ORIGINAL RESEARCH ARTICLE

DNA Methylation Inhibitors, 5-azacytidine and ZebularinePotentiate the Transdifferentiation of Rat Bone MarrowMesenchymal Stem Cells into Cardiomyocytes

Nadia Naeem,1 Kanwal Haneef,1 Nurul Kabir,1 Hana’a Iqbal,1 Siddiqua Jamall2 & Asmat Salim1

1 Dr. Panjwani Center for Molecular Medicine and Drug Research (PCMD), International Center for Chemical and Biological Sciences (ICCBS), University

of Karachi, Karachi, Pakistan

2 Department of Biochemistry, University of Karachi, Karachi, Pakistan

Keywords

5-azacytidine; Cardiomyocytes; Demethylating

agent; Mesenchymal stem cells;

Transdifferentiation; Zebularine.

Correspondence

Asmat Salim, Dr. Panjwani Center for

Molecular Medicine and Drug Research,

International Center for Chemical and

Biological Sciences, University of Karachi,

Karachi-75270, Pakistan.

Tel.: +(92-21) 34824930;

Fax: +(92-21) 34819018;

E-mail: asmat.salim@iccs.edu

doi: 10.1111/j.1755-5922.2012.00320.x

SUMMARY

Background: Mesenchymal stem cells (MSCs) have immense self-renewal capability.

They can be differentiated into many cell types and therefore hold great potential in

the field of regenerative medicine. MSCs can be converted into beating cardiomyo-

cytes by treating them with DNA-demethylating agents. Some of these compounds are

nucleoside analogs that are widely used for studying the role of DNA methylation in

biological processes as well as for the clinical treatment of leukemia and other carci-

nomas. Aims: To achieve a better therapeutic option for cardiovascular regeneration, this

study was carried out using MSCs treated with two synthetic compounds, zebularine and

5-azacytidine. It can be expected that treated MSCs prior to transplantation may increase

the likelihood of successful regeneration of damaged myocardium. Methods: The opti-

mized concentrations of these compounds were added separately into the culture medium

and the treated cells were analyzed for the expression of cardiac-specific genes by RT-PCR

and cardiac-specific proteins by immunocytochemistry and flow cytometry. Treated MSCs

were cocultured with cardiomyocytes to see the fusion capability of these cells. Results:

mRNA and protein expressions of GATA4, Nkx2.5, and cardiac troponin T were observed in

the treated MSCs. Coculture studies of MSCs and cardiomyocytes have shown improved

fusion with zebularine-treated MSCs as compared to untreated and 5-azacytidine-treated

MSCs. Conclusion: The study is expected to put forth another valuable aspect of certain

compounds, that is, induction of transdifferentiation of MSCs into cardiomyocytes. This

would serve as a tool for modified cellular therapy and may increase the probability of

better myocardial regeneration.

Introduction

Myocardial infarction is now the leading cause of death world-

wide. In South Asia, heart diseases are increasing at a rate

greater than in other regions of the world [1]. Current phar-

maco-therapies for treating heart failure such as neurohormonal

inhibition with angiotensin-converting enzyme inhibitors and

b-blockers that improve clinical outcomes as well as interven-

tional and surgical procedures are limited in preventing ventric-

ular remodeling because of their inability to repair or replace

damaged myocardium. Therefore, owing to the high morbidity

and mortality rates associated with heart failure, novel methods

to improve cardiac function after myocardial infarction are

desirable. Regenerative medicine has been introduced as a

potential therapy for treating myocardial infarction. Stem cell

therapy offers a new perspective for cardiac regeneration in

which the scar tissue may be replaced with viable contracting

muscle cells and blood vessels. Mesenchymal stem cells (MSCs)

have self-renewal capability and can differentiate into different

cell types. MSCs, therefore, have immense potential in the

emerging field of regenerative medicine. In cardiac regenera-

tion, MSCs have shown the ability to transdifferentiate into car-

diomyocytes, both in vitro and in vivo [2,3]. Recently, it was

shown that some small molecules such as dexamethasone,

ascorbic acid, 5-azacytidine or all-trans retinoic acid are capable

of inducing the differentiation of stem cells into different cells

types [4]. Demethylating agents have gained interest because of

their epigenetic reprogramming ability during somatic and stem

cell differentiation [5]. In vitro treatment with 5-azacytidine, a

DNA-demethylating agent, was able to induce differentiation of

adult mesenchymal stem cells into cardiac muscle-like cells [6–

8]. Nitric oxide (NO), a free radical signaling molecule, and its

intermediates can also induce increased expression of cardiac-

specific genes in bone marrow mesenchymal stem cells [9].

