Direct Comparison of Distinct Cardiomyogenic ... - Springer Link

Tissue Engineering and Regenerative Medicine, Vol. 9, No. 6, pp 311-319 (2012) DOI 10.1007/s13770-012-0336-6

｜Original Article｜

Direct Comparison of Distinct Cardiomyogenic Induction Methodologies in Human Cardiac-Derived c-Kit Positive Progenitor Cells Sung Hyun Choi1†, Seok Yun Jung2†, Takayuki Asahara3,4, Wonhee Suh5, Sang-Mo Kwon2*, and Sang Hong Baek1* 1

Laboratory of Cardiovascular Regeneration, Division of Cardiovascular Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea School of Medicine, Seoul, Korea 2 Laboratory of Vascular Medicine and Stem Cell Biology, Department of Physiology, Pusan National University School of Medicine, Yangsan, Korea 3 Stem Cell Translational Research, Institute of Biomedical Research and Innovation/RIKKEN Center of Developmental Biology, Kobe, Japan 4 Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Japan 5 College of Pharmacy, Ajou University, Suwon, Korea (Received: February 17th, 2012; Revision: October 9th, 2012; Accepted: October 10th, 2012)

Abstract : Cardiac stem/progenitor cells can be differentiated into cardiomyocytes in vitro using several differentiation methodologies. However, the methodology of cardiomyogenic induction in human c-kit positive progenitor cells (hCPCsc-kit+) was not fully demonstrated. Thus, the purpose of our study was to directly evaluate each cardiomyocyte induction system using hCPCsc-kit+. In this study, cardiomyocyte induction methodologies were divided into the following three groups; treatment with dexamethasone, 5-azacytidine, and co-treatment with 5-azacytidine and Transforming Growth Factor Beta 1 (TGF-β1), using different serum concentrations [2% or 10% fetal bovine serum (FBS)]. GATA4 and Nkx2-5, cardiac-specific transcription factors, were expressed in our hCPCsckit+. However, the GATA4 and Nkx2-5 expressions were significantly decreased in 10% FBS/cardiomyogenic induction system (p < 0.01), whereas the GATA4 and Nkx2-5 expressions were preserved in 2% FBS/cardiomyogenic induction system (p > 0.05). GATA4 and Nkx2-5 is crucial roles in cardiac development, thus we considered the low serum conditions more affected in our cardiomyogenic induction system. In addition, c-kit expression decreased significantly during cardiomyogenic differentiation. Importantly, we demonstrated that co-treated with 5-azacytidine and TGF-β1 led to an earlier expression pattern of alpha-sarcomeric actin (α-SA), implying that this cardiomyocyte induction system facilitates early cardiomyocyte differentiation of hCPCsc-kit+. Thus, the present study provides a pivotal cardiomyogenic differentiation methodology using hCPCsc-kit+ for basic or clinical research. Key words: 5-azacytidine, cardiomyocyte differentiation, dexamethasone, hCPCsc-kit+, TGF-β1

positive sorting.3 These hCPCs are also capable of differentiation into cardiomyocytes, ECs and SMCs in vivo or in vitro, and might provide high regenerative potential for cardiac patients. Although hCPCs might be a potential source of stem cell therapy for cardiovascular disease, the in vitro differentiation methodologies for hCPCs have not yet been elucidated. Establishment of in vitro differentiation protocols for cardiomyocyte from hCPCs could provide important clues to understand cardiovascular regeneration and the mechanical cascade of cardiomyogenic differentiation. Several studies have demonstrated that in co-culture with cardiomyocytes4, 5, or with the proper stimulation using chemical reagents such as 5-azacytidine6, and dexamethasone3, a variety of stem cells are capable of undergoing cardiomyocytes differentiation. In fact, cardiomyocytes differentiation highly associated with

1. Introduction The recent discovery of resident cardiac progenitor cells (CPCs) in the human myocardial niche suggests that the heart is not a terminally differentiated organ.1 Messina et al. reported that isolated human and mouse cardiac sphere derived stem cells differentiate into cardiomyocytes as well as endothelial cells (ECs) and smooth muscle cells (SMCs).2 Bearzi and their colleagues further demonstrated that human CPCs (hCPCs) could be isolated from a human heart biopsy specimen by c-kit†

