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Clin Transl Sci. 2008 September; 1(2): 116–125.
Published online 2008 September 10. doi:  10.1111/j.1752-8062.2008.00034.x
PMCID: PMC2863128
NIHMSID: NIHMS193402

BMP‐2 and FGF‐2 Synergistically Facilitate Adoption of a Cardiac Phenotype in Somatic Bone Marrow c‐kit+/Sca‐1+ Stem Cells

Brent R. DeGeorge, Jr., B.S., 1 , * Marc Rosenberg, M.D., 2 , * Volker Eckstein, Ph.D., 3 Erhe Gao, M.D., Ph.D., 4 Nicole Herzog, B.S., 2 Hugo A. Katus, M.D., 2 Walter J. Koch, Ph.D., 1 Norbert Frey, M.D., 2 and Patrick Most, M.D. 4

Abstract

The aim of this study was to explore the effect of bone morphogenetic protein‐2 (BMP‐2) and fibroblast growth factor‐2 (FGF‐2)— paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)—on murine bone marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent protein (GFP) expressing hematopoietic lineage negative (lin‐) c‐kit ligand (c‐kit) and stem cell antigen‐1 (Sca‐1) positive (GFP‐lin‐/c‐kit+/sca+) mBMSC were co‐cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP‐2 and FGF‐2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co‐culture at day 10. The addition of BMP‐2 in concert with FGF‐2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5‐fold (~20%), whereas the addition of anti‐BMP‐2 and/or anti‐FGF‐2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP‐2/FGF‐2 compared to untreated co‐cultures. BMP‐2 and FGF‐2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding protein 4; GATA‐4) and late (myosin light chain kinase [MLC‐2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co‐culture. In addition, re‐cultured fluorescence‐activated cell sorting (FACS)‐purified BMP‐2/FGF‐2‐treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L‐type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine‐sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long‐term self‐renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross‐talk within an appropriate cardiac microenvironment.

Keywords: mBMSCs, BMP‐2, FGF‐2, paracrine signaling

Introduction

Given the varying degrees of success of the clinical studies utilizing intracoronary delivery of somatic stem cells, an uncertainty has arisen regarding which cell type will best supplement the process of cardiac repair. 1 , 2 , 3 Various cell types have shown the potential to transdifferentiate into cardiac myocytes, including endothelial progenitor cells, 4 mesenchymal stem cells, 5 embryonic stem (ES) cells, 6 as well as islet 1‐positive (ISL1+) cardioblasts, 7 Sca‐1+ cells, 8 and c‐kit+ cells. 9 However, there is still controversy as to whether somatic bone marrow stem cells (BMSCs) truly possess a cardiomyogenic potential in v ivo. 10 An improved knowledge of whether BMSCs can function as a potential in vivo cardiac progenitor and the molecular pathways that facilitate this process will aid in the understanding of the therapeutic potential of this cell type.

The focus of recent efforts has shifted to developing our understanding of the paracrine signaling that occurs between resident cardiac cells and exogenous stem cells. The factors involved in embryonic cardiogenesis, which relies heavily on paracrine signaling, have been intensely studied as potential candidates to facilitate the differentiation of stem cells toward a cardiac phenotype. Several growth factors, including bone morphogenetic proteins (BMPs)—members of the transforming growth factor‐β (TGF‐β) superfamily—and fibroblast growth factors (FGFs), which are both secreted from the endodermal tissue surrounding the precardiac mesoderm, have been implicated in inducing the expression of cardiac‐restricted transcription factors, such as Nkx2.5, Myocyte Enhancer Factor‐2c (Mef‐2C), and GATA‐4, in ES cells. 11 , 12

Stem cell and cardiomyocyte co‐culture studies have provided a means to study both the physical and the paracrine interactions between the stem cells and a cardiac microenvironment. Studies using this methodology have demonstrated the capability of TGF‐β, FGF‐2, and BMP‐2 in enhancing cardiac differentiation in ES cells; 13 , 14 however, a similar phenomenon in adult somatic stem cells has not yet been investigated. Additionally, TGF‐β and FGF‐2 have been shown to be upregulated in the myocardium following an ischemic injury, possibly acting as paracrine mediators in the process of infarct healing. 15 Due to the potential similarities at the molecular level between the processes of embryonic cardiogenesis and ischemic cardiac repair, we hypothesized that paracrine factors upregulated in an injured myocardium could facilitate the differentiation of somatic stem cells capable of self‐renewal in a manner consistent with that seen in the developing embryo.

