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(Circulation. 2004;110:2658-2665.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Differentiation of Bone Marrow Stromal Cells Into the Cardiac Phenotype Requires Intercellular Communication With Myocytes

Meifeng Xu, MD, PhD; Maqsood Wani, PhD; Yan-Shan Dai, MD, PhD; Jiang Wang, MD, PhD; Mei Yan, MD, PhD; Ahmar Ayub, DVM; Muhammad Ashraf, PhD

From the Department of Pathology and Laboratory Medicine (M.X., J.W., M.Y., A.A., M.A.), University of Cincinnati Medical Center, and the Department of Pediatrics (M.W., Y.-S.D.), University of Cincinnati, Children’s Hospital Medical Center, Cincinnati, Ohio.

Correspondence to Muhammad Ashraf, PhD, Department of Pathology and Laboratory Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0529. E-mail Muhammad.Ashraf{at}uc.edu

Received December 30, 2003; de novo received April 15, 2004; revision received June 1, 2004; accepted June 3, 2004.

	Abstract

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Background— Bone marrow stromal cells (BMSCs) have the potential to differentiate into various cells and can transdifferentiate into myocytes if an appropriate cellular environment is provided. However, the molecular signals that underlie this process are not fully understood. In this study, we show that BMSC differentiation is dependent on communication with cells in their microenvironment.

Methods and Results— BMSCs were isolated from green fluorescent protein (GFP)–transgenic mice and cocultured with myocytes in a ratio of 1:40. Myocytes were obtained from neonatal rat ventricles. The differentiation of BMSCs in coculture was confirmed by immunohistochemistry, electron microscopy, and reverse transcription–polymerase chain reaction. Before coculturing, the BMSCs were negative for -actinin and exhibited a nucleus with many nucleoli. After 7-day coculture with myocytes, some BMSCs became -actinin–positive and formed gap junctions with native myocytes. However, BMSCs separated from myocytes by a semipermeable membrane were still negative for -actinin. Transdifferentiated myocytes from BMSCs were microdissected from cocultures by laser captured microdissection to determine the changes in gene expression. BMSCs cocultured with myocytes expressed mouse cardiac transcription factor GATA-4.

Conclusions— When cocultured with myocytes, BMSCs can transdifferentiate into cells with a cardiac phenotype. Differentiated myocytes express cardiac transcription factors GATA-4 and myocyte enhancer factor-2. The transdifferentiation processes rely on intercellular communication of BMSCs with myocytes.

Key Words: myocytes • stem cells • signal transduction • genetics

	Introduction

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Stem cells have the ability to respond to environmental demands. However, their scarcity within tissue and the difficulty of isolating pure populations make it difficult to perform biochemical and molecular studies. The bone marrow, in addition to blood-forming progenitors, contains cells that have the properties of stem cells. These cells are referred to as bone marrow stromal or bone mesenchymal stem cells (BMSCs). BMSCs are multipotent and can differentiate into several distinct cell types, including skeletal muscle,¹ liver cells² and neural cells³ after their transplantation into these tissues. It has also been demonstrated that BMSCs can differentiate into cardiac cell components and develop into functional phenotypes of myocardial cells within the microenvironment of the heart.^4–6 Human mesenchymal stem cells cocultured with myocytes express myosin heavy chain, ß-actin, and troponin T.⁷ BMSCs can not only differentiate into myocytes but also improve left ventricular performance⁸ and cause a significant reduction of infarct size when injected into the infarcted hearts or border zones of rats or mice.^5,6,9 Moreover, BMSCs can be delivered into the coronary artery, because they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.¹⁰ These studies indicate that BMSCs respond to signals from the host tissue microenvironment and differentiate into mature cells, which might contribute to regeneration of the infarcted myocardium. The addition of functional myocytes transdifferentiated from BMSCs could be a very promising and innovative approach to salvage myocardium and reduce cardiac remodeling associated with postmyocardial infarction.

Although BMSCs have the ability to differentiate into new myocytes, the requirements for transformation into mature, functional cardiac myocytes are not clear. In this study, we tested the hypothesis that BMSCs differentiate into myocytes in response to signals from neighboring myocytes, which may result from intercellular communication through formation of gap junctions.