ª 2012 John Wiley & Sons Ltd Cardiovascular Therapeutics 31 (2013) 201–209 201

Similarly, difluoromethylornithine (DFMO) can also induce

early differentiation of MSCs to the cells of myocardial lineage

[10]. Recently, combination of 5-azacytidine and cytokines that

play important roles in cellular development have been used to

induce differentiation of bone marrow mesenchymal cells into

cardiomyocytes. This includes cardiotrophin-1, a cardiac hyper-

trophic factor and endothelin-1 that show increased expression

during myocardial infarction [11,12].

In this study, cardiomyogenic differentiation potential of rat

bone marrow mesenchymal stem cells by 5-azacytidine and zeb-

ularine was explored. The treated mesenchymal stem cells were

analyzed for the expression of cardiac markers at the genetic

and protein levels. The objective is to come up with a better

compound that would be highly potent in terms of differentiat-

ing the mesenchymal stem cells into cardiac cells while being

least toxic to biological systems. The results obtained from this

study would serve as an attempt in the therapeutic march

toward the regeneration of injured cardiomyocytes due to

coronary artery diseases.

Materials and Methods

Animals

All animal procedures were carried out in accordance with the

international guidelines for the care and use of laboratory animals

and local ethical committee approval. Sprague Dawley (SD) rats of

both sexes weighing 200–300 g were allowed to acclimatize for a

period of 3–4 days prior to the start of the experiment. The ani-

mals were provided with sterile water and food ad libitum, in a

temperature (21 ± 1°C) and humidity-controlled (55 ± 5%) room

with 12-h light: 12-h dark cycle.

Cell Culture

Mesenchymal stem cells (MSCs) were isolated from tibia and

femur of SD rats under aseptic conditions. Whole bone marrow

was cultured in Dulbecco’s modified Eagle’s medium (DMEM;

GIBCO-Life Technologies, Carlsbad, CA, USA) supplemented

with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and

100 lg/mL streptomycin. Cultures were maintained at 37°C in a

humidified atmosphere containing 5% CO2. Nonadherent hema-

topoietic cells were removed from the adherent mesenchymal

stem cells. Culture medium was changed every 3 days. The cells

were subcultured when they reached 70–80% confluence. MSCs

of 1st or 2nd passages were used throughout the study.

Characterization of Mesenchymal Stem Cells

Immunocytochemistry

Cultured mesenchymal stem cells were analyzed for the presence

of surface markers by immunostaining using antibodies for CD44

(BD Biosciences Pharmingen, San Diego, CA, USA), CD90 (Cedar-

lane, Ontario, Canada), and CD29 (Chemicon-Millipore, Billerica,

MA, USA). Cells were fixed using 4% paraformaldehyde for

30 min. After blocking with phosphate-buffered saline (PBS) con-

taining 2% bovine serum albumin (BSA) and 2% normal goat

serum, cells were incubated with primary antibodies at 1:100 dilu-

tion in blocking solution overnight at 4°C. Cells were treated with

Alexa fluor 546 goat anti-mouse secondary antibody (Molecular

Probes-Life Technologies, Carlsbad, CA, USA) for 1 h at room

temperature. The cells were counterstained with DAPI and exam-

ined under fluorescent microscope (Nikon, Tokyo, Japan).

Flow Cytometry

Cultured MSCs were analyzed for the expression of surface mark-

ers by flow cytometry (FACSCalibur, Becton Dickinson, San Jose,

CA, USA). The cells were detached with cell dissociation solution

and washed with FACS solution (PBS containing 1% BSA, 1 mM

EDTA, and 0.1% sodium azide). After blocking nonspecific bind-

ing sites with buffer containing 1% BSA, the cells were incubated

with primary antibodies for rat CD29 (Chemicon) and CD90 (Ce-

darlane) for 30 min at 4°C. After washing with FACS solution, the

cells were treated with Alexa fluor 546 goat anti-mouse secondary

antibody (Molecular Probes-Life Technologies). Unlabeled cells

and cells labeled only with secondary antibody were used as

controls.

Treatment of MSCs with 5-azacytidine andZebularine

Concentrations of 5-azacytidine and zebularine were optimized

prior to treatments. Four concentrations (3, 10, 25, and 50 lM)

of both compounds were used for optimization using the Cyto-

Tox 96 nonradioactive cytotoxicity assay kit (Promega Inc.,

Madison, WI, USA) according to manufacturer’s instructions.

The assay measures a stable cytosolic enzyme, lactate dehydro-

genase (LDH) that is released upon cell lysis. Released LDH was

measured following conversion of a tetrazolium salt into a red

formazen product. An equal volume of culture medium and

reconstituted substrate was mixed and incubated for 30 min at

room temperature. The absorbance was measured at 490 nm

and % cytotoxicity was determined.