The aim of this study was to explore the effect of BMP‐2 and FGF‐2—paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)—on murine BMSC (mBMSC) differentiation in an ex vivo cardiac microenvironment. To investigate this, we used a co‐culture of GFP‐lin‐/c‐kit+/Sca‐1+ mBMSCs onto cardiac myocytes to assess the cardiomyogenic potential of mBMSCs as defined by engraftment into the beating cardiomyocyte monolayer, the presence of cardiac early lineage markers, Nkx2.5 and GATA‐4, and markers of later stages of cardiogenesis, including myosin light chain kinase (MLC‐2v) and connexin 43 (Cx43), and calcium cycling.

Methods

Myocardial infarction

All procedures were performed in accordance with our institutional guidelines for animal research conforming to the NIH guidelines. The induction of MI was carried out by a left anterior descending (LAD) coronary artery ligation in 8‐ to 10‐week‐old mice as previously described. 16 2,3,5‐triphenyl tetrazolium chloride (TTC) staining was performed as described elsewhere to assess the MI size. 17

Isolation of neonatal cardiac myocytes

Ventricular cardiomyocytes from 1‐ to 2‐day‐old neonatal rat hearts (NVCMs) were prepared as previously described. 18 Briefly, NVCMs were cultured in Dulbecco's modified Eagle's medium (DMEM; Biochrom, Cambridge, UK) supplemented with penicillin/streptomycin (100 units/mL), L‐glutamine (2 mM), and 10% fetal calf serum (FCS Gold; PAA Laboratories GmbH, Pasching, Austria) at 37°C in a 95% air/5% CO2‐humidified atmosphere for 24 hours to receive subconfluent contracting monolayers.

Isolation, purification, and adenoviral infection of Lin– c‐kit+ Sca‐1+ mBMSCs

BM cell suspensions were generated by flushing the shafts of the femurs and tibias of 6‐ to 10‐week‐old C57/B6 mice using a 26‐gauge syringe with ice‐cold phosphate buffered saline (PBS) supplemented with 0.5% FCS. The cell suspension was then gently disaggregated, filtered, and washed. The cell were then subjected to magnetic cell sorting using the MACS magnetic bead system (Miltenyi Biotech, Bergisch Gladbach, Germany) for “lineage antigens” (CD5, CD45R, CD11b, Gr‐1, 7–4, and Ter‐119)‐generating Lin– and Lin+ cell populations following the manufacturer's protocol. The Lin– fractions were further enriched by FACS for the presence of both c‐kit and Sca‐1 using phycoerythrin (PE)‐conjugated rat anti‐mouse c‐kit and fluorescein isothiocyanate (FITC)‐conjugated rat anti‐mouse Sca‐1, respectively (BD Pharmingen, San Jose, CA, USA). The labeled cells were sorted using an EPICS Altra cell sorting system (Beckman Coulter, Fullerton, CA, USA). The initial Lin– c‐kit+/Sca‐1+ population was re‐sorted for the presence of c‐kit and Sca‐1 to confrm the purity of this cell population. An adenoviral infection of Lin–c‐kit+/Sca‐1+‐mBMSCs with an eGFP‐containing adenovirus was carried out in the StemPro‐34 SFM medium (Invitrogen, GIBCO, Carlsbad, CA, USA), with a multiplicity of infection (MOI) of 10 plaque‐forming units (pfu) per cell to differentiate between mBMSCs‐derived eGFP+ cells and NVCMs following co‐culture. After 4 hours of incubation at 37°C in a 95% air/5% CO2‐humidified atmosphere, the cells were carefully washed and left in 10% FCS‐supplemented media

Co‐culture experiments

Lin– c‐kit+/Sca‐1+ mBMSCs infected with an eGFP‐containing adenovirus (MOI 10 pfu) were mixed and plated with NVCMs in a ratio of 1:10, respectively, and then co‐cultured in the presence of a vehicle or BMP‐2 (20 ng/mL) alone, FGF‐2 (20 ng/mL) alone, or BMP‐2 and FGF‐2 for up to 10 days in the presence of inhibitors, as indicated in the fgure legends, in 10% FCS‐supplemented media. The integration of mBMSCs to the adherent monolayer was determined by fluorescent microscopy and FACS sorting for the presence of mBMSC‐derived GFP+ cells within the monolayer following enzymatic dissociation and filtering to obtain a single‐cell suspension. The sorted mBMSC‐derived GFP+ cells were either subjected to RNA isolation or re‐cultured for up to 10 days to assess the expression of cardiac markers and permanence of the potential cardiac phenotype with respect to calcium transients, respectively, as described below.

