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O R I G I N A L A R T I C L E

Byung Sun Yoon . Seung Jun Yoo . Jeoung Eun Lee .

Seungkwon You . Hoon Taek Lee . Hyun Soo Yoon

Enhanced differentiation of human embryonic stem cells intocardiomyocytes by combining hanging drop culture and5-azacytidine treatment

Received November 1, 2005; accepted in revised form January 23, 2006

Abstract Cell replacement therapy is a promising ap-proach for the treatment of cardiac diseases. It is, how-ever, challenged by a limited supply of appropriate cells.Therefore, we have investigated whether functional card-iomyocytes can be efficiently generated from human em-bryonic stem cells (hESCs). In this study, we developedan efficient protocol for the generation of functionalcardiomyocytes from hESCs by combining hanging dropculture and 5-azacytidine, a well-known demethylatingagent, and then evaluated the expression of cardiac-spe-cific markers. hESCs were cultured both in the mediumwithout or with 0.1, 1, or 10 mM of 5-azacytidine under ahanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. Toverify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron micros-

copy and electrophysiological recording. The efficiencyof beating cell generation was significantly improved inthe hanging drop culture compared with that in suspen-sion culture. Treatment of hESCs with 0.1mM o f 5 -azacytidine for 13 days significantly increased thenumber of beating cells and simultaneously enhancedthe expression of cardiac-specific markers. Transmissionelectron microscopy and electrophysiological recordingshowed that hESC-derived cardiomyocytes acquiredstructural and functional properties of cardiomyocytes.In conclusion, these results suggest that differentiation ofhESCs into cardiomyocytes can be enhanced by the

combination of hanging drop culture and 5-azacytidinetreatment. Also the methylation status of genes related tocardiomyocyte development may play an important rolein the differentiation of hESCs into cardiomyocytes.

Key words human embryonic stem cell cardiomyocyte 5-azacytidine hanging drop culture

Introduction

Human embryonic stem cells (hESCs) have been suc-cessfully derived from the inner cell mass (ICM) of

blastocysts and can be maintainedin vitro

for prolongedperiods (Thomson et al., 1998; Reubinoff et al., 2000;Baharvand et al., 2004). Embryonic stem (ES) cells canform embryoid bodies (EBs, strictly endoderm sur-rounding an inner core of ectoderm) that can give riseto cells found in all three embryonic germ layers(ectoderm, mesoderm, and endoderm), which are em-bryonic sources of all the cells in the body (Itskovitz-Eldor et al., 2000). A number of studies suggest thathESCs are pluripotentthey can differentiate into a

variety of cell types, including pancreaticb-cells, neuralcells, cardiomyocytes, and blood cells. These features of

Byung Sun Yoon Hoon Taek LeeDepartment of Animal ScienceKonkuk University1 Hwayang-dong, Gwangjin-guSeoul 143-701, Korea

Seung Jun Yoo Seungkwon YouDivision of Biotechnology and Genetic EngineeringCollege of Life and Environmental SciencesKorea UniversitySeoul, Korea

Jeoung Eun Lee Hyun Soo Yoon ( .*)Department of Anatomy & Cell BiologyCollege of MedicineHanyang University17 Haengdang-dong, Seongdong-guSeoul 133-792, KoreaTel: 182-2-2220-0602Fax: 182-2-2281-7841E-mail: hsyoon@hanyang.ac.kr

U.S. Copyright Clearance Center Code Statement:03014681/2006/7404149 $ 15.00/0

Differentiation (2006) 74: 149159 DOI: 10.1111/j.1432-0436.2006.00063.xr 2006, Copyright the AuthorsJournal compilation r 2006, Interntaional Society of Differentiation

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hESCs suggest that they have the potential to providean unlimited supply of various cell types for cell re-placement therapy.

The pluripotency of hESCs has the great advantagethat it could be used in cell replacement therapy forcardiac failure as well as for the study on the mecha-nisms of heart development in vitro (Klug et al., 1996;

Johkura et al., 2003). Cardiomyocytes have alsobeen successfully derived from various stem cells, suchas bone marrow stem cells (Orlic et al., 2001),hematopoietic stem cells (Jackson et al., 2001), and em-bryonic carcinoma cells (Rudnicki et al., 1990).Recent studies have suggested that hESCs can formEBs in vitro, some of which initiate spontaneous beating(Kehat et al., 2001, 2002). Based on the expressionof cardiac-specific genes, extracellular electricalactivity, and cellular ultrastructure, these beating cellscontain cardiomyocytes (Kehat et al., 2001; Snir et al.,2003). The potential of hESCs to differentiate intocardiomyocytes was first reported by Kehat et al.

(2001), followed by other groups (He et al., 2003;Snir et al., 2003). This differentiation of hESCsinto cardiomyocytes is aided by co-culture withEND2 visceral endoderm-like cells (Mummery et al.,2003).

Several groups have reported that 5-azacytidine, ademethylating agent, induced the differentiation of

mesenchymal stem cells into cardiomyocytes in vitro(Makino et al., 1999; Hakuno et al., 2002; Fukuda,2003). Xu et al. (2002) reported that 5-azacytidineinduced the differentiation of hESCs into card-iomyocytes. This compound can cause extensive de-methylation of 5-methylcytosine and reduce DNA

methyltransferase activity in the cell (Haaf and Sch-mid, 2000). Recently, 5-azacytidine was reported to re-verse the differentiation status of EBs back to ES cells(Tsuji-Takayama et al., 2004). 5-Azacytidine has beenuseful for studying the roles of DNA methylation in themechanisms of gene activation and cell differentiation.

