International Journal of

Radiation Oncology

biology physics

www.redjournal.org

Biology Contribution

Mesenchymal Stem Cells Retain Their DefiningStem Cell Characteristics After Exposure toIonizing RadiationNils H. Nicolay, MD, PhD,*,y Eva Sommer, DVM,y Ramon Lopez, MSc,y

Ute Wirkner, PhD,y Thuy Trinh,*,y Sonevisay Sisombath,y Jurgen Debus, MD, PhD,*Anthony D. Ho, MD,z Rainer Saffrich, PhD,z and Peter E. Huber, MD, PhD*,y

*Department of Radiation Oncology, Heidelberg University Hospital, Heidelberg, Germany; yDepartment of Molecular andRadiation Oncology, German Cancer Research Center, Heidelberg, Germany; and zDepartment of Hematology andOncology, Heidelberg University Hospital, Heidelberg, Germany

Received Jun 18, 2013, and in revised form Aug 16, 2013. Accepted for publication Sep 3, 2013.

Summary

The sensitivity of bonemarrow-derived mesen-chymal stem cells (MSCs) toionizing radiation (IR) islargely unknown. In ouranalysis, MSCs exhibiteda relative radioresistance;even high doses of IR did notsignificantly affect thedefining stem cell character-istics of MSCs, which werefound to be still able toadhere, proliferate, anddifferentiate into functionaltissue cells.

Reprint request to: Nils H. Nicolay, MD, Ph

ular and Radiation Oncology, German Cancer R

Int J Radiation Oncol Biol Phys, Vol. 87, No. 5

0360-3016/$ - see front matter � 2013 Elsevie

http://dx.doi.org/10.1016/j.ijrobp.2013.09.003

Purpose: Mesenchymal stem cells (MSCs) have the ability to migrate to lesion sites andundergo differentiation into functional tissues. Although this function may be important fortissue regeneration after radiation therapy, the influence of ionizing radiation (IR) on cellularsurvival and the functional aspects of differentiation and stem cell characteristics of MSCs haveremained largely unknown.Methods and Materials: Radiation sensitivity of human primary MSCs from healthy volunteersand primary human fibroblast cells was examined, and cellular morphology, cell cycle effects,apoptosis, and differentiation potential after exposure to IR were assessed. Stem cell geneexpression patterns after exposure to IR were studied using gene arrays.Results: MSCs were not more radiosensitive than human primary fibroblasts, whereas therewere considerable differences regarding radiation sensitivity within individual MSCs. Cellularmorphology, cytoskeletal architecture, and cell motility were not markedly altered by IR. Evenafter high radiation doses up to 10 Gy, MSCs maintained their differentiation potential.Compared to primary fibroblast cells, MSCs did not show an increase in irradiation-inducedapoptosis. Gene expression analyses revealed an upregulation of various genes involved inDNA damage response and DNA repair, but expression of established MSC surface markersappeared only marginally influenced by IR.Conclusions: These data suggest that human MSCs are not more radiosensitive than differenti-ated primary fibroblasts. In addition, upon photon irradiation, MSCs were able to retain theirdefining stem cell characteristics both on a functional level and regarding stem cell markerexpression. � 2013 Elsevier Inc.

D, Department of Molec-

esearch Center (dkfz), Im

Neuenheimer Feld 280, 69120 Heidelberg, Germany. Tel: (49) 6221-42-

2587; E-mail: n.nicolay@dkfz.de

Conflict of interest: none.

, pp. 1171e1178, 2013

r Inc. All rights reserved.

Nicolay et al. International Journal of Radiation Oncology � Biology � Physics1172

Introduction

Mesenchymal stem cells (MSCs) were first isolated from adult bonemarrow but can be detected in other tissues, including adiposetissue, kidney, skin, umbilical cord, and placenta (1, 2). MSCs arecharacterized by a fibroblast-like appearance, their ability to adhereto plastic surfaces and proliferate in culture, and their osteogenic,adipogenic, and chondrogenic differentiation potential (2). Incontrast to hematopoietic stem cells, MSCs form a heterogeneouspopulation and are therefore often addressed as multipotentmesenchymal stromal cells (3, 4). To date, no generally acceptedpattern of unique surface markers has been described, althoughdifferent cell surface proteins have been discussed as potentialmeans of prospectively identifying MSCs (4, 5).