Calcium transient recordings

Intracellular Ca2+ transients of co‐cultured GFP+ mBMSCs and enzymatically dissociated GFP+ cells in the presence or absence of BMP‐2 and FGF‐2 were measured as previously described. 19 Nifedipine (15 μM) and caffeine (10 mM) were added as indicated. To identify GFP+ cells, the FURA2‐AM loaded monolayer was excited at 488 nm and the emission was collected at 510 nm, and additional measurements were taken below and above the GFP excitation spectrum to exclude confounding autofluorescent cells.

RNA isolation, reverse transcription, and semiquantitative reverse transcription polymerase chain reaction (RT‐PCR)

Total RNA isolation was performed using the TRIZOL method, according to the manufacturer's protocol (Invitrogen, GIBCO, Carlsbad, CA, USA) as described previously. 16 Following RNA isolation, cDNA was synthesized from 1 μg of total RNA using the iScript cDNA synthesis kit (Bio‐Rad Laboratories, Hercules, CA, USA). A quantitative PCR was carried out on cDNA diluted 100‐fold using the iQ SYBR Green Supermix (Bio‐Rad Laboratories, Hercules, CA, USA), as described previously. 16 18S, c‐kit, Sca‐1, BNP, TGF‐β, BMP‐2, BMP‐4, FGF‐2, Nkx2.5, GATA‐4, MLC‐2v, and Cx‐43 sequences are available upon request.

Statistical analysis

Data are expressed as mean ± standard error of mean (SEM). An unpaired two‐tailed Student's t‐test and two‐way repeated analysis of variance (ANOVA) were performed for between‐group comparisons. For all tests, a p value of <0.05 was considered significant.

Results

Purification and characterization of Lin– c‐kit+/Sca‐1+ mBMSCs

To obtain largest cellular plasticity, we purified Lin– c‐kit+/Sca‐1+ somatic BM cells (mBMSC) from adult C57/B6 mice. 20 The average cellular yield for BM obtained from the tibiae and fbulae is 4.98 ± 0.5 × 107 per adult mouse (n= 60; see Figure 1A ). Approximately 0.8% of these cells (4.03 ± 0.5 × 105) can be isolated in a lineage‐depleted fraction per mouse (see Figure 1F ). Tis Lin– fraction consists of approximately 47.9% of c‐kit+ cells (see Figure 1H ), 12.5% of Sca‐1+ cells (see Figure 1I ), and 2.8% of Lin– c‐kit+/Sca‐1+ mBMSCs (see Figure 1J ). To facilitate further experiments, the Lin– c‐kit+/Sca‐1+ fractions from 10 to 15 mice were pooled (~1.0–1.5 × 105 cells) for each co‐culture experiment (n= 4). FACS‐based re‐sorting for the presence of both c‐kit and Sca‐1 confrmed highest purity of this subpopulation (see Figure 1L ).

Figure 1
Isolation and purification of somatic Lin– c‐kit+/Sca‐1+ mBMSC for use in co‐culture onto cardiac myocytes. (A–L) Representative FACS analysis: A–E depict the characterization of (A) total (unsorted) bone ...

Semiquantitative gene expression analysis of TGF‐β, BMP‐2, and FGF‐2 in infarcted murine hearts

The expression of cardiogenic factors was analyzed in infarcted hearts exhibiting an MI of approximately 45% of the left ventricle compared to sham (see Figure 2A ). The postischemic activation of the fetal gene program is illustrated by the increase in brain natriuretic peptide (BNP) expression in both the MI region (MI) and the residual myocardium (MR) regions as compared to sham (see Figure 2B ). The factors involved in embryonic cardiogenesis such as TGF‐β, BMP‐2, and FGF‐2 were increased by approximately 7‐fold, 14‐fold, and 8‐fold in the MI region, and by approximately 7‐fold, 6‐fold, and 4‐fold in the MR region, respectively (see Figure 2C–E ).