In the present study, therefore, we developed theprotocol for the enhanced differentiation of hESCs intofunctional cardiomyocytes. We also evaluated the effectof hanging drop culture and 5-azacytidine on the dif-ferentiation of hESCs into cardiomyocytes as well as the

gene expression of cardiac-specific markers.

Materials and methods

Culture of hESCs

Two hESC lines (Miz-hES2 and HSF-6) were maintained on mouseembryonic fibroblasts (MEFs) established from day-13.5-post-coitum fetuses of CF1 mice. Feeder cells were cultured inDulbeccos modified Eagles medium (DMEM) (high glucose, In-vitrogen, Carlsbad, CA), supplemented with 10% FBS (HyClone,Logan, UT), 0.1 mMb-mercaptoethanol, and 0.1mM non-essentialamino acids. They were treated with 10mg/ml mitomycin C (Sigma,

St. Louis, MO) for 1.5 hr to arrest mitosis and replated at a con-centration of 6.1 104 cells/well in gelatin-coated 4-well culturedishes. The hESCs were cultured in DMEM/F12 (without pyruv-ate) (DMEM/F12), supplemented with 20% knock-out serum re-placement (SR) (Gibco/BRL, Invitrogen, Carlsbad, CA), 0.1 mMb-mercaptoethanol, 1% non-essential amino acids (Gibco/BRL),100 U/ml penicillin G, 100mg/ml streptomycin, and 4 ng/ml humanrecombinant basic fibroblast growth factor (Invitrogen). The hESCcolonies were transferred onto newly prepared CF1 feeder layers

and mechanically dissociated using a micropipette every 57 days.We used hESCs with passage numbers between 50 and 70.

Differentiation of hESCs into cardiomyocytes

To induce differentiation of cardiomyocytes, hESCs were mechan-ically dissociated into small clumps. EBs were formed using EBmedium, which consisted of DMEM/F12 with 20% fetal bovineserum (FBS), 1 mM glutamine, 0.1mM b-mercaptoethanol, and1% non-essential amino acids, and were cultured by using a hang-ing drop method (Maltsev et al., 1994). The effect of 5-azacytidine(Sigma) on the differentiation of hESCs was examined by eithersuspension culture or hanging drop culture following differentia-tion for 13 days. After 3 days of differentiation in the hangingdrop or suspension culture, EBs were plated on gelatin (0.1%, Si-gma)-coated 4-well plates (Nunc, Roskilde, Denmark) with four tosix EBs per well. Daily microscopic observations were conducted todetect beating EBs and to determine the beating rate. Contractingareas were mechanically dissected using a micropipette and werecontinuously subcultured.

Reverse transcription (RT)-polymerase chain reaction (PCR)

Total RNAs were prepared by using Trizol reagent (Invitrogen).Standard RT was performed using 500 ng of total RNA, oligod(T)1218 primer (Invitrogen), and AMV reverse transcriptase(Roche Molecular Biochemicals, Nutley, NJ). The RT-PCR was

carried out with 1 ml of cDNA template, 10 pmol of primers, andPCR premix (1 U Tag DNA polymerase, 250mM dNTPs, 10 mMTris-HCl, 40 mM KCl, and 1.5mM MgCl2; Bioneer, Korea). Prim-er sequences for PCR are shown in Table 1. All primer sets had acalculated annealing temperature of 621C. The PCR was carriedout in a GeneAmp 9600 (Perkin Elmer, Boston, MA) using thefollowing: a 5 min denaturation at 941C; 30 cycles of 941C for30 sec, 621C for 30 sec and 721C for 30 sec; and a final extension for10 min at 721C.

Real-time quantitative PCR

Real-time PCR using the iCycler Optical Detection System (Bio-Rad) was carried out in a final reaction volume of 25 ml with SYBR

Green (Bio-Rad, Hercules, CA). Briefly, 500 ng of total RNA wastranscribed to cDNA. One microliter of cDNA template was thenadded to 12.5ml of the 2 SYBR green PCR master mix and10 pmol of each primer. The temperature profiles were the same asthose for RT-PCR with the exception of the annealing temperature,which was 651C. The thermal denaturation protocol was run at theend of the PCR to determine the amplification of the specificproducts. The cycle number at which the reaction crossed an ar-bitrarily placed threshold (Ct) was determined for each gene. Datawere analyzed by using the 2DDCt method to obtain the relativeexpression level and by using that of b-actin as a normalizationcontrol in each sample. The relative amount of target5 2DDCt ,where Ct is the threshold cycle for target amplification,DCt Cttarget gene Ctinternal reference , and DDCt DCtsample DCtcalibrator .