These authors contributed equally to this work *Tel: +82-2-2258-6030; Fax: +82-2-591-3614 e-mail: [email protected] (Sang Hong Baek) *Tel: +82-51-510-8070; Fax: +82-51-510-8076 e-mail: [email protected] (Sang-Mo Kwon)

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Table 1. Proportion of isolated hCPCsc-kit+. Proportions of c-kit positive cells were calculated by three independent experiments. Number of total cardiac cells

Number of c-kit positive cells

Percentage of c-kit positive cells

1st

1×107

1.5×105

1.5

nd

1.5×10

2×10

1.3

3rd

8×106

1×105

Means

1.1×10 ±3.6×10

2

7

7

5

6

1.3

1.5×10 ±5×10 5

4

1.4±0.13 (%)

10 mL of phosphate-buffered saline (PBS), centrifuged at 1,500 rpm (Hanil Science), and then incubated in culture media at 37oC in a humidity chamber. The hCPCsc-kit+ culture media, which was based on Ham’s F12 media (Hyclone Laboratories, USA), contained 10% fetal bovine serum (FBS, Gendepot, USA), 1× penicillin streptomycin (Hyclone Laboratories), 2.5 U of human erythropoietin (R&D systems Inc., USA), 0.2 mM glutathione (Sigma-Aldrich, USA), and 5 µg of human recombinant fibroblast growth factor basic (FGF-basic, PeproTech, USA). When the expanded single cardiac cells reached 60%–70% confluence, they were splint into 10 dishes (Passage 1 cells; P1). When the P1 cells expanded and reached 70%–80% confluence, the cells were conjugated c-kit primary antibody (1:10, Santa Cruz Biotechnology, USA), and hCPCsc-kit+ were isolated by using magnetic-activated cell sorting (MACS). The sorted hCPCc-kit+ were used for the experiments or sorted or in liquid nitrogen.3

expressions of GATA4 and Nkx2-5 as cardiomyocyte-specific transcription factors.7 The GATA4, a membrane of a family of zinc finger transcription factors8, expressed in the heart of various vertebrates9, 10 and expressed during early cardiac development.11, 12 Moreover, some mice experiments suggested that elimination of Nkx2-5 observed severe growth retardation, perturbed cardiac morphogenesis, and embryonic lethality.13, 14 Stimulation by dexamethasone in adult stem cells15 and embryonic stem cells16 has been reported to promote expressions of sarcomeric actin (SA) and cardiac myosin heavy chain (MHC). Recently, multiple reports have clearly shown that treatment with transforming growth factor-beta 1 (TGF-β1) induces the cardiomyogenic differentiation potential of stem cell antigen-1 positive (Sca-1+) CPCs or bone marrow-derived stem cell growth factor receptor positive (c-kit+) stem cells in vitro.4, 17 Other research suggested that the use of ex vivo experiment of TGF-β1 preprogrammed c-kit+ stem cells enhanced the potential for recovering injured myocardium and improving cardiac function.18, 19 Despite these promising results, in vitro cardiomyocytes differentiation methodologies for hCPCs are still not clearly defined. The purpose of our study was to comparisons of adequate protocols for cardiomyogenic differentiation in vitro using human cardiac-derived c-kit positive progenitor cells (hCPCsc-kit+).

2.2 Characterization of Isolated hCPCsc-kit+ The isolated hCPCsc-kit+ were analyzed by microscopy and immunofluorescence staining. For the morphology comparison, we used an inverted microscope (Olympus, CKX41, Japan) under x4 and magnifications. For the characterization of cellular markers, conjugated antibodies against c-kit (1:200, Santa Cruz Biotechnology), GATA4 (1:200, Santa Cruz Biotechnology), and Nkx2-5 (1:200, Santa Cruz Biotechnology) were used directly. The cell stains were then visualized by the appropriate fluorescence dye conjugated secondary antibodies as Alexa 488 (1:200, Molecular probes, Invitrogen, USA) and Alex 594 (Molecular probes). Florescence images were collected at room temperature using a laser-scan confocal microscope (λabs; 495 nm and 590 nm, λem; 595 nm and 617 nm, Olympus, Flow View, FV1000) under x20 magnification.

