* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Stable benefit of embryonic stem cell therapy in - AJP
Survey
Document related concepts
Cytokinesis wikipedia , lookup
Cell growth wikipedia , lookup
Extracellular matrix wikipedia , lookup
Tissue engineering wikipedia , lookup
Cell culture wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
List of types of proteins wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cellular differentiation wikipedia , lookup
Hematopoietic stem cell wikipedia , lookup
Somatic cell nuclear transfer wikipedia , lookup
Stem cell controversy wikipedia , lookup
Transcript
Am J Physiol Heart Circ Physiol 287: H471–H479, 2004; 10.1152/ajpheart.01247.2003. CALL FOR PAPERS Cellular Plasticity in the Cardiovascular System Stable benefit of embryonic stem cell therapy in myocardial infarction Denice M. Hodgson,1 Atta Behfar,1 Leonid V. Zingman,1 Garvan C. Kane,1 Carmen Perez-Terzic,1,2 Alexey E. Alekseev,1 Michel Pucéat,1,3 and Andre Terzic1 1 Division 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 3 Centre de Recherches de Biochimie Macromoléculaire, CNRS FRE 2593, 34293 Montpellier, France Submitted 31 December 2003; accepted in final form 22 March 2004 engraftment; xenotransplant; plasticity; cardiac differentiation; remodeling; heart 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. leads to cardiomyocyte loss, ventricular remodeling, and consequent impairment of myocardial function, whereas the mitotic capacity of cardiomyocytes is too limited to support adequate myocardial regeneration (5, 7, 39). Moreover, current therapeutic modalities attenuate disease progression without contributing significantly to myocardial repair (16). In this regard, an alternative approach to conventional strategies is emerging with advances in the manipulation of nonterminally differentiated cells that maintain the potential to form cardiomyocytes and, thus, the propensity to replace diseased myocardium (27, 28, 30, 36, 42). Although multiple candidate cell types have been isolated from cardiac or noncardiac sources and shown to display Embryonic stem cells. 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- Address for reprint requests and other correspondence: A. Terzic, Guggenheim 7, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: [email protected]). 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. MYOCARDIAL INJURY http://www.ajpheart.org METHODS 0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society H471 Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 Hodgson, Denice M., Atta Behfar, Leonid V. Zingman, Garvan C. Kane, Carmen Perez-Terzic, Alexey E. Alekseev, Michel Pucéat, and Andre Terzic. Stable benefit of embryonic stem cell therapy in myocardial infarction. Am J Physiol Heart Circ Physiol 287: H471–H479, 2004; 10.1152/ajpheart.01247.2003.—Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to -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. H472 SUSTAINED STEM CELL-BASED CARDIAC REPAIR AJP-Heart Circ Physiol • VOL calculate EF as follows: EF ⫽ 100 䡠 (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 0.5m-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-m 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. RESULTS Cardiogenic potential of embryonic stem cells and delivery into infarcted heart. 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. Sustained functional benefit of stem cell- vs. sham-treated infarcted hearts. 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, 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 emission scanning electron microscopy, cells were fixed in phosphatebuffered 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 fieldemission 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 lowviscosity 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 hangingdrop 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 l of Glasgow MEM) without cells (sham) or CGR8 embryonic stem cells (3 ⫻ 105 in 20 l 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 SUSTAINED STEM CELL-BASED CARDIAC REPAIR H473 stem cell-treated infarcted hearts demonstrated a significant positive inotropic response. Pharmacological stress testing by injection of the -adrenergic agonist isoproterenol (3 g/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 