	Methods

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Laboratory Animals
All animals were treated in accordance with the Guidelines for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, revised 1996).

Isolation of BMSCs and Coculture With Myocytes
BMSCs were isolated according to the method described by Makino et al,¹¹ with some modifications. In brief, femurs and tibias from green fluorescent protein (GFP)–transgenic mice developed by Hadjantonakis et al¹² were removed. Muscle and extraossial tissue were trimmed. Bone marrow cells were flushed. Cells were cultured with Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 20% fetal bovine serum and penicillin (100 U/mL)/streptomycin (100 µg/mL) at 37°C in humid air with 5% CO₂. After being seeded for 2 days, BMSCs adhered to the bottom of culture plates, and hematopoietic cells remained suspended in the medium. The culture medium was changed every 3 days, and the nonadherent hematopoietic cells were completely washed out after 4 changes of media. Myocytes were isolated from the hearts of neonatal rats (1 to 3 days old) with use of the neonatal cardiomyocyte isolation system (Worthington Biochemical Co) as previously described.¹³

Immunocytochemistry
Immunocytochemistry was performed as described previously by our laboratory, with some modifications.¹⁴ In brief, cells cultured on glass coverslips were fixed in 4% paraformaldehyde or methanol and then incubated with the mouse monoclonal anti-sarcomeric -actinin (Sigma), rabbit polyclonal anti–myocyte enhancer factor (MEF)-2, anti-connexin43, anti–c-kit, anti-CD34, anti–VE-cadherin, goat anti–GATA-4 (Santa Cruz), and DiI-labeled acetylated low-density lipoprotein (Dil acLDL) (Molecular Probes). After thorough washing, secondary antibodies of goat anti-mouse IgG (fluorescein isothiocyanate [FITC] or rhodamine conjugate) and/or goat anti-rabbit IgG (FITC or rhodamine conjugate) or rabbit anti-goat IgG (rhodamine conjugate) were applied. Nuclei were stained with 4',6-diamino-2-phenylindole (DAPI) when necessary. Fluorescent images were obtained with an Olympus BX 41 microscope equipped with a digital camera (Olympus). Confocal images were obtained with a Leitz DMRBE fluorescence microscope equipped with a TCS 4D confocal scanning attachment (Leica, Inc).

Transmission Electron Microscopy
Cultured cells were rinsed with buffer and immersed in 2.5% buffered glutaraldehyde for 4 hours, rinsed in 0.1 mol/L sodium cacodylate buffer (pH 7.3), and postfixed for 1 hour in 1% buffered OsO₄. The cells were embedded in Epon resin and cut into 60-nm-thick sections with a Sorvall MTB2 ultramicrotome. The sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-600 electron microscope at 75 kV.

Microdissection of BMSCs and Extraction of RNA
After BMSCs were cocultured with myocytes for 7 days, the cells were fixed in 70% ethanol and double-stained for myofibers and nuclei. Mouse monoclonal anti–-actinin and goat anti-mouse IgG peroxidase conjugate were applied to label the myofibers. The nuclei were stained with hematoxylin 2 (Richard-Allan Scientific). The myocytes transdifferentiated from BMSCs showed a nucleus with multiple nucleoli and were positive for -actinin. Approximately 200 cells were targeted and microdissected with a laser capture microdissection system (Arcturus). RNA was extracted from the captured cells with a PicoPure RNA isolation kit (Arcturus). Total RNA from cultured cells was isolated by lysing the cells in Trizol reagent (Invitrogen).

Reverse Transcription–Polymerase Chain Reaction
RNA was used for cDNA synthesis with SuperScript III RNase H^– reverse transcriptase (Invitrogen) in a 20-µL reaction mixture. An aliquot of the cDNA was amplified with Taq DNA polymerase (2.5 U, Invitrogen) in the presence of sense and antisense primers (1 µmol/L) for nonspecific GATA-4 and mouse-specific GATA-4. The sequences of primers were as follows: nonspecific GATA-4, 255 bp, 5'-CTG TCA TCT CAC TAT GGG CA and 5'-CCA AGT CCG AGC AGG AAT TT; mouse-specific GATA-4, 197 bp, 5'-CCT CTC CCA GGA ACA TCA AA and 5'-ACC CAT AGT CAC CAA GGC TG; and mouse-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 372 bp, 5'-CTC TTG CTC TCA GTA TCC TTG and 5'-GCT CAC TGG CAT GGC CTT CCG. RNA of rat myocytes was used as a negative control, and RNA of mouse heart was used as a positive control to amplify mouse-specific GATA-4. PCR was performed for 35 cycles, with each cycle consisting of denaturation at 96°C for 50 seconds, annealing at 53°C to 60°C for 45 seconds, and extension at 72°C for 45 seconds, with autoextension for 10 minutes at 72°C after completion of the last cycle. The PCR products were size-fractionated by 1.5% agarose gel electrophoresis.