2. Experimental Method 2.1 Isolation of hCPCsc-kit+ The hCPCsc-kit+ were isolated from human cardiac-derived biopsy specimens obtained from patients heart during cardiac surgery. The Ethics Review Board at the Hospital of Pusan National University in YangSan, Korea approved the protocols, and the experimental study was conducted in accordance with the Declaration of Helsinki. Briefly, the cardiac tissues were minced and digested with 0.2% collagenase type II solution (Worthington, USA), filtered (0.2 µm of mesh; BD Bioscience, USA), and then digested single cardiac cells were centrifuged at 1,500 rpm for 5 min (Hanil Science, Korea). Single cardiac cells were washed 3 times with

2.3 Induction of Cardiomyocyte Differentiation For comparison, the groups were divided as follows. The cardiac derived stem/progenitor cells were seeded at 50,000 cells/well into a 6-well culture plate (SPL Life Science, Korea). The non-treated group served as the negative control group. The 5-azacytidine group (5AZA) was treated with 1 to 3 µM of 5-azacytidine

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Cardiomyogenic Induction Methodologies in hCPCsc-kit+t

(Sigma-Aldrich) on first 3 days.6 The 5-azacytidine and TGFβ1 co-induction group (5AZA+T) was treated with both 1– 3 µM of 5-azacytidine and 1 ng/mL TGF-β1 (PeproTech).17 In addition, treatment with 10 nM dexamethasone (Sigma-Aldrich) was used as a positive control for induction.3 Differentiation condition media was based on minimum essential medium eagle (Hyclone Laboratories), and contained 2% or 10% FBS (Gendepot) and 1× penicillin streptomycin.

Table 2. Primer list of cardiomyogenic related gene. Gene

Sequence

Gata4-F

GAC GGG TCA CTA TCT GTG GAA C

Gata4-R

AGA CAT CGC ACT GAC TGA GAA C

Nkx2-5-F CTT CAA GCC AGA GGC CTA CG Nkx2-5-R CCG CCT CTG TCT TCT CCA GC ACTC1-F TCT ATG AGG GCT ACG CTT TG ACTC1-R GCC AAT AGT GAT GAC TTG GC

2.4 Cardiomyocyte Differentiation Capacities Cardiomyocyte differentiation capacity was confirmed morphologically and molecularly using an inverted microscope (Olympus, CKX41), reverse transcriptase – polymeric chain reaction (RT-PCR) and immunofluorescence staining, respectively.

MHC-F

GGG GAC AGT GGT AAA AGC AA

MHC-R

TCC CTG CGT TCC ACT ATC TT

β-actin-F

CCC AAG GCC CAA CCG CGA GAA GAT

β-actin-R GTC CCG GCC AGC AGG TCC AG

Size 475 bp 488 bp 260 bp 543 bp 656 bp

then used for amplification by PCR (Bioer Technology, LifePro, China) with specific primers for cardiac-related genes (Table 2). After images were taken by (iNNOPLEX, LSG1000, Korea) image document, and images were collected at room temperature.

2.4.1 Morphological Analysis Morphological analysis to confirm differentiation was performed under an inverted microscope (Olympus, CKX41) under x4 magnification. Morphological images were taken every week for 4 weeks during differentiation at room temperature.

2.4.3 Immunofluorescence Staining After cardiomyocyte induction, the presence of c-kit (1:200, Santa Cruz), GATA4 (1:200, Santa Cruz), Nkx2-5 (1:200, Santa Cruz), α-SA (1:1000, Sigma-Aldrich), cardiac MHC (1:200, Abcam, Dawin Bio inc, Korea), cardiac troponin I (1:200, Abcam), and cardiac troponin T (1:200, Abcam) were confirmed by immunofluorescence staining. The hCPCsc-kit+ were washed 3 times with 1× PBS, fixed with 4% paraformaldehyde (USB corporation, USA) at room temperature for 5 to 7 min, and then treated with 3% bovine serum albumin (BOVOGEN, Australia) solution in PBS to block non-specific antibody binding. The