MSCs are believed to facilitate tissue repair because of theirability to integrate into organ tissues and differentiate into func-tional cells and also through growth factor and cytokine secretion(6). Preclinical data and early clinical trials have shown beneficialeffects of tissue damage treatment with MSCs for myocardial scars,cartilage injuries, and lung, skin, and nerve tissue defects (7, 8).

Radiation therapy is a mainstay in tumor treatment. Ionizingradiation (IR) targets cells by inducing DNA damage; depending onthe type and amount of damage, cells fail to recognize or repair thelesions, resulting in cell cycle arrest, apoptosis, or mutagenesis. Indi-vidual tissues reveal considerably different sensitivities to IR. Intrinsiccellular radiation sensitivities have been attributed to differences in theability to performDNA repair (9). It is well known that hematopoieticstem cells are exquisitely sensitive to IR (10), but the radiation sensi-tivity of other somatic stem cells is largely unknown.

In this study, we examined the radiation response of humanMSCs compared to primary human differentiated fibroblasts inrespect to cellular survival, mobility, differentiation potential, andcell cycle effects. In addition, gene expression was analyzed beforeand after exposure to IR to assess for potential gene regulationphenomena underlying the radiation response of MSCs.

Methods and Materials

Cells and cultures

Human MSC283, MSC284, and MSC285 cells were harvestedfrom the bone marrow of 3 healthy volunteer donors and isolated

Fig. 1. Mesenchymal stem cells (MSCs) exhibit similar radiationshowing differences in radiation sensitivity among 3 individual MSCsdifferent primary fibroblast cell types. Error bars show standard erro***P<.001.

as published previously (11). Cells were grown in MSC basalmedium (Cambrex, Wiesbaden, Germany), supplemented withSingleQuots (Cambrex). Untransformed normal primary humandermal fibroblasts (NHDFs) and HS68 human fibroblast cells werepurchased from Promocell (Heidelberg, Germany) and AmericanType Culture Collection (Manassas, VA), respectively. NHDFswere maintained in endothelial cell basal medium (Promocell)containing 500 mg/L gentamycin and 0.01mg/L amphotericin B.HS68 cells were grown in Dulbecco’s modified Eagle medium(Biochrom, Berlin, Germany) containing 10% fetal calf serum and3.5 g/L glucose. All donor-derived cell samples were harvestedafter written consent was obtained from volunteers according toethics guidelines, and this study was approved by the HeidelbergUniversity Hospitals ethics board.

Clonogenic survival assays

Cells were plated (500 cells for 0 and 2 Gy; 2500 cells for 4 Gy)and allowed to attach before IR treatment. Radiation experimentswere performed at room temperature, using a 6-MeV linearaccelerator (dose rate of 3 Gy/min). After treatment, cells wereallowed to form colonies (6-10 days for fibroblasts, 21 days forMSCs). Colonies were fixed with 25% acetic acid (v/v) in meth-anol and stained using crystal violet solution. Colonies containingmore than 50 cells were counted using a light microscope. Allclonogenic assays were performed in triplicate. The survivingfraction was calculated according to the following formula: (no. ofcolonies/no. of plated cells)treated/(no. of colonies/no. of platedcells)untreated.

Cell morphology analysis

Cell morphology after treatment was analyzed using unstainedcells at �200 magnification. To assess treatment effects on thecytoskeleton, we irradiated log-phase cells grown on cover slipswith 10 Gy and fixed after 24 hours with 3.7% paraformaldehydesolution for 10 minutes before washing and treatment with acetonefor 5 min at �20�C. Actin filaments were then stained with 5 mMAlexa Fluor-633-coupled phalloidin (Invitrogen, Karlsruhe,Germany) diluted in phosphate-buffered saline containing 5%methanol and visualized using a fluorescence microscope at 400�magnification.

