Serial changes in the proliferation and differentiation of adipose-derived stem cells after ionizing radiation

Abstract

Background: Adipose-derived stem cells (ASCs) are important to homeostasis and the regeneration of subcutaneous fat. Hence, we examined the proliferation and differentiation capacity of irradiated ASCs over time.

Methods: Two female pigs received a single 18 Gy dose of ionizing radiation to an 18 × 8 cm area on the dorsal body skin via a 6 MeV electron beam. After irradiation, the ASCs were cultured from adipose tissue harvested from a non-irradiated area and an irradiated area at 2, 4, and 6 weeks. The proliferation capacity of ASCs was evaluated by a colony-forming units-fibroblasts (CFUs-Fs) assay, a cholecystokinin (CCK) test with 10 % fetal bovine serum (FBS), and a 1 % FBS culture test. The senescence of ASCs was evaluated through morphological examination, immunophenotyping, and β-galactosidase activity, and the multipotent differentiation potential of ASCs was evaluated in adipogenic, osteogenic, and chondrogenic differentiation media.

Results: Irradiated ASCs demonstrated significantly decreased proliferative capacity 6 weeks after irradiation. As well, the cells underwent senescence, which was confirmed by blunted morphology, weak mesenchymal cell surface marker expression, and elevated β-galactosidase activity. Irradiated ASCs also exhibited significant losses in the capacity for adipocyte and chondrocyte differentiation. In contrast, osteogenic differentiation was preserved in irradiated ASCs.

Conclusions: We observed decreased proliferation and senescence of irradiated ASCs compared to non-irradiated ASCs 6 weeks after irradiation. Furthermore, irradiated ASCs demonstrated impaired adipocyte and chondrocyte differentiation but retained their osteogenic differentiation capacity. Our results could shed light on additional pathogenic effects of late irradiation, including subcutaneous fibrosis and calcinosis.

Keywords: Cell differentiation; Cell proliferation; Mesenchymal stromal cells; Radiation; Senescence; Swine.

Fig. 1

The simulation of irradiation level using simulation software. The 18 Gy dose of radiation level is delineated by the red line

Fig. 2

Proliferation capacity. a Macroscopic and microscopic view of colony-forming units (CFUs) determined by crystal violet staining. The number of viable ASC colonies formed was significantly more abundant in the N groups than in the 6R group. b Quantitative analysis of CFUs. CFU formation in the 6 N group was 2.5-fold higher than in the 6R group (^* p < 0.05). c Cell Counting Kit-8 (CCK-8) assay with 10 % fetal bovine serum (FBS). The cell numbers in the 6R group were significantly lower than numbers in the N group after day 7 (^** p < 0.01). d CCK-8 assay with 1 % FBS to analyze cellular growth under stress conditions. The cell numbers in the 6R group were higher than in the other groups throughout the entire experimental period. However, statistically significant differences were not obtained

Fig. 3

Senescence of irradiated ASCs. a Crystal violet staining. Cell morphology was inhomogeneous, smaller, and blunter in the 6R group compared to the other groups. b β-galactosidase immunostaining. Positive β-galactosidase staining (arrowhead) was rarely observed in the N groups, but was readily apparent in the 6R group. c Immunophenotyping by flow cytometry. All groups demonstrated reduced expression of the hematopoietic surface markers CD 31 and CD41. The 6R groups demonstrated lower CD90 expression than the other groups

Fig. 4

Adipogenic differentiation. a Bright field view. Adipogenic differentiation was not observed in the 6R group. b Oil Red O staining. Loss of adipogenic differentiation in the 6R group was confirmed by Oil Red O staining. c Leptin analysis. The secretion of leptin hormone from adipocytes was significantly lower in the 6R group than in the 6 N group (^* p < 0.05). d RT-PCR for PPAR-γ and aP2. The levels of PPAR-γ and aP2 mRNA were significantly lower in the 6R group than in the 6 N group (^* p < 0.05)

Fig. 5

Chondrogenic differentiation. a Bright field view. All of the groups exhibited chondrogenic differentiation. b H&E staining (first row) demonstrated that cultured cartilage cells are prominently increased in the 2 N group in the absence of necrosis or apoptosis. However, cartilage cells are decreased and substituted for necrotic cells and apoptosis in the 4R and 6R groups. Alcian blue staining (second row) likewise demonstrated that cultured cartilage cells declined remarkably as the weeks after exposure to radiation progressed. Immunohistochemistry for type II collagen (third row) demonstrated positivity for cytoplasmic localization of the cultured cartilage cells in the 2 N group. Viable cartilage cells are markedly reduced and replaced with necrotic cells with negative collagen type II antibody expression in the 4R and 6R groups. c Sulfated glycosaminoglycan (sGAG) assay. The level of sGAG was significantly lower in the 6R group than in the 6 N group (^* p < 0.05). d RT-PCR for aggrecan and type II collagen did not reveal any statistically significant differences

Fig. 6

Osteogenic differentiation. a Bright field view. Calcium deposition (black dots) was scattered throughout the entire area after osteogenic differentiation in all groups. Calcium deposition was condensed and became a mineralized spot (brown and black area) in all groups. b Alkaline phosphatase (AP) activity. All of the groups demonstrated AP activity. Although the 6R group demonstrated the small and round shape morphology of senescent cells, AP activity was also detected in the senescent cells. c Alizarin Red S staining. Mineralization of osteogenically differentiated ASCs was confirmed in all groups by Alizarin Red S staining. d RT-PCR for osteocalcin and type I collagen. No statistically significant differences in osteocalcin type I collagen mRNA levels were detected between the groups