2.4.2 RT-PCR After 2 weeks of cardiomyogenic induction with 5-azacytidine, dexamethasone, or 5-azacytidine and TGF-β1, total RNA was isolated with TRIzol reagent (TAKARA, Japan) from induced hCPCsc-kit+. The mRNA from the lysed cells was precipitated by isopropanol and finally the mRNA pellet was washed with 70% ethanol and dissolved in diethylpyrocarbonate (DEPC) water. The cDNA was prepared using reverse transcriptase enzyme (Elpis Biotech, Korea) and 100 ng of mRNA. This cDNA was

Figure 1. Characterization of isolated hCPCsc-kit+. Direct morphology of isolated hCPCsc-kit+ under microscopy (A; left, x4 magnification; right, x16 digital crop images). Molecular analysis of c-kit, GATA4, and Nkx2-5 expression in isolated hCPCsc-kit+ (B). c-kit (red, Alexa 594), GATA4 (green, Alexa 488), Nkx2-5 (green, Alexa 488) and nucleus (blue, DAPI).

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primary antibodies (c-kit, GATA4, Nkx2-5, and α-SA) were incubated at 4oC overnight. The cell stains were then visualized by the appropriate fluorescence dye conjugated secondary antibodies as Alexa 488 (Molecular probes) and Alex 594 (Molecular probes). Images were taken with a confocal microscope (λabs; 495 nm and 590 nm, λem; 595 nm and 617 nm, Olympus, FV1000) under x200 magnification and images were collected at room temperature.

(Olympus, FV ASW, Ver 2.0, Japan). The quantification of fluorescence intensity was performed using open-source image analysis software (ImageJ;http://rsb.info.nih.gov/ij). All results are presented as the mean±S.E.M. Statistical significance between each group was determined using Student’s t-test. Differences with a p value of < 0.05 were considered statistically significant.

3. Results 2.5 Statistical Analysis GATA4 and Nkx2-5 gene expressions were analyzed by counting fluorescence-positive cells on microscopic images

3.1 Characterization of isolated hCPCsc-kit+ The hCPCsc-kit+ isolated by MACS, which was approximately

Figure 2. GATA4 and Nkx2-5 expression in different serum conditions during cardiomyocytes differentiation. Representative GATA4 expression was measured by confocal microscope (A) and the quantification of GATA4 fluorescence intensity is shown (B). In addition, representative Nkx2-5 expression was measured by confocal microscope (C) and the quantification of Nkx2-5 fluorescence intensity is shown (D). *p < 0.01 compared to starvation; #p < 0.01 compared to the non-treated group. GATA4 and Nkx2-5 (red, Alexa 594) and nucleus (blue, DAPI).

314


1.4% in total cardiac cells (Table 1). Our isolated hCPCsc-kit+ had spindle-shaped morphology similar to that of cardiac fibroblasts (Fig1A). These cells strongly expressed the stem cell marker ckit (Fig 1B), as well as cardiac-specific transcription factors such as GATA4 and Nkx2-5 (Fig 1B).

3.2 Differential Expression of GATA4 and Nkx2-5 Between High and Low Serum Conditions To determine the cardiomyogenic induction/maintenance conditions, we checked for an adequate percentage of FBS during cardiomyogenic differentiation by examining the expression of the cardiac-specific transcription factors such as GATA4 and Nkx2-5 in same time; 4weeks after differentiation induction (Fig 2 and 3). We were observed that our hCPCsc-kit+ expressed GATA4 in 10% FBS; however, expression was significantly reduced in all other cardiomyocytes differentiation induction groups (DEXA, 5AZA1, 5AZA3, 5AZA1+T, and 5AZA3+T) (p < 0.01, Fig 2A, 2B). Similarly, we observed that the expression of Nkx2-5 was also significantly reduced in all cardiomyocytes differentiation induction groups (p < 0.01, Fig 2C, 2D). Importantly, we were demonstrated that GATA4 and Nkx2-5 expression was preserved in 2% FBS even during cardiomyocyte differentiation (Fig 2A, 2B). Additionally, expression of the cardiac-specific transcription factors was also preserved in the differentiation induction groups in the presence of 2% FBS (p > 0.05, Fig 2B, 2D).

