Effects of acute exposure to low-dose radiation on the characteristics of human bone marrow mesenchymal stromal/stem cells

Abstract

Background: In recent years, increasing attention has been paid to the effects of low-dose irradiation on human health. We examined whether low-dose irradiation affected the functions of mesenchymal stromal/stem cells (MSCs), which are tissue/organ-supportive stem cells, derived from bone marrow (BM).

Methods: Normal human BM-MSCs from five healthy individuals were used in this study. Culture-expanded BM-MSCs were exposed to 0.1 gray (Gy) of γ-radiation (Cesium-137) at a rate of 0.8 Gy/min (Ir-MSCs), and their expansion, multi-differentiation, and hematopoiesis-supportive capabilities were investigated.

Results: The expansion of BM-MSCs was transiently delayed after low-dose γ-irradiation compared with that of non-irradiated BM-MSCs (non-Ir-MSCs) in two out of five lots. Adipogenic and osteogenic differentiation capabilities were not significantly affected by low-dose irradiation, although one lot of BM-MSCs tended to have transiently reduced differentiation. When human BM hematopoietic stem/progenitor cells (HPCs) were co-cultured with Ir-MSCs, the generation of CD34⁺CD38⁺ cells from HPCs was enhanced compared with that in co-cultures with non-Ir-MSCs in two out of five lots. The mRNA expression level of interleukin (IL)-6 was increased and those of stem cell factor (SCF) and fms-related tyrosine kinase 3 ligand (Flt3L) were decreased in the affected lots of Ir-MSCs. In the other three lots of BM-MSCs, a cell growth delay, enhanced generation of CD34⁺CD38⁺ cells from HPCs in co-culture, and a combination of increased expression of IL-6 and decreased expression of SCF and Flt3L were not observed. Of note, the characteristics of these affected Ir-MSCs recovered to a similar level as those of non-Ir-MSCs following culture for 3 weeks.

Conclusions: Our results suggest that acute exposure to low-dose (0.1 Gy) radiation can transiently affect the functional characteristics of human BM-MSCs.

Keywords: Bone marrow mesenchymal stromal/stem cells; Human; Low-dose irradiation.

Fig. 1

Surface marker expression of human BM-MSCs. Flow cytometric analysis shows that lot A of BM-MSCs is positive for mesenchymal stem cell-associated surface markers (CD73, CD90, and CD105) and is negative for hematopoietic cell-associated surface markers (CD14, CD19, CD34, and CD45). Filled histograms indicate control staining. Numbers in each histogram indicate the percentage of cells. The same surface marker expression profiles were confirmed in lots B–E of BM-MSCs

Fig. 2

Expansion of Ir-MSCs. (a–e) BM-MSCs (lots A–E) were exposed to (Ir-MSCs, dotted lines) or not exposed to (non-Ir-MSCs, solid lines) 0.1 Gy γ-radiation on day 0. They were then cultured until confluent. The number of viable cells was counted weekly after γ-irradiation using 0.5% trypan blue. Data are mean values ± SD. n = 5 per group (a–c) or n = 3 per group (d, e). *, p < 0.05

Fig. 3

Characteristics of H-MSCs. BM-MSCs (lots A and B) were exposed to 4 Gy (H-MSCs) or 0.1 Gy (Ir-MSCs) γ-radiation, or were not exposed to γ-radiation (non-Ir-MSCs). (a, b) Expansion of lots A and B of H-MSCs (dashed lines) and non-Ir-MSCs (solid lines). The number of viable cells was counted weekly after γ-irradiation using 0.5% trypan blue. (c) Morphology of lots A and B of H-MSCs, Ir-MSCs, and non-Ir-MSCs. Representative phase contrast images are shown. Yellow arrows indicate H-MSCs. Bars, 20 μm. (d, e) Expression of adipogenic and osteogenic markers in lots A and B of H-MSCs (gray bars), Ir-MSCs (open bars), and non-Ir-MSCs (solid bars), as assessed by quantitative real-time PCR. Expression in H-MSCs and Ir-MSCs is shown relative to that in non-Ir-MSCs. Data are mean values ± SD (a, b, d, e). n = 4 per group (a, d) or n = 5 per group (b, e). *, p < 0.05; **, p < 0.01

Fig. 4

Microarray analysis of Ir-MSCs. (a) Venn diagram of the number of genes whose expression levels were downregulated in lots A, B, C, and D of Ir-MSCs compared with non-Ir-MSCs. The purple, green, gray, and orange ellipses represent genes in lots A, B, C, and D, respectively. (b) Pathway analysis was performed of 1495 genes that were downregulated in both lots A and C of Ir-MSCs, but not in lot B or D of Ir-MSCs. The pathway “G1 to S cell cycle control” was significantly enriched. (c) GSEA of genes from lots A, B, C, and D of Ir-MSCs and non-Ir-MSCs using the gene set AMUNDSON_POOR_SURVIVAL_AFTER_GAMMA_RADIATION_2G. NES normalized enrichment score, FDR false discovery rate