Figure 2
Effect of myocardial infarction on mRNA levels of TGF‐β, BMP‐2, and FGF‐2 in adult murine hearts. (A) Representative pictures of a sham (top) and an infarcted (bottom) dissected 2,3,5‐triphenyl tetrazolium chloride ...

mRNA expression of BMP and FGF isoforms in mBMSCs following co‐culture with NVCMs0

To investigate if the co‐culture of mBMSCs with NVCMs might have an impact on the expression of distinct BMP and FGF isoforms in both cell types, GFP‐transfected mBMSCs (GFP+ mBMSCs) were co‐cultured with a 10‐fold excess of NVCMs for 10 days. mBMSCs prior to co‐culture exhibit a spherical morphology which grow in suspension (data not shown). The incorporation of GFP+ mBMSCs into the contracting monolayer was assessed by both fluoroscence microscopy and FACS following enzymatic dissociation of the contracting monolayer. Approximately 4% of mBMSC‐derived GFP+ cell were integrated into the beating NVCM monolayer, exhibited a flattened, fibroblast‐like morphology, and beat along with the surrounding cardiac myocytes (see Figure 3A–C ). The nonintegrated mBMSC‐derived GFP+ cells remained in the suspension and possessed a spherical morphology consistent with that of mBMSCs prior to co‐culture.

Figure 3
Effect of mBMSCs/NVCMs co‐culture on mRNA levels of distinct BMP and FGF isoforms. (A–C) Representative fluorescent microscopy pictures of mBMSCs co‐cultured onto 10‐fold excess of NVCMs for 48 hours demonstrate a rare ...

Following 48 hours of co‐culture, the monolayer was enzymatically dissociated, further separated by FACS, and GFP+ mBMSCs and NVCMs were subjected to RNA isolation. Control experiments were performed in which pure cultures of mBMSCs or NVCMs were cultured alone for 48 hours, and then subjected to RNA isolation. The co‐culture of mBMSCs with NVCMs resulted in a significant increase in BMP‐2, BMP‐4, and FGF‐2 mRNA expression levels in NVCMs, whereas only FGF‐2 mRNA expression levels were significantly increased in mBMSCs (see Figure 3D–F ).

Effect of BMP‐2 and FGF‐2 on mBMSC incorporation following co‐culture with NVCMs

In order to assess whether cardiogenic factors, which were upregulated following MI and co‐culture, could enhance mBMSC incorporation, purified FGF‐2 and BMP‐2 were added to the co‐culture medium. As previous studies mostly focused on the impact of BMP‐2 on cardiac commitment in stem cells, we utilized this factor in further experiments. The addition of BMP‐2 (20 ng/mL) in concert with FGF‐2 (20 ng/mL) significantly enhanced the amount of integrated GFP+ mBMSC by approximately 5‐fold (21%) compared to untreated co‐cultures (see Figure 4A–B ). Decreasing BMP‐2 and FGF‐2 concentrations below 2 ng/mL abolished the synergistic effect (data not shown) so that further experiments were performed with the combined use of 20 ng/mL BMP‐2 and FGF‐2. However, the addition of FGF‐2 (20 ng/mL) alone or BMP‐2 (20 ng/mL) alone did not significantly increase the percentage recovery of mBMSCs from the monolayer (see Figure 4A and B ). In addition, anti‐BMP‐2 and/or anti‐FGF‐2 antibodies completely abolished GFP+ mBMSC integration (see Figure 4B ). Taken together, these data indicate that FGF‐2 in concert with BMP‐2 seems to facilitate the incorporation of mBMSCs into the cardiac microenvironment provided by the NVCMs.

Figure 4
Effect of BMP‐2 and FGF‐2 on mBMSC incorporation following co‐culture with NVCMs. (A) Representative pictures of co‐cultured GFP+ mBMSCs with a 10‐fold excess of NVCMs in the absence (control) and presence of BMP‐2/FGF‐2. ...