Figure 3. Morphological changes and mRNA expression during cardiomyocytes differentiation. Morphological analysis of hCPCsc-kit+ during cardiomyocyte differentiation under an inverted microscope. All images were taken at the same x10 magnification (A, Leica), and mRNA expression of cardiomyogenic associated factors is shown (B, GATA4, 475 bp [upper band]; Nkx2-5, 488 bp [lower band]).

factors were expressed at a low level in hCPCsc-kit+; but, GATA4 mRNA was strongly induced compared to its expression in the non-treated group. In contrast, Nkx2-5 expression did not differ between the non-treated group and the cardiomyogenic induction groups. Importantly, we demonstrated that stimulation by 5-azacytidine and TGF-β1 significantly increased ACTC-1 expression (Fig 3B).

3.3 Morphological Changes During Cardiomyocyte Differentiation We observed that our hCPCsc-kit+ had spindle-shaped morphology and were highly attached to non-coated dishes (Fig 3A). Importantly, we demonstrated that the morphologies of cardiomyogenic induction groups treated with only 5azacytidine (5AZA) or co-stimulated with 5-azacytidine and TGF-β1 (5AZA+T) were markedly changed into a sarcomeric structure at 4 weeks after induction; however, the group treated with dexamethasone did not show any morphological changes 4 weeks after induction (Fig 3A).

3.5 Expression of c-Kit During Cardiomyocyte Differentiation To examine whether or not each of the cardiomyogenic induction methods affected the stemness of the patient-derived CPCs, we measured the expression of a stemness marker, c-kit, 2 weeks after cardiomyocyte differentiation. As shown in Fig 4A, c-kit expression in the control group was not changed; however, expression of c-kit was significantly reduced in the other cardiomyocyte differentiation induction groups (p < 0.01, Fig 4). Interestingly, the co-stimulated with both 5-azacytidine and TGF-β1 group had markedly reduced c-kit expression compared to that of the control group (p < 0.05, Fig 4).

3.4 Expression of Cardiomyogenic-Related Genes During Cardiomyocyte Differentiation To examine the expression of cardiomyogenic-related factors, cardiomyogenic transcription factors (GATA4, Nkx25), and cardiac structural factor as alpha cardiac actin-1 (ACTC-1, α-sarcomeric actin subunits), we performed RTPCR analysis after induction of cardiomyogenic differentiation. As shown in Fig 3B, we observed that cardiomyogenic-related

315


Figure 4. c-kit expression during cardiomyocyte differentiation. Representative c-kit expression was measured using confocal microscopy (A). Quantification of c-kit fluorescence intensities (B). *p < 0.05, compared to starvation; #p < 0.05, compared to the non-treated group; **p < 0.05, compared to 2 weeks after cardiomyocyte differentiation induction. c-kit (red, Alexa 594) and nucleus (blue, DAPI).

expressed cardiac-specific transcription factors including GATA4 and Nkx2-5 (Fig 1B). Paradoxically, recent researches reported that hCPCs isolated from adult heart was not expressed GATA4 and Nkx2-5 in undifferentiated state. 3, 20 Some opposite evidences have shown that epicardium-derived ckit+ cells and postnatal isl-1+ cells were expressed GATA4 and Nkx2-5 in undifferentiated state as similar as our observation.21, 22 These different expression patterns were probably attributed to our cardiac stem cells isolated from infants' heart, and molecular expression diversity able to exist according to the different part of cardiac although cardiac stem cells were isolate using by same markers. Expressions of GATA4 and Nkx2-5 play essential for cardiomyocytes differentiation, because expressions of cardiomyocytes-related structural proteins (α-SA, MHC and Troponin-I, etc.) were required to activation of GATA4 and Nkx2-5.7 According to the evidences, GATA4 and Nkx2-5 expressions were increased in hCPCsc-kit+ by treatment with dexamethasone in MEM contained with 10% FBS.3 Contrast in our observation, hCPCs c-kit+ did not differentiated into cardiomyocytes by 10% FBS condition, because expressions of GATA4 and Nkx2-5 were reduced during cultured in MEM contained with 10% FBS condition (Fig 2 and 3), but expressions of GATA4 and Nkx2-5 were retained by 2% FBS

3.6 Differential Changes in Cardiomyogenic-Related Factors During Cardiomyocyte Differentiation To determine the cardiomyogenic differentiation efficacy of each induction system, we performed immunocytochemistry analyses using specific antibodies against MHC, cardiac troponin I, cardiac troponin T (data not shown), and α-SA. As shown in Fig 5, we observed that the expression of α-SA was significantly induced by co-stimulation with 5-azacytidine and TGF-β1; however, the cardiac structural proteins, cardiac MHC, cardiac troponin I, and cardiac troponin T were not expressed. In the group co-treated with 5-azacytidine and TGFβ1, α-SA expression increased significantly 2 weeks after cardiomyocyte differentiation (p < 0.05, Fig 5). However, we observed that treatment with either 5-azacytidine or dexamethasone did not affect α-SA expression at 2 weeks, but significantly increased α-SA expression 4 weeks after stimulation (p > 0.05, Fig 5).