Statistical Analysis
Quantitative data were obtained from 2 coverslips each time from 3 separate experiments. Five random fields per coverslip were analyzed. All data were expressed as mean±SEM.

	Results

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Characterization of BMSCs in Culture
The BMSCs were isolated from transgenic mice expressing GFP as described in Methods. BMSCs in primary culture expressed GFP at various levels. The morphology and size of these BMSCs varied and had the tendency to grow in clusters (Figure 1A). As shown in Figure 1B, the nucleus of each BMSC had >1 nucleolus. To characterize the BMSCs, we performed immunostaining with antibodies against various markers. BMSCs were positive for c-kit, a stem cell marker. Some BMSCs expressed CD34, a marker of endothelial progenitor cells, and VE-cadherin, Flk-1, and acLDL, markers of endothelial cells (Figure 2). The results indicated that cultured, adherent bone marrow cells contained mesenchymal stem cells, fibroblasts, and endothelial progenitor cells, as well as endothelial cells.

View larger version (22K): [in this window] [in a new window]   Figure 1. BMSCs from GFP transgenic mice. A, Primary cultured GFP-positive BMSCs. Mainly 2 types of BMSC can be seen: small spindle- or triangle-shaped cells. B, Nuclei are stained with DAPI. All nuclei show multiple nucleoli. Abbreviations are as defined in text.

View larger version (25K): [in this window] [in a new window]   Figure 2. Expression of different cell markers in BMSCs. Cell markers are colored red. A1, c-kit: all BMSCs are positive for c-kit (arrows); A2, same as A1, except stained with DAPI. B1, CD34-positive BMSCs (arrows); B2, same as B1, except stained with DAPI. C1, Flk-1-positive BMSCs (arrow); C2, same as C1, except stained with DAPI. D, acLDL-positive BMSCs (arrows). E1, VE-cadherin–positive BMSCs (arrow); E2, same as E1, except stained with DAPI. All nuclei were stained with DAPI because green fluorescence in some BMSCs was not very strong. Each nucleus is shown with multiple nucleoli. F, Percentage of cells as identified by different markers. Abbreviations are as defined in text.

Transdifferentiation of BMSCs Into Cardiac Myocytes
BMSC-derived cardiomyocytes were characterized by immunostaining and electron microscopy. Native myocytes were isolated from neonatal rat ventricles as previously described. After being cultured for 24 hours, myocytes began to beat spontaneously, and immunostaining showed that all myocytes were positive for -actinin. Myofibers were seen with clear Z-lines in sarcomeres, and myocytes had physical contacts with neighboring myocytes. Nuclei were centrally located and homogeneous. However, BMSCs were negative for -actinin and had a nucleus with multiple nucleoli. To induce differentiation, BMSCs were cocultured with rat myocytes in a ratio of 1:40. When BMSCs were cocultured with myocytes for 7 days, 14% to 32% of BMSCs differentiated into cardiac phenotypes. Synchronous contractions of differentiating BMSCs with native myocytes were observed under the inverted microscope. These cells were positive for -actinin staining (Figure 3). Differentiation was also confirmed by transmission electron microscopy. The ultramicroscopic details of differentiated BMSCs showed a cardiomyocyte-like ultrastructure, including sarcomeres, abundant glycogen granules, and a number of mitochondria, but these cells contained nucleus with multiple nucleoli, as observed in the BMSC nucleus (Figure 4).