MSCs were divided into five groups: (1) untreated control, and

treated with (2) 3 lM 5-azacytidine, (3) 10 lM 5-azacytidine, (4)

3 lM zebularine, and (5) 10 lM zebularine. The compounds were

added to the culture medium and MSCs were incubated for 24 h

at 37°C in a humidified atmosphere containing 5% CO2. The cells

were then washed twice and the medium was replaced with nor-

mal DMEM. The experiments were terminated after 15 days.

Expression of Cardiac Markers

RT-PCR

Total RNA from MSCs of treated and control groups were iso-

lated using the RNeasy Mini kit (Qiagen Inc, Velencia, CA,

USA) according to the manufacturer’s protocol. One microgram

of RNA was reverse transcribed using the Superscript RT kit

(Invitrogen) and amplified using oligonucleotide primers for rat

cardiac-specific genes. Rat glyceraldehyde 3-phosphate dehydro-

genase (GAPDH) gene was used as an internal standard. The

primer sequences used in this study along with the expected

product sizes and PCR annealing temperatures are listed in

202 Cardiovascular Therapeutics 31 (2013) 201–209 ª 2012 John Wiley & Sons Ltd

Differentiation of MSCs by Methylation Inhibitors N. Naeem et al.

Table 1. The products of reverse transcription reaction were

denatured for 1 min at 94°C, followed by 35 cycles of amplifica-

tion (denaturation at 94°C, annealing at 50–60°C and extension

at 72°C for 1 min each) and a final extension at 72°C for

10 min. Each PCR product was electrophoretically resolved on

1% agarose gel. Neonatal rat cardiomyocytes were used as posi-

tive controls.

Flow Cytometry and Immunocytochemistry

Treated MSCs were analyzed by flow cytometry for cardiac pro-

tein expression. The primary antibodies used were anti-NKx2.5,

anti-GATA4, and anti cardiac troponin T (SantaCruz Biotechnol-

ogy, Santa Cruz, CA, USA). Zebularine-treated MSCs were also

analyzed for the expression of GATA4 and cardiac troponin T by

immunocytochemistry using methods as described for the

expression of mesenchymal surface markers. The blocking buffer

for intracellular staining also contained 0.2% Nonidet-P40

(NP40).

Analysis of Fusion Potential of Treated MSCsand CMs

Coculture Studies

Rat neonatal cardiomyocytes were cocultured with both pretreat-

ed and normal mesenchymal stem cells according to the groups

described in previous sections. MSCs and CMs were grown in

DMEM supplemented with antibiotics and 10% FBS. When MSCs

reach 70–80% confluence, they were treated with 5-azacytidine

and zebularine in both 3 and 10 lm concentrations. After

15 days, treated and untreated MSCs were co-cultured with CMs

in a ratio of 2:1 for 5 and 15 days in the humidified CO2 incuba-

tor. Prior to coculture, CMs and MSCs were labeled with PKH26

(Sigma-Aldrich, St. Louis, MO, USA) and PKH67 (Sigma) cell

labeling dyes, respectively, according to the manufacturer’s

instructions. After the completion of the coculture period, med-

ium was removed and cells were washed twice with PBS. Samples

were analyzed for the presence of double positive cells in flow cy-

tometer (FACSCalibur, BD). The excitation and emission maxima

of PKH26 were 551 and 567 nm, while that of PKH67 were 490

and 502 nm, respectively. Untreated MSCs cocultured with car-

diomyocytes were used as control. Data were evaluated using BD

cell Questpro software.

Results

Isolation, Propagation, and Characterization ofMSCs

MSCs were isolated from bone marrow of SD rats on the basis of

their adherent property. Adherent MSCs in culture showed a

spindle-shaped fibroblast like morphology (Figure 1). MSCs were

characterized by specific cell surface antigens that are not

expressed in the hematopoietic stem cells. We used immunocyto-

chemistry and flow cytometry techniques to determine the pres-

ence of cell surface–specific markers on MSCs. Cell surface

antigen expression was observed to be positive for CD29, CD44,

and CD90 in case of immunocytochemistry, while CD29 and

CD90 were found to be more than 80% positive in case of flow

cytometry (as shown in the supplementary material).

Optimization of Concentrations of 5-azacytidneand Zebularine

Fourdifferent concentrations (3, 10, 25, and50 lM)of 5-azacytidine

and zebularine were used for cytotoxicty assessment to optimize

their concentrations for treatment. It was observed that concentra-

tions of 25 and 50 lM (P < 0.01) were toxic to cells in case of both

the compounds, whereas that of 3 and 10 lM showed nonsignifi-

cant LDH release and therefore were not considered as cytotoxic.

These concentrations were then used in all the treatments. Cyto-

toxicity assay results are shown in supplementarymaterial.

