Cytokinesis-Block Micronucleus Assay Adaptedfor Analyzing Genomic Instability

of Human Mesenchymal Stem Cells

Deborah Afonso Cornelio,1 Joana Cristina Medeiros Tavares,2 Thais Valeria Costa de Andrade Pimentel,1

Geraldo Barroso Cavalcanti Jr.,3 and Silvia Regina Batistuzzo de Medeiros1

Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of theproblems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain asuitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measuresgenetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclearbuds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodologyto analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The bestprotocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration(4.0mg/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). Theseadaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. Thefrequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alter-ations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, ahigh frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitroculture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, theCBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safeclinical use of hMSCs in cell therapy.

Introduction

Human mesenchymal stem cells (hMSCs), also knownas multipotent stromal cells [1], have been described as

being extremely important to the success of regenerativemedicine procedures [2,3]. They are multipotent adult stemcells with the capacity to self-renew, multilineage in vitrodifferentiation, secrete bioactive molecules, and regulate im-mune responses [4,5]. They are easily isolated from a numberof different organs and tissues, including bone marrow, skin,lung, liver, adipose tissue, placenta, and the umbilical cord [6].Moreover, they are easily expanded in in vitro culture, wherethey demonstrate excellent proliferation capacity, making theex vivo expansion the strategy of choice for producing a largenumber of cells to use in cell therapy—considering that only avery small number of hMSCs can be isolated from bonemarrow and other tissues, an insufficient amount for clinicalapplications in certain regenerative medicine procedures [7].

A number of authors have shown that human stem cellsremain morphologically and genetically stable after in vitro

culture [8–17]. However, one of the main problems that canarise in stem cell lines isolated from different sources is thepossibility of genetic instability during cell expansion [18,19].Although the occurrence of karyotypic instability in culturedhMSCs has been documented, it remains controversial[19–29], and only two groups demonstrated the occurrence ofin vitro spontaneous transformation of hMSCs isolated frombone marrow [30,31]. Some researchers defend the hypothesisthat human stem cells derived from bone marrow do nottransform spontaneously in vitro and that aneuploidy canoccur without leading to malignant transformation of hMSCs,possibly being only a sign of senescence [32,33].

To draw a firm conclusion regarding the biosecurity ofthese cells, a larger number of hMSC cultures must be an-alyzed and more genetic tests need to be included in order toassess genetic integrity before any treatment involving celltherapy. The cytokinesis-block micronucleus (CBMN) assayconsists of analyzing nuclear alterations in binucleated cellsthat were impeded from completing cytokinesis through theuse of cytochalasin-B (cyt-B), enabling the accumulation of

1Departamento de Biologia Celular e Genetica, Centro de Biociencias, Universidade Federal do Rio Grande do Norte, Natal, Brazil.2Faculdade de Ciencias da Saude do Trairi (FACISA)–UFRN, Santa Cruz, Brazil.3Departamento de Analises Clınicas e Toxicologicas, Centro de Ciencias da Saude, UFRN, Natal, Brazil.

STEM CELLS AND DEVELOPMENT

Volume 23, Number 8, 2014

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2013.0383

823

nearly all the cells that divided in the binucleated stage [34].Although CBMN analysis is less resolutive and less infor-mative than metaphasic chromosome analysis using the Gbanding technique, it is much easier and faster [35], since itallows analysis of a larger number of cells and applicationof statistical tests, and the detection and classification ofnuclear alterations do not require as extensive personneltraining as human karyotype analysis.

The CBMN assay can assess a number of nuclear alter-ations, such as micronucleus (MN), nucleoplasmic bridges(NPBs), and nuclear buds (NBUDs). The MN is the result ofchromosomal breakage and/or loss caused by errors in DNArepair or in chromosomal segregation. Therefore, MNcharacterizes whole chromosomes or acentric fragments notincluded in the main nucleus that are surrounded individu-ally by a nuclear membrane [36]. Analysis of MN in in-terphasic cells of cell smears has been used to detectchromosomal alterations that arise due to the aneugenic andclastogenic effects caused by a variety of compounds. It isbeing used in the analysis of exfoliated cells to detect pre-cancerous and cancerous lesions, determining high-riskgroups for tumors in the oral cavity, urinary tract (bladder),cervix, and esophagus [37,38]. The frequency of micro-nucleated cells increases in tissues exposed to carcinogensbefore any clinical symptom emerges. Therefore, the MN testin exfoliated epithelial cells is a biomarker of occupationalexposure to genotoxic chemical agents [39] and a possibleindicator of early signs of carcinogenesis. The NPBs indi-cate the occurrence of rearrangements in which chromatidsor chromosomes are pulled to opposite poles during theanaphase: dicentric chromosomes, ring chromosomes, andchromosomes involved in telomeric associations. In CBMN,binucleated cells with NPBs are easily observed because ofcytokinesis inhibition by cyt-B, thereby avoiding the breakageof anaphase bridges from which NPBs derive. Therefore, thenuclear membrane forms around the bridge that links the twonuclei, making it possible to detect these chromosomal al-terations [40]. The NBUDs are morphologically similar to theMN, except that they remain connected to the nucleus. Theyare alterations that indicate amplified DNA removed from thecell nucleus and therefore a marker of gene amplification [36].NBUDs can also form when an NPB between two nucleibreaks and the remnants shrink back toward the nuclei. Fur-ther, NBUDs can occur temporarily after the NPB breaks [40].Recent data have demonstrated that NPBs and NBUDs arealso useful biomarkers for monitoring genetic damage, sincethey are easily recognizable nuclear alterations that allow ra-pid analysis and counting. MN, NPBs, and NBUDs are nuclearalterations characteristic of chromosomally unstable cells of-ten detected in tumor cells and peripheral blood of patientswith cancer [41]. The frequency of nuclear alterations ob-served by CBMN is a relevant biomarker for the risk of lungcancer and melanoma, and can be used for early detection ofthe disease, making it a useful tool in clinical and epidemio-logical studies [42,43]. The CBMN method also allows as-sessment of the impact of micronutrients in the diet and thecombination of micronutrients on DNA damage, revealing thecarcinogenic or protective potential of certain diets [44,45].

CBMN is a reliable method for detecting and quantifyingDNA damage and chromosomal instability in human cells,initially developed to analyze peripheral blood lymphocytecultures [34], but adapted for applications in established cell

lines, solid-tumor cultures, bone marrow, and even in re-constructed human epidermis [46–50].

Most genotoxicity tests have been conducted in humancell lines immortalized in vitro; however, they do not rep-resent normal cell types. Some authors have suggested theuse of human embryonic stem cells (hESCs) and inducedpluripotent stem cells (iPSCs) [51] in toxicological studiesand drug research as a new strategy for determining thesafety of new chemicals [52–56]. However, there are anumber of ethical, religious, and political issues related tothe use of hESCs, and the iPSCs derive from somatic cellsthat need to be genetically handled to return to a state ofpluripotency [51]. hMSCs are primary multipotent cells thatrepresent normal cell types; that is, they are isolated andcultured with no genetic handling whatsoever and, therefore,can be used as a cell model in drug research in order todiscover the most efficient chemicals and make therapeuticbiomonitoring safer. To that end, genotoxicity tests could becarried out with these cells exposed to chemicals, but thereare no studies of CBMN using hMSCs for that purpose.

Accordingly, for the first time, the present study describesCBMN assay methodology with the necessary adaptations toanalyze hMSCs isolated from the umbilical vein and assessthe genetic instability of these hMSCs in vitro expanded.

Materials and Methods

hMSC isolation and cell culture

The procedure used in this study was approved by theUFRN Ethics Committee under protocol No. FR132464,following Brazilian guidelines (Resolution 196/96). Writteninformed consent was obtained from mothers to collect freshhuman umbilical cord specimens from three full-termhealthy newborns (two boys, originating primary cell cul-tures hMSC1 and hMSC2; and one girl, originating pri-mary cell culture hMSC3). The hMSCs for this study wereisolated from umbilical veins and expanded in vitro as previ-ously described [57]. Briefly, the umbilical vein was cathe-terized, cleaned until it was free of excess blood, and filledwith a 0.5% collagenase type IV solution (Worthington).After enzymatic disaggregation, the cell suspension was col-lected after gentle massaging of the cord and by washingwith phosphate-buffered saline (PBS). The cell pellet was re-suspended in alpha-minimum essential medium (a-MEM;Gibco–Invitrogen) supplemented with 20% fetal bovine serum(FBS; HyClone) and 1% antibiotic solution (prepared with10,000 U/mL penicillin G sodium and 10,000mg/mL strepto-mycin sulfate; Gibco–Invitrogen). The cells were plated ontoT25 tissue culture flasks (TPP) and cultures were kept at 37�Cin a humidified atmosphere containing 5% CO2. hMSC cul-tures reaching 60%–70% confluence were harvested for ex-pansion using 0.25% trypsin/1 mM EDTA (Gibco–Invitrogen);cells were counted using the trypan blue (Corning–CellGro)exclusion test and replated at 3,000–4,000 viable cells/cm2

with a-MEM supplemented with 10% FBS and 1% antibioticsolution. This procedure was equivalent to one passage andwas repeated every three days approximately. The number ofdoublings in cell populations (PD) was calculated according tothe following formula PD = 3.32 · (log10Nt - log10No), whereNo is number of viable cells at seeding and Nt is number ofviable cells at harvest. The medium culture was changed andnonadherent cells were removed at least twice a week.

824 CORNELIO ET AL.

Characterization of hMSCs: determinationof the cell-surface antigen profileand multilineage differentiation potential

In accordance with the minimum requirements for definingmultipotent mesenchymal stromal cells established by theInternational Society for Cellular Therapy [1], hMSCs cul-tured in this study were phenotypically characterized usingflow cytometry and induced to differentiate into osteoblasts,adipocytes, and chondroblasts under differentiation condi-tions and protocols that we previously reported [57]. Tophenotypically characterize hMSCs and define their purity,cells were labeled with monoclonal antibodies to analyze thecell-surface expression of typical marker proteins: CD105FITC

and CD90PE-Cy5 (purchased from Bioscience), and CD73PE,CD34PE, HLA-DRFITC, CD45FITC, and CD14PE (from BectonDickinson). For each test, isotype-matched monoclonal anti-body was used as a negative control (IgG 1-FITC, PE, andPE-Cy5; Becton Dickinson). Flow cytometer analysis wasconducted with a fluorescence activated cell analyzer(FACScan) using Cell Quest Software (Cell QuestTM Soft-ware; Becton Dickinson Immunocytometry Systems).