View larger version (42K): [in this window] [in a new window]   Figure 3. Expression of myocyte-specific marker in BMSCs after coculture with myocytes. A, BMSCs express GFP. B, Same field as A, in which cells were stained with anti–-actinin antibody to demonstrate myofilaments (red). C, Overlay of A and B. Nuclei are stained with DAPI (blue). White arrow shows green BMSCs that have differentiated into cardiac phenotypes. White arrowhead shows undifferentiated BMSCs. Green arrow points to native myocyte with clear Z-lines. Abbreviations are as defined in text.

View larger version (148K): [in this window] [in a new window]   Figure 4. Electron microscopic images of myocytes, BMSCs, and differentiated myocytes from BMSCs. A, Typical myocyte has myofibers with clear sarcomeres (S), central nucleus with single nucleolus (arrow), and uniformly dispersed chromatin material in nucleoplasm. B, BMSC has no myofibers, and nucleus has multiple nucleoli (arrows). C, Differentiated myocyte is confirmed by fragmentation of nucleoli (arrow) and appearance of sarcomeres (S, shown as arrowhead) with abundant mitochondria (M) and endoplasmic reticulum (E). All other abbreviations are as defined in text.

Communication With Native Myocytes
To determine whether communication between BMSCs and native myocytes is required in the transdifferention of the former, cocultured cells were simultaneously stained with anti–-actinin and anti-connexin43 antibodies and DAPI. Cells were fixed with methanol, which faded the green fluorescence of GFP. Observations showed the clear formation of gap junctions between differentiating myocytes from BMSCs (positive for -actinin and having a nucleus with multiple nucleoli) and native myocytes. The gap junctions were similar to those formed between native rat myocytes (Figure 5A–5C). The formation of gap junctions was also observed by electron microscopy (Figure 5D). This was further confirmed by confocal microscopy to ensure that nuclei of both myocytes and BMSCs were in the same plane (Figure 5E and 5F). To establish that physical contact between BMSCs and myocytes is needed for BMSC transdifferentiation, preparations of these 2 cell types were cultured in 2 chambers separated by a semipermeable membrane. This system allows the diffusion of secreted factors but prevents physical contact between the 2 cell populations. Three dual-chamber setups were used with different seeding in the upper and lower chambers. They were as follows: (1) BMSCs in the upper chamber and BMSCs in the lower; (2) myocytes in the upper and BMSCs in the lower; and (3) myocytes in the upper and myocytes in the lower. After 7 days in culture, the cells in the lower chambers were double-stained with DAPI and anti–-actinin antibody. BMSCs in the lower chambers of setups 1 and 2 were negative for anti–-actinin (data not shown). This result suggests that cell-to-cell contact is necessary for transdifferentiation of BMSCs.

View larger version (90K): [in this window] [in a new window]   Figure 5. BMSCs and myocytes form gap junction in coculture. A, DAPI-stained nuclei of cocultured myocytes and BMSCs (arrow shows nucleus with multiple nucleoli). B, Same as A, except stained with anti–-actinin. BMSC (star indicates location of nucleus) is positive for -actinin staining, which means that this BMSC has differentiated into myocyte. C, Simultaneous staining of -actinin (green), connexin43 (red), and DAPI (blue). Gap junctions (arrows) are observed between BMSC (blue, nucleus with multiple nucleoli) and myocytes. Arrowheads show gap junctions between native myocytes. D, Electron photomicrograph of BMSC shows close apposition with neighboring myocytes (arrow). E and F, Connexin43 immunostaining by confocal microscopy. E, Cocultured GFP BMSCs with myocytes observed under FITC filter (green). Edge of green BMSC is shown by arrows. F, Same as E, except stained with connexin43 antibody (red). Gap junctions (arrows) are clearly shown between green BMSC (arrowhead) and myocyte (*). Abbreviations are as defined in text.