sensitivities as adult fibroblasts. (A) Clonogenic survival assays. (B) Clonogenic assays comparing pooled MSC survival with 2r of the mean. Statistical analysis performed by Student t test;

Fig. 2. Ionizing irradiation (IR) treatment does not significantly alter mesenchymal stem cell (MSC) morphology and ability to migrate.(A) Unstained samples of 3 MSCs and 2 adult fibroblast cell types showing no marked difference of cellular morphology at differentradiation doses (�20 objective; bar Z 100 mm). (B) Immunocytochemistry images indicating similar actin filament structures betweenuntreated and irradiated cells (�40 objective; bar Z 50 mm). (C) Absolute (upper panel) and relative (lower panel) velocity of stem cellsand adult fibroblasts at different radiation doses. Error bars represent standard error of the mean.

Volume 87 � Number 5 � 2013 MSCs keep functions after irradiation 1173

Electrical impedance sensing

Cellular resistance in culture was assessed by electrical cellsubstrate impedance sensing. Cells were irradiated with a singledose of 10 Gy and resuspended in growth medium before beingplated in multiwell plates containing gold electrodes 6 hours aftertreatment. Electrodes were connected to a phase-sensitive lock-inamplifier, and an AC current was applied to the electrodes ata frequency of 45,000 Hz. Impedance at the surface between cellsand growth medium was measured over a period of 45 hours.

Cell differentiation experiments

Log-phase cells were plated in 24-well plates and left to attachbefore being irradiated with 10 Gy. At 24 hours after treatment,differentiation medium was added to each well, and cells weregrown for another 2 weeks. Osteogenic differentiation was inducedwith Dulbecco’s modified Eagle medium supplemented with 10%fetal calf serum, 2 mM L-glutamine, 100 nM dexamethasone, 200mM L-ascorbic acid-2-phosphate, 10 mM b-glycerophosphate, and100 U/mL penicillin/streptomycin (12). To differentiate adipocytes,cells were grown in Dulbecco’s modified Eagle medium containing

10% fetal calf serum, 2 mM L-glutamine, 1 mM dexamethasone,500 mM 1-methyl-3-isobutylxanthine, 10 mg/mL insulin, and 100 U/mL penicillin/streptomycin (2). Differentiation medium wasexchanged twice weekly. Osteogenic differentiation was assessedusing von Kossa staining. After washing and fixation with ethanoland acetone for 30 min at 4�C, cells were stained with AgNO3 for10 min before exposure to UV light and refixation with 5% sodiumthiosulfate solution. Hematoxylin staining was then performed for90 min. Differentiation of adipocytes was demonstrated by Oil RedO staining. Briefly, after washing and fixation with 4% para-formaldehyde, cells were treated with 60% isopropanol for 5 minbefore staining with Oil Red dissolved in 60% isopropanol. Cellswere exposed to hematoxylin for 2 min and covered in glycerol.Pictures were taken using a light microscope at 100�magnification.

Cell cycle analysis

To analyze cell cycle profiles, cells were harvested and washedbefore fixation with ice-cold 70% ethanol. Cell pellets were incu-bated with 10 mg/mL propidium iodide solution containing 200 mg/mL RNase A. Fluorescence-assisted cell sorting was performed

Fig. 3. Irradiation does not influence the ability of mesenchymal stem cells (MSCs) to adhere to plastic surfaces. (A) Electrical cellsubstrate impedance sensing curves of MSCs and adult fibroblasts up to 45 hours after plating. (B) Quantitative analysis of impedancesensing revealed no significant differences between irradiated and untreated cells for both stem cells and adult fibroblasts. Error barsrepresent standard error of the mean.

Nicolay et al. International Journal of Radiation Oncology � Biology � Physics1174

with a FACScan system (Becton-Dickinson, Heidelberg, Germany).A total of 10,000 events were counted for each experiment, and cellcycle profiles were modeled using Cell Quest Pro software (BDBiosciences, San Jose, CA).