Cells were seeded and cultured into six-well plates (TPP)to induce osteogenic differentiation. When cultures weresubconfluent (80%), hMSCs were incubated in osteogenicmedium: a-MEM with 10% FBS/1% antibiotic solutionsupplemented with 50mM ascorbate 2-phosphate (Sigma-Aldrich), 0.01mM dexamethasone (Sigma-Aldrich), and10 mM b-glycerolphosphate (Fluka Sigma-Aldrich). Adipo-genic differentiation induction occurred when subconfluent(80%) cultures of hMSCs that were seeded into six-wellplates (TPP) were treated with adipogenic medium: Dulbec-co’s modified Eagle’s medium high glucose (DMEM-HG;Gibco–Invitrogen) and 15% FBS/1% antibiotic solutionsupplemented with 5mg/mL insulin from bovine pancreas(Sigma-Aldrich), 1mM dexamethasone (Sigma-Aldrich), and60mM indomethacin (Cayman Chemical Company). Forchondrogenic differentiation, 2 · 105 hMSCs were centri-fuged to form a cell pellet that was not disturbed and wascultured for 21 days in DMEM-HG and 1% FBS/1% anti-biotic solution supplemented with 0.5mg/mL insulin frombovine pancreas (Sigma-Aldrich), 10 ng/mL transforminggrowth factor beta 1 (Sigma-Aldrich), and 50mM ascorbate 2-phosphate (Sigma-Aldrich). After 21 days of treatment inspecific differentiation medium, cells treated with osteogenicand adipogenic medium were fixed, and the chondrogenicpellets were fixed with 10% buffered formalin and embeddedin paraffin. Osteoblast differentiation was demonstrated bystaining the mineralized extracellular matrix with AlizarinRed S (Sigma-Aldrich), adipocyte differentiation was dem-onstrated by staining lipid-containing vacuoles with Oil RedO (Sigma-Aldrich), and chondroblast differentiation wasdemonstrated by Alcian Blue (Sigma-Aldrich) staining of theextracellular matrix rich in sulfated glycosaminoglycans.

CBMN assay

CBMN assay was carried out according to methodologydescribed by Fenech [36] with a number of modificationsadapted to the primary hMSC culture. The cells were submittedto CBMN assay at passage 10 (a total of 24 PD, consideringthat the mean cell yield was related to a number of about 2.4

PD per passage). If cultures were healthy—consisting of small,adherent, spindle-shaped fibroblastoid cells—*60% confluentwith frequent refractile doublets of newly dividing cells, andwith all cells viable (detected by trypan blue test), then weproceeded with the protocol to obtain binucleated cells. In-itially, 5 · 104 umbilical cord 1 cells (hMSC1) were seeded intoeach well of the six-well plate (TPP) in order to conduct pre-liminary experiments to determine cyt-B concentration andincubation time to achieve a significant number of dividingcells blocked at the binucleated stage. The following cyt-B(Sigma Chemical Company) concentrations were checked: 1.5,2, 2.5, 3, 3.5, 4, 4.5, and 6mg/mL, after 12 and 24 h of incu-bation at 37�C in a humidified atmosphere containing 5% CO2.These experiments fared poorly because it was observed thatthe well area of the six-well plate hindered obtaining an ade-quate number of binucleated cells for CBMN analysis (see‘‘Results’’ section).

The experiments were repeated with T25 tissue cultureflasks (TPP), resulting in a sufficient number of binucleatedcells for CBMN analysis, which was performed in triplicateon hMSCs isolated from three donors (hMSC1, hMSC2, andhMSC3).

In the hMSC culture undergoing active cell division(*60% confluent), cells were detached with 0.25% trypsin/EDTA solution and replated at 8 · 104 cells per T25 flask.After cell adhesion (16–24 h), the medium was changed innegative control flasks, and, in the positive control flasks,cells were challenged with fresh medium containing mito-mycin-C (mit-C; final concentration 0.2 mg/mL). After 24 hof mit-C treatment, the culture medium in all flasks waschanged again and cyt-B was added to each sample, re-sulting in a final concentration of 4.0 mg/mL. The culturemedium was collected 24 h after the addition of cyt-B, andcells were detached from the plastic substratum with 0.25%Trypsin/EDTA solution and transferred to centrifuge tubes,which were centrifuged gently (300 g) for 5 min. The super-natant culture medium was then removed, leaving *100mL offluid on top of the cell pellets. In this step, the procedure wastested without using a hypotonic solution, according to Fenech[36], but failed. Thus, we tested a new hypotonic treatment inhMSCs. The hMSCs were exposed to 200mL of hypotonicsolution developed in our laboratory: 0.075 M KCl (Merck)mixed to solution 9 distilled water:1 culture medium with FBSat a final proportion of 1:1. The hypotonic solution was slowlyadded for 3 min and mixed with the cells at room temperature.The hypotonization process was immediately interrupted byadding 100mL of cold methanol:acetic acid (9:1; Merck) fix-ative solution freshly made, and the cell suspension was re-suspended to prevent clump formation. Before defining thefixative concentration (9 methanol:1 acetic acid), the cyto-plasmic area of the cells was evaluated following treatmentwith fixative containing the proportions of methanol/aceticacid 3/1 [36], 9/1, and 14/1 at the end of hypotonic treatment,the best ratio being 9 methanol:1 acetic acid fixative. The tubeswere centrifuged immediately (300 g) for 6 min; the superna-tant was removed and replaced slowly with 2 mL of fixativeconsisting of cold methanol/acetic acid (9/1). It is important todisperse cells gently before adding the fixative. Prior to slidepreparation, the tubes were centrifuged, the supernatant wasremoved, and cells were resuspended in 100–150mL of freshfixative depending on cell pellet size; 25–35mL of cell sus-pension (depending on cell concentration) was evenly

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 825

distributed onto a clean glass slide placed above the hot waterbath (40–60�C) for 10–20 s and left to air dry. Slides were agedin a 60�C oven for 30 min, and stained using 10% Giemsa stainin potassium phosphate buffer solution for 8 min.

Slides were prepared in duplicate from each triplicate cul-ture and scores were obtained from two different scorers. Foreach replicate of hMSC cultures, at least 600 binucleated cellswere analyzed, for a total of at least 1,800 binucleated cells ineach experimental group. The cells were analyzed for thepresence of MN (biomarkers of chromosome breakage orloss), NPBs (biomarker of asymmetrical chromosome re-arrangement), and NBUDs (biomarkers of gene amplifica-tion). Slides were examined at 400 and 1,000 magnificationusing a bright-field microscope (OLYMPUS CX31). Binu-cleated cells were evaluated according to the criteria describedby Fenech [36] with one exception; in this study, the two mainnuclei in a binucleated cell could not touch or overlap eachother, and a cell with two overlapping nuclei could not bescored (even if the nuclear boundaries of each nucleus weredistinguishable). Since the nuclei of hMSCs are very large andmay conceal NPBs that can occur between them, it is im-portant that the nuclei be well spread out in the cytoplasm andseparated from one another.

Five hundred viable cells were scored for each replicate ofindividual hMSC culture in order to obtain the nuclear divi-sion index (NDI). The NDI provides an estimated measure ofthe nuclear division status and cell division kinetics. NDI wascalculated as follows: NDI = (M1 + 2M2 + 3M3 + 4M4)/N;where M1–M4 represents the number of viable cells with oneto four nuclei and N is the total number of viable cells scored(excluding necrotic and apoptotic cells) [36].

CBMN in other cell lines

CBMN was used for the first time in hMSCs. To determinea possible influence of the methodological procedures usedon the results obtained in these cells, the CBMN assay wasperformed in two cell lines that are routinely cultivated in ourlaboratory, XP4PA and A549, using the same protocol es-tablished for hMSCs, except that in the A549 cell line thehypotonic solution was not used. The XP4PA cell line [58](kindly supplied by Dr. Alain Sarasin) consists of an im-mortalized human fibroblast xeroderma pigmentosum (XP),group C. A549 is a lung adenocarcinoma cell line establishedfrom culturing cancerous lung tissue from a 58-year-oldCaucasian man [59]. Cells were cultivated in DMEM sup-plemented with 10% FBS and 1% antibiotics in a humidifiedincubator at 37�C containing 5% CO2. The CBMN assay wasperformed in triplicate for each cell line, and the slide anal-ysis was done in the same way described for hMSCs.

Detection of natural occurrence of anaphasebridges between hMSCs on in vitro culture

CBMN analysis revealed a large number of NPBs in allhMSC treatments, including the negative control (see de-scription of ‘‘Results’’ section). To observe whether thereare anaphase bridges between hMSCs undergoing divisionin vitro without the addition of cyt-B, proliferative cellswere removed from T25 flasks and processed in a 15-mLtube following the same procedure described for CBMN (re-moving the cyt-B). After the material was fixed, the slideswere prepared and stained with Giemsa. hMSC anaphases

were observed under 40 · and 100 · lenses using an OlympusCX31 microscope.

Statistical analysis

The frequencies of binucleated cells with normal nucleiand with nuclear alterations (NPBs, NBUDs, or MN) foreach hMSC culture in both standard culture medium withoutmit-C (SC) group and with mit-C consisting of the positivecontrol group were compared using the t-test. A P val-ue < 0.05 was considered significant.

The frequencies of binucleated cells with normal nucleiand with nuclear alterations (NPBs, NBUDs, or MN) for eachhMSC culture group, A549 and XP4PA cell lines grown instandard culture medium, were compared using two-wayanalysis of variance. A P value < 0.05 was considered sig-nificant and data are shown as mean – SD for all figures.

Results

Isolation, expansion, and characterizationof hMSCs

hMSCs were obtained from the umbilical vein of threedonors, originating primary cultures hMSC1, hMSC2, andhMSC3. Healthy cultures of hMSCs exhibited a lot ofsmall fusiform cells and a high division rate (Fig. 1A), and,when cells grew in a dispersed manner, morphologicallydistinguishable cell types could be identified (Supple-mentary Fig. S1; Supplementary Data are available onlineat www.liebertpub.com/scd). After isolation and establish-ment of adherent cells on plastic culture vessels, hMSCsshowed to be extremely sensitive to environmental alterations(pH of the medium, temperature, and CO2 concentration) andto excessive cell confluence. In this work, it was establishedthat the hMSC culture must not exceed 70% confluence andthat hMSCs must be seeded at a density of 3,000 to 4,000cells/cm2.

The capacity for in vitro differentiation of hMSCs in os-teoblasts was confirmed by Alizarin Red S staining, showinga calcium-rich extracellular matrix (Fig. 1B). After 21 days ofexposition to chondrogenic medium in the micromass culturesystem, the cartilaginous pellet formed by hMSCs showedextracellular matrix acid-rich polysaccharides stained withAlcian Blue (Fig. 1C). Adipogenic differentiation was de-tected by the presence of fat vacuoles within cells stainedwith Oil Red O (Fig. 1D). Cell cultures that were grown inmedium without the differentiation supplements did not ex-hibit spontaneous osteogenic, chondrogenic, or adipogenicdifferentiation even after 21 days of cultivation.