Expression of Transcription Factors
Cardiac transcription factors include MEF-2 and GATA-4. All native myocytes expressed MEF-2, which was located in the nucleus (Figure 6A). However, only 58.1% of BMSCs weakly expressed MEF-2 (Figure 6C). The native myocytes were positive for GATA-4, whereas BMSCs were negative for GATA-4. When cocultured with myocytes for 7 days, some BMSCs were positive for GATA-4 (Figure 7A). RT-PCR analysis indicated that the differentiated BMSCs, which were microdissected from coculture, expressed GATA-4, whereas the undifferentiated BMSCs did not (Figure 7B). However, the nonspecific PCR primers for GATA-4 are conserved between mouse and rat species. To rule out the possibility of contamination from rat cardiomyocytes in captured BMSCs, we designed a pair of mouse GATA-4 primers that are not conserved in rat GATA-4. We also designed mouse GAPDH primers for PCR amplification of mouse GAPDH as a loading control. As shown in Figure 7C, PCR only amplified mouse GATA-4 in mouse heart and cocultures of rat myocytes with BMSCs. Cultures of BMSC or rat myocytes alone were negative for GATA-4 by PCR. These results indicate that the GATA-4 expression observed in cocultures was directly derived from transdifferentiated myocytes from mouse BMSCs.

View larger version (31K): [in this window] [in a new window]   Figure 6. Expression of MEF-2 in cardiac myocytes and BMSCs. A and B, All myocytes express MEF-2. C and D, Some BMSCs express MEF-2 but are weaker than in myocytes (exposure time was 4 times longer than that in myocytes). Abbreviations are as defined in text.

View larger version (68K): [in this window] [in a new window]   Figure 7. Expression of GATA-4 in BMSCs cocultured with myocytes. A1–A4, Immunostained for GATA-4. A1, Seven green BMSCs (arrow and arrowheads); A2, same as A1, except stained with DAPI; A3, Only 1 green BMSC expressing GATA-4 in nucleus (red, arrow), and other BMSCs are negative for GATA-4 (compare with A2); A4, overlay of GFP-positive cells and GATA-4 presence (arrow). B and C, RT-PCR analysis of expression of GATA-4. B, BMSCs microdissected from cocultured cells expressed nonspecific GATA-4, but BMSCs cultured alone did not. C, RT-PCR amplified mouse GATA-4 in both cocultured cells and positive control mouse heart. Abbreviations are as defined in text.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Mouse BMSCs can differentiate into cardiomyocytes in vitro in the presence of myocytes. Cardiomyocyte transcription factors, such as GATA-4 and MEF-2, are expressed during BMSC transdifferentiation. Contact with native myocytes is required when BMSCs differentiate into myocytes.

Immunostaining has shown that the cultured bone marrow cells are positive for c-kit, a marker of stem cells. The various shapes and expression of different cell markers indicate that cultured bone marrow adherent cells consist of mesenchymal stem cells, fibroblasts, endothelial progenitor cells, and endothelial cells.

In this study, we confirmed that BMSCs can differentiate into myocytes in the cardiac environment. Our results are consistent with other in vivo and in vitro studies.^4,5,7 The transdifferentiation of BMSCs into myocytes was evaluated with immunohistochemical markers, electron microscopy, and RT-PCR. -Actinin was used as a marker to identify the myocytes, because monoclonal anti–-actinin (sarcomeric) is specific for -skeletal and -cardiac muscle actinin. The differentiated BMSCs contained cardiac sarcomeres that reacted with anti–-actinin antibody. The bone marrow cell–derived cardiac myocytes also exhibited typical developing sarcomeres that contained Z-bands and contractile filaments that were observed by electron microscopy. BMSCs from transgenic mice expressing GFP¹² were easily distinguished from native rat myocytes by green fluorescence. In this study, differentiating BMSCs were also analyzed by the presence of multiple nucleoli in their nuclei, a distinctive feature of stem cells. Therefore, the combination of green fluorescence and nuclei with multiple nucleoli provided a measure of identification of BMSCs. By these criteria, a differentiated myocyte was a green cell with multiple nucleoli that was positive for -actinin.

It has been previously reported that myocytes differentiated from stem cells display action potential activity in line with excitation-contraction coupling¹⁵ and cardiac-specific ion currents identical to those described for adult myocytes.¹⁶ Furthermore, Makino et al¹¹ previously isolated a cardiomyogenic cell line from murine BMSCs and demonstrated that they exhibit at least 2 types of distinguishable morphological action potentials: sinus node–like potential and ventricular myocyte-like potential. Taken together, these differentiated myocytes more likely represent structural phenotypes of adult myocytes.