Effect of BMP‐2 in concert with FGF‐2 on calcium cycling in mBMSCs following co‐culture with NVCMs

One of the hallmarks of cardiac myocytes is active calcium cycling characterized by calcium‐induced calcium release from internal stores in the sarcoplasmic reticulum (SR). To examine the possible presence of this phenomenon in mBMSCs before and after co‐culture with cardiac myocytes, we probed calcium cycling in mBMSC‐derived GFP+ cells co‐cultured with NVCMs for 10 days. Electric field stimulation resulted in the presence of sporadic and low‐amplitude calcium transients in mBMSC‐derived GFP+ cells (4/20 cells from four different experiments) within the NVCM monolayer (see Figure 5 ), which could not be detected in mBMSCs cultured without the presence of cardiomyocytes (data not shown). The presence of BMP‐2 and FGF‐2 in the co‐culture medium did not significantly afect the calcium transient amplitude of the NVCMs, conversely mBMSC‐derived GFP+ cells co‐cultured in the presence of BMP‐2 and FGF‐2 demonstrated robust calcium transient amplitudes (18/20 cells from four different experiments) (see Figure 5 ). We further assessed the decay constant τ in NVCMs and engrafted BMP‐2/FGF‐2‐treated mBMSC‐derived GFP+ cells yielding significantly faster cytosolic calcium removal in NVCMs (241 ± 21 milliseconds) than mBMSC‐derived GFP+ cells (478 ± 17 milliseconds, p < 0.05 vs. NVCMs). As τ reflects the net removal of cytosolic calcium due to different molecular mechanisms including SR calcium reuptake and sarcolemmal calcium extrusion, it is tempting to speculate that a combined BMP‐2/FGF‐2 treatment might have promoted the development of these systems in GFP+ Lin– c‐kit+/Sca‐1+ cells, and due to their different developmental stages their relative contribution might differ from NVCMs. GFP+ mBMSCs treated with BMP‐2 and FGF‐2 in the absence of cardiomyocytes did not exhibit detectable calcium transients (data not shown). These data indicated that co‐culture of mBMSCs with NVCMs in the presence of BMP‐2 and FGF‐2 apparently results in robust calcium cycling, suggesting electrical coupling to surrounding cells and expression of calcium handling proteins.

Figure 5
Effect of combined BMP‐2/FGF‐2 treatment on calcium cycling in mBMSCs during NVCM co‐culture. (Top) Representative tracings of field‐stimulated calcium transients in co‐cultured NVCM and mBMSCs. Note that BMP‐2/FGF‐2 ...

Induction of genes associated with cardiac differentiation in co‐cultured mBMSCs following BMP‐2 and FGF‐2 treatment

To assess whether mBMSCs express markers of cardiogenesis prior to co‐culture, we performed RT‐PCR for the presence of the cardiac transcription factors Nkx2.5 and GATA‐4, as well as Cx‐43 and MLC‐2v. High levels of these cardiac markers were present in NVCMs; however, as expected, there were no detectable levels of these genes in mBMSCs prior to co‐culture.

After 10 days, the co‐culture was enzymatically dissociated and sorted into GFP+ and GFP– fractions, at which point, RNA was isolated for RT‐PCR analysis. The co‐culture resulted in a significant expression of early, Nkx2.5 and GATA‐4 (see Figure 6A and B ), and late, Cx‐43 and MLC‐2v (see Figure 6C and D ), cardiac markers in mBMSC‐derived GFP+ cells. In addition, mRNA expression of both c‐kit and Sca‐1 were significantly reduced following co‐culture, indicating that the mBMSCs have lost their undifferentiated phenotype, as evaluated by the presence of stem cell markers (see Figure 6E and F ). GFP+ mBMSCs cultured independently for 10 days in the absence of a cardiac environment, even in the presence of BMP‐2 and FGF‐2 (data not shown), displayed no detectable levels of cardiac‐associated genes and retained levels of c‐kit and Sca‐1 (see Figure 6A–F ). Of note, GFP mRNA levels did not differ in GFP+ cells prior to and after isolation from the co‐culture (data not shown).

Figure 6
Analysis of cardiac marker expression in mBMSC in response to NVCM co‐culture and BMP‐2/FGF‐2 treatment. (A–F) Quantitative analysis of Nkx2.5, GATA‐4, MLC‐2v, Cx‐43, c‐kit, and Sca‐1 ...