The cells isolated and expanded in this study were alsocharacterized by immunophenotyping. They were positivefor the endoglin receptor CD105, extracelular matrix proteinCD90, and surface enzyme CD73. No contamination ofhematopoietic cells was detected, since flow cytometryanalysis was negative for hematopoietic stem cell markers(CD14, CD34, CD45, and HLA-DR) (Fig. 2).

Modifications in the CBMN assayfor analyzing hMSCs

The well-established protocol for CBMN analysis in pe-ripheral blood cells [36] was modified to measure nuclear

826 CORNELIO ET AL.

alterations in primary hMSC culture. The major adaptationswere (1) cell concentration and area of culturing, (2) cyt-Bconcentration, and (3) hypotonic and fixative solutions.

Concerning the first point, a number of aspects hinderedthe assay conducted in six-well plates, as follows: thehMSCs adhere and proliferate preferentially in the center ofthe well, where they form a region of high confluence,thereby increasing cell contact between hMSCs. In thiscondition, cells presented high sensitivity to cyt-B, consid-ering that 3.5 mg/mL up to 6 mg/mL of cyt-B drastically al-tered cell morphology, resulting in significant cell death.The cells became branched and arborized (Fig. 3) after only10 h of treatment with cyt-B. After 24 h of treatment withcyt-B in six-well plate, binucleated cells appeared in the

culture, but most detached from the well surface, and therewas considerable cellular debris floating in the culture me-dium. Thus, there were few cells to analyze, and it is notrecommended the use of six-well plate for conducting theCBMN assay with hMSCs. Cells were therefore seeded inT25 flasks in order to conduct the CBMN assay. In thisgrowth area, there was no cell aggregation throughout theentire 72-h incubation period, such that the cells proliferatedas a monolayer homogeneously spread over the surface ofthe flask. It was also established that 8 · 104 cells seeded toperform the CBMN is the ideal initial density for the T25flask to be 60%–70% confluent at the harvest time, therebyavoiding clot formation during cell processing. According tothe replicative potential of each hMSC culture (which can

FIG. 2. Different expressionlevels of hMSC markers ana-lyzed by flow cytometry. Pa-nels (A, B) negatively expresshematopoietic markers CD14,CD45, CD34, and HLA DR.Panels (C–E) strongly expressmesenchymal antigens CD90,CD73, and CD105. (F) Dot-plot histogram showing thephysical characteristics ofcells (size and complexity).The R1 marked region con-tains the cells analyzed.

FIG. 1. Culture and differentiationof human mesenchymal stem cells(hMSCs) isolated from the umbilicalvein. (A) Healthy cell culture with fi-broblastoid cells spread over the flasksurface; arrows indicate hMSC divi-sion (10 · lenses). (B) Osteogenicdifferentiation obtained after 21 daysof culture in induction medium; arrowsindicate the calcium-rich extracellularmatrix with Alizarin Red S staining. (C)Chondrogenic differentiation obtainedafter 21 days of culture in inductionmedium; the arrow indicates staining ofthe acid-polysaccharide-rich extracellu-lar matrix with Alcian Blue staining.(D) Adipogenic differentiation obtained21 days after culture in the inductionmedium; the arrow indicates fat vacu-oles with Oil Red O staining.

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 827

be donor dependent), the initial number of cells to be seededmust be adjusted.

The second important point in the CBMN protocol wasconcerning the cyt-B concentration. Among all cyt-B con-centrations tested, the 4.0mg/mL of cyt-B for 24 h in T25flasks was the best one. It did not cause cell death, did notdrastically affect hMSC morphology, and allowed obtainingan adequate number of binucleated cells for CBMN analysis.

The last modification in the original CBMN protocol wasthe experiment with the hypotonic treatment. In the absenceof the hypotonization process, hMSCs remained collapsed,precluding clear visualization of the nuclei. To improvedispersion of the nuclei within the cytoplasm, we used ahypotonic solution developed in our laboratory and rou-tinely employed to eliminate the cytoplasm and enhancechromosome spreading and G banding pattern for cytoge-netic analysis in hMSCs. The solution consists of a mixturein equal parts of a solution of 0.075 M KCl with a solutionof 9 parts water:1 part FBS-enriched medium, which mustbe added slowly for 3 min at room temperature for theCBMN assay. The hypotonic solution must not enter incontact with the cells for more than 3 min, the time required

to increase cytoplasm swelling, without cell membrane lysis.To preserve the cytoplasm, a requirement for recognizing celldelimitations, the hypotonic action must be interrupted by thefixative solution immediately after the 3 min of hypotoniza-tion and before centrifugation. At the end of hypotonictreatment, when cell preparation was fixed at 9 parts metha-nol:1 part acetic acid, more cells with preserved cytoplasmwere observed (Fig. 4). The larger the proportion of aceticacid in the fixative solution (3 methanol:1 acetic acid) thegreater the number of cells with cytoplasmic degeneration,and when the proportion of acetic acid was lower (14methanol:1 acetic acid) the cytoplasm was obscured and thenuclei did not spread correctly within the cell, hindering theirvisualization (Fig. 4). Further, when using the 9 methanol:1acetic acid fixative solution, it was not necessary to wash thecells with two additional fixative exchanges, just in that caseswith a very obscured cytoplasmic background, which ham-pered clear visualization of the nuclei.

These adaptations for hMSCs could also be used in im-mortalized cell lines, such as XP4PA and A549.

Characterization of nuclear alterations in hMSCs

To perform the nuclear alteration analysis, it is importantthat cells are in division; a measure of the cell proliferativestatus can be obtained by the NDI. The lowest NDI valuepossible is 1.0 (all of the viable cells have failed to divideduring the cytokinesis-block period) and the highest NDIvalue for CBMN analysis is 2.0, considering that all cellscompleted one nuclear division and are therefore all binu-cleated (36). The NDIs for the three hMSCs were 1.56, 1.95,and 1.67 under standard culture condition comparing to1.43, 1.71, and 1.55 for each positive control, from hMSC1,hMSC2, and hMSC3, respectively.

Binucleated cells with MN, NPBs, and NBUDs wereobserved in the three primary hMSC cultures as well as inthe two immortalized cell lines (XP4PA and A549) (Fig. 5).The mean frequencies of nuclear alterations are shown inFig. 6. The mean frequency of binucleated hMSCs with MN(3.41 – 3.94) was low even in the mit-C treatment(12.93 – 6.37). However, the mean frequency of NPBs(153.59 – 122.73) was high in all hMSCs analyzed, in-creasing in the presence of mit-C (379.27 – 127.08). Afterthe treatment with mit-C, the frequency of binucleated cellswith MN was lower than binucleated cells with NPBs(t34 = 12.22; P < 0.001), and binucleated cells with NBUDs(26.84 – 16.84) (t34 = 3.28; P < 0.005). The mean frequency

FIG. 3. Culture of hMSCs in the six-well plate in thepresence of cyt-B (3.5 mg/mL). Cell morphology with al-tered branching patterns resulting in an arborized aspect.Arrows indicate the presence of cell debris floating in theculture medium.

FIG. 4. Preservation differ-ences in the cytoplasm ofhMSCs fixed with differentconcentrations of metha-nol:acetic acid. (A) Cytoplasmdestruction with fixative solu-tion of 3 methanol:1 aceticacid. (B) Cytoplasm preservedwith fixative solution of 9methanol:1 acetic acid. (C)Obscure cytoplasm and littlenuclear separation with fixa-tive solution of 14 methanol:1acetic acid.

828 CORNELIO ET AL.

of MN was higher in the XP4PA line (16.88 – 2.46) than inthe A549 line (3.82 – 4.64) and hMSC cultures (F4,29 =22.53; P < 0.05) under normal culture conditions, and verysimilar to mean MN frequencies found in hMSCs treatedwith mit-C (F3,23 = 2.72; P > 0.05).

Cells isolated from cord 1 under normal culture condi-tions exhibited fewer NPBs when compared with hMSC2(t = 26.57; P < 0.0001) and hMSC3 (t = 6.34; P < 0.0001), butthe frequency of hMSC1 with NPBs is higher when com-pared with A549 (t = 8.44; P < 0.0001) and XP4PA (t = 7.44;P < 0.0001) (Fig. 6). The mean frequencies of binucleatedcells with NPBs were lower in lines A549 and XP4PA(A549 = 2.16 – 1.89 binucleated cells with NPB/1,000 bi-nucleated cells; XP4PA = 5.41 – 4.00 binucleated cells withNPB/1,000 binucleated cells) when compared with meanfrequencies observed in hMSC cultures (F4,29 = 502.67;P < 0.05) (Fig. 6), but there was no difference in the NPBfrequency between A549 and XP4PA cells (t = 1.8; P = 0.1).

To determine whether the emergence of NPBs was due tothe use of cyt-B, an experiment was conducted without theaddition of cyt-B. The profile of in vitro hMSC division,without the addition of cyt-B, showed the spontaneous oc-currence of anaphase bridges and binucleated cells withNPBs during in vitro culture without mit-C (Fig. 7).

Analysis of nuclear alterations (NPBs and NBUDs) usingthe CBMN assay shows interindividual differences in hMSCresponse (Fig. 6 and Table 1) to cell culture under standardconditions (mean frequencies of different NPBs F2,17 = 374.11;P < 0.05; mean frequencies of different NBUDs F2,17 = 16.17;P < 0.05) and treatment with mit-C (mean frequencies of dif-ferent NPBs F2,17 = 102.88; P < 0.05; mean frequencies ofdifferent NBUDs F2,17 = 26.35; P < 0.05). Cells isolated fromcord 1 exhibited fewer alterations, and those from cord 2showed a large number of cells binucleated with NPBs andNBUDs. However, the mean frequency of cells with MN didnot differ among the three different hMSC cultures in the

FIG. 5. Binucleated cellsanalyzed by the cytokinesis-block micronucleus (CBMN)assay; micronucleus (MN),nucleoplasmic bridges (NPBs),and nuclear buds (NBUDs)were observed in the three pri-mary hMSC cultures and celllines A549 and XP4PA. (A, E,I, M, Q) The normal binucle-ated cells observed in A549,XP4PA, hMSC1, hMSC2, andhMSC3, respectively. (B, F, J,N, R) Binucleated cells withNPBs observed in A549,XP4PA, hMSC1, hMSC2, andhMSC3, respectively. (C, G,K, O, S) Binucleated cells withNBUDs observed in A549,XP4PA, hMSC1, hMSC2, andhMSC3, respectively. (D, H, L,P, T) Binucleated cells withMN observed in A549,XP4PA, hMSC1, hMSC2, andhMSC3, respectively.