Fig. 5

Adipogenic and osteogenic differentiation of Ir-MSCs. (a) Quantitative measurement of the adipogenic differentiation of lot A of BM-MSCs that were exposed to (white bar) or not exposed to (black bar) 0.1 Gy γ-radiation, as assessed by Oil Red O staining. (b) Representative images are shown. Yellow arrows indicate lipid-laden fat cells. Bars 250 μm. (c) Quantitative measurement of the osteogenic differentiation of lot A of BM-MSCs that were exposed to (white bar) or not exposed to (black bar) 0.1 Gy γ-radiation, as assessed by Alizarin Red S staining. (d) Representative images are shown. Bars 250 μm. (e–g) Adipogenic and osteogenic differentiation of Ir-MSCs at the late phase. (e) Schema of culture of BM-MSCs after γ-irradiation. (f, g) Quantitative measurement of the adipogenic (f) and osteogenic (g) differentiation of lot A of BM-MSCs that were exposed to (white bars) or not exposed to (black bars) 0.1 Gy γ-radiation and then cultured for a further 3 weeks (Ir-MSCs at late phase), as assessed by Oil Red O staining and Alizarin Red S staining, respectively. (a, c, f, g) Data are mean values ± SD. n = 5 per group

Fig. 6

Adipogenic and osteogenic differentiation of three different lots of Ir-MSCs. (a–c) Quantitative measurement of the adipogenic differentiation of three different lots of BM-MSCs (lots B, C, and D) that were exposed to (white bars) or not exposed to (black bars) 0.1 Gy γ-radiation, as assessed by Oil Red O staining. (d–f) Quantitative measurement of the osteogenic differentiation of three different lots of BM-MSCs (lots B, C, and D) that were exposed to (white bars) or not exposed to (black bars) 0.1 Gy γ-radiation, as assessed by Alizarin Red S staining. Lot E of BM-MSCs was unavailable for this analysis due to an inadequate number of cells. Data are mean values ± SD. n = 4 per group (a, d) or n = 3 per group (b, c, e, f). *, p < 0.05

Fig. 7

Co-culture of HPCs with Ir-MSCs. Flow cytometric analysis showing the generation of hematopoietic cells from HPCs after 10 days of co-culture with lot A (a, b) and lot B (c, d) of BM-MSCs that were exposed to (white bars) or not exposed to (black bars) 0.1 Gy γ-radiation. The numbers of CD45⁺ cells, CD34⁺ cells, CD34⁺CD38⁻ cells, and CD34⁺CD38⁺ cells are shown in (a, c). Data are mean values ± SD. n = 5 per group. *, p < 0.05. Representative dot plots of CD34 versus CD38 expression are shown in (b, d). Numbers in each box indicate the percentage of cells

Fig. 8

Generation of hematopoietic cells from HPCs in co-culture with Ir-MSCs. (a–c) The numbers of CD45⁺ cells, CD34⁺ cells, CD34⁺CD38⁻ cells, and CD34⁺CD38⁺ cells in co-culture with three different lots of BM-MSCs (lots C, D, and E) that were exposed to (white bars) or not exposed to (black bars) 0.1 Gy γ-radiation are shown. Data are mean values ± SD. n = 5 per group. *, p < 0.05

Fig. 9

Expression of hematopoiesis-associated molecules in Ir-MSCs. (a, b) mRNA expression of various hematopoiesis-associated molecules in lot A of BM-MSCs that were exposed to (Ir-MSCs, white bars) or not exposed to (non-Ir-MSCs, black bars) 0.1 Gy γ-radiation, as assessed by quantitative real-time PCR analysis. Molecules whose expression levels differed between Ir-MSCs and non-Ir-MSCs are shown in (a), whereas those whose expression levels did not differ between Ir-MSCs and non-Ir-MSCs are shown in (b). Expression in Ir-MSCs is shown relative to that in non-Ir-MSCs. Data are mean values ± SD. n = 5 per group. *, p < 0.05; **, p < 0.01. (c) mRNA expression of molecules affected in lot A of BM-MSCs was examined in lot B of BM-MSCs that were exposed to (Ir-MSCs, white bars) or not exposed to (non-Ir-MSCs, black bars) 0.1 Gy γ-radiation, as assessed by quantitative real-time PCR analysis. Data are mean values ± SD. n = 5 per group

Fig. 10

Graphical summary. Acute exposure to low-dose γ-radiation (0.1 Gy) could affect the functional characteristics of human BM-MSCs at the early phase. However, these changes were transient and recovered at the late phase