AJP-Heart Circ Physiol • VOL 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 shamtreated 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 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 1. Validation of cardiogenic potential and myocardial delivery of embryonic stem cells. A and B: field emission scanning (A) and transmission (B) electron microscopy of undifferentiated CGR8 embryonic stem cells in culture. In B, N denotes nucleus; n, nucleolus; and m, mitochondria. C: cardiomyocytes, derived in vitro from the CGR8 stem cell colony, express cardiac-specific transcription factor MEF2C and contractile protein ␣-actinin demonstrated by immunofluorescence. 4⬘,6-Diamidino-2-phenylindole (DAPI) staining is used as a nuclear marker. D: stem cell-derived cardiomyocytes exhibit action potential activity under current-clamp mode (top); current-voltage relation was obtained under voltage-clamp mode in response to a ramp stimulus (rate of 1.2 V/s) and displays characteristic peaks corresponding to Na⫹ and Ca2⫹ inward conductances (bottom). Em, membrane potential. E: Ca2⫹ transients were probed by fluo 3-assisted laser confocal microscopy from a stem cell-derived cardiomyocyte (inset) and recorded at 35 ⫾ 2°C. AU, arbitrary units. F: local retention of CGR8 stem cells after intramyocardial delivery into mouse heart (inset). Cryosection (at ⫻40 magnification) of myocardium with an overlay of fluorescing stem cells. G: lack of stem cell dispersion into noncardiac tissues with absence of fluorescence or cell hyperproliferation in mouse brain (left), kidney (center), and liver (right) shown at low (top) and ⫻40 magnification (bottom). H474 SUSTAINED STEM CELL-BASED CARDIAC REPAIR Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 2. Myocardial infarction and stem cell therapy. A and B: infarction of rat hearts confirmed by the presence of anterior and lateral Q waves on 12-lead electrocardiogram (A) and by visual inspection after thoracotomy (B). In A, arrowheads indicate location of Q waves on electrocardiography. In B, arrowheads indicate sites of stem cell injection at the base, midventricle, and apex in the peri-infarct zone. Inset in B shows injection of stem cells into infarcted heart. baseline and with stress and was sustained on follow-up without evidence of proarrhythmia in this model. Stem cell engraftment associated with de novo cardiogenesis and normalized myocardial architecture. 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 AJP-Heart Circ Physiol • VOL Fig. 3. Stem cell therapy of infarcted hearts is associated with improved left ventricular function and response to stress. A: at 3 wk after injection, left ventricular ejection fraction, measured by M-mode echocardiography, was significantly greater in stem cell-treated (n ⫽ 4) than in sham-treated (n ⫽ 3) infarcted hearts (⫺stress; P ⬍ 0.05). After stress induced by isoproterenol (3 g/kg ip), left ventricular ejection fraction increased in the stem cell- but not the sham-treated group (⫹stress; P ⬍ 0.05). *Significant difference between stem cell and sham groups; **significant difference between stem cell and sham groups as well as significant difference in the stem cell group with and without stress. B: representative M-mode image under stress reveals a hypokinetic-to-akinetic anterior wall and left ventricular dilation in the sham-treated heart in contrast to normal anterior wall motion and vigorous contractile function in the stem cell-treated heart. Dashed and solid lines indicate diastolic and systolic left ventricular dimensions, respectively. MI, myocardial infarction. 287 • AUGUST 2004 • www.ajpheart.org SUSTAINED STEM CELL-BASED CARDIAC REPAIR H475 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 AJP-Heart Circ Physiol • VOL 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. DISCUSSION The present study of myocardial infarction shows a stable favorable impact of embryonic stem cell therapy. This manifested as a sustained benefit on cardiac contractile performance and ventricular remodeling associated with documented cardiogenesis in the infarct zone from injected stem cells. These findings indicate that the advantage of embryonic stem cell delivery occurs early, as first evidenced in the present design at 3 wk after therapy, and