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Cellular Plasticity in the Cardiovascular System
1Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology, and Experimental Therapeutics, and 2Physical Medicine and Rehabilitation, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and 3Centre de Recherches de Biochimie Macromoléculaire, CNRS FRE 2593, 34293 Montpellier, France
Submitted 31 December 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to -adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.
engraftment; xenotransplant; plasticity; cardiac differentiation; remodeling; heart
Although multiple candidate cell types have been isolated from cardiac or noncardiac sources and shown to display varying degrees of cardiogenicity (4, 22, 29, 31, 37), embryonic stem cells derived from the inner cell mass of the blastocyst possess the most recognized capacity to yield cardiomyocytes (6, 12, 23, 34, 38). Because of their ability to indefinitely proliferate in vitro, embryonic stem cells can generate large colonies and supply a reservoir for extensive tissue regeneration (15, 35, 45). Indeed, in vitro differentiation of embryonic stem cells produces cells that recapitulate the cardiac phenotype expressing characteristic cardiac markers and demonstrating functional excitation-contraction coupling (6, 13, 24, 32, 34). Furthermore, when transplanted into injured hearts, embryonic stem cells generate cardiomyocytes that repopulate significant regions of dysfunctional myocardium and result in improved contractile performance with reduced mortality (3, 25, 26). This transition of embryonic stem cells to cardiomyocytes occurs under the direction of host paracrine signaling that supports cardiac-specific differentiation (3). Although understanding of the molecular mechanisms of stem cell commitment and integration with host myocardium is increasing, limited information is available on the natural history of stem cell therapy outcomes.
We serially monitored the effect of embryonic stem cell therapy in a model of myocardial infarction and demonstrated improvement in myocardial function sustained over a 12-wk follow-up period. The presence of embryonic stem cell-derived cardiomyocytes within the infarct regions was associated with preserved left ventricular structure and diminished scar. Thus embryonic stem cells, through myocardial regeneration and an impact on ventricular remodeling, can provide stable long-term benefit after myocardial infarction.
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METHODS |
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The CGR8 murine embryonic stem cell line was propagated in baby hamster kidney (BHK21) cells or Glasgow MEM supplemented with pyruvate, nonessential amino acids, mercaptoethanol, 7.5% fetal calf serum, and the leukemia inhibitory factor (24, 32). A CGR8 cell clone was engineered to express the yellow fluorescent protein or the enhanced cyan fluorescent protein (ECFP) under the control of the cardiac-specific 
-actin promoter subcloned upstream of ECFP using XhoI and HindIII restriction sites of the promoterless pECFP vector (Clontech). This -actin promoter construct was linearized using XhoI and electroporated into CGR8 stem cells as described elsewhere (3, 24). To image stem cells by field-emission scanning electron microscopy, cells were fixed in phosphate-buffered saline containing 1% glutaraldehyde and 4% formaldehyde (pH 7.2), dehydrated with ethanol, and dried in a critical point dryer. Cells, coated with platinum with use of an indirect argon ion-beam sputtering system (Ion Tech, VCR Group) operating at accelerating voltages of 9.5 kV and 4.2 mA, were then examined on a field-emission scanning microscope (model 4700, Hitachi) (33). For transmitted scanning electron microscopy, stem cells were postfixed in phosphate-buffered 1% OsO4, stained en bloc with 2% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in low-viscosity epoxy resin. Thin (90-nm) sections were stained with lead citrate, and micrographs were taken on an electron microscope (JEOL 1200 EXII) (14).
Stem cell-derived cardiomyocytes.
