Telomere attrition occurs during ex vivo expansion of human dental pulp stem cells

Abstract

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

Figure 1

Phase contrast micrograph of DPSCs in culture. (a) Small colonies (CFU-F) formed by DPSCs appeared in primary culture after 3 days in vitro. (b) Spindle-shaped morphology of cells in culture reaching 70% confluence. Scale bars 50 μm.

Figure 2

The growth kinetics of DPSC lines. (a) Cumulative population doublings of 11 DPSCs lines are shown. The cultures were derived from individual patients and passaged as described in Section 2. The number of population doublings was calculated as follows: PD = (log₁₀n_f−log₁₀n_i)/0.301, where  n_i is the initial number of cells and n_f is the final number of cells at each passage. (b, c) Data of doubling time reached by DPSCs from male (b) and female (c) donors at different passages in culture. The linear regression line best fitting the data is shown, R = 0.71, P < .0001. The doubling time was calculated from the formula DT = log₁₀2T/log₁₀n_f−log₁₀n_i, where T is time in hrs. R = 0.56, P < .0001. Filled circles indicate males and open circles indicate females.

Figure 3

Flow cytometric analysis of cell surface marker expression in DPSCs at different passages. Results are expressed as arithmetic mean ± SEM.

Figure 4

Immunocytochemical detection of analyzed markers in cultured DPSCs. (a) STRO-1 expression was readily detectable in all cells. (b) Immunostaining of vimentin revealed a regular filamentous cytoskeletal network within the cells with signal accentuation in perinuclear region. (c) Nestin expression appeared in the cell body, however, in cytoplasmic outgrowths the signal was lower but still distinguishable. Intensive signal is localized in dividing cells. (d) CXCR4 was localized mainly in the cytoplasm, namely, in its perinuclear area; some signal was also distinguishable within the cell nuclei. (e) Sox2 was distributed mainly in cell nuclei; signal in perinuclear cytoplasm likely reflects synthesis of this transcription factor in endoplasmic reticulum. (f) Nucleostemin was observed in nodular structures in cell nuclei; in mitotic cells or cells that had just finished cytokinesis (arrowheads) immunoreactivity was lost from the nucleus and distributed diffusely. (g) CD90 immunofluorescence showed homogenous dot-like membrane staining pattern. (h) CD166 was localised in membrane of all DPSCs contouring the cell edges. Nuclei were counterstained with DAPI. Arrows indicate dividing cells. Scale bars 50 μm.

Figure 5

Osteogenic and chondrogenic potential of DPSCs. (a) Phase contrast micrograph of osteogenic nodule after 4 week-cultivation in differentiation medium. (b) Histological examination revealed multilayered structure consisting of osteoblast-like cells surrounded by extracellular matrix containing collagen fibres; Ladewig blue trichrome. (c) Immunoperoxidase histochemistry of osteonectin confirmed the presence of this bone-specific glycoprotein that links minerals to collagen within the micromass pellets. (d) Chondrogenic micromass bodies were composed of cells and extracellular matrix. Alcian blue staining confirmed the presence of sulphated acidic glycosaminoglycans, a principal component of cartilaginous ground substance. Scale bars (a)-(b) 100 μm, (c) 200 μm, (d) and 50 μm.

Figure 6

A standard curve generated from a reference DNA sample serially diluted by dilution factor 2 to produce six concentrations of DNA ranging from 0.625 to 20 ng of DNA per reaction. Shown inside are the correlation regression coefficients (R²) of Ct versus the amount of DNA. Filled circles indicate telomeres and open circles indicate 36B4 control.

Figure 7

Plot of average telomere length (relative T/S) against passage number and doubling time versus T/S. (a) Relative telomere length expressed as ratio of T/S by quantitative real-time PCR analysis shows a decrease in T/S with increasing passage numbers. (b) Plot of doubling time in hours against relative telomere length (T/S) and regression line. The correlation coefficients of the linear regression line best fitting the data are shown. Filled circles indicate males (n = 5 individuals) and open circles indicate females (n = 6).