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 829

standard cultivation (F2,17 = 1.68; P = 0.22) nor in the presenceof mit-C (F2,17 = 2.21; P = 0.14).

Discussion

The present study describes for the first time the meth-odological adaptation of CBMN assay for hMSCs isolatedfrom the umbilical vein.

Although CBMN assay is a well-known technology, itmust be optimized to hMSC nuclear alteration analysis. It is

much easier and faster technique and does not require ex-tensive personnel training as human karyotype analysis [35],allowing counting of a larger number of cells for assessingthe genetic instability. So, the modifications described inthis methodology will be important for using CBMN assayroutinely in biosafety evaluation of the cell therapy.

In the present study, stem cell isolation, expansion, andcharacterization were conducted successfully as describedby our group [57] and other authors who also isolatedhMSCs from the umbilical vein [60–66]. The hMSC

FIG. 6. (A–C) Frequencies (number of binucleated cells with determinate nuclear alteration/total number of binucleatedcells analyzed) of each nuclear alteration detected by the CBMN assay in the three hMSC cultures cultivated in standardculture and with the addition of mitomycin C (positive control = PC): (A) BNP, (B) NBUD, and (C) MN. The resultsrepresent the mean – SD of three replicates of each hMSC culture in vitro cultivated without and with mitomycin C. Thet-test for unpaired data (two-tailed) was used to assess the probability of significant differences between untreated andtreated samples (*P < 0.05). (D–F) Frequencies (number of binucleated cells with determinate nuclear alteration/totalnumber of binucleated cells analyzed) of each nuclear alteration detected by the CBMN assay in the five cell lines analyzed:(D) BNP, (E) NBUD, and (F) MN. The results represent the mean – SD of three replicates of each cell line cultured undernormal conditions of in vitro expansion using analysis of variance. The t-test for unpaired data (two-tailed) was used toassess the probability of significant differences between each pair of cell lines tested (*P < 0.05). (G, H) Frequencies(number of binucleated cells with determinate nuclear alteration/total number of binucleated cells analyzed) of each nuclearalteration detected by the CBMN assay in the three hMSC cultures cultivated in standard culture (G) and with the additionof mitomycin C (H) showing that the frequencies of hMSCs with NPB is higher than the frequencies of other nuclearalterations (NBUD and MN). The results represent the mean – SD of three replicates of each hMSC culture in vitrocultivated without and with mitomycin C. The t-test for unpaired data (two-tailed) was used to assess the probability ofsignificant differences between the frequencies of nuclear alterations (*P < 0.05).

830 CORNELIO ET AL.

morphology was consistent with the guidelines established byMontesinos et al. [67]. The results obtained from the flowcytometry analysis and differentiation studies are in accor-dance with International Society for Cellular Therapy Guide-lines [1], proving that the cells isolated from the umbilical veinare hMSCs.

CBMN analysis must be carried out in healthy cultures ofhMSCs with high viability and high proliferation rates. Wedetermined that seeding the cells in T25 flask is better forCBMN assay, avoiding extensive cell confluence (a factor thatstresses hMSCs [68]), so the concentration of 4.0mg/mL of cyt-B was not cytotoxic for hMSCs. Initially, the cyt-B concen-tration tested in the hMSCs seeded into six-well plate causeddrastic alterations in cell morphology. The region of highconfluence formed in the center of the well may have increasedthe cellular stress and cell sensitivity to cyt-B. Previous studieshave showed that cytochalasin D alters actin cytoskeleton ofMSCs, thereby affecting cell morphology [69,70].

Tests were conducted to determine whether hypotonictreatment of hMSCs was necessary, since, according toFenech [36], it is not advisable to use the hypotonic treat-ment in CBMN, because this procedure can destroy necroticand apoptotic cells, which must be included in analyses ofthe action mechanism and measure of cell sensitivity tochemical compounds or radiation. However, CBMN assaywithout the hypotonization step failed in hMSCs because thenuclear boundaries of each nucleus within binucleated cellswere not distinguishable. It is believed that this occurredbecause most hMSCs have thin fusiform bodies with largernucleus-to-cytoplasm ratio than differentiated cells. Also thecytoplasm of hMSCs is very dense, resulting in intensivesynthetic and metabolic activity [71]. Considering thehMSC characteristics described previously, the hypotonictreatment of hMSCs is necessary in a short time of exposureand cannot be skipped. The hMSC cytoplasm has to swelland the two nuclei have to spread, but the cytoplasmic

FIG. 7. Anaphase bridges(A–C) and binucleated cellswith nucleoplasmic bridges(D–F) observed under normalconditions of hMSC culturewithout addition of cytocha-lasin B. AB, anaphase bridge;NPB, nucleoplasmic bridge.

Table 1. Pairwise Analysis of the Frequencies of Binucleated Cells

with Nuclear Alterations NPBs, NBUDs, and MN, Using the t-Test

Nuclear alterations

NPBs NBUDs MN

Comparison T value P value T value P value T value P value

hMSC1 SC vs. hMSC1 PC 20.45 < 0.0001 1.86 0.0921 3.39 0.0068hMSC2 SC vs. hMSC2 PC 11.59 < 0.0001 5.37 0.0003 4.67 0.0009hMSC3 SC vs. hMSC3 PC 10.42 < 0.0001 7.72 < 0.0001 4.16 0.002A549 vs. hMSC1 8.44 < 0.0001 0.29 0.077 1.75 0.11A549 vs. hMSC2 34.25 < 0.0001 3.62 0.0047 1.15 0.28A549 vs. hMSC3 12.55 < 0.0001 1.03 0.33 0.02 0.99XP4PA vs. hMSC1 7.44 < 0.0001 0.62 0.55 15.14 < 0.0001XP4PA vs. hMSC2 33.49 < 0.0001 5.32 0.0003 12.76 < 0.0001XP4PA vs. hMSC3 11.94 < 0.0001 0.14 0.89 5.54 < 0.0002XP4PA vs A549 1.80 0.10 0.91 0.38 6.09 < 0.0001hMSC1 vs. hMSC2 26.57 < 0.0001 4.12 0.0021 1.36 0.20hMSC1 vs. hMSC3 6.34 < 0.0001 0.74 0.48 1.53 0.16hMSC2 vs. hMSC3 17.97 < 0.0001 5.51 0.0003 1.01 0.34

A P value < 0.05 was considered significant.SC, hMSCs cultivated with standard culture medium without mit-C; PC, positive control consisting of hMSCs treated with mitomycin-C.hMSCs, human mesenchymal stem cells; NPBs, nucleoplasmic bridges; NBUDs, nuclear buds; MN, micronucleus.

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 831

membrane of binucleated cells must be intact and clearlydistinct from adjacent cells for optimal CBMN analysis.Thus, it is necessary that any damage to the hMSC cyto-plasmic membrane does not occur during the procedure ofharvest [72]. Considering these facts, it was determined thathMSC preparation must be fixed at 9 parts methanol:1 partacetic acid for the hMSC membrane preservation. Theseresults corroborate those of other studies that show that al-terations in hypotonic treatment and different proportions ofmethanol–acetic acid fixative solution influence preserva-tion of the cytoplasmic area depending on the cell type [72–74]. It is therefore important to standardize this step forCBMN analysis in hMSCs.

The methodology described here can be used to obtainbinucleated cells originating from other sources; it wassuccessfully tested in hMSCs isolated from three umbilicalcord donors and in two cell lines A549 and XP4PA. Thisstandardization favors future studies that will assess thegenomic integrity of hMSCs after in vitro culture and de-termine the response of hMSCs to therapeutically importantchemicals, reinforcing the use of hMSCs as an in vitromodel of multipotent cells without genetic manipulation ingenotoxicity assays.

After CBMN assay methodology was established, nuclearalterations indicative of genetic instability in hMSCs ex-panded under standard in vitro conditions without mit-C andtreated with mit-C were analyzed. NDI determination isalways the first step in CBMN analysis, since NDI providesa measure of the proliferative status of cell culture, an in-dicator of the cytostatic effects of the conditions beingtested [36]. All three hMSC cultures showed a decline inproliferative potential in the presence of mit-C (positivecontrol), but the drug was not cytostatic in the concentrationused, since it led to the appearance of a sufficient number ofbinucleated cells for CBMN analysis in the positive controltreatment. The fact that hMSC2 exhibited a high replicationrate (NDI = 1.95), resulting in a large number of trinucleatedand tetranucleated cells, in addition to the binucleated,justified the reduced exposure time to cyt-B [72], since itmeans that several viable cells completed more than onenuclear division in 24 h. However, as we were in the stan-dardization phase of the protocol, we considered it moreadvisable to use the same incubation time with cyt-B (24 h)in the treatment of hMSCs isolated from the three umbilicalcords.

Nuclear alterations in hMSCs were more frequent in thepositive control (except NBUDs in hMSC1), where cellswere treated with 0.2 mg/mL of mit-C. This indicates that thehMSCs responded to treatment with mit-C, primarily owingto the increased frequency of binucleated cells with NPBs.Mit-C is considered a potent clastogenic agent because itproduces several types of DNA lesions, which induce theemergence of double-strand breaks (DSBs) due to the rep-lication fork barrier [75]. Interestingly, the mean frequencyof binucleated cells with MN in culture treated with mit-Cwas very low, indicating that the hMSCs isolated from theumbilical cord likely contain mechanisms to avoid a highlevel of chromosomal breaks. The XP4PA line exhibitsDNA repair deficiency [76], thereby justifying the high MNindex, and suggests that the low frequency of MN in hMSCstreated with mit-C is related to its greater repair capacity.Vinoth et al. [52] observed lower levels of chromosomal

alterations in hESCs after treatment with mit-C than fibro-blasts from the IMR-90 cell line, suggesting that this factmay be related to the presence of more efficient DNA repairmechanisms in hESCs. Increased DNA repair capacity hasbeen frequently detected in stem cells (iPSCs, embryonic, oradult stem cells from humans or mice) when compared withdifferentiated cells, indicating that stem cells normallyprotect their genome through an increase in DNA repairmachinery [77–79]. It has been demonstrated that the repaircapacity of DSBs is greater in human hematopoietic stemcells isolated from umbilical cord blood and decreasesduring cell maturation [80]. The authors concluded that stemcells are protected by extensive DNA repair, whereas cellsalready compromised by cell differentiation, when dam-aged, can be eliminated by apoptosis. Chen et al. [81]demonstrated that hMSCs isolated from bone marrow havegreater antioxidant response and DSB repair capacity, whichfavor the characteristic resistance to radiation of these stemcells. The role of DNA damage response in the resistance ofmouse MSCs to ionizing radiation was assessed by Sugrueet al. [82], who showed that MSCs have efficient DSB re-pair, expressing high DNA damage response protein (ATM,Chk2, and DNA Ligase IV) and anti-apoptotic protein levels[Bcl-2 and Bcl-(XL)]. A recent study demonstrated thatDSB repair is faster in hMSCs than in differentiated oste-oblasts after g-irradiation-induced DNA damage [83]. Theyalso found that hMSCs induced to osteogenic or adipogenicdifferentiation undergo high levels of apoptosis after g-irradiation, in contrast to undifferentiated hMSCs, which arehighly resistant to treatment and exhibit 95% DSB repair.

