First Insights into the Effect of Low-Dose X-Ray Irradiation in Adipose-Derived Stem Cells

Abstract

(1) Background: Emerging interest of physicians to use adipose-derived stem cells (ADSCs) for regenerative therapies and the fact that low-dose irradiation (LD-IR ≤ 0.1 Gy) has been reported to enhance the proliferation of several human normal and bone-marrow stem cells, but not that of tumor cells, lead to the idea of improving stem cell therapies via low-dose radiation. Therefore, the aim of this study was to investigate unwanted side effects, as well as proliferation-stimulating mechanisms of LD-IR on ADSCs. (2) Methods: To avoid donor specific effects, ADSCs isolated from mamma reductions of 10 donors were pooled and used for the radiobiological analysis. The clonogenic survival assay was used to classify the long-term effects of low-dose radiation in ADSCs. Afterwards, cytotoxicity and genotoxicity, as well as the effect of irradiation on proliferation of ADSCs were investigated. (3) Results: LD (≤ 0.1 Gy) of ionizing radiation promoted the proliferation and survival of ADSCs. Within this dose range neither geno- nor cytotoxic effects were detectable. In contrast, greater doses within the dose range of >0.1-2.0 Gy induced residual double-strand breaks and reduced the long-term survival, as well as the proliferation rate of ADSCs. (4) Conclusions: Our data suggest that ADSCs are resistant to LD-IR. Furthermore, LD-IR could be a possible mediator to improve approaches of stem cells in the field of regenerative medicine.

Keywords: X-ray; adipose-derived stem cells; low-dose radiation; mesenchymal stem cells; radiation therapy; regenerative medicine.

Figure 1

Clonogenic survival of pooled adipose-derived stem cells (pADSCs) after X-ray irradiation. pADSCs of 10 different donors were exposed to low (0.01, 0.05, and 0.1 Gy) and moderate (0.5, 1.0, and 2.0 Gy) doses of X-rays. Twenty days later, the cells were stained by crystal violet to visualize formed colonies. The cell survival fractions were normalized to those of sham irradiated cells. (a) Data from five independent experiments are presented as mean values ± SEM of the survival fraction. The significances refer to the 0 Gy control. Asterisks illustrate significances: ** p < 0.01, *** p < 0.002 (one sample t-test). (b) enlarged view of the low-dose area.

Figure 2

Lactate dehydrogenase (LDH) release as marker for cytotoxicity in pADSCs 24 and 48 h after X-ray irradiation. LDH activity was measured using the colorimetric LDH assay. The LDH release was normalized to this of the positive control. Data from three independent experiments are presented as mean values ± SEM (Mann–Whitney U Test).

Figure 3

Capacity of pADSCs to repair DNA double strand breaks induced by irradiation (IR). The number of phosphorylated H2AX per nucleus was used to determine the corresponding number of DNA double strand breaks. To identify the DNA damaging effect of IR and the capacity of pADSCs to repair them, the cells were fixed 0.5 and 24 h after IR and incubated thereafter with anti-γH2AX antibody and IgG1 (red), as well as the DNA counterstaining with 4,6-diamidino-2-phenylindole (DAPI, blue). (a) Exemplary images of immune-cytochemistry staining after IR. (b) Results are illustrated as the mean number of γH2AX foci per cell ± standard deviation (SD) of three independent experiments; asterisks illustrate significant differences to * shame irradiated cells (control): * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed t-test and two-way ANOVA with Bonferroni post-test).

Figure 4

Alteration of the proliferation rate in pADSCs within 24 (a) and 48 h (b) following the irradiation procedure. Proliferation was determined by a colorimetric BrdU ELISA assay. Results are illustrated as mean ± standard deviation (SD) of five independent experiments. Asterisks illustrate significant differences referring to sham irradiated cells: * p < 0.02; ** p < 0.01; *** p < 0.002 (one sample t-test and two-way ANOVA with Bonferroni post-test).

Figure 5

Content of matrix metalloproteinase-2 (MMP-2) in the supernatant of adipose-derived stem cells (ADSCs) 6, 24, and 48 h after irradiation. Results are illustrated as mean ± standard deviation (SD) of three independent experiments. Asterisks illustrate significant differences referring to sham irradiated cells: * p < 0.05; *** p < 0.005 (two-tailed t-test and two-way ANOVA with Bonferroni post-test).