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Immunofluorescence and confocal microscopy

Beating areas were mechanically dissected using a micropipette.These beating cells were then enzymatically dispersed using0.05% trypsin-EDTA for 15 min at 371C. The hESC-derived card-iomyocytes and enzymatically dispersed cells were fixed for 30 minat room temperature in phosphate-buffered saline (PBS) with Ca21

and Mg21 containing 4% paraformaldehyde and then permeabili-zed for 1 hr at room temperature with PBS containing 0.1% TritonX-100. The cells were then blocked with 3% bovine serum albuminand incubated with the corresponding primary antibodies for 1 hrat room temperature. Primary antibodies (1:100 dilutions) includedanti-rabbit antibody against human GATA-4, anti-goat antibodyagainst human cardiac myosin light chain, and anti-goat antibodyagainst human atrial natriuretic peptide (ANP), anti-mouse anti-body against human desmin (Santa Cruz Biotechnology, SantaCruz, CA) and anti-mouse antibody against human a-actinin(Sigma). After washing three times with PBS, cells were exposed for45 min at room temperature to the corresponding fluorescentisothiocyanate- or rhodamine-conjugated secondary antibodies at adilution of 1:200 and then observed under fluorescence and con-focal microscopes.

Transmission electron microscopy (TEM)

For TEM, the beating cells were mechanically dissected and fixedfor 3 hr in 2.5% glutaraldehyde, post-fixed in PBS containing 1%osmium tetroxide, dehydrated in ascending concentrations of et-hanol, and embedded in Epon 812. Semi-thin sections were ob-tained using a glass knife and thin sections with a diamond knife on

an ultramicrotome (LKB, Kent City, MI). Semi-thin sections wereplaced on glass slides, stained with 1% toluidine blue, and exam-

ined under a light microscope. Thin sections were placed on coppergrids, stained for 10 min with 2% uranyl acetate and lead citrate,and examined under an H-7600 transmission electron microscope(Hitachi, Tokyo, Japan).

Electrophysiological recording

Cells were isolated from beating hESC-derived cardiomyocytes bytreating them with 0.05% trypsin-EDTA for 15 min at 371C. Afterdissociation, cells were replated on glass coverslips for 57 days.Electrical activities of dissociated single cells were measured at 371Cby whole-cell patch-clamp techniques. In the voltage-clamp mode,whole-cell currents were recorded by the application of a family ofvoltage steps (from 90 to 40mV with a series of 10mV incre-

ments) for 40 ms. The initial holding potential was 70 mV. In thecurrent-clamp mode, the action potentials were elicited by a seriesof current injections (from 0.1 to 3.10 nA in 0.5 nA increments) for9 ms. The initial current was held at 0.15 nA to generate a mem-brane potential of approximately 90 mV. The bath solution con-sisted of 147mM NaCl, 5 mM KCl, 10mM HEPES, 1.5mM CaCl2,1mM MgCl2, and 5 mM glucose (pH 7.4). The internal pipettesolution contained 155 mM KCl, 100 mM aspartic acid, 2 mMMgCl2, 7 mM Na2ATP, 10mM EGTA, 20mM HEPES, and10 mM phosphocreatine (pH 7.2). Patch pipettes resistances were58MO. The sampling rates were 10 kHz (voltage-clamp mode) and3.3 kHz (current-clamp mode). The data were acquired using anAxopatch 1D patch, a Digidata 1200B interface, and pCLAMP 6software (Axon instruments, Union City, CA).

Table 1 Primer sequence information for RT-PCR

Gene Accession no. Primer sequence (50 ! 30) Annealingtemperature (1C)

Productsize (bp)

b-actin BC002409 Forward: AGCAAGCAGGAGTATGACGA 62 260

Reverse: TGTGAACTTTGGGGGATG

GATA-4 NM_002052 Forward: CTCCCCTGGCAAAACAAGAG 62 422

Reverse: TGCCGTGTCTTAGCAGTCGT

Nkx2.5 AB021133 Forward: GGTCTATGAACTGGAGCGGC 62 322Reverse: ATAGGCGGGGTAGGCGTTAT

MLC-2A BC027915 Forward: GCTCTTTGGGGAGAAGCTCA 62 239

Reverse: CGTCTCCATGGGTGATGATG

MLC-2V BC031006 Forward: GGCGCGTGAACGTGAAAAAT 62 200

Reverse: CAGCATTTCCCGAACGTAAT

a-MHC NM_002471 Forward: GTCATTGCTGAAACCGAGAATG 62 413

Reverse: GCAAAGTACTGGATGACACGCT

b-MHC X06976 Forward: AGATGGATGCTGACCTGTCC 62 396

Reverse: GGTTTTTCCTGTCCTCCTCC

ANP NM_006172 Forward: GAACCAGAGGGGAGAGACAGAG 62 406

Reverse: CCCTCAGCTTGCTTTTTAGGAG

cTnT BC002653 Forward: GGCAGCGGAAGAGGATGCTGAA 62 152

Reverse: GAGGCACCAAGTTGGGCATGAACGA

cTnI X54163 Forward: CCTGCGGAGAGTGAGGATCT 62 218

Reverse: TAGGCAGGAAGGCTCAGCTC

bIII-tubulin AF427491 Forward: CTCAAGATGTCCTCCACCTTCAT 62 246Reverse: CTCCTCGTCGTCTTCGTACATCT

MAP2 BC038857 Forward: TAGCAGTCCTGAAAGGTGAACAAG 62 256

Reverse: GACTTTGTGCTACCCTGTAAAGCA

Oct-4 NM_002701 Forward: AAGAACATGTGTAAGCTGCGGCCC 62 455

Reverse: GGAAAGGCTTCCCCCTCAGGGAAAGG

Nanog NM_024865 Forward: ATAGCAATGGTGTGACGCAG 62 219

Reverse: GATTGTTCCAGGATTGGGTG

a-MHC,a-myosin heavy chain; b-MHC,b-myosin heavy chain; ANP, atrial natriuretic peptide; cTnT, cardiac troponin T; cTnI, cardiactroponin I.