CGR8 embryonic stem cells were differentiated in vitro using the previously established hanging-drop method to generate embryoid bodies (3, 20). After enzyme dissociation of embryoid bodies, Percoll gradient was used to isolate a highly enriched population of stem cell-derived cardiomyocytes as described elsewhere (32). The presence of cardiac markers in purified cells was probed by laser confocal microscopy (LSM 510 Axiovert, Zeiss) using anti-MEF2C (Cell Signaling Technology) and anti--actinin (Sigma) antibodies. Membrane electrical activity was determined by patch-clamp recording in the whole cell configuration using the current- or voltage-clamp mode (Axopatch 1C, Axon Instruments). Action potential profiles and voltage-current relation were acquired and analyzed with Bioquest software from cells superfused with Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (with pH adjusted to 7.4 with NaOH) using patch pipettes (5–10 M
) containing (in mM) 140 KCl, 1 MgCl2, 10 HEPES, and 5 EGTA and supplemented with 5 mM ATP (with pH adjusted to 7.2 with KOH). Electrophysiological measurements were performed at 31 ± 1°C with a temperature controller (model HCC-100A, Dagan) equipped with a Peltier thermocouple (47). To assess intracellular Ca2+ dynamics, cells were loaded with the Ca2+-fluorescent probe fluo 3-AM (Molecular Probes), line scanned with a Zeiss laser confocal microscope, and analyzed using imaging software (LSM Image Browser, Zeiss) as described elsewhere (32, 46).
Myocardial infarction model.
Myocardial infarction was induced at 6 wk of age by ligation of the left anterior descending coronary artery in male Sprague-Dawley rats, resulting in an established model with an 
30% infarcted left ventricle (Charles River). Consequently, left ventricular ejection fraction (EF) was depressed from 75 ± 2% at baseline to 47 ± 3% after infarct. Infarcted animals were randomized into sham- and embryonic stem cell-treatment groups. At 8 wk after infarct, animals were anesthetized with isoflurane (3% induction, 1.5% maintenance), 12-lead electrocardiography was performed, and the heart was exposed by thoracotomy. Medium (20 µl of Glasgow MEM) without cells (sham) or CGR8 embryonic stem cells (3 x 105 in 20 µl of medium), engineered to express ECFP under control of the cardiac-specific actin promoter, were injected through a 28-gauge needle at three sites (at the left ventricular base just below the left atrium, in the midanterior region, and at the apex) along the border of the left ventricular infarcted areas.
Electrocardiography.
Twelve-lead electrocardiography was performed under isoflurane anesthesia with subcutaneous needle electrodes (Grass Instruments) and a differential electrocardiographic amplifier (model RPS312, Grass Instruments). Standard and augmented limb leads (I, II, III, aVR, aVL, and aVF) as well as precordial leads (V1–V6) were recorded before sham treatment or stem cell injection and serially thereafter.
Echocardiography.
Under isoflurane anesthesia, two-dimensional M-mode echocardiographic images were obtained from the parasternal short-axis view with a 5-MHz probe at the ventricular base (Vingmed System FiVe, GE Medical Systems). The leading-edge convention of the American Society of Echocardiography was used to calculate EF as follows: EF = 100·(D2 – S2)/D2, where D is the end-diastolic cavity diameter and S is the end-systolic cavity diameter (3, 14).
Histopathology.
On autopsy, gross pathological examination was performed on 4% formalin-fixed transverse-cut hearts, and then 0.5-µm-thick paraffin sections stained with hematoxylin and eosin were examined by light microscopy (46). The fraction of chamber circumference with residual postinfarction left ventricular scar was determined in standardized sections through the left ventricular base at the midlevel of the infarct. Fluorescent microscopy (Zeiss) was performed on unstained paraffin sections. For transmitted electron microscopy, myocardial specimens were postfixed in phosphate-buffered 1% OsO4, stained en bloc with 2% uranyl acetate, dehydrated in ethanol and propylene oxide, and embedded in low-viscosity epoxy resin. Thin (90-nm) sections were cut on an ultramicrotome (Reichert Ultracut E), placed on 200-µm mesh copper grids, and stained with lead citrate. Micrographs were taken on an electron microscope (JEOL 1200 EXII) operating at 60 kV (14).
Statistics.
Values are means ± SE. Embryonic stem cell- vs. sham-treated groups were compared using Student's t-test, with P < 0.05 considered significant. Wilcoxon's log-rank test was used for nonparametric evaluation of randomization.
All protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee.
