (r=-0.77; p=0.01), which was expected and simply reflects the fact that senescent cells lose their ability to proliferate. Subsequently, we examined the correlation between the fraction of Ki67+ cells and the four clonogenic endpoints (Figure 3B). We found the highest statistical degree of inverse correlation between the Ki67+ fraction and the fraction of diffuse colonies (r=-0.85, p=0.02) (Figure 3B). In contrast, for the rest of the clonogenic endpoints (ECO-f, the fraction of dense colonies and the fraction of mixed colonies), a strong positive correlation was observed (r=0.71, р=0.02; r=0.59, р=0.07; r=0.72, р=0.02, respectively). Further we examined correlations between the colony phenotypes and other endpoints related to fibroblasts proliferation or their degree of aging (Figure 3C). Statistically significant (p=0.0001) inverse correlation between the fraction of diffuse colonies and the clonogenic capacity was found, whereas the first endpoint demonstrated a significant positive correlation with the fraction of SA-βgal+ cells in both mass culture and dense colonies (р=0.013 and p=0.002, respectively). Dense colonies are in essence a monolayer of cells and the percentage of SA-βgal+ cells in them may be reflective of an overall aging status of a mass culture. This data suggests that the fraction of diffuse colonies may be informative for evaluating the degree of aging of fibroblast cultures. Lastly, we examined whether the fraction of Ki67+ cells in mass cultures correlated with the same four endpoints (Figure 3D). Mirror correlation patterns to those for diffuse colonies (Figure 3D) were observed, with one exception for the fraction of dense colonies which was not statistically significant (р=0.07).

Fibroblast isolation and culturing

All human donors provided written consent for this study. Ethics approval for the study was granted by the the Russian Healthcare Regulation Authority and the Ethics Committee and Academic Council of the Central Research Institute of Dental and Maxillofacial Surgery (#4/276). A skin biopsy was obtained with a disposable punch from behind the ear auricle under local infiltrative anesthesia with the 2% lidocaine solution. The isolated skin fragment (appx. 4 mm³) was immediately transferred to the labeled sterile container with shipment medium (DMEM/F12). Thereafter the biomaterial was transferred to a Petri dish under laboratory sterile conditions and washed three times with Hank’s solution supplemented with antibiotic (gentamycin) in Gibco^® Versene solution (0.2 g EDTA(Na₄₎ per liter of Phosphate Buffered Saline (PBS), pH 7.4) manufactured by Thermo Fisher Scientific, USA. The tissue homogenate was prepared by sequential dispersing with a sterile scalpel, the addition of the disaggregating 0.1% collagenase solution, and the incubation of disaggregating solution at 37° C for 1-1.5 hours.

After the incubation, the tissue suspension was vigorously mixed by the pipette and centrifuged at 300 g for 10 minutes. The supernatant was discarded, and the pellet was resuspended again in the tissue culture medium (DMEM/F12, 1:1) containing 10% defined bovine fetal serum (FBS) (HyClone, USA) and 40 ug/ml of gentamicin (TCM). The resulting suspension was transferred into a culture vial and incubated under the 5% CO₂ atmosphere at 37° C in CO₂-incubator. The culture medium was changed every 3-4 days.

At 70% of confluency, the cells were washed with Gibco^® Versene solution and detached from the surface of a culture flask by use of Gibco^® Versene solution and 0.25% trypsin at 37°C for 3-5 minutes. The cell suspension was diluted in a culture medium and explanted into a larger cultural flask for subsequent cultivation.

Clonal fibroblast analysis

At second cell passage, the cell monolayer was washed three times with Gibco^® Versene solution and trypsinized at 37°C, 5% CO₂ for 10 min. The homogenate was centrifuged at 300 g for 10 minutes. The supernatant fluid was discarded, cells were resuspended in Hank’s solution, and counted in Gorjaev’s chamber.

The cells were diluted by TCM to obtain final concentration 100 cells/ml. The cell suspension was split into three 100 mm Petri dishes to obtain a clonal inoculum density of 1.5 cells/cm².

The Petri dishes were incubated in a CO₂-incubator at the saturated humidity conditions at 37°C in the 5% CO₂ atmosphere for 14 days. Thereafter, the culture dishes with the pre-formed colonies were washed three times with PBS (рH 7.4 and fixed with 70% alcohol for 15 minutes at room temperature. Then alcohol residuals were removed by triple washing with distilled water, and the colonies were stained with a KaryoMAX® Giemsa Stain Stock Solution produced by Gibco, USA, for 20 minutes at 37°C. The dishes with the stained colonies were thoroughly washed from excessive stain and dried at room temperature for 5-7 hours.

Thereafter, the analysis of the colonies was carried out. All of the colonies were ranked in three groups: dense, diffuse and mixed colonies. The colony forming effectiveness of the fibroblasts (ECO-f) was determined according to Fridenshtein’s equation for stromal progenitor cells: ECO-f = a ratio between the number of pre-formed colonies and the number of explanted cells multiplied by 100% [7,33]. The only counted colonies include more than 20 cells of the total number of explanted cells. The clones of fibroblasts which include fewer cells were not considered as colonies and, accordingly, were not included in the count.

Analysis of β-galactosidase positive cells

To quantify the proportion of β-galactosidase positive cells the commercial kit “Cellular Senescence Assay» (EMD Millipore, USA, Catalog Number: KAA002) was used. 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 (Figure 2). 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.

Immunocytochemical analysis of Ki67 positive cells

For immunocytochemical analysis cells were seeded at the density of 5 × 10³ cells/cm² in 500 μL of culture medium onto coverslips placed inside 35 mm Petri dishes (Corning, USA). To improve adhesion of cells additional volume of culture medium (1.5 mL) was added into Petri dishes 15 minutes after seeding. Cells seeded on coverslips were incubated at 37°C and 5% CO₂ for at 48 h prior to fixation.

Cells were fixed on coverslips in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature followed by two rinses in PBS and permeabilization for 40 min with 0.3% Triton-X100 (in PBS, pH 7.4) supplemented with 2% bovine serum albumin (BSA) to block non-specific antibody binding. Cells were then incubated for 1 hour at room temperature with primary mouse monoclonal antibody against Ki67 protein (dilution 1:400, clone Ki-S5, Merck-Millipore, USA) which were diluted in PBS with 1% BSA. Following several rinses with PBS, cells were incubated for 1 hour at room temperature with secondary antibodies IgG (H+L) goat anti-mouse (Alexa Fluor 488 conjugated, dilution 1:600; Merck-Millipore, USA) with 1% BSA. Coverslips were then rinsed several times with PBS and mounted on microscope slides with ProLong Gold medium (Life Technologies, USA) with DAPI for DNA counter-staining. Cells were viewed and imaged using Nikon Eclipse Ni-U microscope (Nikon, Japan) equipped with a high definition camera ProgRes MFcool (Jenoptik AG, Germany). Filter sets used were UV-2E/C (340–380 nm excitation and 435–485 nm emission) and B-2E/C (465–495 nm excitation and 515–555 nm emission). At least 200 cells per data point were counted.

Statistical analysis

Statistical and mathematical analyses of the data were conducted using the Statistica 8.0 software (StatSoft). Data points in Figures are mean values obtained from three independent experiments; error bars are standard errors. Statistical significance was tested using the Student t-test at p < 0.05.