Re‐culture of mBMSCs enzymatically dissociated from the contracting monolayer of NVCMs and assessment of calcium handling in mBMSCs independent of cardiac myocytes

To investigate the relative permanence of the cardiac‐like phenotype induced in mBMSC‐derived GFP+ cells following co‐culture with NVCMs, we re‐cultured the FACS‐isolated GFP+ cell fraction following 10 days of co‐culture. After 3 days in culture in the absence of a cardiac microenvironment, these cells were assessed for the presence of active calcium cycling in response to electric field stimulation. Isolated mBMSC‐derived GFP+ cells co‐cultured in the absence of BMP‐2 and FGF‐2 demonstrated sporadic and rudimentary calcium transients in response to field stimulation that could be inhibited by the LTCC inhibitor nifedipine (15 μM), thus indicating that the calcium cycling was due to LTCC current, as in the cardiac myocytes (see Figure 7C and D ). Isolated mBMSC‐derived GFP+ cells co‐cultured in the presence of BMP‐2 and FGF‐2 displayed robust calcium transients that could also be abolished using nifedipine (see Figure 7C and D ). Moreover, these cells exhibited robust caffeine‐sensitive enhancement of the intracellular FURA‐2 signal, indicating ryanodine receptor 2 (RyR2)‐gated release of calcium from intracellular stores (see Figure 7C ). Taken together, these data indicate that co‐culture in the presence of BMP‐2 and FGF‐2 results in a stable functional cardiac‐like phenotype including sarcolemmal LTCC and RyR2‐mediated SR calcium release.

Figure 7
Assessment of calcium handling in re‐cultured mBMSC‐derived GFP+ cells. Representative pictures of re‐cultured mBMSC‐derived GFP+ cells co‐cultured in the absence (A/A′) and presence of BMP‐2/FGF‐2 ...

Discussion

Cell‐based therapy has been proposed as a promising treatment strategy for regeneration of the myocardium. 21 However, recent efforts have focused on developing our understanding of the paracrine signaling between resident cardiac cells and exogenous stem cells to facilitate improved stem cell engraftment, proliferation, fusion, and/or differentiation in a damaged myocardium. 13 , 22 , 23 To this end, we utilized a cardiomyocyte co‐culture to study the cardiogenic effect of BMP‐2 and FGF‐2 on somatic BMSCs. The rationale for the investigation into the role BMP‐2 and FGF‐2 in cardiogenesis derives from their cooperative function in the process of embryonic cardiogenesis, 24 as well as their co‐expression in the infarcted myocardium and induction of BMP‐2 and FGF‐2 co‐expression in stem cell‐cardiomyocyte co‐culture, both of which we have described for the first time here. Using the co‐culture, we report the novel finding that BMP‐2 provided in concert with FGF‐2 acts on adult stem cells retaining self‐renewal potential to facilitate the adoption of a cardiac‐like phenotype, with resultant morphological, biochemical, and functional features of nascent cardiomyocytes. It is important to note that the Lin– c‐kit+/Sca‐1+ population of mBM utilized in this study possesses both long‐ and short‐term self‐renewal potential and have been depleted of cells committed to the hematopoietic lineage. 20 This cell population possesses multilineage differentiation potential, including commitment to endothelial, fibroblast, glial, and muscular cell lineages. 25 , 26 , 27 , 28 , 29

The presence of a cardiac microenvironment potentially providing intercellular structural and paracrine signaling appears to be a prerequisite for the synergistic effect of BMP‐2 and FGF‐2 as these growth factors did not induce cardiac gene expression or calcium cycling in the absence of a co‐culture. Furthermore, the nascent cardiac phenotype induced by BMP‐2 in synergy with FGF‐2 displayed a relative permanence, as assessed by isolating mBMSCs following co‐culture and then re‐culturing the mBMSCs independent from the cardiac myocytes and investigating their calcium handling properties. The re‐cultured mBMSCs treated with BMP‐2 and FGF‐2 adopted robust nifedipine‐ and caffeine‐sensitive calcium cycling, indicating an L‐type calcium channel‐driven transsarcolemmal influx and the presence of RyR‐operated intracellular calcium stores. These molecular factors are a hallmark of functional cardiac myocytes, indicating that although a cardiac microenvironment is necessary for the induction of the phenotypic changes in BMSCs, the relative maintenance of this phenotype is independent of either direct or paracrine signaling through the cardiac myocytes. Further studies have to investigate the excitation–contraction process in these cells in greater detail.