is not compromised by spontaneous failure of stem cell-derived cardiomyocytes and/or by rejection of this allogenic transplant by the host. The lack of diminishing effect over time suggests the potential for therapeutic use of 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 4. Stable benefit of stem cell therapy in infarcted hearts over 12-wk follow-up. A: representative M-mode images at 12 wk demonstrate left ventricular dilation and lower ejection fraction in sham- than in stem cell-treated infarcted hearts. Dashed and solid lines indicate diastolic and systolic left ventricular dimensions, respectively. B: serial echocardiographic assessments at 3, 6, 9, and 12 wk after therapy indicate that the benefit of stem cell therapy over sham therapy occurs as early as 3 wk and is stable over the entire period of follow-up. C: at 12-wk, electrocardiographic evidence of myocardial necrosis as manifested by Q waves was decreased compared with initial electrocardiograms in the stem cell- but not the sham-treated group (P ⬍ 0.05). *Significant difference between stem cell-treated and shamtreated groups. D and E: representative electrocardiograms from sham-treated (D) and stem cell-treated (E) rats at 12 wk indicate fewer Q waves in stem cell-treated hearts, suggesting less extensive area of infarct. Arrowheads indicate location of Q waves. H476 SUSTAINED STEM CELL-BASED CARDIAC REPAIR Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org H477 SUSTAINED STEM CELL-BASED CARDIAC REPAIR embryonic stem cells in the chronic management of myocardial injury. 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 cellderived 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 Fig. 5. Infiltration of stem cell-derived cardiomyocytes into infarct zone diminishes scar and preserves left ventricular architecture. The presence of cardiomyocytes derived from injected embryonic stem cells can be demonstrated through expression of enhanced cyan fluorescent protein under control of the ␣-actin promoter. Fluorescence is not seen in untreated noninfarcted or sham-treated infarcted hearts (1st row) but is seen within the infarct zone of stem cell-treated hearts (4th row; ⫻10 magnification). Compared with noninfarcted hearts, gross specimens and hematoxylin-and-eosin-stained cross sections at the base of each heart within the sham-treated group demonstrate dilated left ventricular cavities and prominent anterolateral scar with aneurysms (2nd and 3rd rows). In contrast, left ventricular cavity size and wall thickness are largely preserved in the stem cell-treated hearts, which also demonstrate less gross evidence of scar (5th and 6th rows). AJP-Heart Circ Physiol • VOL 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 6. In vivo stem cell-derived cardiomyocytes integrate with host myocardium. A: representative hematoxylin-and-eosin-stained myocardial sections without interstitial infiltration of small round basophilic mononuclear cells consistent with absence of lymphocytes and chronic inflammation in control (top) or stem cell-injected (bottom) infarcted hearts. B and C: high-magnification fluorescence confocal microscopy of stem celltreated hearts reveals fluorescent cardiomyocytes indicating expression of enhanced cyan fluorescent protein under control of the ␣-actin promoter and, thus, injected embryonic stem cell origin. Stem cell-derived cardiomyocytes organize to form regular fibers (B), oriented along the same axis as host cardiomyocytes (B and C), display typical sarcomeric striations, and form junctions with nonfluorescent host cardiomyocytes (C). D and E: representative transmission electron micrographic images of biopsies taken from the infarct zone reveal acellular regions with pronounced collagen deposition in shamtreated heart (D) compared with cellular areas with normal subcellular architecture in stem cell-treated heart (E). H478 SUSTAINED STEM CELL-BASED CARDIAC REPAIR GRANTS This study was supported by National Institutes of Health Grants HL64822, HL-07111, GM-65841, and GM-08685, the American Heart Association, Marriott Foundation, Miami Heart Research Institute, Mayo-Dubai Healthcare City Research Project, Mayo Clinic CR20 Program, and Association Francaise contre les Myopathies and Fondation de France. M. Pucéat is an Established Investigator of Institut National de la Santé et de la Recherche Médicale. A. Terzic is an Established Investigator of the American Heart Association. REFERENCES 1. Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, and Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 9: 1370 –1376, 2003. 2. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, and Penn MS. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362: 697–703, 2003. 3. Behfar A, Zingman LV, Hodgson DM, Rauzier J, Kane GC, Terzic A, and Puceat M. Stem cell differentiation requires a paracrine pathway in the heart. FASEB J 16: 1558 –1566, 2002. 4. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, and Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114: 763–776, 2003. AJP-Heart Circ Physiol • VOL 5. Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami CA, and Anversa P. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 344: 1750 –1757, 2001. 6. Boheler KR, Czyz J, Tweedie D, Yang HT, Anisimov SV, and Wobus AM. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 91: 189 –201, 2002. 7. Braunwald E and Bristow MR. Congestive heart failure: fifty years of progress. Circulation 102: IV14 –IV23, 2000. 8. Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, Vogl TJ, Martin H, Schachinger V, Dimmeler S, and Zeiher AM. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 108: 2212–2218, 2003. 9. Drukker M, Katz G, Urvach A, Schuldiner M, Markel G, ItskovitzEldor J, Reubinoff B, Mandelboim O, and Benvenisty N. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci USA 99: 9864 –9869, 2002. 10. Erdo F, Buhrle C, Blunk J, Hoehn M, Xia Y, Fleischmann B, Focking M, Kustermann E, Kolossov E, Hescheler J, Hossmann KA, and Trapp T. Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke. J Cereb Blood Flow Metab 23: 780 – 785, 2003. 11. Frandrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DG, Parwaresch R, Zavazava N, and Binas B. Preimplantation-stage stem cells induce long-term allogenic graft acceptance without supplementary host conditioning. Nat Med 8: 171–178, 2002. 12. Gepstein L. Derivation and potential applications of human embryonic stem cells. Circ Res 91: 866 – 876, 2002. 13. He JQ, Ma Y, Lee Y, Thomson JA, and Kamp TJ. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 93: 32–39, 2003. 14. Hodgson DM, Zingman LV, Kane GC, Perez-Terzic C, Bienengraeber M, Ozcan C, Gumina RJ, Pucar D, O’Coclain F, Mann DL, Alekseev AE, and Terzic A. Cellular remodeling in heart failure disrupts KATP channel-dependent stress tolerance. EMBO J 22: 1732–1742, 2003. 15. Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF, Boiani M, and Scholer HR. Derivation of oocytes from mouse embryonic stem cells. Science 300: 1251–1256, 2003. 16. Hunt SA, Baker DW, Chin MH, Cinquegrani MP, Feldman AM, Francis GS, Ganiats TG, Goldstein S, Gregoratos G, Jessup ML, Noble RJ, Packer M, Silver MA, Stevenson LW, Gibbons RJ, Antman EM, Alpert JS, Faxon DP, Fuster V, Gregoratos G, Jacobs AK, Hiratzka LF, Russell RO, and Smith SC Jr. ACC/AHA guidelines for the evaluation and management of chronic heart failure in the adult. Circulation 104: 2996 –3007, 2001. 17. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, and Langer R. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 99: 4391– 4396, 2002. 18. Lila N, Amrein C, Guillemain R, Chevalier P, Latremouille C, Fabiani JN, Dausset J, Carosella ED, and Carpentier A. Human leukocyte antigen-G expression after heart transplantation is associated with a reduced incidence of rejection. Circulation 105: 1949 –1954, 2002. 19. Lin Q, Schwarz J, Bucana C, and Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science 276: 1404 –1407, 1997. 20. Maltsev VA, Wobus AM, Rohwedel J, Bader M, and Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res 75: 233–244, 1994. 21. Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, and Dzau VJ. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 9: 1195–2001, 2003. 22. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, and Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 41: 1078 –1083, 2003. 23. Mery A, Papadimou E, Zeineddine D, Menard C, Behfar A, Zingman LV, Hodgson DM, Rauzier JM, Kane GC, Perez-Terzic C, Terzic A, and Puceat M. Commitment of embryonic stem cells toward a cardiac 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 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. SUSTAINED STEM CELL-BASED CARDIAC REPAIR 24. 