Expression of Cardiac Markers in Treated MSCs

After separate treatments with 5-azacytidine and zebularine at 3

and 10 lM concentrations, MSCs continued to proliferate and dif-

ferentiate. After 15 days in culture, the cells showed extended

cytoplasmic processes (Figure 1B,C). RT-PCR analysis for the

expression of cardiac-specific genes showed that treated cells

expressed GATA4, Nkx2.5, and cardiac troponin T (Figure 2;

Table 1 Forward and reverse primers, their expected product sizes and annealing temperatures

PCR primersa Gene IDs Sequences

Annealing

temperatures (°C)

Product

sizes (BP)

GAPDH (F)

GAPDH (R)

BC09593 5′-GGAAAGCTGTGGCGTGATGG-3′

5′-GTAGGCCATGAGGTCCACCA-3′

60 414

CTT (F)

CTT (R)

NM_012676 5′-TTCGACCTGCAGGAAAAGTT-3′

5′-GTGCCTGGCAAGACCTAGAG-3′

57 206

Nkx 2.5 (F)

Nkx 2.5 (R)

NM_053651 5′-ACCGCCCCTACATTTTATCC-3′

5′-GACAGGTACCGCTGTTGCTT-3′

57 230

GATA4 (F)

GATA4 (R)

NM_144730 5′-TCTCACTATGGGCACAGCAG-3′

5′-CCGAGCAGGAATTTGAAGAG-3′

60 245

aPrimers: GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; CTT, Cardiac Troponin T.

ª 2012 John Wiley & Sons Ltd Cardiovascular Therapeutics 31 (2013) 201–209 203

N. Naeem et al. Differentiation of MSCs by Methylation Inhibitors

Panel A). Cardiac-specific genes were not expressed in untreated

MSCs. Immunocytochemical analysis of zebularine-treated MSCs

showed expression of cardiac troponin T and GATA4 (Figure 2;

Panel B), while flow cytometry results have shown that cells

treated with 5-azacytidine and zebularine were positive for

GATA4, Nkx2.5, and cardiac troponin T (Figure 3).

Analysis of fusion potential of treated MSCs andCMs

The 5 day coculture of MSCs and CMs have shown increase in the

number of fused cells when treated MSCs were used as compared

to the untreated MSCs. Number of fused cells were 10.86% in case

of untreated MSCs, while in case of 5-azacytidine and zebularine-

treated MSCs, the number of fused cells increased to 17.34%

(3 lM), 18.33% (10 lM), 21.67% (3 lM), and 17.64% (10 lM),

respectively (Figure 4; Panel A). Best fusion was seen in case of

3 lm zebularine (Figure 4; Panel C).

Similar results were obtained with 15 day coculture of MSCs

and CMs. There was an increase in the number of fused cells when

treated MSCs were used as compared to the untreated MSCs.

Number of fused cells were 17.18% in case of untreated MSCs,

while in case of 5-azacytidine and zebularine-treated MSCs, the

number of fused cells increased to 22.9% (3 lM), 18.39%

(10 lM), 25.95% (3 lM), and 22.62% (10 lM), respectively (Fig-

ure 4; Panel B). Best fusion was seen in case of 3 lM zebularine

(Figure 4; Panel C).

Discussion

DNA methylation is one of the epigenetic mechanisms that regu-

late chromatin organization and gene expression. It is also a

reversible process that promotes expression of genes associated

with differentiation. Therefore, the relationship between patterns

of DNA methylation and gene regulation during stem cell differ-

entiation should be thoroughly investigated to improve our

understanding of the cellular changes that occur during differenti-

ation. This, in turn, may facilitate the development of procedures

that will allow the control of stem cell differentiation into desired

cell types [13].

5-azacytidine is an important DNA-demethylating agent. It can

be incorporated into DNA to form covalent adducts with DNA

methyltransferase-1. This results in decrease in enzyme activity

that causes demethylation of the genomic DNA [14]. It has been

documented that hypomethylation caused by 5-azacytidine can

increase the expression of a number of genes, such as the tumor

suppressor genes, by epigenetic modification of the cellular DNA

[15–17]. Although the effects of 5-azacytidine on bone marrow

stem cells have been widely studied, there is little evidence of

5-azacytidine being directly involved in stem cell differentiation

[8, 18, 19]. According to the protein profiling results obtained

from the effect of 5-azacytidine on mesenchymal stem cells

(MSCs), it has been observed that cardiac-specific proteins were

expressed in the treated MSCs. These proteins may play an impor-

tant role in the process of induced differentiation [20–22].