4. Discussion In the present study, we were isolated hCPCsc-kit+ and examined their cardiomyogenic potential using several previously reported differentiation protocols. 2, 3 Our isolated hCPCsc-kit+ not only expressed c-kit, but also

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Figure 5. α-SA expression during cardiomyocyte differentiation. Representative α-SA expression measured using confocal microscopy (A) Quantification of α-SA fluorescence intensity (B). *p < 0.05, compared to starvation; #p < 0.05, compared to the non-treated group; **p < 0.05, compared to 2 weeks after cardiomyocyte differentiation induction. α-SA (green, Alexa 488) and and nucleus (blue, DAPI).

claimed that 5-azacytidine promoted BM-MSCs differentiation into adipocytes and chondrocytes or induced uncontrollable expression of various genes.29, 30 Furthermore, treatment of stem cells with TGF-β1 also facilitates differentiation into cardiomyocyte-like cells; thus, the mechanisms of cardiomyogenic induction in stem cells are well established. According to the results of Li and colleagues, treatment of BM-MSCs with TGF-β1 triggered differentiation into cardiomyocyte-like cells via phosphorylation of SMAD2 and SMAD3.4 In addition, treatment of Sca-1+ CPCs with TGFβ1 induced differentiation into cardiomyocyte-like cells via phosphorylation of SMAD2.18 Interestingly, in the present study, we demonstrated that costimulation with 5-azacytidine and TGF-β1 significantly induced α-SA, a cardiac structural factor, at an early period of myocardial lineage differentiation (Fig 5). These results suggest that co-stimulation with 5-azacytidine and TGF-β1 might synergistically affect the expression of cardiomyogenicassociated proteins more potently than treatment with 5azacytidine and/or dexamethasone alone. Therefore, based on our report and those of others, co-stimulation with 5-azacytidine and TGF-β1 is proposed as a potential inducer of cardiac

condition during cardiomyogenic induction (Fig 2 and 3). Although the underlying mechanisms are not clearly understood, the high-serum condition may hinder the induction of the differentiation signal via the induction of proliferation.23 In spite of this effect, the expression of GATA4 and Nkx2-5 played a crucial role in cardiomyocyte differentiation; thus, we consider that it is important to maintain the expression of GATA4 and Nkx2-5 during cardiomyogenic induction.7 Several studies have suggested that stem cells differentiate into cardiomyocytes upon treatment with chemical reagents such as 5-azacytidine.24-26 According to the results of Choi and colleagues, treatment of P19 cells with 5-azacytidine caused the cells to differentiate into cardiomyocyte-like cells through demethylation.24 In addition, Qian and colleagues found that treatment of MSCs with 5-azacytidine triggered differentiation into cardiomyocyte-like cells through ERK activation.26 However, the in vitro data regarding the effect of 5-azacytidine on stem cells is controversial. Several groups have reported the ability of these cells to differentiate into cardiomyocytes, whereas others have reported a failure of 5-azacytidine to induce a cardiomyogenic phenotype in bone marrow-derived mesenchymal stem cells (BM-MSCs) in vitro.27, 28 In addition, other reports