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RESULTS |
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The CGR8 embryonic stem cell colony demonstrated typical features of undifferentiated cells, including a high nucleus-to-cytosol ratio, prominent nucleoli, and mitochondria with few cristae (Fig. 1, A and B). The cardiogenic capacity of this embryonic stem cell line was probed by in vitro differentiation, with cells readily derived that express the cardiac transcription factor MEF2C (19), the cardiac contractile protein -actinin, and sarcomeric striations (Fig. 1C). Consistent with proper differentiation toward cardiac lineage, stem cell-derived cardiomyocytes demonstrated action potential activity associated with prominent inward Na+ and Ca2+ currents (Fig. 1D), critical for excitation-contraction coupling manifested as rhythmic intracellular Ca2+ transients (Fig. 1E). Injection of CGR8 cells into the myocardium resulted in local retention of these embryonic stem cells (Fig. 1F) without detectable dispersal into noncardiac tissues (Fig. 1G). To determine the outcome of stem cell therapy for cardiac repair in myocardial infarction, rats were randomly assigned to stem cell- or sham-treatment groups. At 8 wk after left anterior descending coronary artery ligation, infarction was confirmed by electrocardiographic evidence of myocardial necrosis (Fig. 2A) as well as by direct visual inspection of the myocardium after thoracotomy (Fig. 2B). Embryonic CGR8 stem cells from the pretested colony or acellular preparations (sham controls) were then injected into the peri-infarct zone (Fig. 2B) for assessment of functional and structural impact over time.
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Cardiac contractile function, assessed by echocardiography at 3 wk after injection, was superior in embryonic stem cell- compared with sham-treated infarcted hearts (Fig. 3A). On average, left ventricular EF was 0.80 ± 0.05 vs. 0.52 ± 0.05 in the stem cell- vs. the sham-treated group (P < 0.05). Moreover, although sham-treated infarcted hearts failed to augment function under inotropic challenge, stem cell-treated infarcted hearts demonstrated a significant positive inotropic response. Pharmacological stress testing by injection of the -adrenergic agonist isoproterenol (3 µg/kg) produced a 12% increase in the EF of stem cell-treated infarcted hearts vs. no significant response in the sham-treated group (Fig. 3A). M-mode imaging under stress further demonstrated that, in contrast to the hypokinetic or akinetic anterior left ventricular walls in sham-treated infarcted hearts, stem cell-injected infarcted hearts exhibit dynamic anterior wall motion with vigorous ventricular function (Fig. 3B). Long-term follow-up found no decay in the contractile advantage of stem cell therapy (Fig. 4). Indeed, the contractile performance benefit of stem cell- vs. sham-treated infarcted hearts was maintained at 3, 6, 9, and 12 wk after injection, such that at 3 mo after cell delivery left EF was 83 ± 4% and 62 ± 4%, respectively (P < 0.05; Fig. 4, A and B). On M-mode images 3 mo after therapy, the left ventricular dilation and the anterior regional wall motion abnormalities persisted in the sham-treated group but were not seen in the stem cell-treated group (Fig. 4A). Furthermore, electrocardiography performed at 3 mo after therapy revealed in the stem cell-treated group a 33% decrease in the total number of anterior and lateral leads, with Q waves reflecting net reduction in myocardial necrosis (P < 0.05) not seen in the sham-treated group (Fig. 4, C–E). Throughout the follow-up period, serial electrocardiograms did not document ventricular ectopy, and no animal experienced sudden cardiac death. Thus delivery of embryonic stem cells into infarcted hearts was associated with a functional benefit at baseline and with stress and was sustained on follow-up without evidence of proarrhythmia in this model.