Zebularine is a cytidine analog containing a 2-pyrimidinone

ring. It was originally developed as a cytidine deaminase inhibi-

tor. It lacks an amino group at position 4 of the pyrimidine ring

[23–26]. Unlike 5-azacytidine, zebularine is stable in aqueous

solution of pH up to 12 [27,28]. In vitro experiments have shown

that synthetic oligonucleotides containing zebularine form tight

complexes with bacterial methyltransferases leading to potent

(A)

(B)

(C)

Figure 1 Normal and treated mesenchymal stem cells (MSCs): (A) Normal

untreated MSCs; MSCs treated with (B) 5-azacytidine and (C) zebularine.

Extended cytoplasmic processes are seen in the treated MSCs.

204 Cardiovascular Therapeutics 31 (2013) 201–209 ª 2012 John Wiley & Sons Ltd

Differentiation of MSCs by Methylation Inhibitors N. Naeem et al.

inhibition of DNA methylation [29]. The ability of zebularine to

inhibit DNA methylation was demonstrated in microbial system as

well as mammalian cell lines [30–32]. Because of better stability

and less cytotoxic potential of zebularine, it could be a better

replacement for 5-azacytidine for differentiation of MSCs into

cardiomyocytes.

In the present study, rat bone marrow-derived MSCs were

treated separately with 5-azacytidine and zebularine for 24 h in

two optimized concentrations. The changes in the mRNA levels of

cardiac-specific proteins in treated MSCs were determined by

RT-PCR, while protein expression was analyzed by flow cyto-

metry and immunocytochemistry. After 2 weeks of treatment,

myotube-like structures were formed between adjoining cells. The

characteristic appearance of “stick-like” cells may be attributed to

the expression of proteins responsible for maintaining the cyto-

skeleton. The immunocytochemical and flow cytometry analysis

showed that the treated MSCs expressed cardiac-specific proteins,

such as GATA4, Nkx2.5, and cardiac troponin T, proving that the

treated cells had attained cardiomyogenic phenotype. We, how-

ever, obtained two nonspecific bands along with the GATA4 band

(A)

(B)(a) (b)

(c) (d)

Figure 2 Expression of cardiac-specific genes and proteins in treated mesenchymal stem cells (MSCs): (A) Reverse Transcriptase (RT) PCR analysis of

treated MSCs with 3 and 10 lM 5-azacytidine and zebularine showing the mRNA expression of Nkx2.5, cardiac troponin T (CTT), and GATA4. Untreated

MSCs showed no expression, while cardiomyocytes (CMs) showed positive expression. Also shown are the expression of theses genes relative to

glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal control. Data are presented as mean ± standard error of the mean (SEM) and

calculated using Microsoft Excel. Statistical significance (*P < 0.05) was determined by analysis of variance (ANOVA) with Bonferroni correction using

SPSS software. (B) Immunocytochemical analysis showing expression of cardiac-specific proteins (a-b) GATA4 and (c-d) cardiac troponin T in MSCs treated

with 3 and 10 lM zebularine.

ª 2012 John Wiley & Sons Ltd Cardiovascular Therapeutics 31 (2013) 201–209 205

N. Naeem et al. Differentiation of MSCs by Methylation Inhibitors

(A)

(B)

(C)

(D)

(E)

Figure 3 Cardiac-specific proteins in treated mesenchymal stem cells (MSCs): Flow cytometry analysis showing expression of cardiac-specific proteins,

Nkx2.5, GATA4, and cardiac troponin T in MSCs treated with (B) 3 lM and (C) 10 lM 5-azacytidine and (D) 3 lM and (E) 10 lM zebularine, while (A)

unstained MSCs, untreated MSCs stained only with secondary antibody and untreated MSCs stained with primary antibodies to Nkx2.5, GATA4, and

cardiac troponin T were used as controls. FSC is selected as the threshold parameter and threshold is set to a value of 52 which eliminate small debris.

206 Cardiovascular Therapeutics 31 (2013) 201–209 ª 2012 John Wiley & Sons Ltd

Differentiation of MSCs by Methylation Inhibitors N. Naeem et al.

in the treated MSCs. Very low intensity bands of the same size

were also observed in untreated MSCs. These bands can be consid-

ered insignificant because of the small molecular size, that is, less

than 150 bp.

Coculture studies were performed using CMs and treated MSCs

to see the fusion potential of these cells. Flow cytometric analysis

of treated MSCs cocultured with CMs showed an increased num-

ber of fused cells as compared to that of untreated MSCs with

CMs. We obtained highest number of fused cells in case of 3 lMzebularine-treated MSCs. Number of fused cells were more in case

of 15 day coculture. Interestingly, in case of both 5-azacytidine

and zebularine, lesser concentration (3 lM) of the compound

(A)

(C)

(B)

(a) (b)

(c) (d)

(e) (f)

(a) (b)

(c) (d)

(e) (f)

Figure 4 Coculture analysis by flow cytometry: (A) 5 day coculture of either untreated or treated mesenchymal stem cells (MSCs) (labeled with PKH 26)

with CMs (labeled with PKH 67) as analyzed. (a) Unlabeled cells, (b) CMs and untreated MSCs, (c) CMs and MSCs treated with 3 lM 5-azacytidine, (d) CMs

and MSCs treated with 10 lM 5-azacytidine, (e) CMs and MSCs treated with 3 lM zebularine and (f) CMs and MSCs treated with 10 lM zebularine.