Gene expression analysis

Irradiation effects on MSCs were analyzed using a whole-humangenome microarray 4x44k (product no. G4112F; Agilent Tech-nologies, Boblingen, Germany). Cells were proliferated toa density of 70% before irradiation with 10 Gy or mock irradia-tion. At 6 hours after treatment, cells were harvested, and RNAwas extracted using an RNeasy mini-kit (Qiagen, Hilden,Germany). Data were extracted with Agilent, version 9.1, featureextraction software and analyzed. Statistical analysis was per-formed using paired Student t test.

Results

MSCs and adult fibroblasts exhibit similarsensitivities to IR

To compare radiation sensitivities of MSCs and adult humanfibroblasts, clonogenic assays of bone marrow-derived MSCs from3 different donors and 2 adult fibroblast cell types were per-formed. The 3 MSC samples showed considerable differencesregarding survival after exposure to IR, with MSC283 being themost sensitive type at 4 Gy (Fig. 1A; P<.005, Student t test).

To rule out sample-specific effects, results for all 3 stem cellpopulations were pooled to compare with adult fibroblasts. In thisanalysis, MSCs did not exhibit a significant difference regardingsensitivity to IR compared to finite life-span primary skin fibro-blasts (NHDFs) and the HS68 fibroblast cell line (PZ.18 for MSCsvs HS68; PZ.19 for MSCs vs NHDFs; Student t test) (Fig. 1B).

IR influences cellular morphology and motility ofMSCs

At 4 Gy, cell morphology for both MSCs and adult fibroblast cellsappeared unchanged (Fig. 2A). After exposure to 10 Gy, cells

increased in size, with reduction in the number of mitoses. Nomorphological signs of increased apoptosis were detected at 24hours after irradiation, using light microscopy.

To assess IR-induced changes in the cytoskeletal architecture,mock-treated and irradiated cells were stained for actin filamentsat 24 hours after IR using Alexa Fluor-633-coupled phalloidin.Irradiated cells exhibited no visible differences regarding theircytoskeleton compared to untreated cells (Fig. 2B).

Cell motility was assessed over a period of 62 hours afterradiation. MSCs exhibited a lower velocity over time than HS68and NHDF cells for both mock-treated and irradiated samples.Irradiation with both 10 Gy and 20 Gy did result in a small butnonsignificant reduction in absolute and relative velocities foreach cell line, and there was no significant difference betweenMSCs and adult fibroblasts regarding cellular motility (Fig. 2C).

IR does not impede MSC adherence

Cellular adherence was measured using electrical cell substrateimpedance sensing. Comparing MSC285 and NHDF cells afterirradiation with 10 Gy, impedance increased for several hours afterplating for both cell lines, reflecting the process of initial adher-ence (Fig. 3A). Over the total time course of 45 hours, there wasno significant difference in electrical impedance between irradi-ated and mock-treated cells. Impedance levels of MSC285 andNHDF cells were comparable throughout the duration of theexperiment, suggesting that even high doses of irradiation do notaffect the inherent ability of MSCs to adhere to plastic surfaces(Fig. 3B).

IR does not affect the differentiation potential ofMSCs

The potential for osteogenic and adipogenic differentiation isa hallmark of MSCs. In order to assess whether this intrinsicability to differentiate was still present after exposure to IR, MSCswere treated with a single radiation dose of 4 Gy or 10 Gy, anddifferentiation features were studied by immunocytochemistry asdescribed above.

Irradiation did not abrogate the ability of MSCs to producecalcium phosphate crystals as assessed by von Kossa staining(Fig. 4A). Similarly, the potential for adipogenic differentiation

Fig. 4. Mesenchymal stem cells (MSCs) retain their osteogenic and adipogenic differentiation abilities after exposure to ionizingradiation (IR). (A) Von Kossa staining for osteogenic differentiation in 2 MSC samples. (B) Oil red O staining of adipogenically differ-entiated MSC283 and MSC285. All pictures were taken at �100 magnification.

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after radiation treatment with 4 and 10 Gy was tested bymeasuring lipid inclusions using Oil Red O staining. Even afterhigher radiation doses, all tested MSC samples kept the potentialfor adipogenic differentiation after culturing in differentiationmedium for 14 days (Fig. 4B).

Taken together, these data suggest that the ability of MSCs todifferentiate is not inhibited by exposure to IR.