25. 26. 27. 28. 30. 31. 32. 33. 34. 35. 36. AJP-Heart Circ Physiol • VOL 37. Thompson RB, Emani SM, Davis BH, van den Bos EJ, Morimoto Y, Craig D, Glower D, and Taylor DA. Comparison of intracardiac cell transplantation: autologous skeletal myoblasts versus bone marrow cells. Circulation 108: II264 –II271, 2003. 38. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, and Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147, 1998. 39. Towbin JA and Bowles NE. The failing heart. Nature 415: 227–233, 2002. 40. Tzukerman M, Rosenberg T, Ravel Y, Reiter I, Coleman R, and Skorecki K. An experimental platform for studying growth and invasiveness of tumor cells within teratomas derived from human embryonic stem cells. Proc Natl Acad Sci USA 100: 13507–13512, 2003. 41. Vassilopoulos G, Wang PR, and Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 422: 901–904, 2003. 42. Verfaillie CM. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 12: 502–508, 2002. 43. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, AlDhalimy M, Lagasse E, Finegold M, Olson S, and Grompe M. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422: 897–901, 2003. 44. Yang Y, Min J, Rana JS, Ke Q, Cai J, Chen Y, Morgan JP, and Xiao Y. VEGF enhances functional improvement of postinfarcted hearts by transplantation of ECS-differentiated cells. J Appl Physiol 93: 1140 –1151, 2002. 45. Ying QL, Nichols J, Chambers I, and Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115: 281–292, 2003. 46. Zingman LV, Hodgson DM, Bast PH, Kane GC, Perez-Terzic C, Gumina RJ, Pucar D, Bienengraeber M, Dzeja PP, Miki T, Seino S, Alekseev AE, and Terzic A. Kir6.2 is required for adaptation to stress. Proc Natl Acad Sci USA 99: 13278 –13283, 2002. 47. Zingman LV, Hodgson DM, Bienengraeber M, Karger AB, Kathmann EC, Alekseev AE, and Terzic A. Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sensitive K⫹ channels. J Biol Chem 277: 14206 –14210, 2002. 287 • AUGUST 2004 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on May 3, 2017 29. lineage: molecular mechanisms and evidence for a promising therapeutic approach for heart failure. J Muscle Res Cell Motil 24: 269 –274, 2003. Meyer N, Jaconi M, Landopoulou A, Fort P, and Puceat M. A fluorescent reporter gene as a marker for ventricular specification in ES-derived cardiac cells. FEBS Lett 478: 151–158, 2000. Min J, Yang Y, Converso KL, Liu L, Huang Q, Morgan JP, and Xiao Y. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 92: 288 –296, 2002. Min J, Yang Y, Sullivan MF, Ke Q, Converso KL, Chen Y, Morgan JP, and Xiao Y. Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells. J Thorac Cardiovasc Surg 125: 361–369, 2003. Murry CE, Whitney ML, Laflamme MA, Reinecke H, and Field LJ. Cellular therapies for myocardial infarct repair. Cold Spring Harb Symp Quant Biol 67: 519 –526, 2002. Nir SG, David R, Zaruba M, Franz WM, and Itskovitz-Eldor J. Human embryonic stem cells for cardiovascular repair. Cardiovasc Res 58: 313–323, 2003. Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y, Pocius J, Michael LH, Behringer RR, Garry DJ, Entman ML, and Schneider MD. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 100: 12313–12318, 2003. Olson EN and Schneider MD. Sizing up the heart: development redux in disease. Genes Dev 17: 1937–1956, 2003. Orlic D, Hill JM, and Arai AE. Stem cells for myocardial regeneration. Circ Res 91: 1092–1102, 2002. Perez-Terzic C, Behfar A, Mery A, van Deursen JM, Terzic A, and Puceat M. Structural adaptation of the nuclear pore complex in stem cell-derived cardiomyocytes. Circ Res 92: 444 – 452, 2003. Perez-Terzic C, Gacy AM, Bortolon R, Dzeja PP, Puceat M, Jaconi M, Prendergast FG, and Terzic A. Directed inhibition of nuclear import in cellular hypertrophy. J Biol Chem 276: 20566 –20571, 2001. Sachinidis A, Fleischmann BK, Kolossov E, Wartenberg M, Sauer H, and Hescheler J. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res 58: 278 –291, 2003. Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17: 435– 462, 2001. Solloway MJ and Harvey RP. Molecular pathways in myocardial development: a stem cell perspective. Cardiovasc Res 58: 264 –277, 2003. H479