(B) 15 day coculture of either untreated or treated MSCs (labeled with PKH 26) with CMs (labeled with PKH 67). (a) Unlabeled cells, (b) CMs and untreated

MSCs, (c) CMs and MSCs treated with 3 lM 5-azacytidine, (d) CMs and MSCs treated with 10 lM 5-azacytidine, (e) CMs and MSCs treated with 3 lM

zebularine, and (f) CMs and MSCs treated with 10 lM zebularine. (C) Data of (a) 5 and (b) 15 day coculture are presented as mean ± standard error of the

mean (SEM) and calculated using Microsoft Excel. Statistical significance (*P < 0.05; **P < 0.01) was determined by analysis of variance (ANOVA) with

Tukey’s correction using SPSS software.

ª 2012 John Wiley & Sons Ltd Cardiovascular Therapeutics 31 (2013) 201–209 207

N. Naeem et al. Differentiation of MSCs by Methylation Inhibitors

seemed to influence the fusion potential of MSCs. In 5 day cocul-

ture, 5-azacytidine-treated MSCs showed no significant difference

in 3 lM or 10 lM concentrations as the number of fused cells

were 17.34% and 18.32%, respectively. In case of 15 day cocul-

ture, fused cells were 22.9% and 18.39%, respectively, in case of

3 and 10 lM concentrations, respectively. Zebularine-treated

MSCs showed decreased number of fused cells from 21.67% to

17. 64% and from 25.95% to 22.62% when the concentration of

the compound was increased from 3 to 10 lM in 5 and 15 day co-

culture, respectively. The results suggest that lower concentration

of zebularine is more effective with respect to enhancing the

fusion potential of MSCs with CMs. Experiments carried out with

zebularine to explore its efficacy as antitumor agent, demonstrate

that zebularine treatment decreased cell proliferation paralleled

with the up-regulation of pro-apoptotic genes in a concentration,

time and dose-dependent manner [33,34]. It has the capacity to

re-express epigenetically silenced genes in human breast cancer

cells at low doses. Zebularine is therefore considered as an effec-

tive methyl transferase inhibitor and demethylating agent in

human breast cancer cell lines. This action of zebularine has been

associated with increased expression of p21, decreased expression

of cyclin-D, and induction of S-phase arrest. At high doses, zebul-

arine induced changes in apoptotic proteins in a cell line specific

manner manifested by alteration in caspase-3, Bax, Bcl2, and

PARP cleavage [33]. In another study, it has been demonstrated

that different doses of zebularine has opposite roles in immunoge-

nicity. Low dose treatment with zebularine resulted in the

enhancement of immunogenicity of rat colon cancer cells,

whereas high dose resulted in suppression of immunogenicity

[35].

Previous studies have demonstrated that treatment of MSCs

isolated from different sources with 5-azacytidine resulted in the

expression of cardiac-specific proteins in these cells in vitro

[8,20,36]. The present study demonstrates that demethylating

agents, 5-azacytidine and zebularine can induce ultra-structural

changes in MSCs leading to their differentiation in vitro and direct

them toward the cardiomyogenic lineage. To assure the quality of

the final therapeutic product, it is, however, important to evaluate

the telomere length and telomerase activity and check their effect

in vivo. There is an association between telomerase activity and

differentiation [37]. In cells with short telomere length, telomere-

associated DNA damage is apparent while these effects were not

present in cells with long telomeres [38]. It would be worthwhile

to investigate whether the treatment of multipotent stem cells

with demethylating agents prior to transplantation can direct

them toward specific cardiomyogenic lineage in the in vivo envi-

ronment. This would enhance the rate of bone marrow stem cell

differentiation into mature cardiomyocytes in the injured heart.

Acknowledgments

This study was supported by Institutional Grant from ICCBS.

Conflict of Interest

The authors declare no conflict of interest.

References

1. Saleheen D, Zaidi M, Rasheed A, et al. Risk of

Myocardial Infarction Study: a resource for the

study of genetic, lifestyle and other

determinants of myocardial infarction in South

Asia. Eur J Epidemiology 2009;24: 329–338.

2. Li X, Yu X, Lin Q, Deng C, Shan Z, Yang M, Lin

S. Bone marrow mesenchymal stem cells

differentiate into functional cardiac phenotypes

by cardiac microenvironment. J Mol Cell Cardiol

2007;42: 295–303.