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Statistical analysis

A w2-test was used for statistical analyses, and, in all cases, a valuewith a probability of po0.05 was considered as an indication ofstatistical significance. Results were expressed as the mean stand-ard error of the mean (SEM).

Results

Effects of hanging drop culture and 5-azacytidinetreatment on the differentiation of hESCs intocardiomyocytes

The EBs were formed by dissociating hESC colonies(Fig. 1A) and by growing them in a hanging drop cul-ture (Fig. 1B). Differentiated cardiomyocytes were cul-tured up to 4050 days (Figs. 1C, 1D, respectively). Weobserved that efficiency of beating cell generation washigher in hanging drop culture than in a suspensionculture. We then used a demethylating agent, 5-azacyti-dine, which has been shown to induce the differentia-tion of bone marrow-derived mesenchymal stem cells

and hESCs into cardiomyocytes (Makino et al., 1999;Hakuno et al., 2002; Xu et al., 2002; Fukuda, 2003).Differentiation of hESCs (Miz-hES2 and HSF-6) intocardiomyocytes was enhanced by the combination ofhanging drop culture and 5-azacytidine treatment (Fig.2A). The effect of 5-azacytidine on hESC differentiationinto cardiomyocytes was examined by treating EBseither in suspension or in hanging drop cultures for 3days with different doses (0.1, 1, and 10 mM). We foundthat beating cells appeared following 4 days of differ-

entiation and remained viable for several months inculture. Treatment with 0.1 mM of 5-azacytidine for 13

days significantly increased the rate of differentiationinto cardiomyocytes (Miz-hES2: 41.88% for treatedcells versus 22.37% for untreated control cells, HSF-6:34.89% for treated group versus 18.55% for controlgroup). On the other hand, high doses of 5-azacytidine(1 and 10 mM) decreased the rate of differentiation intocardiomyocytes (Table 2). In HSF-6 cell line, the cells

were all lysed with the treatment of 10 mM 5-azacyti-dine. The generation of beating cells in the EBs gener-

ally increased with time in culture and treatment with0.1mM of 5-azacytidine significantly increased the per-centage of beating cells when they were in the tissueculture plates (Fig. 2B).

Expression of cardiomyocyte-specific markers fromhESC-derived cardiomyocytes

The expression of several cardiac-specific markers wasevaluated by RT-PCR in hESC-derived card-

iomyocytes, undifferentiated hESCs, EBs after 3 daysof differentiation, and differentiated non-beating cells.Complementary DNA generated from commerciallyavailable human fetal heart RNA was used as a positivecontrol. As shown in Fig. 3A, hESC-derived card-iomyocytes expressed the cardiac transcription factorsGATA-4 and Nkx2.5, as well as the cardiac-specificgenes;a-myosin heavy chain (a-MHC),b-MHC, cardi-ac troponin T (cTnT), cardiac troponin I (cTnI), ANP,myosin light chain-2A (MLC-2A), and MLC-2V. Theexpression of cardiac-specific genes was, however, bare-ly detectable in the control group (undifferentiatedhESCs, EBs and non-beating cells). Furthermore, we

found cTnI expression, which was used as a cardiac-specific markers (Kehat et al., 2004), in the undifferen-tiated hESCs and non-beating cells (non-card-iomyocytes), as well as cardiomyocytes. To clarify itsexpression, the amplified cTnI RT-PCR product wassequenced and was verified as human cTnI (data notshown). Undifferentiated markers, such as Oct-4 andNanog, and neuronal markers, MAP2 and bIII-tubulin,were not expressed in hESC-derived cardiomyocytes ei-ther. Immunofluorescence and confocal microscopywere performed to confirm the presence of cardiac-spe-cific proteins in beating cells. Figure 3B shows that

hESC-derived cardiomyocytes were positively immuno-stained with anti-ANP (a1), anti-desmin (b), anti-MLC(c), and anti-a-actinin (d). We found that these cardiac-specific proteins were localized only in the beating re-gions. Most of the differentiated cells expressed markersof cardiomyocytes. The hESC-derived cardiomyocytesalso specifically expressed the cardiac transcriptionfactor, GATA-4 (Fig. 3Ba2). Additionally, a-actininand GATA-4 expression was also detected in single cellsisolated from beating EBs (Figs. 3Be, f1, f3). Undiffer-entiated hESCs were used as a negative control and theydid not react with cardiac muscle-specific antibodies

Fig.1 Differentiation of human embryonic stem cells (hESCs) intocardiomyocytes. (A) Undifferentiated hESCs. (B) Embryoid bodies(EBs) after 3 days of hanging drop culture. (C) Differentiatedcardiomyocytes from hESCs on day 40. (D) Expansion of thebeating area in EBs from hESCs at day 50. Scale bars5 100mm.