Communication between BMSCs and native myocytes is required in the transdifferention of BMSCs. Gap junctional channels that couple myocytes mediate conduction phenomena in the heart. These channels are hexamers of transmembrane proteins belonging to the connexin family.¹⁷ We not only observed the formation of gap junctions between differentiated myocytes from BMSCs and native myocytes but also showed that BMSCs remained unchanged when cultured with myocytes in a dual-chamber culture system separated by a semipermeable membrane, even though the medium contained soluble factors released from cardiomyocytes. Previously, it was reported that myoblasts can also engraft into normal and infarcted myocardium by direct injection. Myoblasts contract and relax like myocytes only when they are externally stimulated, but myoblasts do not transdifferentiate into myocytes, because they are unable to develop gap junctions.^18,19 Chedrawy et al²⁰ showed that the physical barrier between BMSCs and cardiomyocytes is nonconducive for BMSCs to transdifferentiate, despite the presence of a myocardial environment. The study of Rangappa et al⁷ indicated that in addition to soluble signaling molecules, direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of adult stem cells to cardiomyocytes. To form a functional unit, however, these cells must be interconnected to maintain electrical coupling. Moreover, early expression of connexin43 was observed before BMSCs were able to transdifferentiate into myocytes.²⁰ In our study, we showed that formation of gap junctions between myocytes and BMSCs was observed as early as 3 days in coculture and preceded the process of BMSC differentiation (data not shown). Such supracellular structural integration was enhanced by fiber stretching during cardiac contractions, sending signals for cellular reorientation and incorporation. Cadherins may mediate this crucial cell-to-cell contact.²¹

Cardiac transcription factors MEF-2 and GATA-4 govern the intricate process of cardiogenesis by regulating cardiac-specific gene expression. MEF-2 transcription factors are involved in cardiac morphogenesis.²² Our studies showed that BMSCs partially expressed MEF-2 and that transdifferentiation of BMSCs into myocytes was related to the expression level of MEF-2 (data not shown). However, before coculture, BMSCs did not show the phenotype of myocytes, even though some BMSCs expressed MEF-2. As reviewed by Molkentin,²³ GATA-4 controls cardiac structural and regulatory gene expression. Our studies showed that GATA-4 immunoreactivity was found only in nuclei of some BMSCs that were cocultured with myocytes. RT-PCR also indicated that GATA-4 was highly expressed in BMSCs cocultured with myocytes but not in BMSCs in cultures without myocytes. This indicates that GATA-4 is associated with cardiomyocyte differentiation.

Our results suggest that BMSCs in coculture generated a myocyte phenotype as a result of transdifferentiation. Terada et al²⁴ demonstrated that bone marrow cells can fuse spontaneously with embryonic stem cells in culture that contains interleukin-3. Spontaneously fused bone marrow cells can subsequently adopt the phenotype of the recipient cells. Wang and colleagues²⁵ also showed that hepatocytes derived from bone marrow arise from cell fusion. A recent study showed that bone marrow–derived cells fuse in vitro with neural progenitors and in vivo with hepatocytes in liver, Purkinje neurons in the brain, and cardiac muscle in the heart, resulting in the formation of multinucleated cells.²⁶ However, cell fusion appears to be a very low-frequency event, occurring perhaps once in 10 000 to 100 000 cells, whereas transdifferentiation has been reported to occur at as high a rate as 55% in coculture assays.^27–29 In our study, 14% to 32% of BMSCs transdifferentiated into myocytes when cocultured with rat myocytes, but we could not discern any cell fusion. Cocultured BMSCs expressed mouse-specific GATA-4, which also indicates that the new myocytes were differentiated from stromal cells originally derived from mouse bone marrow. However, we could not completely exclude the rare possibility of fusion in BMSCs cocultured with myocytes.

In conclusion, we found that BMSCs have the ability to transdifferentiate into the cardiac phenotype in the presence of an appropriate microenvironment. The transdifferentiation of BMSCs is dependent on the formation of intercellular connections with native myocytes. Such communication may induce signals that may ultimately result in changes in gene expression, such as that of MEF-2 and GATA-4.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL074272 and HL70062. We thank Dr Jeffrey Brown for insightful discussion and critical review of the manuscript. We thank Richard Montione for his help in confocal microscopy.

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