A combined BMP‐2 and FGF‐2 administration (20 ng/mL each) appears to act in a dose‐dependent compulsory synergy as BMP‐2 or FGF‐2 administered alone do not significantly afect BMSC engraftment, gene expression, or calcium handling properties following co‐culture with NVCMs in the presence of presumably lower, endogenously produced FGF‐2 and BMP‐2, respectively. Moreover, the effects of the combined BMP‐2 and FGF‐2 application appeared to be dose‐dependent as decreasing BMP‐2 and FGF‐2 concentrations below 2 ng/mL abolished their synergistic effect (data not shown). Nevertheless, even locally produced FGF‐2 and BMP‐2 are likely to play a role in the low rate of BMSC engraftment under control conditions as anti‐BMP‐2 and anti‐FGF‐2 antibody administration completely abolished this process. These observations are in line with the cooperative action of BMP‐2 and FGF‐2 in cardiomyocyte formation. 24 Tis seems to be in contrast to a recent report identifying cardiomyogenic differentiation of ES cells in response to either BMP‐2 or FGF‐2 alone. 14 However, this finding does not negate our findings in adult BMSCs because ES cells have been shown to release TGF‐β superfamily members as well as members of the FGF family in response to a variety of stressors. 30 , 31 Therefore, given the rather low concentration either of FGF‐2 or of BMP‐2 that has been reported to promote cardiac differentiation of ES cells, 14 they may retain the ability to produce sufcient amounts of FGF‐2 that could signal in an autocrine fashion in response to BMP‐2 treatment or vice versa, therefore allowing ES cells to directly compensate for the lack of either BMP‐2 or FGF‐2. Alternatively, ES cells could be producing some other soluble factor that could induce the release of paracrine factors from the co‐cultured cardiac myocytes. Although, BMSCs demonstrate a requirement for higher concentrations of BMP‐2 in concert with FGF‐2, both ES cells and adult BMSCs demonstrate enhanced integration, as well as biochemical and functional evidence of cardiac‐like differentiation in response to the paracrine factors.

Lagostena et al. report that a population of c‐kit+ BM cells exhibited expression of cardiac transcription factors, Mef2C, GATA‐4, and α‐sarcomeric actin, and Na+ and Ca2+ currents following co‐culture with NVCMs; however, they failed to identify any evidence of electromechanical coupling or calcium cycling. 32 In addition, the authors co‐cultured this population of c‐kit+ BM cells with NVCMs in the presence of TGF‐β and BMP‐4 and reported a hyperpolarized membrane potential and increased inward rectifying potassium currents, resembling a molecular mechanism underlying membrane potential stabilization in nascent cardiac myocytes. However, they reported limited evidence of cardiac differentiation. As we investigated a BM population depleted of lineage markers and enriched in the stem cell markers c‐kit and Sca‐1, our findings might expand those of Lagostena et al. 32 who studied c‐kit+ BMSCs—shown to consist largely of hematopoietic lineage‐committed cells. 33 Lin– c‐kit+ Sca‐1+ mBMSCs in the present report represent less than 2.8% of all BM cells, hence the populations being studied vary significantly, especially with respect to the relative plasticity of the cells in the population. 20 In addition, the paracrine factors utilized by Lagostena et al. 32 , TGF‐β and BMP‐4, differ from the combination used in the present study; therefore, the specific factors or the sequence to which they are applied may not meet the full requirements for the full adoption of a cardiac phenotype.

One mitigating factor in the present study is the potential for antiapoptotic effects induced by signaling through BMP‐2. 34 , 35 , 36 , 37 BMP‐2 has been shown to prevent apoptosis in a variety of cell types, including NVCMs, by increasing the mRNA expression of B‐cell lymphoma extra large (Bcl‐XL) through a Smad1 (homologs of both the C. elegans protein SMA and Drosophila protein “mothers against decapentaplegic” MAD)‐dependent signaling pathway. Although this is beyond the scope of the present work, future investigation can be designed to elucidate whether our combination of BMP‐2 and FGF‐2 alters the integration of BMSCs into the contracting layer of NVCMs by way of improving the survival of BMSCs to facilitate a longer period of interaction between these cell types. This could thereby allow time for alterations in the cell surface characteristics of both cell types, 38 such as integrin and cadherin receptor expression required for cardiogenesis. 39 However, the synergistic effects of BMP‐2 and FGF‐2 in cardiac embryogenesis cannot be accounted for solely by an improved survival; therefore, it is unlikely that the antiapoptotic actions of these factors are responsible for the structural, biochemical, and functional phenotypes that we have reported.