3. Jackson L, Jones D, Scotting P, Sottile V. Adult

Mesenchymal Stem cells, differentiation

potential and therapeutic application. J Postgrad

Med 2007;53: 121–127.

4. Ding S, Schultz PG. A role of chemistry in stem

cell biology. Nat Biotechnol 2004;22: 833–840.

5. Dimarakis I, Levicar N, Nihoyannopoulos P,

Gordon MY, Habib NA. In vitro stem cell

differentiation into cardiomyocytes. Part 2:

chemicals, extracellular matrix, physical stimuli

and coculture assays. J Cardiothoracic-renal Res

2006;1: 115–121.

6. Antonitsis P, Ioannidou-Papagiannaki E,

Kaidoglou A, et al. Cardiomyogenic potential of

human adult bone marrow mesenchymal stem

cell in vitro. Thorac Cardiov Surg 2008;56: 77–82.

7. Ye NS, Chen J, Luo GA, Zhang RL, Zhao YF,

Wang YM. Proteomic profiling of rat marrow

mesenchymal stem cells induced by 5-

azacytidine. Stem Cells Dev 2006;15: 665–676.

8. Antonitsis P, Ioannidou-Papagiannaki E,

Kaidoglou A, Papakonstantinou C. In vitro

cardiomyogenic differentiation of adult human

bone marrow mesenchymal stem cells. The role

of 5-acacytidine. Interact Cardiovasc Thorac Surg

2007;6: 593–597.

9. Rebelatto CK, Aguiar AM, Senegaglia AC, et al.

Expression of cardiac function genes in adult

stem cells is increased by treatment with nitric

oxide agents. Biochem Biophys Res Commun

2009;378: 456–461.

10. Muscari C, Bonafe F, Carboni M, et al.

Difluoromethylornithine stimulates early cardiac

commitment of mesenchymal stem cells in a

model culture with cardiomyocytes. J Cell

Biochem 2008;103: 1046–1052.

11. Xinyun C, Zhi Z, Bin Z et al. Effects of

cardiotrophin-1 on differentiation and

maturation of rat bone marrow mesenchymal

stem cells induced with 5-azacytidine in vitro.

Int J Cardiol 2010;143: 171–177.

12. Dong HY, Zhang ZM, Zhou ZX. Effects of

endothelin-1 on differentiation of cardiac

myocyte induced from rabbit bone marrow

stromal cells. Chin Med J 2006;119: 832–

839.

13. Zhou G-S, Zhang X-L, Wu J-P, Zhang R-P,

Xiang L-X, Dai L-C, Sha J-Z. 5-Azacytidine

facilitates osteogenic gene expression and

differentiation of mesenchymal stem cells by

alteration in DNA methylation. Cytotechnology

2009;60: 11–22.

14. Juttermann R, Li E, Jaenisch R. Toxicity of

5-aza-2’-deoxycytidine to mammalian cells is

mediated primarily by covalent trapping of DNA

methyltransferase rather than DNA

demethylation. Proc Natl Acad Sci USA 1994;91:

11797–11801.

15. Fang JY, Lu YY. Effects of histone acetylation and

DNA methylation on p21 (WAF1) regulation.

World J Gastroenterol 2002;8: 400–405.

16. Egger G, Aparicio AM, Escobar SG, Jones PA.

Inhibition of histone deacetylation does not

block resilencing of p16 after 5-aza-20-

deoxycytidine treatment. Cancer Res 2007;67:

346–353.

17. Wu YH, Tsai Chang JH, Cheng YW, Wu TC,

Chen CY, Lee H. Xeroderma pigmentosum

group C gene expression is predominantly

regulated by promoter hypermethylation and

contributes to p53 mutation in lung cancers.

Oncogene 2007;26: 4761–473.

18. Liu Y, Song J, Liu W, Wan Y, Chen X, Hu C.

Growth and differentiation of rat bone marrow

stromal cells: does 5-azacytidine trigger their

cardiomyogenic differentiation. Cardiovasc Res

2003;58: 460–468.

19. Burlacu A, Rosca AM, Maniu H, Titorencu I,

Dragan E, Jinga V, Simionescu M. Promoting

effect of 5-azacytidine on the myogenic

differentiation of bone marrow stromal cells.

Eur J Cell Biol 2008;87: 173–184.

20. Ye NS, Zhang RL, Zhao YF, Feng X, Wang YM,

Luo GA. Effect of 5-azacytidine on the protein

208 Cardiovascular Therapeutics 31 (2013) 201–209 ª 2012 John Wiley & Sons Ltd

Differentiation of MSCs by Methylation Inhibitors N. Naeem et al.

expression of porcine bone marrow

mesenchymal stem cells in vitro. Geno Prot

Bioinfo 2006;4: 18–25.