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(Fig. 3Bg1). As an additional negative control for an-tibody specificity, we incubated cells with only second-

ary antibodies to monitor the non-specific binding ofsecondary antibodies to the cells (Fig. 3Bh1).

Ultrastructural analysis of hESC-derivedcardiomyocytes

TEM was performed to visualize the ultrastructure ofhESC-derived cardiomyocytes. TEM revealed that

hESC-derived cardiomyocytes showed the typical stri-ation pattern of the sarcomeres (Figs. 4A4D).

Notably, the hESC-derived cardiomyocytes containedZ-band (arrow, Z), which is a major characteristic ofcardiac muscles containing fascia adherens (arrow, FA),desmosomes (arrow, D), gap junctions (arrow, G) (Fig.4Aa), and granules of ANP (A) (Fig. 4Ad).

Electrophysiology of hESC-derived cardiomyocytes

To further determine whether hESC-derived card-iomyocytes possess the electrical properties of maturecardiomyocytes, we performed electrophysiologicalstudies using a whole-cell patch-clamp technique.

All electrical activities were obtained from dissociatedsingle cells. Whole-cell currents elicited by the applica-tion of voltage steps showed active voltage-dependentresponses (Fig. 4Ba). Voltage dependence of whole-cellcurrents is common to all excitable cells includingcardiac muscle cells. Typical inward sodium currentswere evident, and outward currents were also detected,such as A-type and delayed potassium channels,which suggest the existence of voltage-gated inwardand outward channels (data not shown). The activities

of these voltage-gated sodium and potassiumchannels were more evident when the action potentials

Fig. 2 Enhanced differentiation of human embryonic stem cells(hESCs) into cardiomyocytes. (A) Efficiency of beating embryoidbody (EB) generation in the presence or absence of 5-azacytidine ina hanging drop culture and in a suspension culture. denotes sta-tistical significance at po0.05. denotes statistical significance at

po0.01. (B) Cumulative percentage of cardiomyocytes derivedfrom hESCs with various doses of 5-azacytidine. Days of platingindicates the number of days that the cells were cultured on a tissueculture plate after the cells had been taken from the hanging dropculture. Left panel: Miz-hES2; right panel: HSF-6.

Table 2 Efficiency of beating EB generation at various doses of 5-azacytidine

Cell lines 5-azacytidine(mM)

Number ofEB

Number of beating(%) (mean SEM)

Miz-hES2 0 206 45 (22.37 1.15)

0.1 217 91 (41.88 0.92)

1 201 30 (15.25 1.34)

10 168 10 (5.44 0.55)

HSF-6 0 199 36 (18.55 0.59)

0.1 192 67 (34.89 1.29)

1 192 14 (7.29 0.66)

10 155 0 (0 0)

po0.05 by w2-test. (n5 6).EB, embryoid body; SEM, standard error of the mean.

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were evoked by current injections. Currents thatwere sufficiently strong to overcome a threshold poten-tial (around 45 mV) generated the action potentials(Fig. 4Bb). Furthermore, the complete depletion ofthe action potential by the application of tetrodotoxin,which is a sodium channel blocker, in the bath solutionindicates that voltage-gated sodium channels are re-

sponsible for the rising phase of the actionpotential (Fig. 4Bc). Therefore, it appears that hESC-derived cardiomyocytes have at least some func-tional electrical properties of the differentiated card-iomyocytes.

Real-time quantitative PCR

Real-time PCR experiments showed that 5-azacytidineup-regulated the expression of cardiac-specific markers.The highest expression of the cardiomyocyte transcrip-tion factors GATA-4 and Nkx2.5 was detected in thegroup treated with 1 mM of 5-azacytidine: the cardiac-specific markers were most highly expressed in the0.1mM-treated groups (Table 3: Miz-hES2). The higherpercentage of beating cells in the 0.1 mM-treated groupwas correlated with the higher expression of cardiac-specific markers rather than that of the cardiac tran-

Fig.3 Characterization of cardiomyocytesderived from human embryonic stem cells(hESCs). (A) Expression of cardiac-specif-ic markers in the hESC-derived cardio-myocytes. Lane 1, undifferentiated hESCs;lane 2, embryoid bodies (EBs) at day 3;lane 3, non-beating cells; lane 4, beat-ing cells; lane 5, human fetal heart as apositive control; lane 6, negative control(no RT). (B) Confocal microscopy forcardiac-specific markers in hESC-derivedcardiomyocytes. The hESC-derived card-iomyocytes (a) were stained with antibod-ies against atrial natriuretic peptide (ANP)(a1) and GATA-4 (a2). The hESC-derivedcardiomyocytes stained with anti-desmin(b), anti-myosin light chain (c), and anti-a-actinin antibodies (d). Single cells wereisolated from beating cells and werestained with anti-a-actinin (e) and anti-GATA-4 antibodies (f1, f3). (g) Undiffer-entiated hESCs. (g1) Undifferentiated hE-SCs stained with anti-ANP antibody. As anegative control for antibody specificity,the hESC-derived cardiomyocytes werestained only with secondary antibodies(h1). Scale bars5 100mm (e, f25 25mm).