The present study detected a high occurrence of binu-cleated cells with NPBs in all three cords analyzed. NPBshave been validated as biomarkers of DNA damage andchromosomal rearrangement [84]. The formation of NPBs isincreased by a wide range of agents, including endogenousoxidative agents, ionizing radiation, polycyclic aromatichydrocarbons, patulin and folate, and selenium deficiency[40,85,86]. Duan et al. [87] demonstrated that frequencies ofNPBs and NBUDs were more sensitive measures than MNfor assessing the genotoxicity of polycyclic aromatic hy-drocarbons. Interestingly, in the present study, the NPBfrequencies were high in hMSCs cultured under standard invitro culture patterns, without the addition of any genotoxicagent. The cell lines A549 and XP4PA have showed lowerfrequencies of NPBs, suggesting that these bridges thatappear in hMSCs do not result from the methodology usedand are characteristic of hMSCs isolated from the umbilicalvein and in vitro expanded. In another previous study ofCMBN using CHOK1 cells, we detected that high fre-quencies of NPBs do not occur in all conditions tested [46].

Ebert et al. [88] showed that hMSCs isolated from bonemarrow and in vitro cultured with a low amount of seleniumexhibit symptoms of oxidative stress and a high frequencyof MN, and that selenium supplementation in the culturemedium decreases the number of MN by at least 58%. Sincethe authors did not analyze MN in binucleated cells, therewas no description of NPB occurrence. Researchers under-scored the importance of preserving the in vitro genomicintegrity of hMSCs, concluding that selenium supplemen-tation in the culture medium for hMSC expansion would bean excellent conduct for cell preparations used in tissueengineering, thereby improving the quality of hMSCs

832 CORNELIO ET AL.

ex vivo manipulated. Two other studies described MNanalysis in binucleated cells using murine stem cell as amodel [89,90]; however, the authors did not analyze theoccurrence of other nuclear alterations such as NPBs andNBUDs, precluding comparison with data obtained in thepresent study.

The occurrence of anaphase bridges and spontaneous bi-nucleated cells with NPBs was detected during expansionunder normal conditions of in vitro culture of hMSCs iso-lated from the umbilical vein. Anaphase bridges and NPBsare signs of genomic instability [84]. This fact is relevant,since the ex vivo expansion process of hMSCs is the strat-egy selected to produce a large number of cells for use incell therapy. This procedure, necessary for the clinical ap-plication of hMSCs, is associated to the potential risk of cellimmunogenicity, with issues of biosecurity regarding com-ponents of the culture medium and FBS, as well as thepossibility of in vitro transformation of hMSCs [7]. Highsusceptibility to malignant transformation of MSCs has beendescribed in animal models using stem cells isolated fromthe bone marrow of mice and monkeys, some becominghighly tumorigenic after in vitro culture, since they causedtumors in mice at sites where these cells were infused [91–98]. A recent study reported that differentiated hMSCsisolated from human bone marrow became tumorigenic indiabetic mice. The authors believe that this occurred due tospontaneous transformation of hMSCs after prolonged invitro culture [31]. Genetic alterations have been described incultured hMSCs [19–29], but the biological significance ofthese alterations remains unclear and it is an issue that raisescontroversy [29,33]. Given this lack of consensus, morestudies on the genetic stability of hMSCs are needed tocome to a firm conclusion regarding this issue and theCBMN assay can be useful.

In this respect, the present study may contribute to thediscussion of this subject, since the anaphase bridges de-scribed here in hMSCs in vitro expanded are characteristicalterations in tumor cells [99]. They emerge due to thepresence of dicentric chromosomes formed by the fusion oftwo deficient telomeres and indicate genetic instabilitywithin cells, a common characteristic in a wide range ofcancers [100–102]. Anaphase bridges often break [101,103],resulting in chromosomal alterations, such as amplifications,translocations, or deletions, which are biomarkers of geno-mic instability in malignant cells and used for diagnosticpurposes in oncology [104]. After anaphase bridge breakageand fusion of uncapped chromosome ends, in the next in-terphase, breakage–fusion–bridge cycles initiate and prop-agate through several divisions, generating more alterations,such as aneuploidy, polyploidy, and genetic mutations [105–108]. Calado et al. [109] provided direct clinical evidencethat telomere shortening in hematopoietic progenitor cells,followed by breakage–fusion–bridge cycles, increases pre-disposition to malignant transformation in patients withaplastic anemia. Moreover, another study revealed that themechanism of chromosomal instability in leukemia pro-genitor cells occurs due to the continuous generation ofunstable chromosomal alterations, which undergo repeatedbreakage–fusion–bridge cycles and subsequent lesion repair[110]. In the present study we detected a high frequency ofNPBs in three hMSC cultures; these nuclear alterations caninitiate repeated breakage–fusion–bridge cycles during ex

vivo expansion of the progenitor cells and cause a highdegree of genetic instability in these cells.

On the other hand, a new type of anaphase bridge wasrecently described: ultrafine anaphase bridges (UFBs)[111,112]. These are ultrafine structures that form bridgesbetween daughter cells in the anaphase (DNA bridges thatform between sister chromatids during mitosis); they areBrdU stained, but cannot be evidenced by other DNA stains,such as Giemsa and DAPI [113]. UFBs are common char-acteristics of cultured cells, and considered physiologicalstructures of normal cells that undergo in vitro replication[114]. Exposure to in vitro expansion causes an increase indecatenated DNA structures and late-replicating intermedi-ates [115]. A number of UFBs occur in the centromeres andmay play the important physiological function of unitingDNA structures, acting as a signaling platform for thespindle assembly checkpoint. The spindle assembly check-point is activated when sister kinetochores are not biorienteddue to errors in microtubule ligation or loss of tension, and itwas recently demonstrated that spindle assembly checkpointpersists for longer in cells deficient in resolving the Hollidayjunction during DSB repair in DNA, with a higher frequencyof anaphase bridges and aneuploidy [116]. Another UFBsubtype originates in telomeric DNA and its frequency iscorrelated with defects in telomere replication. UFBs areimmunohistochemically stained with the BTR complex[formed of BLM (Bloom syndrome, RecQ helicase-like),topoisomerase IIIa, and RecQ-mediated genome instability1 and 2 proteins (RMI1 and RMI2)] and PICH (Plk1-interacting checkpoint helicase). BLM has a role in sup-pressing and/or resolving UFBs, likely contributing tomaintaining the telomere acting on late-replicating inter-mediate resolution [115,117]. There are also UFBs not de-rived from centromeres characterized by colocalization ofPICH with Fanconi anemia proteins FANCD2 and FANCI[118,119]. FANCD2 and FANCI proteins are specificallyassociated to fragile site loci, marking abnormally inter-twined DNA structure induced by replication stress. Theassociation between PICH and the BTR complex and Fan-coni anemia proteins suggests that the processing and res-olution of UFBs are very important for maintaining genomicstability [120]. The presence of UFBs in anaphases of nor-mal-cultured cells with intact genome shows that the cellsare able to resolve late-replicating intermediate sequences orcatenane structures, ensuring adequate chromosomal segre-gation without the need of activating cell cycle checkpointsand avoiding a decline in in vitro proliferation efficiency[115].

Since the number of NPBs is higher in the hMSC2, whichexhibited high mitotic index, it can be assumed that thebridges frequently observed in the hMSCs of this studycould be in some way related to these ultrafine bridges,although the bridges detected in this study were stained withGiemsa. Therefore, anaphase bridges (characterized asNPBs in CBMN) may be a normal characteristic of in vitrohMSC growth, or on the other hand, they might characterizea pathological condition introduced or enhanced by themanner in which they are cultured, as discussed previously.These issues are important and need to be clarified, since ifNPBs are signs of instability generated by the culture con-dition, then other methodologies for in vitro expansionshould be established.

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 833

Another factor that must be discussed regarding analysisof the genetic stability of cultured hMSCs is the differencein nuclear alteration frequencies exhibited by distinct do-nors. Analysis of the gene expression of stem cells isolatedfrom different individuals also exhibited interindividualdifferences due to genetic variability resulting in a uniquegene expression signature [121,122]. The same situationoccurs with the gene expression of the DNA repair system,and, in apoptosis induction after cytotoxic treatment in he-matopoietic stem cells isolated from umbilical cord blood[80], cells displayed different levels of donor-specific re-sponses. It has also been established that MN formation canbe influenced by certain gene polymorphisms involved inDNA repair pathways, in the xenobiotic metabolism, and infolate transport and metabolism [123]. However, the presentstudy found no differences in mean MN frequencies in thethree hMSC cultures studied in the absence or presence ofmit-C.

Our laboratory is conducting experiments to clarify thegenetic significance of a low frequency of binucleated cellswith MN in the presence of mit-C and the elevated presenceof NPBs in hMSCs even in the absence of genotoxic agents.Spectral karyotyping (SKY) analysis along with CBMN forhMSCs in vitro expanded will help to define whether there isany particular association between NPBs and any specificchromosome. Further, it is important to measure the rate ofconcurrent gene mutations, chromosomal aberrations, andalterations in gene expression at same cell culture time andconditions in that hMSC CBMN analysis is realized.Afterward, the CBMN could be included in routine analysesof hMSCs as a biomarker in order to improve predictions ofrisk factors, making it an important tool associated withother genetic analyses to ensure the safe clinical use ofhMSCs in cell therapy.

Acknowledgments

The authors would like to thank Mauro Pichorim for theassistance in the statistical analysis, also Gideao Wagner W.Felix da Costa and Carolina Corado for the assistance withthe figures. We also would like to thank the CNPq andCAPES for financial support and the scholarship awarded toDeborah Afonso Cornelio, Thais Pimentel, and Silva ReginaBatistuzzo de Medeiros.

Author Disclosure Statement

No competing financial interests exist.