21. Zhang Y, Chu Y, Shen W, Dou Z. Effect of

5-azacytidine induction duration on

differentiation of human first-trimester fetal

mesenchymal stem cells towards cardiomyocyte-

like cells. Interact Cardiovasc Thorac Surg 2009;9:

943–946.

22. Celebi B, Elcin AE, Elcin YM. Proteome analysis

of rat bone marrow mesenchymal stem cell

differentiation. J Proteome Res 2010;9: 5217–5227.

23. Kim CH, Marquez VE, Mao DT, Haines DR,

McCormack JJ. Synthesis of pyrimidin-2-one

nucleosides as acid-stable inhibitors of cytidine

deaminase. J Med Chem 1986;29: 1374–1380.

24. Laliberte J, Marquez VE, Momparler RL. Potent

inhibitors for the deamination of cytosine

arabinoside and 5-aza-2′-deoxycytidine by

human cytidine deaminase. Cancer Chemother

Pharmacol 1992;30: 7–11.

25. Gonzalgo ML, Hayashida MLT, Bender CM,

et al. The role of DNA methylation in expression

of the p19/p16 locus in human bladder cancer

cell lines. Cancer Res 1998;58: 1245–1252.

26. Driscoll JS, Marquez VE, Plowman J, Liu PS,

Kelley JA, Barchi JJ Jr. Antitumor properties of

2(1H)-pyrimidinone riboside (zebularine) and

its fluorinated analogues. J Med Chem 1991;34:

3280–3284.

27. Barchi JJ Jr, Musser S, Marquez VE. The

decomposition of 1-(beta-D-ribofuranosyl)-1,2-

dihydropyrimidin-2-one (zebularine) in alkali:

mechanism and products. J Org Chem 1992;57:

536–541.

28. Kelley JA, Driscoll JS, McCormack JJ, Roth JS,

Marquez VE. Furanose-pyranose isomerization

of reduced pyrimidine and cyclic urea ribosides.

J Med Chem 1986;29: 2351–2358.

29. Hurd PJ, Whitmarsh AJ, Baldwin GS, et al.

Mechanism-based inhibition of C5-cytosine

DNA methyltransferases by 2-H pyrimidinone. J

Mol Biol 1999;286: 389–401.

30. Irelan JT, Selker EU. Cytosine methylation

associated with repeat-induced point mutation

causes epigenetic gene silencing in Neurospora

crassa. Genetics 1997;146: 509–523.

31. Selker EU. Trichostatin A causes selective loss of

DNA methylation in Neurospora. Proc Natl Acad

Sci USA 1998;95: 9430–9435.

32. Cheng JC, Matsen CB, Gonzales

FAet al.Inhibition of DNA methylation and

reactivation of silenced genes by zebularine. J

Natl Cancer Inst 2003;95: 399–409.

33. Billam M, Sobolewski MD, Davidson NE. Effects

of a novel DNA methyltransferase inhibitor

zebularine on human breast cancer cells. Breast

Cancer Res Treat 2010;20: 581–592.

34. Savickiene J, Treigyte G, Jonusiene V, Bruzaite

R, Borutinskaite VV, Navakauskiene R.

Epigenetic changes by zebularine leading to

enhanced differentiation of human

promyelocytic leukemia NB4 and KG1 cells. Mol

Cell Biochem 2012;359: 245–261.

35. Liu H, Xue ZT, Sjogren HO, Salford LG,

Widegren B. Low dose Zebularine treatment

enhances immunogenicity of tumor cells. Cancer

Lett 2007;257: 107–115.

36. Rosca AM, Burlacu A. Effect of 5-azacytidine:

evidence for alteration of the multipotent ability

of mesenchymal stem cells. Stem Cells Dev

2011;20: 1213–1221.

37. Armstrong L, LakoM, Lincoln J, Cairns PM, Hole

N. mTert expression correlates with telomerase

activity during the differentiation of murine

embryonic stem cells.Mech Dev 2000;97:

109–116.

38. Phatak P, Dai F, Butler M et al. KML001-

cytotoxic activity is associated with its binding

to telomeric sequences and telomere erosion in

prostate cancer cells. Clin Cancer Res 2008;14:

4593–4602.

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Figure S1. Positive expression of MSC specific cell surface anti-

gens: Panel A showing the immunocytochemistry staining of rat

mesenchymal stem cells (MSCs) with positive expressions of

CD29, CD90, and CD44.

Figure S2. Cytotoxicity measurement of MSCs using various con-

centrations of 5-azacytidine and zebularine: Four concentrations

(3, 10, 25 and 50 mM) of each compound were used for optimiza-

tion.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.

ª 2012 John Wiley & Sons Ltd Cardiovascular Therapeutics 31 (2013) 201–209 209

N. Naeem et al. Differentiation of MSCs by Methylation Inhibitors