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scription factors, compared with that of normalizedcontrol with no 5 azacytidine treatment group. In HSF-6 cell line, the higher expression of both cardiac tran-scription factors and cardiac-specific markers were,

however, detected in the 0.1 mM-treated group. Duringcardiac development, the expression of cardiac tran-scription factors and cardiac-specific markers increasedcontinuously until 15 days of differentiation and de-creased slightly thereafter, compared with that ofnormalized control with day 5 differentiation group(Table 4.).

Discussion

The generation of cardiomyocytes from hESCs has sev-eral potential applications, including transplantationfor curing heart failure. Cardiomyocytes derived fromhESCs have been successfully transplanted to generatepacemaker cells in a swine model of atrioventricularblock. In this case, the source of the new ventricularectopic rhythm was confirmed to be the site of cell

transplantation (Kehat et al., 2004). When a typicalmyocardial infarction occurs, more than 25% of thecells in the heart are lost, which leads to heart failure.For example, the left ventricle of the human heart con-

tains approximately 5.8109 myocytes (Kajstura et al.,1998). Therefore, a large-scale culture is needed to sup-ply enough cells for clinical applications. Generationof a number of cells can be achieved with an efficient,directed-differentiation system coupled with an enrich-ment method.

Kehat et al. (2001) first reported that hESCs could

be differentiated into cardiomyocytes with 8.1%efficiency. Xu et al. (2002) reported that the additionof 5-aza-20-deoxycytidine (5-aza-dC) to developing EBsat later days of differentiation (68 days) has beenshown to be more effective than the addition at earlierdays (15 days). It was also found that the 5-aza-dC-treated beating unit contained 44 2% cTnI-positivecells, which were not clearly associated with thefunctional cardiomyocytes (Messner et al., 2000; Xuet al., 2002).

In the present study, we sought to determine anefficient protocol for the generation of functional

Fig. 4 Ultrastructural and electrophysiological analyses of humanembryonic stem cell (hESC)-derived cardiomyocytes on day 70. ( A)Transmission electron microscopy (TEM) image of card-iomyocytes. (a) TEM image showing the presence of a desmosome(D), gap junction (G), and fascia adherens (FA). (bd). A, granuleof atrial natriuretic peptide; N, nucleus; Z, Z-bands; S, sarcomere.Scale bars5 0.5mm (panels a, c, and d) or 10 mm (panel b). (B)Representative whole-cell currents and evoked action potentialsobtained from a single hESC-derived cardiomyocyte. (a) Whole-cell

current traces were measured by the application of a series of volt-age steps. Active voltage responses and inward sodium currents areevident by depolarizing potential steps. (b) The action potentialswere evoked by a series of current injections. Only currents enoughto overcome the threshold potential (approximately 45mV inthis figure) can generate the action potentials. (c). The action po-tential was blocked by the application of tetrodotoxin (200 nM) inbath solution. An action potential trace from B(b) was superim-posed for comparison.

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cardiomyocytes using different lines of hESCs. In com-parison with previous reports (Kehat et al., 2001; Xuet al., 2002), we found that the hanging drop culturewas more efficient than the suspension culture for thegeneration of beating cells. Also, there was no effectwhen 5-azacytidine was treated after differentiation day4 in our study (data not shown). These apparent dif-

ferences in the efficiency of differentiation may be dueto differences in the methods used for EB formation andthe culture conditions for the differentiated EBs. In ourstudy, EBs were formed by mechanical dissociation ofhESCs into clumps and then cultured clumps in hangingdrops containing DMEM/F12 with 20% FBS. We ob-served that EBs in suspension culture formed a lowerpercentage of beating cardiomyocytes than EBs inhanging drop culture. Therefore, we used the hangingdrop culture to form the EBs, and attached the EBsonto plates for cardiomyocyte differentiation after 3-day culture of EBs in hanging drop instead of 710 daysculture of EBs in suspension, as previously described

(Kehat et al., 2001). In addition, we applied a single EBper 20 ml of hanging drop containing DMEM/F12 me-dium with 20% FBS. We also addressed the importanceof the number of EBs per well for the differentiationinto cardiomyocytes. We found that five EBs per1.9 cm2/well were more optimal for the cardiomyocytedifferentiation (data not shown). With the differentia-

tion protocol, by combining hanging drop culture and5-azacytidine treatment, we were able to enhance therate of beating EBs generation by up to 41.88 0.92%(Miz-hES2) and 34.89 1.29% (HSF-6), which is muchmore efficient than the 8.1% originally reported byKehat and coworkers (2001) [9].

Furthermore, various hESC lines (HSF-6 and Miz-hES1, 2, 4, and 6) (Park et al., 2003; Kim et al., 2005)can be efficiently induced to differentiate into card-iomyocytes in vitro with 5-azacytidine treatment. Al-though spontaneous beating has never been observed inMiz-hES1, 4, and 6 hESC cell lines, we could, however,generate beating cells from Miz-hES1, 4, and 6 hESCcell lines by combining hanging drop culture and 5-azacytidine treatment up to 17.6%, 11.1%, and 5%,respectively.