Future studies will need to determine whether BMP‐2 and FGF‐2 effects are driven through differentiation or cell fusion (suggested by the recent works of Nygren et al. 40 and Matsuura et al. 41 ); this caveat is not necessarily at odds with the present findings, considering that fusion and transdifferentiation are processes that may not be as clearly demarcated as previously believed. 40 , 41 Furthermore, the calcium cycling data presented in this work appear to argue against cell fusion as a potential mechanism to explain the phenotypic changes seen in the BMSCs. The presence of calcium transients that are responsive to caffeine and nifedipine indicates the presence of RyR2‐operated sarcoplasmic reticulum being responsive to LTCC‐dependent transsarcolemmal calcium influx L‐type calcium channel‐dependent sarcoplasmic reticulum calcium stores that are characteristic of NVCMs. However, the calcium transient amplitude reported in the BMSCs following BMP‐2 and FGF‐2 treatment are approximately 50% that of NVCMs. If the fusion of BMSCs and NVCMs was responsible for the presence of calcium transients, one would predict the calcium transient amplitude seen in GFP+ fused cells to be equivalent to that of NVCMs, given the more mature calcium handling properties and the larger cell volume of the NVCMs—thus, cell fusion cannot explain the calcium handling phenomenon that we report here. Similarly, if cell fusion would be the underlying major mechanism, we would expect similar calcium transient amplitudes merely occurring in a higher number of GFP+ cells in BMP‐2/FGF‐2‐treated co‐cultures compared with control co‐cultures. However, different calcium transient amplitudes were observed between groups rather pointing toward different developmental stages of calcium handing that, to our understanding, cannot simply be explained by a higher rate of cell fusion. However, it is also important to point out that none of these arguments clearly disprove the enhanced cell fusion in our experimental setting, and future studies need to address this important question in greater detail. Nevertheless, this study might represent a stepping stone toward elucidating the specific intercellular or paracrine signaling networks occurring between cardiac myocytes and somatic BM‐derived stem cells and how to modulate this process toward a cardiac‐like phenotype. Although we have identifed that a combined application of BMP‐2 and FGF‐2 provides evidence for the differentiation of BMSCs, we have not explored the specific pathways implicated in the signaling of these paracrine mediators due to the limited cell number remaining following the co‐culture experiments.

However, this ex vivo approach could be used to expand the present findings of BMP‐2 and FGF‐2 synergy in other cell populations of interest in the field of stem cell biology. 4 , 5 , 7 , 8 , 9 , 42 An improved understanding of this area of cellular communication could bolster our ability to design studies to target and embed stem cells to the damaged myocardium with the goal of improving the cardiac function through paracrine signaling; however, in vivo application of BMP‐2 and FGF‐2‐treated BMSCs is beyond the scope of the current study.

Conceivably, BMP‐2 and FGF‐2, which are upregulated in the injured myocardium following MI, may be facilitating stem cell engraftment and adoption of a cardiac phenotype invivo, as suggested by our present ex vivo data. However, this process might still not be potent enough to see dramatic effects in vivo in light of insufcient cardiac homing of BMSC, which can be enhanced by increasing myocardial concentrations of stem cell factor (SCF) as recently shown by Frey and co‐workers. 43 Enhancing local levels of BMP‐2 and FGF‐2 in a spatiotemporally specific manner in the heart following MI may prove to be a very relevant methodology to enhance endogenous cardiac repair and will be tested in further in vivo studies.

Conclusion

In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. Tis study elucidates the first evidence that mBMSCs with long‐term self‐renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross‐talk with the cardiac milieu.

Acknowledgments

Tis work was supported in part by the Deutsche Forschungsge‐meinschaft grant 1066/1–1 and 562/1–1 (Dr. Patrick Most), the NIH (RO1HL092130–01 to Dr. Patrick Most), and the Forschungsfoerderungsprogramm Medizinische Universitaet Heidelberg (61/2003, Dr. Patrick Most), the NIH (R01HL56205 to Dr. Walter J. Koch, W.W. Smith Professor of Medicine), and in part by a grant by the Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions.

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