In vitro polydeoxyribonucleotide effects on human pre-adipocytes

Abstract

Objectives: Adipose tissue is the most abundant and accessible source of adult stem cells. Human processed lipoaspirate contains pre-adipocytes that possess one of the a characteristic pathways of multipotent adult stem cells and are able to differentiate in vitro into mesenchymal and also neurogenic lineages. Because stem cells have great potential for use in tissue repair and regeneration, it would be significant to be able to obtain large amounts of these cells in vitro. As demonstrated previously, purine nucleosides and nucleotides mixtures can act as mitogens for several cell types. The aim of this study was to evaluate the effects of polydeoxyribonucleotides (PDRN), at appropriate concentrations, on human pre-adipocytes grown in a controlled medium, also using different passages, so as to investigate the relationship between the effect of this compound and cellular senescence, which is the phenomenon when normal diploid cells lose the ability to divide further.

Materials and methods: Human pre-adipocytes were obtained by liposuction. Cells from different culture passages (P6 and P16) were treated with PDRN at different experimental times. Cell number was evaluated for each sample by direct counting after trypan blue treatment. DNA assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test were also carried out in all cases.

Results and conclusions: PDRN seemed to promote proliferation of human pre-adipocytes at both passages, but cell population growth increased in pre-adipocyte at P16, after 9 days as compared to control. Our data suggest that PDRN could act as a pre-adipocyte growth stimulator.

Figure 1

Senescence‐associated β‐galactosidase assay in pre‐adipocytes at the 6th (a) and 16th (b) passages in vitro. The blue‐green staining represents senescent cells (original magnification ×100).

Figure 2

BrdU staining in human pre‐adipocytes at the 6th (P6) and 16th (P16) passages in vitro. The axis of graphics illustrate propidium iodide (PI) versus secondary antibody Alexa Fluor 488 F(ab′) fragment of goat antimouse IgG (H + L); 1% pre‐adipocytes at P6 and 2% pre‐adipocytes at P16 are in S phase.

Figure 3

CFSE staining in human pre‐adipocytes at the 6th (P6) and 16th (P16) passages at different times (1, 2, 5 and 9 days).

Figure 4

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 16th passage in vitro. Cells were cultured in the experimental conditions in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Values are expressed as an arbitrary unit (the absorbance was measured spectrophotometrically at 570 nm), and represent the mean ± SEM of three separate experiments in quadruplicate. *P < 0.05, compared to control by analysis of variance followed by Dunnett's test.

Figure 5

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 6th passage in vitro. Cells were cultured in the experimental conditions in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Values are expressed as an arbitrary unit (the absorbance was measured spectrophotometrically at 570 nm) and represent the mean ± SEM of three separate experiments in quadruplicate.

Figure 6

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 16th passage in vitro. Cells were cultured in the experimental conditions in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Cell counting was measured by Trypan blue staining. Values are expressed as a cell number and represent the mean ± SEM of three separate experiments in quadruplicate.

Figure 7

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 6th passage in vitro. Cells were cultured in the experimental conditions in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Cell counting was measured by Trypan blue staining. Values are expressed as a cell number and represent the mean ± SEM of three separate experiments in quadruplicate.

Figure 8

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 16th passage in vitro. Cells were cultured in the experimental conditions in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Cell proliferation was estimated by referring fluorescence units to a linear standard curve for DNA fluorescence versus cell number, and represent the mean ± SEM of two separate experiments in duplicate. *P < 0.05, compared to control by analysis of variance followed by Dunnett's test.

Figure 9

Effect of PDRN on the growth of cultured human pre‐adipocytes at the 6th passage in vitro. Cells were cultured in the experimental conditions, in the absence and in the presence of 80 µg/mL PDRN for 3, 6, 9, 12 and 15 days. Cell proliferation was estimated by referring fluorescence units to a linear standard curve for DNA fluorescence versus cell number, and represent the mean ± SEM of two separate experiments in duplicate. *P < 0.05, compared to control by analysis of variance followed by Dunnett's test.

Figure 10

Immunostaining with Ki‐67 antibody: (a) in untreated pre‐adipocytes (original magnification ×600) and (b) in pre‐adipocytes treated with PDRN (100 µg/mL) (original magnification ×600), as shown by the arrows.