MSCs show no increase in apoptosis following IR

Cell cycle profiles of MSCs and adult fibroblasts were analyzedafter IR treatment using fluorescence-activated cell sorting. More

than 80% of untreated MSCs were found in G1 phase of the cellcycle compared to 55% of HS68 and 48% of NHDF cells, cor-responding to their considerably longer doubling time (Fig. 5A).For MSCs, IR treatment with 10 Gy led to a small but significantincrease in the percentage of G2 phase cells (P<.05, Studentt test). In comparison, both adult fibroblast cell lines exhibiteda large increase in G2 phase, suggesting a radiation-induced G2arrest. An increase in the percentage of G1-phase cells in theuntreated controls over the time course of the experiment is likelydue to an increased cell density on the culture plates, leading toa reduction in growth speed (Figs. 5B and 5C).

The sub-G1 population was examined as a readout for IR-induced apoptosis. All 3 MSC samples showed no increase in

Fig. 5. Irradiation of mesenchymal stem cells (MSCs) results in G2 phase arrest but no increase in apoptosis. Cell cycle profiles of MSCs(A) and adult fibroblasts (B, C) after treatment with 10 Gy. NHDF, normal primary human dermal fibroblasts. (D) Percentage of apoptoticcells after 10 Gy irradiation as assessed by sub-G1 population. Error bars show standard error of the mean. Statistical analysis performed byStudent t test; *P<.05.

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the percentage of sub-G1cells after exposure to 10 Gy IR. Incomparison, the HS68 cell line had a small but significantincrease in sub-G1 cells after exposure to 10 Gy IR starting at48 hours (0.7% vs 3.8%; P<.05; Student t test). This effect waseven more pronounced in primary NHDF cells which exhibiteda 30-fold increase in sub-G1 cells at 96 hours after IR treatmentwith 10 Gy (0.7% vs 30.0%; P<.01; Student t test) (Fig. 5D).

IR affects gene expression pattern of MSCs

Gene expression was analyzed after MSC283 and MSC285 cellswere treated with a single IR dose of 10 Gy, using a whole-humangenome microarray. IR led to significant changes in the expressionof 664 of 23,643 assessed genes (P<.005; Student t test) (Fig. 6A).The expression levels of stem cell markers and growth factorreceptors were measured in untreated MSCs and in stem cells 6hours after treatment with 10 Gy IR. As expected, the MSCsurface markers CD13, CD29, CD44, CD73, CD90, CD105, andCD106/VCAM-1 were all highly expressed in both MSC283 andMSC285 cells, whereas the hematopoietic surface markers CD31,CD34, CD45, and CD116 were not significantly expressed(Fig. 6B). Radiation led to small alterations in some of theestablished MSC markers as shown in Figure 6B, although for all

markers, the regulation levels were well below 2-fold. Mostgrowth factors relevant for the upkeep of the MSC-like propertieswere expressed. The BMP-6 and RUNX-2 genes, as markers forosteogenic differentiation, were highly expressed but were notfound to be significantly altered following IR treatment; similarly,PPARG as the marker for adipogenic differentiation was stablyexpressed after irradiation.

Discussion

This study examined the impact of radiation on the functionalproperties of human MSCs in comparison to those of differenti-ated fibroblast cells. In this analysis, bone marrow-derived MSCsshowed a considerable level of resistance to IR, comparable to thatof differentiated fibroblasts.

Radiation sensitivity is influenced by the ability of the cell tocope with the deleterious effects of IR on its genomic information.Therefore, failure to repair radiation-induced damage may result inthe loss of proliferative ability and eventually in cellular death. Wefound that even after high doses of IR, MSCs did not reveal anincrease in apoptosis levels. It has been suggested previously thatMSCs undergo retinoblastoma protein 1-induced prematuresenescence rather than apoptosis following irradiation (13, 14).

Fig. 6. Mesenchymal stem cell (MSC) surface marker genes are stably expressed after irradiation. (A) Heat map showing the top 50genes that were found either downregulated (green panel) or upregulated (red panel) upon irradiation with 10 Gy. (B) Relative expressionpatterns of positive and negative stem cell surface marker genes of 2 MSC samples after ionizing irradiation treatment.

