Differential Effects of X-Rays and High-Energy ⁵⁶Fe Ions on Human Mesenchymal Stem Cells

Purpose

Stem cells hold great potential for regenerative medicine, but they have also been implicated in cancer and aging. How different kinds of ionizing radiation affect stem cell biology remains unexplored. This study was designed to compare the biological effects of X-rays and of high–linear energy transfer (LET) ⁵⁶Fe ions on human mesenchymal stem cells (hMSC).

Methods and Materials

A multi-functional comparison was carried out to investigate the differential effects of X-rays and ⁵⁶Fe ions on hMSC. The end points included modulation of key markers such as p53, cell cycle progression, osteogenic differentiation, and pathway and networks through transcriptomic profiling and bioinformatics analysis.

Results

X-rays and ⁵⁶Fe ions differentially inhibited the cell cycle progression of hMSC in a p53-dependent manner without impairing their in vitro osteogenic differentiation process. Pathway and network analyses revealed that cytoskeleton and receptor signaling were uniquely enriched for low-dose (0.1 Gy) X-rays. In contrast, DNA/RNA metabolism and cell cycle regulation were enriched for high-dose (1 Gy) X-rays and ⁵⁶Fe ions, with more significant effects from ⁵⁶Fe ions. Specifically, DNA replication, DNA strand elongation, and DNA binding/transferase activity were perturbed more severely by 1 Gy ⁵⁶Fe ions than by 1 Gy X-rays, consistent with the significant G2/M arrest for the former while not for the latter.

Conclusions

⁵⁶Fe ions exert more significant effects on hMSC than X-rays. Since hMSC are the progenitors of osteoblasts in vivo, this study provides new mechanistic understandings of the relative health risks associated with low- and high-dose X-rays and high-LET space radiation.

Introduction

Stem cells hold great potential for regenerative medicine because they can self-renew and differentiate along tissue-specific lineages. Mesenchymal stem cells (MSCs), the multipotent stromal cells from bone marrow, play important roles in the maintenance and repair of various tissues, including bone, cartilage, and muscle (1). The differentiation of MSCs into osteoblasts is controlled by the transcription factor Runx2 (2). Stem cells also respond to genotoxic stresses, for example, ionizing radiation (IR), which may lead to the induction of mutations and chromosome aberrations, transient cell-cycle arrest, apoptosis, mitotic catastrophe, cellular senescence, or malignant transformation. Abnormalities in stem cell biology have been implicated in cancer and aging, but the mechanisms are poorly understood (3). For example, MSCs have been shown as a target for neoplastic transformation following manipulations 4, 5. However, major challenges still remain to define the normal molecular signaling and tumorigenic pathways in stem cells.

Because of their high tissue-penetrating property, IR such as X-rays/gamma rays is used in CT scans, cancer radiotherapy, and nuclear medicine. However, IR is also a well-known DNA-damaging agent, clastogen, and carcinogen 6, 7. During deep space missions, space crews encounter an elevated radiation environment that includes high-energy–charged (HZE) particles, such as ⁵⁶Fe ions from the galactic cosmic rays (8). Such densely ionizing radiation induces more severe clustering of damage on DNA compared to X-rays, and gives rise to complex DNA double-strand breaks (DSBs) that are more prone to be misrepaired and cause permanent genomic changes (9). Another complicating factor during space travel is the possibility of synergistic relationships with other risk factors, such as microgravity. The possibility that HZE radiation affects the differentiation process of human mesenchymal stem cells (hMSC) into osteoblasts, important for bone regeneration and loss in space because of microgravity, has not been investigated.

Advances in systems biology have opened up new possibilities for elucidating the molecular mechanisms underlying cellular responses to IR. Global gene expression changes in response to IR have been reported for Saccharomyces cerevisiae(10), murine brain (11), normal human skin fibroblasts (12), and human lung fibroblasts (13). These studies suggested that IR regulates gene expression at the transcriptional level in a dose-dependent manner and depending on radiation quality, for example, X-rays vs. HZE particles. On the other hand, proteomic and transcriptomic studies of hMSC have provided important insights into their proliferation and differentiation 14, 15. However, no direct correlations between phenotypic radioresponses and pathway regulations have been reported for hMSC, particularly in the context of osteogenic differentiation.

Here we describe the first comprehensive study of the radioresponses of hMSC to X-rays and high energy ⁵⁶Fe ions. We show that neither X-rays nor ⁵⁶Fe ions impaired the in vitro osteogenic differentiation process of hMSC. We further show that they differentially regulated the cell cycle progression of hMSC in a dose-dependent and p53-dependent manner. Pathway and network analyses of transcriptomic profiles revealed the underlying mechanisms.