References

1. Dominici M, K Le Blanc, I Mueller, I Slaper-Cortenbach, FMarini, D Krause, R Deans, A Keating, DJ Prockop and EHorwitz. (2006). Minimal criteria for defining multipotentmesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy 8:315–317.

2. Meirelles Lda S and NB Nardi. (2009). Methodology,biology and clinical applications of mesenchymal stemcells. Front Biosci 14:4281–4298.

3. Ren G, X Chen, F Dong, W Li, X Ren, Y Zhang and YShi. (2012). Concise review: mesenchymal stem cells andtranslational medicine: emerging issues. Stem Cells TranslMed 1:51–58.

4. Caplan AI and D Correa. (2011). The MSC: an injurydrugstore. Cell Stem Cell 9:11–15.

5. Di Trapani M, G Bassi, M Ricciardi, E Fontana, F Bifari,L Pacelli, L Giacomello, M Pozzobon, F Feron, et al.(2013). Comparative study of immune regulatory proper-ties of stem cells derived from different tissues. StemCells Dev 22:2990–3002.

6. Hass R, C Kasper, S Bohm and R Jacobs. (2011). Dif-ferent populations and sources of human mesenchymalstem cells (MSC): a comparison of adult and neonataltissue-derived MSC. Cell Commun Signal 14:9–12.

7. Bernardo ME, AM Cometa, D Pagliara, L Vinti, F Rossi,R Cristantielli, G Palumbo and F Locatelli. (2011). Exvivo expansion of mesenchymal stromal cells. Best PractRes Clin Haematol 24:73–81.

8. Spees JL, CA Gregory, H Singh, HA Tucker, A Peister, PJLynch, SC Hsu, J Smith and DJ Prockop. (2004). Inter-nalized antigens must be removed to prepare hypoimmu-nogenic mesenchymal stem cells for cell and genetherapy. Mol Ther 9:747–756.

9. Caisander G, H Park, K Frej, J Lindqvist, C Bergh, KLundin and C Hanson. (2006). Chromosomal integritymaintained in five human embryonic stem cell lines afterprolonged in vitro culture. Chromosome Res 14:131–137.

10. Bernardo ME, N Zaffaroni, F Novara, AM Cometa, MAAvanzini, A Moretta, D Montagna, R Maccario, R Villa,et al. (2007). Human bone marrow–derived mesenchymalstem cells do not undergo transformation after long-termin vitro culture and do not exhibit telomere maintenancemechanisms. Cancer 67:9142–9149.

11. Zhang ZX, LX Guan, K Zhang, S Wang, PC Cao, YHWang, Z Wang and LJ Dai. (2007). Cytogenetic analysisof human bone marrow-derived mesenchymal stem cellspassaged in vitro. Cell Biol Int 31:645–648.

12. Choumerianou DM, H Dimitriou, C Perdikogianni, GMartimianaki, M Riminucci and M Kalmanti. (2008).Study of oncogenic transformation in ex vivo expandedmesenchymal cells, from paediatric bone marrow. CellProlif 41:909–922.

13. Fariha MM, KH Chua, GC Tan, AE Tan and AR Hayati.(2011). Human chorion-derived stem cells: changes instem cell properties during serial passage. Cytotherapy13:582–593.

14. Poloni A, G Maurizi, L Babini, F Serrani, E Berardinelli,S Mancini, B Costantini, G Discepoli and P Leoni.(2011). Human mesenchymal stem cells from chorionicvilli and amniotic fluid are not susceptible to transfor-mation after extensive in vitro expansion. Cell Trans-plant 20:643–654.

15. Roubelakis MG, V Bitsika, D Zagoura, O Trohatou, KIPappa, M Makridakis, A Antsaklis, A Vlahou and NPAnagnou. (2011). In vitro and in vivo properties of distinctpopulations of amniotic fluid mesenchymal progenitorcells. J Cell Mol Med 15:1896–1913.

16. Fong CY, A Subramanian, A Biswas, K Gauthaman, PSrikanth, MP Hande and A Bongso. (2010). Derivationefficiency, cell proliferation, freeze-thaw survival, stem-cell properties and differentiation of human Wharton’sjelly stem cells. Reprod Biomed Online 21:391–401.

17. Gauthaman K, CY Fong, CA Suganya, A Subramanian, ABiswas, M Choolani and A Bongso. (2012). Extra-em-bryonic human Wharton’s jelly stem cells do not inducetumorigenesis, unlike human embryonic stem cells. Re-prod Biomed Online 24:235–246.

834 CORNELIO ET AL.

18. Goldring CE, PA Duffy, N Benvenisty, PW Andrews, UBen-David, R Eakins, N French, NA Hanley, L Kelly,et al. (2011). Assessing the safety of stem cell therapeutics.Cell Stem Cell 8:618–628.

19. Binato R, T de Souza Fernandez, C Lazzarotto-Silva, BDu Rocher, A Mencalha, L Pizzatti, LF Bouzas and EAbdelhay. (2013). Stability of human mesenchymal stemcells during in vitro culture: considerations for cell ther-apy. Cell Prolif 46:10–22.

20. Fietz T, WE Berdel, H Rieder, B Reufi, H Hopp, E Thieland WU Knauf. (1999). Culturing human umbilical cordblood: a comparison of mononuclear vs CD34 + selectedcells. Bone Marrow Transplant 23:1109–1115.

21. Kim JW, SY Kim, SY Park, YM Kim, JM Kim, MH Leeand HM Ryu. (2004). Mesenchymal progenitor cells in thehuman umbilical cord. Ann Hematol 83:733–738.

22. Bochkov NP, ES Voronina, NV Kosyakova, T Liehr, AARzhaninova, LD Katosova, VI Platonova and DV Gol’d-shtein. (2007). Chromosome variability of human multi-potent mesenchymal stromal cells. Bull Exp Biol Med143:122–126.

23. Meza-Zepeda LA, A Noer, JA Dahl, F Micci, O Mykle-bost and P Collas. (2008). High-resolution analysis ofgenetic stability of human adipose tissue stem cells cul-tured to senescence. J Cell Mol Med 12:553–563.

24. Buyanovskaya OA, NP Kuleshov, VA Nikitina, ES Vor-onina, LD Katosova and NP Bochkov. (2009). Sponta-neous aneuploidy and clone formation in adipose tissuestem cells during different periods of culturing. Bull ExpBiol Med 148:109–112.

25. Sareen D, E McMillan, AD Ebert, BC Shelley, JA John-son, LF Meisner and CN Svendsen. (2009). Chromosome7 and 19 trisomy in cultured human neural progenitorcells. PLoS One 4:e7630.

26. Takeuchi M, K Takeuchi, Y Ozawa, A Kohara and HMizusawa. (2009). Aneuploidy in immortalized humanmesenchymal stem cells with non-random loss of chro-mosome 13 in culture. In Vitro Cell Dev Biol Anim 45:290–299.

27. Ben-David U, Y Mayshar and N Benvenisty. (2011).Large-scale analysis reveals acquisition of lineage-specificchromosomal aberrations in human adult stem cells. CellStem Cell 9:97–102.

28. Ueyama H, T Horibe, S Hinotsu, T Tanaka, T Inoue, HUrushihara, A Kitagawa and K Kawakami. (2012).Chromosomal variability of human mesenchymal stemcells cultured under hypoxic conditions. J Cell Mol Med16:72–82.

29. Ben-David U, Y Mayshar and N Benvenisty. (2012).Significant acquisition of chromosomal aberrations inhuman adult mesenchymal stem cells: response to Sen-sebe et al. Cell Stem Cell 10:10–11.

30. Wang Y, DL Huso, J Harrington, J Kellner, DK Jeong, JTurney and IK McNiece. (2005). Outgrowth of a trans-formed cell population derived from normal human BMmesenchymal stem cell culture. Cytotherapy 7:509–519.

31. Tang DQ, Q Wang, BR Burkhardt, SA Litherland, MAAtkinson and LJ Yang. (2012). In vitro generation offunctional insulin-producing cells from human bone mar-row-derived stem cells, but long-term culture running riskof malignant transformation. Am J Stem Cells 1:114–127.

32. Tarte K, J Gaillard, JJ Lataillade, L Fouillard, M Becker,H Mossafa, A Tchirkov, H Rouard, C Henry, et al.; So-ciete Francaise de Greffe de Moelle et Therapie Cellulaire.

(2010). Clinical-grade production of human mesenchymalstromal cells: occurrence of aneuploidy without transfor-mation. Blood 115:1549–1553.

33. Sensebe L, K Tarte, J Galipeau, M Krampera, I Martin,DG Phinney, Y Shi; MSC Committee of the InternationalSociety for Cellular Therapy. (2012). Limited acquisitionof chromosomal aberrations in human adult mesenchymalstromal cells. Cell Stem Cell 10:9–10.

34. Fenech M. (2000). The in vitro micronucleus technique.Mutat Res 455:81–95.

35. Narin A and O Tuncay. (2012). Relationships betweenmalignant melanoma and chromosome damage in humanperipheral blood lymphocytes. Asian Pac J Cancer Prev13:5229–5232.

36. Fenech M. (2007). Cytokinesis-block micronucleus cy-tome assay. Nat Protoc 2:1084–1104.

37. Aires GM, JR Meireles, PC Oliveira, JL Oliveira, ELAraujo, BC Pires, ES Cruz, NF Jesus, CA Pereira and EMCerqueira. (2011). Micronuclei as biomarkers for evalu-ating the risk of malignant transformation in the uterinecervix. Genet Mol Res 10:1558–1564.

38. Gayathri B, R Kalyani, A Hemalatha and B Vasavi.(2012). Significance of micronucleus in cervical in-traepithelial lesions and carcinoma. J Cytol 29:236–240.

39. Singaraju M, S Singaraju, R Parwani and S Wanjari.(2012). Cytogenetic biomonitoring in petrol station at-tendants: a micronucleus study. J Cytol 29:1–5.

40. Fenech M, M Kirsch-Volders, AT Natarajan, J Surralles,JW Crott, J Parry, H Norppa, DA Eastmond, JD Tuckerand P Thomas. (2011). Molecular mechanisms of micro-nucleus, nucleoplasmic bridge and nuclear bud formationin mammalian and human cells. Mutagenesis 26:125–132.

41. Bonassi S, R El-Zein, C Bolognesi and M Fenech. (2011).Micronuclei frequency in peripheral blood lymphocytesand cancer risk: evidence from human studies. Mutagen-esis 26:93–100.

42. El-Zein R, A Vral and CJ Etzel. (2011). Cytokinesis-blocked micronucleus assay and cancer risk assessment.Mutagenesis 26:101–106.