Volume 87 � Number 5 � 2013 MSCs keep functions after irradiation 1177

However, in those datasets, very high single doses up to 20 Gy wereused; here we found that cellular viability was unaffected even atthese high radiation levels. For lower doses up to 10 Gy, we foundthat MSCs were still able to proliferate after exposure to IR.

As described for a variety of other cells lines, irradiationresulted in an increase of G2 phase cells for both MSCs andprimary fibroblasts (15, 16). However, the overall cell cycledistribution of MSCs varied significantly from that of primaryfibroblasts, and the cell doubling time of MSCs in this analysisappeared almost 3 times longer than that of primary fibroblasts.Previous data have suggested a correlation between prolonged celldoubling time and a relative increase in radiation resistance,leaving cells more time to efficiently repair IR-induced DNAdamage (17, 18). Additionally, it has been shown that MSCsexhibit increased activity of the nonhomologous end-joiningproteins Ku70 and DNA-PK upon irradiation, further linking theobserved radiation resistance to an increased level of DNA repair(19). Accordingly, using gene array data from irradiated MSCs,we found an upregulation of genes involved in the repair of DNAlesions caused by IR. For example, PCNA, ATM, and CHK2 wereupregulated upon irradiation. Ataxia teleangiektasia mutated(ATM)-dependent DNA damage recognition has been described asa key pathway in MSCs after exposure to IR, and upregulation of

the DNA damage recognition pathways has been attributed toincreased radiation resistance in MSCs (20, 21).

As MSCs consist of a heterogeneous population of cells,functional characteristics have been published that serve to iden-tify these stem cells: MSCs can adhere to plastic surfaces andexpand in vitro and undergo osteogenic, adipogenic, or chondro-genic differentiation upon induction (2, 4, 22). However, theinfluence of IR on these hallmarks of MSCs has not been eluci-dated. We found that cellular morphology of MSCs was notmarkedly altered and that there were also no apparent effects of IRon the actin cytoskeleton. Additionally, irradiated MSCs did notlose their ability to proliferate and expand in vitro after treatmentwith IR, and we did not detect any visible radiation-induceddifferentiation. Similarly, another study using MSCs that wereirradiated in vivo showed that doses up to 9 Gy had no prolongedeffect on the cells’ proliferative ability (23). These findingsdemonstrate a clear difference between MSCs and primaryfibroblasts, which undergo induced terminal differentiation uponirradiation and permanently lose their ability to divide (24, 25).

The potential for osteogenic or adipogenic differentiation repre-sents a hallmark for MSCs, and the data presented here show thatMSCs could differentiate into osteoblasts or fat cells after exposure tohigh radiation doses. The osteogenic markers BMP-6 and RUNX-2

Nicolay et al. International Journal of Radiation Oncology � Biology � Physics1178

and the adipogenic marker PPARG showed stable expressionfollowing IR. One other study reported a dose-dependent reduction inthe differentiation potential of MSCs after irradiation, although inthat analysis, the stem cells did not completely lose their differenti-ation ability even after treatment with high single doses (26).

In our study, analysis of microarray data demonstrated highexpression of the established positive MSC surface markers CD13,CD29, CD44, CD29, CD73, CD105, and CD106/VCAM-1 and theabsence of the negative markers CD31, CD34, CD45, and CD116both before and after treatment with IR, which in turn may serveas a validation criterion for the analysis. In addition to the phys-iological characteristics of MSCs described above, these markershave been established to differentiate MSCs from other bonemarrow-derived and other stromal cells (2, 27, 28).

In vivo data testing of the response of MSCs to tissue irradi-ation have so far produced conflicting findings. Whereas Singhet al (23) reported no damaging effect on MSCs after in vivoirradiation up to 9 Gy, Cao et al (29) found a significant reductionin cell numbers after IR treatment.

Conclusions

In summary, our data show that bone marrow-derived MSCs arerelatively resistant to IR compared to differentiated fibroblasts. Wehave demonstrated for the first time that even high radiation dosesmay not alter the functional characteristics of MSCs.

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