The number of beating cells significantly increasedwhen hESCs were treated with 0.1 mM of 5-azacytidine

during differentiation days 13. In addition, 0.1 mM of5-azacytidine increased the expression of cardiac-spe-cific transcription factors and marker genes, includingGATA-4, Nkx2.5, ANP, cardiaca/b-MHC, and MLC-2V, compared with those in the no 5-azacytidinetreatment group, while the expression of the cardiactranscription factors was even higher in 1 mM of the5-azacytidine treatment group. Therefore, it appearedthat the concentration of 5-azacytidine was critical inthe differentiation of hESCs into cardiomyocytes. Infact, in the group treated with 1 mM of 5-azacytidine,half of the cells were dispersed to death, and the re-T

able3

Expressionofcardiac-specifictranscriptionfactorsandmarkersinhESC-derivedcardiomyocytesbyreal-tim

ePCR

Cell

lines

5-azacytidine

(mM)

DCt

DDCt

2

DDCt

GATA-4Nkx2.5

a-MHC

b-MHC

MLC-2V

AN

P

GATA-4Nkx2.5a-MHCb-MHC

MLC-2VANPGATA-4Nkx2.5a-MHC

b-MHCMLC-2VANP

Miz-hES2

0

5.20

2.348.13

0.353.53

0.313.70

1.006.93

0.492.07

0.40

0.00

0.00

0.00

0.00

0.00

0.00

1.000

1.000

1.000

1.000

1.000

1.000

0.1

1.43

0.217.17

1.170.60

0.402.17

0.236.50

0.361.23

0.15

3.77

0.97

2.93

1.53

0.43

0.8313.611

1.954

7.639

2.895

1.350

1.782

1

0.60

0.354.80

0.102.33

0.151.47

0.356.83

0.312.17

0.40

4.60

3.33

1.20

2.23

0.10

0.1024.251

10.079

2.297

4.702

1.072

0.933

10

1.57

1.045.40

1.003.40

2.001.87

1.426.30

0.611.93

0.42

3.63

2.73

0.13

1.83

0.63

0.1312.409

6.650

1.097

3.564

1.551

1.097

HSF-6

0

4.07

1.836.90

0.874.00

1.186.77

0.645.67

0.644.23

0.97

0.00

0.00

0.00

0.00

0.00

0.00

1.000

1.000

1.000

1.000

1.000

1.000

0.1

0.80

0.444.07

0.861.70

0.822.77

0.381.77

1.771.07

0.64

3.27

2.83

2.30

4.00

3.90

3.17

9.624

7.127

4.925

16.000

14.929

8.980

1

2.20

1.574.00

2.411.67

1.693.60

0.261.43

0.612.53

1.83

1.87

2.90

2.33

3.17

4.23

1.70

3.647

7.464

5.040

8.980

18.809

3.249

EBsformedfromahangingdropcultureweretreatedwithvariousdosesof5-azacytidine(mM).Expressionsofthe

variousmRNAswerenormalizedbythe

expressionofb-actin.

Denotesstatisticalsignificancewhenpo0.05byw

2-test.

(po0.01).(n5

3).

EB,embryoidbody;hESC,human

embryonicstem

cells;a-MHC,a-myosinheavychain;b-MHC,

b-myosinheavy

chain;MLC-2V,myosinlightchain-2V;ANP,atrialnatriuretic

peptide.

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sulting EBs decreased in size and had a lower chance toform beating EBs. Also, when we treated the EBs with5-azacytidine for a longer time (72 hr), we observed celldeath in the EBs, which resulted in failure of beatingEBs generation. In HSF-6 cell line, most of the cellswere lysed in 1 mM 5-azacytidine treatment and showedless efficiency for beating cell generation. On the other

hand, 10mM 5-azacytidine-treated cells were all lysedand no beating cells were generated. Therefore, thetoxic effect of 5-azacytidine was higher in HSF-6 cellline than in Miz-hES2 cell line. On the other hand,treatment with 0.1mM of 5-azacytidine increased theexpression of both cardiac transcription factors andcardiac-specific markers in HSF-6 cell line.

Therefore, it appeared that the cardiomyogenic effectof 1 mM 5-azacytidine was somewhat compromised byits toxic effect, and the resulting optimal dose of 5-azacytidine for cardiomyogenic effect could be 0.1 mM.In Miz-hES2 cell line, the highest expression of thetranscription factors, GATA-4 and Nkx2.5, was ob-

served in the 1 mM 5-azacytidine treatment group. Onthe other hand, a positive correlation of expression lev-els was observed only between two transcription factors(GATA-4 and Nkx2.5) and b-MHC. Therefore, it ispossible that cardiac-specific markers are regulated byadditional transcription factors, such as myocyte en-hancer factor 2 or T-box-containing transcription fac-

tor-5 (Morin et al., 2000; Hiroi et al., 2001). In HSF-6cell line, however, the expression levels of both cardiactranscription factors and cardiac-specific markers werehigher in 0.1 mM. Electrophysiological profiles of spon-taneously differentiated beating cells showed that thereare three distinct types of cardiomyocytes, including

atrial-, ventricular-, and nodal-like cells (He et al.,2003). In a recent report, high-resolution activationmaps using microelectrode array (MEA) suggested thepresence of a functional syncytium in the hESC-derivedcardiomyocytes with stable focal activation and con-duction properties (Kehat et al., 2002).