43. McHugh MK, MS Lopez, CH Ho, MR Spitz, CJ Etzel andRA El-Zein. (2013). Use of the cytokinesis-blocked mi-cronucleus assay to detect gender differences and geneticinstability in a lung cancer case-control study. CancerEpidemiol Biomarkers Prev 22:135–145.

44. Bull CF, S Beetstra-Hill, BJ Benassi-Evans, JW Crott, MKimura, T Teo, J Wu and MF Fenech. (2011). Applicationand adaptation of the in vitro micronucleus assay for theassessment of nutritional requirements of cells for DNAdamage prevention. Mutagenesis 26:193–197.

45. Bull CF, G Mayrhofer, D Zeegers, GL Mun, MP Handeand MF Fenech. (2012). Folate deficiency is associatedwith the formation of complex nuclear anomalies in thecytokinesis-block micronucleus cytome assay. EnvironMol Mutagen 53:311–323.

46. Tavares JCM, DA Cornelio, NB Silva, CEB Moura, JDFQueiroz, JC Sa, C Alves Jr and SR Batistuzzo de Me-deiros. (2009). Effect of titanium surface modified byplasma energy source on genotoxic response in vitro.Toxicol 262:138–145.

47. Degrandi TH, IM de Oliveira, GS d’Almeida, CR Garcia,IV Villela, TN Guecheva, RM Rosa and JA Henriques.(2010). Evaluation of the cytotoxicity, genotoxicity andmutagenicity of diphenyl ditelluride in several biologicalmodels. Mutagenesis 25:257–269.

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 835

48. Benassi-Evans B and M Fenech. (2011). Chronic alcoholexposure induces genome damage measured using thecytokinesis-block micronucleus cytome assay and aneu-ploidy in human B lymphoblastoid cell lines. Mutagenesis26:421–429.

49. Gonzalez NV, N Nikoloff, S Soloneski and ML Larra-mendy. (2011). A combination of the cytokinesis-blockmicronucleus cytome assay and centromeric identificationfor evaluation of the genotoxicity of dicamba. ToxicolLett 207:204–212.

50. Andres E, J Molinari, N Remoue, VM Sa-Rocha, C Bar-richello and SP Hurtado. (2012). Successful micronucleustesting with the EPI/001 3D reconstructed epidermismodel: preliminary findings. Mutat Res 743:36–41.

51. Takahashi K, K Tanabe, M Ohnuki, M Narita, T Ichisaka,K Tomoda, S Yamanaka. (2007). Induction of pluripotentstem cells from adult human fibroblasts by defined factors.Cell 131:861–872.

52. Vinoth KJ, BC Heng, A Poonepalli, B Banerjee, L Ba-lakrishnan, K Lu, MP Hande and T Cao. (2008). Humanembryonic stem cells may display higher resistance togenotoxic stress as compared to primary explanted so-matic cells. Stem Cells Dev 17:599–607.

53. Laustriat D, J Gide and M Peschanski. (2010). Humanpluripotent stem cells in drug discovery and predictivetoxicology. Biochem Soc Trans 38:1051–1057.

54. Trosko JE and CC Chang. (2010). Factors to consider in theuse of stem cells for pharmaceutic drug development andfor chemical safety assessment. Toxicology 270:18–34.

55. Vojnits K and S Bremer. (2010). Challenges of usingpluripotent stem cells for safety assessments of sub-stances. Toxicology 270:10–17.

56. Wobus AM and P Loser. (2011). Present state and futureperspectives of using pluripotent stem cells in toxicologyresearch. Arch Toxicol 85:79–117.

57. Duarte DM, DA Cornelio, C Corado, VK Medeiros, LAde Araujo, GB Cavalcanti and SR de Medeiros. (2012).Chromosomal characterization of cryopreserved mesen-chymal stem cells from the human subendothelium um-bilical cord vein. Regen Med 7:147–157.

58. Daya-Grosjean L, MR James, C Drougard and A Sarasin.(1987). An immortalized xeroderma pigmentosum, groupC, cell line which replicates SV40 shuttle vectors. MutatRes 183:185–196.

59. Giard DJ, SA Aaronson, GJ Todaro, P Arnstein, JHKersey, H Dosik and WP Parks. (1973). In vitro cultiva-tion of human tumors: establishment of cell lines derivedfrom a series of solid tumors. J Natl Cancer Inst 51:1417–1423.

60. Covas DT, JL Siufi, AR Silva and MD Orellana. (2003).Isolation and culture of umbilical vein mesenchymal stemcells. Braz J Med Biol Res 36:1179–1183.

61. Panepucci RA, JL Siufi, WA Silva Jr, R Proto-Siquiera, LNeder, M Orellana, V Rocha, DT Covas and MA Zago.(2004). Comparison of gene expression of umbilical cordvein and bone marrow–derived mesenchymal stem cells.Stem Cells 22:1263–1278.

62. Kadivar M, S Khatami, Y Mortazavi, MA Shokrgozar, MTaghikhani and M Soleimani. (2006). In vitro cardio-myogenic potential of human umbilical vein-derivedmesenchymal stem cells. Biochem Biophys Res Commun340:639–647.

63. Romanov YA, VA Svintsitskaya and VN Smirnov.(2003). Searching for alternative sources of postnatal hu-

man mesenchymal stem cells: candidate MSC-like cellsfrom umbilical cord. Stem Cells 21:105–110.

64. Koh SH, KS Kim, MR Choi, KH Jung, KS Park, YG Chai,W Roh, SJ Hwang, HJ Ko, et al. (2008). Implantation ofhuman umbilical cord-derived mesenchymal stem cells asa neuroprotective therapy for ischemic stroke in rats.Brain Res 1229:233–248.

65. Ishige I, T Nagamura-Inoue, MJ Honda, R Harnpra-sopwat, M Kido, M Sugimoto, H Nakauchi and A Tojo.(2009). Comparison of mesenchymal stem cells derivedfrom arterial, venous, and Wharton’s jelly explants ofhuman umbilical cord. Int J Hematol 90:261–269.

66. Li M, SZ Zhang, YW Guo, YQ Cai, ZJ Yan, Z Zou, XDJiang, YQ Ke, XY He, et al. (2010). Human umbilicalvein-derived dopaminergic-like cell transplantation withnerve growth factor ameliorates motor dysfunction in a ratmodel of Parkinson’s disease. Neurochem Res 35:1522–1529.

67. Montesinos JJ, E Flores-Figueroa, S Castillo-Medina, PFlores-Guzman, E Hernandez-Estevez, G Fajardo-Orduna,S Orozco and H Mayani. (2009). Human mesenchymalstromal cells from adult and neonatal sources: compara-tive analysis of their morphology, immunophenotype,differentiation patterns and neural protein expression.Cytotherapy 11:163–176.

68. Ho JH, YF Chen, WH Ma, TC Tseng, MH Chen and OKLee. (2011). Cell contact accelerates replicative senes-cence of human mesenchymal stem cells independent oftelomere shortening and p53 activation: roles of Ras andoxidative stress. Cell Transplant 20:1209–1220.

69. Schiller ZA, NR Schiele, JK Sims, K Lee and CK Kuo.(2013). Adipogenesis of adipose-derived stem cells maybe regulated via the cytoskeleton at physiological oxygenlevels in vitro. Stem Cell Res Ther 4:79.

70. Zhong W, K Tian, X Zheng, L Li, W Zhang, S Wang andJ Qin. (2013). Mesenchymal stem cell and chondrocytefates in a multishear microdevice are regulated by yes-associated protein. Stem Cells Dev 22:2083–2093.

71. Ryu YJ, TJ Cho, DS Lee, JY Choi and J Cho. (2013).Phenotypic characterization and in vivo localization ofhuman adipose-derived mesenchymal stem cells. MolCells 35:557–564.

72. Kalantari H, H Gourabi and H Baharvand. (2012).Adapted cytokinesis-block micronucleus assay (CBMN)for mouse embryonic stem cells. www.nature.com/proto-colexchange/protocols/2355#/introduction.

73. Henegariu O, NA Heerema, L Lowe Wright, P Bray-Ward, DC Ward and GH Vance. (2001). Improvements incytogenetic slide preparation: controlled chromosomespreading, chemical aging and gradual denaturing. Cyto-metry 43:101–109.

74. Deng W, SW Tsao, JN Lucas, CS Leung and AL Cheung.(2003). A new method for improving metaphase chro-mosome spreading. Cytometry A 51:46–51.

75. Moynahan ME, TY Cui and M Jasin. (2001). Homology-directed DNA repair, mitomycin-c resistance, and chro-mosome stability is restored with correction of a Brca1mutation. Cancer Res 61:4842–4850.

76. Chang HC, J Tsai, YL Guo, YH Huang, HN Tsai, PC Tsaiand W Huang. (2003). Differential UVC-induced gadd45gene expression in xeroderma pigmentosum cells. Bio-chem Biophys Res Commun 305:1109–1115.

77. Frosina G. (2010). The bright and the dark sides of DNArepair in stem cells. J Biomed Biotechnol 2010:845396.

836 CORNELIO ET AL.

78. Fan J, C Robert, YY Jang, H Liu, S Sharkis, SB Baylinand FV Rassool. (2011). Human induced pluripotent cellsresemble embryonic stem cells demonstrating enhancedlevels of DNA repair and efficacy of nonhomologous end-joining. Mutat Res 713:8–17.

79. Luo LZ, S Gopalakrishna-Pillai, SL Nay, SW Park, SEBates, X Zeng, LE Iverson and TR O’Connor. (2012).DNA repair in human pluripotent stem cells is distinctfrom that in non-pluripotent human cells. PLoS One7:e30541.

80. Bracker TU, B Giebel, J Spanholtz, UR Sorg, L Klein-Hitpass, T Moritz and J Thomale. (2006). Stringentregulation of DNA repair during human hematopoieticdifferentiation: a gene expression and functional analysis.Stem Cells 24:722–730.

81. Chen MF, CT Lin, WC Chen, CT Yang, CC Chen, SKLiao, JM Liu, CH Lu and KD Lee. (2006). The sensitivityof human mesenchymal stem cells to ionizing radiation.Int J Radiat Oncol Biol Phys 66:244–253.

82. Sugrue T, JA Brown, NF Lowndes and R Ceredig. (2013).Multiple facets of the DNA damage response contribute tothe radioresistance of mouse mesenchymal stromal celllines. Stem Cells 31:137–145.

83. Oliver L, E Hue, Q Sery, A Lafargue, C Pecqueur, F Parisand FM Vallette. (2013). Differentiation-related responseto DNA breaks in human mesenchymal stem cells. StemCells 31:800–807.

84. Thomas P, K Umegaki and M Fenech. (2003). Nucleo-plasmic bridges are a sensitive measure of chromosomerearrangement in the cytokinesis-block micronucleus as-say. Mutagenesis 18:187–194.