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heart, Circulation Research, 95, 911 (2004). 3. C Bearzi, M Rota, T Hosoda, et al., Human cardiac stem cells, Proc Nat Aca Sci Unit Stat America, 104, 14068 (2007). 4. TS Li, T Komota, M Ohshima, et al., TGF-beta induces the differentiation of bone marrow stem cells into immature cardiomyocytes, Biochem Bioph Res Comm, 366, 1074 (2008). 5. Y Zhu, T Liu, K Song, et al., ADSCs differentiated into cardiomyocytes in cardiac microenvironment, Molecular and Cell Biochem, 324, 117 (2009). 6. S Makino, K Fukuda, S Miyoshi, et al., Cardiomyocytes can be generated from marrow stromal cells in vitro, J Clin Invest, 103, 697 (1999). 7. D Durocher, F Charron, R Warren, et al., The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors, EMBO J, 16, 5687 (1997). 8. SH Orkin, GATA-binding transcription factors in hematopoietic cells, Blood, 80, 575 (1992). 9. WY Huang, E Cukerman, CC Liew, Identification of a GATA motif in the cardiac alpha-myosin heavy-chain-encoding gene and isolation of a human GATA-4 cDNA, Gene, 155, 219 (1995). 10. RJ Arceci, AA King, MC Simon, et al., Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart, Mol and Cell Biol, 13, 2235 (1993). 11. S Tamura, XH Wang, M Maeda, et al., Gastric DNA-binding proteins recognize upstream sequence motifs of parietal cellspecific genes, Proc Nati Aca Sci Unit Stat Am, 90, 10876 (1993). 12. F Charron, M Nemer, GATA transcription factors and cardiac development, Seminars in Cell & Develo Biology, 10, 85 (1999). 13. PB Burton, MC Raff, P Kerr, et al., An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes, Develo Biol, 216, 659 (1999). 14. I Lyons, LM Parsons, L Hartley, et al., Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5, Genes & Develo, 9, 1654 (1995). 15. M Farokhpour, K Karbalaie, S Tanhaei, et al., Embryonic stem cellderived cardiomyocytes as a model system to study cardioprotective effects of dexamethasone in doxorubicin cardiotoxicity, Toxicology in vitro : an int J publ asso with BIBRA, 23, 1422 (2009). 16. WS Shim, S Jiang, P Wong, et al., Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells, Biochem Bioph Res Comm, 324, 481 (2004). 17. AM Smits, P van Vliet, CH Metz, et al., Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology, Nature Prot, 4, 232 (2009). 18. MJ Goumans, TP de Boer, AM Smits, et al., TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro, Stem Cell Res, 1, 138 (2007). 19. BC Heng, H Haider, EK Sim, et al., Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro, Cardiovas Res, 62, 34 (2004). 20. H Oh, SB Bradfute, TD Gallardo, et al., Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction, Proc Nat Aca Sci Unit Stat of Am, 100, 12313 (2003).

differentiation. Study limitations: Some limitations of our study are that we observed an increase for only α-SA and not for other cardiac structural proteins in our cardiac induction system, including cardiac troponin I and T and MHC. The difference in the expression patterns of cardiomyogenic factors is thought to be caused by differences in the types of cardiac stem cells, which differ in their stem cell markers.31 Recently, accumulating evidence has suggested that different types of cardiac stem/ progenitor cells with different stem cell markers express different cardiac myocyte genes.31 Isolated c-kit-positive cardiac stem cells expressed α-SA, cardiac myosin, and desmin3, 32, whereas Sca-1+ cardiac stem cells expressed not only α-SA and cardiac myosin but also cardiac troponin I as well as MHC a and b after differentiation.17, 18, 20, 33 However, cardiac side population cells and Islt-1-positive cardiac stem cells were not confirmed α-SA after differentiation.22, 34 In addition, we observed different expression patterns between mRNA and protein during cardiomyogenic induction at the same time point that probably resulted from posttranslational modulation. However, we did not evaluate the mechanisms of posttranslational regulation for GATA4 and Nkx2-5. Therefore, further studies that address celltype-dependent cardiomyocyte-related protein expression patterns and posttranslational regulation of GATA4 and Nkx2-5 are required. Conclusions: In present study, we were observed early induction of expression of α-SA through the combination treatment with 5-azacytidine and TGF-β1 rather than other chemical induction alone, in spite of did not observed other cardiomyocytes-related structural proteins. On the basis of the present results, we conclude that co-stimulation of hCPCsc-kit+ with 5-azacytidine and TGF-β1 led to synergistically increased differentiation into early-stage cardiomyocytes compared to differentiation with other single induction protocols. Acknowledgment: This work was supported by a grant from the Korea Healthcare Technology R & D Project (A100638), Ministry of Health, Welfare, and Family Affairs, Republic of Korea.

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