Mesenchymal stem cells are resistant to carbon ion radiotherapy

Abstract

Mesenchymal stem cells (MSCs) participate in regeneration of tissues damaged by ionizing radiation. However, radiation can damage MSCs themselves. Here we show that cellular morphology, adhesion and migration abilities were not measurably altered by photon or carbon ion irradiation. The potential for differentiation was unaffected by either form of radiation, and established MSC surface markers were found to be stably expressed irrespective of radiation treatment. MSCs were able to efficiently repair DNA double strand breaks induced by both high-dose photon and carbon ion radiation. We have shown for the first time that MSCs are relatively resistant to therapeutic carbon ion radiotherapy. Additionally, this form of radiation did not markedly alter the defining stem cell properties or the expression of established surface markers in MSCs.

Figure 1. Mesenchymal stem cells exhibit different radiation sensitivities to photon and carbon ion irradiation

(A) Clonogenic survival assays for three different MSCs after treatment with photon (upper panel) or carbon ion radiotherapy (lower panel). Error bars represent standard deviation. (B) Images of unstained MSCs showing no measurable difference in morphology after treatment with different doses of photon or carbon ion irradiation (20x objective, scale bar 100μm).

Figure 2. Photon or carbon ion irradiation do not impair the adhesion or migration abilities of MSCs

(A) Relative adhesion rates of MSCs up to 24 hours after treatment with 10 Gy photon or 4 Gy carbon ion irradiation. (B) Average velocity of MSCs after irradiation as assessed by time-lapse microscopy. Error bars show standard deviation.

Figure 3. Photon or carbon ion irradiation do not impair the differentiation potential of MSCs

(A) Oil red O staining for adipogenic differentiation in two MSC samples after irradiation with 10 Gy photon or 4 Gy carbon ion irradiation. Pictures were taken at 100x magnification (B) Alcian blue staining of MSC spheroids after treatment, 40x magnification. (C) Relative expression levels of adipogenic, chondrogenic and osteogenic differentiation markers after radiotherapy. Error bars represent standard deviation.

Figure 4. The expression of MSC surface marker genes is not influenced by photon or carbon ion irradiation

(A) Heat map showing similar patterns of downregulation (green panel) or upregulation (red panel) in MSCs after irradiation with 10 Gy photons or 4 Gy carbon ions. (B) Relative expression levels of positive and negative MSC surface marker genes after irradiation. Error bars represent standard deviation.

Figure 5. Photon and carbon ion irradiation of MSCs leads to G2 phase arrest but no increase in apoptosis

Cell cycle distribution of MSC1 (A) and MSC2 (B) cells after treatment with 10 Gy photon radiation (upper panels) or 4 Gy carbon ion radiation (lower panels). (C, D) Percentage of apoptotic MSC1 and MSC2 cells after 10 Gy photon or 4 Gy carbon ion irradiation as assessed by sub-G1 population and caspase-3 activation. Error bars show standard deviation.

Figure 6. MSCs efficiently repair DNA double strand breaks induced by photon or carbon ion radiotherapy

(A) Number of γH2AX foci in MSCs at various time points after irradiation with different photon and carbon ion doses as measured by immunostaining. *** P<0.001 (B) Sample pictures of γH2AX foci at high photon and carbon doses (400x magnification). (C) Western blot analyses of phosphorylated ATM protein at 2 and 24 hours after irradiation with photons or carbon ions.