85. Glaser N and H Stopper. (2012). Patulin: Mechanism ofgenotoxicity. Food Chem Toxicol 50:1796–1801.

86. Kocaman AY, E Rencuzogulları and M Topaktasx. (2012).In vitro investigation of the genotoxic and cytotoxic ef-fects of thiacloprid in cultured human peripheral bloodlymphocytes. Environ Toxicol. [Epub ahead of print];DOI: 10.1002/tox.21790.

87. Duan H, S Leng, Z Pan, Y Dai, Y Niu, C Huang, P Bin, YWang, Q Liu, W Chen and Y Zheng. (2009). Biomarkersmeasured by cytokinesis-block micronucleus cytome as-say for evaluating genetic damages induced by polycyclicaromatic hydrocarbons. Mutat Res 677:93–99.

88. Ebert R, M Ulmer, S Zeck, J Meissner-Weigl, DSchneider, H Stopper, N Schupp, M Kassem and F Jakob.(2006). Selenium supplementation restores the anti-oxidative capacity and prevents cell damage in bonemarrow stromal cells in vitro. Stem Cells 24:1226–1235.

89. Sartore RC, PB Campos, CA Trujillo, BL Ramalho, PDNegraes, BS Paulsen, T Meletti, ES Costa, L Chicaybam,et al. (2011). Retinoic acid-treated pluripotent stem cellsundergoing neurogenesis present increased aneuploidyand micronuclei formation. PLoS One 6:e20667.

90. Campos PB, RC Sartore, BL Ramalho, ES Costa and SKRehen. (2012). Cycle arrest and aneuploidy induced byzidovudine in murine embryonic stem cells. Mutagenesis27:431–436.

91. Miura M, Y Miura, HM Padilla-Nash, AA Molinolo, BFu, V Patel, BM Seo, W Sonoyama, JJ Zheng, et al.(2006). Accumulated chromosomal instability in murinebone marrow mesenchymal stem cells leads to malignanttransformation. Stem Cells 24:1095–1103.

92. Tolar J, AJ Nauta, MJ Osborn, A Panoskaltsis Mortari, RTMcElmurry, S Bell, L Xia, N Zhou, M Riddle, et al.

(2007). Sarcoma derived from cultured mesenchymalstem cells. Stem Cells 25:371–379.

93. Foudah D, S Redaelli, E Donzelli, A Bentivegna, M Mi-loso, L Dalpra and G Tredici. (2009). Monitoring thegenomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells. Chromosome Res17:1025–1039.

94. Jeong JO, JW Han, JM Kim, HJ Cho, C Park, N Lee, DWKim and YS Yoon. (2011). Malignant tumor formationafter transplantation of short-term cultured bone marrowmesenchymal stem cells in experimental myocardial in-farction and diabetic neuropathy. Circ Res 108:1340–1347.

95. Ren Z, J Wang, W Zhu, Y Guan, C Zou, Z Chen and YAZhang. (2011). Spontaneous transformation of adultmesenchymal stem cells from cynomolgus macaques invitro. Exp Cell Res 317:2950–2957.

96. Tasso R, A Augello, M Carida’, F Postiglione, MG Ti-biletti, B Bernasconi, S Astigiano, F Fais, M Truini, RCancedda and G Pennesi. (2009). Development of sarco-mas in mice implanted with mesenchymal stem cellsseeded onto bioscaffolds. Carcinogenesis 30:150–157.

97. Wislet-Gendebien S, C Poulet, V Neirinckx, B Hennuy,JT Swingland, E Laudet, L Sommer, O Shakova, V Boursand B Rogister. (2012). In vivo tumorigenesis was ob-served after injection of in vitro expanded neural creststem cells isolated from adult bone marrow. PLoS One7:e46425.

98. Xu S, A De Becker, H De Raeve, B Van Camp, K Van-derkerken and I Van Riet. (2012). In vitro expanded bonemarrow-derived murine (C57Bl/KaLwRij) mesenchymalstem cells can acquire CD34 expression and induce sar-coma formation in vivo. Biochem Biophys Res Commun424:391–397.

99. Gisselsson D. (2008). Classification of chromosome seg-regation errors in cancer. Chromosoma 117:511–519.

100. Gisselsson D, L Pettersson, M Hoglund, M Heidenblad, LGorunova, J Wiegant, F Mertens, P Dal Cin, F Mitelmanand N Mandahl. (2000). Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity. ProcNatl Acad Sci U S A 97:5357–5362.

101. Gisselsson D, J Bjork, M Hoglund, F Mertens, P Dal Cin,M Akerman and N Mandahl. (2001). Abnormal nuclearshape in solid tumors reflects mitotic instability. Am JPathol 158:199–206.

102. Gebre-Medhin S, K Broberg, T Jonson, L Gorunova, FVvon Steyern, O Brosjo, Y Jin, D Gisselsson, I Panago-poulos, N Mandahl and F Mertens. (2009). Telomericassociations correlate with telomere length reduction andclonal chromosome aberrations in giant cell tumor ofbone. Cytogenet Genome Res 124:121–127.

103. Hoffelder DR, L Luo, NA Burke, SC Watkins, SM Gollinand WS Saunders. (2004). Resolution of anaphase bridgesin cancer cells. Chromosoma 112:389–397.

104. Heim S and F Mitelman. (2009). Cancer Cytogenetics, 3rdedn. Wiley-Blackwell, New York.

105. Tusell L, J Pampalona, D Soler, C Frıas and A Genesca.(2010). Different outcomes of telomere-dependent ana-phase bridges. Biochem Soc Trans 38:1698–1703.

106. Frias C, J Pampalona, A Genesca and L Tusell. (2012).Telomere dysfunction and genome instability. FrontBiosci 17:2181–2196.

107. Pampalona J, C Frıas, A Genesca and L Tusell. (2012).Progressive telomere dysfunction causes cytokinesis failure

CBMN ANALYSIS OF HUMAN MESENCHYMAL STEM CELLS 837

and leads to the accumulation of polyploid cells. PLoSGenet 8:e1002679.

108. Zakov S, M Kinsella and V Bafna. (2013). An algorithmicapproach for breakage-fusion-bridge detection in tumorgenomes. Proc Natl Acad Sci U S A 110:5546–5551.

109. Calado RT, JN Cooper, HM Padilla-Nash, EM Sloand,CO Wu, P Scheinberg, T Ried and NS Young. (2012).Short telomeres result in chromosomal instability in he-matopoietic cells and precede malignant evolution in hu-man aplastic anemia. Leukemia 26:700–707.

110. Chakraborty S, JM Stark, CL Sun, H Modi, W Chen, TRO’Connor, SJ Forman, S Bhatia and R Bhatia. (2012).Chronic myelogenous leukemia stem and progenitorcells demonstrate chromosomal instability related torepeated breakage-fusion-bridge cycles mediated by in-creased nonhomologous end joining. Blood 119:6187–6197.

111. Chan KL, PS North and ID Hickson. (2007). BLM is re-quired for faithful chromosome segregation and its lo-calization defines a class of ultrafine anaphase bridges.EMBO J 26:3397–3409.

112. Chan KL and ID Hickson. (2011). New insights into theformation and resolution of ultra-fine anaphase bridges.Semin Cell Dev Biol 22:906–912.

113. Baumann C, R Korner, K Hofmann and EA Nigg. (2007).PICH, a centromere-associated SNF2 family ATPase, isregulated by Plk1 and required for the spindle checkpoint.Cell 128:101–114.

114. Rouzeau S, FP Cordelieres, G Buhagiar-Labarchede, IHurbain, R Onclercq-Delic, S Gemble, L Magnaghi-Jaulin,C Jaulin and M Amor-Gueret. (2012). Bloom’s syndromeand PICH helicases cooperate with topoisomerase IIa incentromere disjunction before anaphase. PLoS One 7:e33905.

115. Barefield C and J Karlseder. (2012). The BLM helicasecontributes to telomere maintenance through processing oflate-replicating intermediate structures. Nucleic Acids Res40:7358–7367.

116. Rodrigue A, Y Coulombe, K Jacquet, JP Gagne, C Ro-ques, S Gobeil, G Poirier and JY Masson. (2013). TheRAD51 paralogs ensure cellular protection against mitoticdefects and aneuploidy. J Cell Sci 126(Pt 1):348–359.

117. Larsen NB and ID Hickson. (2013). RecQ Helicases:Conserved Guardians of Genomic Integrity. Adv Exp MedBiol 767:161–184.

118. Chan KL, T Palmai-Pallag, S Ying and ID Hickson. (2009).Replication stress induces sister-chromatid bridging atfragile site loci in mitosis. Nat Cell Biol 11:753–760.

119. Naim V and F Rosselli. (2009). The FANC pathway andmitosis: a replication legacy. Cell Cycle 8:2907–2911.

120. Kaulich M, F Cubizolles and EA Nigg. (2012). On theregulation, function, and localization of the DNA-depen-dent ATPase PICH. Chromosoma 121:395–408.

121. Jaager K, S Islam, P Zajac, S Linnarsson and T Neuman.(2012). RNA-seq analysis reveals different dynamics ofdifferentiation of human dermis- and adipose-derivedstromal stem cells. PLoS One 7:e38833.

122. Schack LM, S Noack, R Winkler, M Jagodzinski, CKrettek, M Buttner and A Hoffmann. (2013). Analysis ofsurface protein expression in human bone marrow stromalcells: new aspects of culture-induced changes, inter-donordifferences and intracellular expression. Stem Cells Dev[Epub ahead of print] PubMed PMID:23927592.

123. Dhillon VS, P Thomas, G Iarmarcovai, M Kirsch-Volders,S Bonassi and M Fenech. (2011). Genetic polymorphismsof genes involved in DNA repair and metabolism influ-ence micronucleus frequencies in human peripheral bloodlymphocytes. Mutagenesis 26:33–42.

Address correspondence to:Silvia Regina Batistuzzo de Medeiros

Departamento de Biologia Celular e GeneticaCentro de Biociencias

Universidade Federal do Rio Grande do NorteAvenida Senador Salgado Filho, 3000

UFRN Campus Universitario, Lagoa NovaNatal 59078-900

Brazil

E-mail: sbatistu@gmail.com

Received for publication September 2, 2013Accepted after revision November 26, 2013

Prepublished on Liebert Instant Online December 13, 2013

838 CORNELIO ET AL.

This article has been cited by:

1. Déborah Afonso Cornélio, Silvia Regina Batistuzzo de Medeiros. 2014. Genetic evaluation of mesenchymal stem cells. RevistaBrasileira de Hematologia e Hemoterapia . [CrossRef]