In the present study, we comprehensively character-ized the cardiomyocytes derived from hESCs by RT-PCR, immunofluorescence, and confocal microscopy aswell as by TEM and electrophysiological recording. Theresults of these studies confirmed that the differentiated

cells were structurally and functionally equivalent to

cardiomyocytes. These cells expressed cardiac-specificgenes, including transcription factors GATA-4 andNkx2.5, which play a significant role in cardiac devel-opment (Grepin et al., 1997; Lien et al., 1999). Inaddition, cardiac-specific genes were also detected, in-cludinga-MHC,b-MHC, cTnT, cTnI, ANP, MLC-2A,and MLC-2V, even though cTnI was expressed inundifferentiated hESC, EBs, and non-beating cells.Furthermore, it has recently been reported that cTnIwas not appropriate for the cardiac muscle specificmarker (Messner et al., 2000). Therefore, it is notenough to conclude that 5-aza-dC had the effect on theT

able4

Expressionofcardiac-specifictranscriptionfactorsandcardiac-specificmarkersduringcardiomyocytedevelopmentbyreal-timePCR

Cell

lines

Daysof

differentiation

DCt

DDCt

2D

DCt

GATA-4Nkx2.5

a-MHC

b-MHC

MLC-2VANP

GATA-4Nkx2.5a-MHCb-MHC

MLC-2VANPGATA-4Nkx2.5a-MHC

b-MHCMLC-2VANP

Miz-hES2

5

3.47

0.514

.33

0.803.87

0.763.20

0.612.20

1.113.03

0.25

0.00

0.00

0.00

0.00

0.00

0.001.000

1.000

1.000

1.000

1.000

1.000

10

2.37

2.804

.10

1.013.20

1.612.70

1.151.87

1.453.47

2.73

1.10

0.23

0.67

0.50

0.33

0.432.144

1.176

1.587

1.414

1.260

0.741

15

1.03

0.873

.30

0.962.00

0.922.10

1.850.40

0.401.63

1.19

2.43

1.03

1.87

1.10

1.80

1.405.401

2.047

3.647

2.144

3.482

2.639

20

1.63

1.172

.63

1.802.03

1.251.43

1.181.77

0.952.13

1.55

1.83

1.70

1.83

1.77

0.43

0.903.564

3.249

3.564

3.403

1.350

1.866

HSF-6

5

4.90

0.3511

.40

0.627.90

0.369.70

0.527.33

0.215.20

0.36

0.00

0.00

0.00

0.00

0.00

0.001.000

1.000

1.000

1.000

1.000

1.000

10

4.47

1.318

.73

0.517.37

0.856.63

0.157.13

1.054.20

2.07

0.43

2.67

0.53

3.07

0.20

1.001.350

6.350

1.447

8.378

1.149

2.000

15

2.23

0.908

.83

0.656.43

0.216.83

0.326.33

1.622.10

0.87

2.67

2.57

1.47

2.87

1.00

3.106.350

5.924

2.764

7.294

2.000

8.574

20

2.17

1.7110

.13

0.746.53

1.366.97

0.814.67

2.062.77

1.42

2.73

1.27

1.37

2.73

2.67

2.436.650

2.406

2.579

6.650

6.350

5.401

Denotesstatisticalsignificancewh

enpo0.05byw

2-test.(n5

3).

ExpressionsofthevariousmRNAswerenormalizedbytheexpressionofb-actin.

a-MHC,a-myosinheavychain;b-MHC,

b-myosinheavychain;MLC-2V,

myosinlightchain-2V;ANP,atrialnatriureticpeptide.

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cardiomyocyte differentiation by performing immuno-staining using antibody against cTnI (Xu et al., 2002).Therefore, identification of hESC-derived cardio-myocytes should be confirmed by performing structur-al and functional analyses, such as electron microscopyand electrophysiology.

In the present study, we performed sequence analysis

of cTnI RT-PCR product to eliminate the false positiveresult and verified that the amplified cTnI RT-PCRproduct was human cTnI and was expressed in undif-ferentiated hESCs, non-beating EBs, and beating EBs(data not shown). TEM and electrophysiological re-cording further verified that our hESC-derived card-iomyocytes had the structural and functional propertiesof cardiomyocytes.

In conclusion, differentiation of hESCs into card-iomyocytes can be enhanced by applying hanging dropculture and 5-azacytidine treatment with the differentefficiencies of cardiomyogenesis among hESC lines test-ed in this study, which suggests that each hESC line

may respond differently to the cardiomyogenic stimuli.Although the mechanism by which 5-azaytidine inducesdifferentiation into cardiomyocytes remains unclear, theresults suggest that the genes required for cardiogenesismay be silenced by methylation. Therefore, the methyl-ation status of genes related to cardiomyocyte develop-ment may play an important role in the differentiation

of hESCs into cardiomyocytes. We are currentlyconducting transplantation of hESC-derived cardio-myocytes into large animal disease models, such as pigsand primates, which will be necessary to confirm thefunction of these cells in vivo. Additional challengeswill be the enrichment of cardiomyocytes by directed

differentiation of hESCs and the elucidation of themechanism by which hESCs differentiate into card-iomyocytes.

Acknowledgments This research was supported by grants from theStem Cell Research Center of the 21st Century Frontier ResearchProgram of the Korean Ministry of Science and Technology(SC2200). This project was also supported by the Korean Ministryof Education and Human Resources (2005).

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