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On pathological examination, the entire group of infarcted hearts treated with stem cells (n = 4) demonstrated a population of cyan fluorescent myocytes dispersed within the nonfluorescent host myocardium (Fig. 5, 1st and 4th rows). This fluorescent population, absent from the sham-injected group (n = 3), indicates the embryonic stem cell origin through expression of the cyan fluorescent protein under control of the cardiac actin promoter (3). In contrast to sham-treated infarcted hearts that demonstrated markedly altered ventricular architecture with thinned free walls and fibrotic scar or aneurysmal areas comprising 34 ± 11% of the ventricle, the presence of stem cell-derived cardiomyocytes was associated with residual adverse remodeling in only 6 ± 4% of the ventricle (P < 0.05) and a myocardial appearance more comparable to that of control noninfarcted heart (Fig. 5, 2nd and 3rd and 5th and 6th rows). Stem cell-injected hearts did not demonstrate inflammatory infiltrates that would otherwise suggest an immune response toward the engrafted cells (Fig. 6A). On high magnification, the fluorescent pattern of stem cell-derived cardiomyocytes revealed distinct sarcomeric striations indicating development of the contractile apparatus (Fig. 6, B and C). Sarcomeres in the infarct area of stem cell-treated hearts showed normal cardiac ultrastructure on electron microscopy, in contrast to acellular infarct areas of sham-treated hearts (Fig. 6, D and E). Thus embryonic stem cells were able to incorporate within host infarct territory, demonstrate cardiogenic differentiation, and contribute to myocardial repair.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Several potential mechanisms may account for the demonstrated benefit of embryonic stem cell therapy. Specifically, embryonic stem cell-derived cardiomyocytes, through electrical and mechanical coupling with native myocardium, could contribute to a net increase in contractile tissue. Here, stem cell-derived cardiomyocytes aligned with and integrated within host myocardial fibers. In fact, the host myocardium has been shown to secrete cardiogenic growth factors that interact in a paracrine fashion with receptors on stem cells, supporting cardiac differentiation with expression of cardiac contractile and gap junction proteins (3, 23). The present failure to observe ectopy is further consistent with electrical integration of stem cell-derived cardiomyocytes and host tissue. The stem cell-derived cardiomyocyte effect on active myocardial properties is moreover evidenced here by improved inotropic response to -adrenergic challenge. A synergistic potential mechanism for functional improvement by stem cell-derived cardiomyocytes is through alteration of myocardial passive mechanical properties (2), as shown here by the limited appearance of scar and less dilation of the left ventricle than in sham-treated infarcted hearts. This may occur through direct repopulation of scar by stem cell-derived cardiomyocytes, as well as limitation of adverse remodeling (8, 21). Moreover, cell fusion after grafting in vivo has been recently documented with adult stem cells in noncardiac tissue, as well as with cardiac progenitor cells in the heart itself (29, 41, 43). Although direct evidence for the propensity of embryonic stem cells to fuse with resident myocardium is lacking, such a possibility could, in principle, further contribute to cardiac repair by lending proliferative capacity to host heart muscle. In this way, cell fusion would complement de novo cardiogenesis occurring with cardiac differentiation of stem cells. As an additional potential mechanism, other cardiovascular cell types could arise directly from injected stem cells or through in situ recruitment, leading to neovascularization and, thus, augmented metabolic support of the host myocardium (1, 17, 44). Although the mechanism of stem cell-based cardiac repair is, thus, likely multifactorial, this study indicates that, rather than a transient reorganization that is short-lived because of rejection or failure of the transplant, the initial reparative benefit of stem cell therapy is stable.
We found no evidence of rejection of the transplanted cells, despite xenotransplantation of murine embryonic stem cells into rat heart. This lack of host vs. graft reaction may be graft and/or host dependent as a result of low expression of immunogenic antigens by stem cells, generation of mixed chimerism, downregulation of host immune response, and/or induction of improved tolerance (9, 11, 18). Finally, we did not observe disorganized differentiation leading to tumor formation, although pluripotent stem cells carry this risk (3, 38) when interacting with the host (10). As previously shown, protection from tumorigenesis is conferred by maintenance of proper host signaling that drives cardiac-specific differentiation of stem cells, thus preventing uncontrolled growth (3, 23).
Thus the stable benefit of embryonic stem cell therapy on myocardial structure and function in this experimental model supports the potential for stem cell-based reparative treatment of myocardial infarction. By regenerating diseased myocardium and promoting cardiac repair, embryonic stem cells provide a unique therapeutic modality that has the potential to reduce the morbidity and mortality of this prevalent heart disease.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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