Bioenergetic and Functional Consequences of Bone Marrow–Derived Multipotent Progenitor Cell Transplantation in Hearts With Postinfarction Left Ventricular Remodeling
Background— The present study examined whether transplantation of adherent bone marrow–derived stem cells, termed pMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractile function in the infarct zone and border zone (BZ) after coronary artery occlusion.
Methods and Results— Permanent left anterior descending artery occlusion in swine caused left ventricular remodeling with a decrease of ejection fraction from 55±5.6% to 30±5.4% (magnetic resonance imaging). Four weeks after left anterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a marked decrease in subendocardial phosphocreatine/ATP (31P magnetic resonance spectroscopy; 1.06±0.30 in infarcted hearts [n=9] versus 1.90±0.15 in normal hearts [n=8; P<0.01]). This abnormality was significantly improved by transplantation of allogeneic pMultistem cells (subendocardial phosphocreatine/ATP to 1.34±0.29; n=7; P<0.05). The BZ protein expression of creatine kinase–mt and creatine kinase–m isoforms was significantly reduced in infarcted hearts but recovered significantly in response to cell transplantation. MRI demonstrated that the infarct zone systolic thickening fraction improved significantly from systolic “bulging” in untreated animals with myocardial infarction to active thickening (19.7±9.8%, P<0.01), whereas the left ventricular ejection fraction improved to 42.0±6.5% (P<0.05 versus myocardial infarction). Only 0.35±0.05% donor cells could be detected 4 weeks after left anterior descending artery ligation, independent of cell transplantation with or without immunosuppression with cyclosporine A (with cyclosporine A, n=6; no cyclosporine A, n=7). The fraction of grafted cells that acquired an endothelial or cardiomyocyte phenotype was 3% and ≈2%, respectively. Patchy spared myocytes in the infarct zone were found only in pMultistem transplanted hearts. Vascular density was significantly higher in both BZ and infarct zone of cell-treated hearts than in untreated myocardial infarction hearts (P<0.05).
Conclusions— Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global left ventricular ejection fraction. These improvements may have resulted from paracrine effects that include increased vascular density in the BZ and spared myocytes in the infarct zone.
- heart failure
- magnetic resonance imaging
- myocardial contraction
- myocardial infarction
Received August 31, 2006; accepted February 9, 2007.
Myocardial infarction (MI) often results in left ventricular (LV) remodeling in which an initial period of relative hemodynamic stability is followed by progressive contractile dysfunction that can eventuate in overt congestive heart failure. Using a swine model of post-MI LV remodeling, we recently reported that myocardial energetic characteristics, as represented by high-energy phosphate content and the phosphocreatine (PCr)/ATP ratio, were markedly abnormal in the peri-infarct border zone (BZ).1 The abnormalities were much greater in the BZ subendocardium (ie, PCr/ATP reduced by ≈50%) than in myocardium remote from the infarct. Similarly, LV contractile function was most depressed in the BZ of infarcted hearts.2,3 These data are in agreement with the hypothesis that bioenergetic and contractile abnormalities of the BZ myocardium result in progressive deterioration of overall LV chamber function. A potentially “curative” therapy would be to replace infarcted or dysfunctional tissue with cardiomyocytes that could prevent LV remodeling and subsequent congestive heart failure.
Clinical Perspective p 1875
Both experimental and clinical studies have demonstrated that cell transplantation can improve LV contractile performance in infarcted hearts,4–15 but the underlying mechanisms are not totally clear. Although improved cardiac function could be due to differentiation of transplanted cells into cardiomyocytes,4–15 it is generally believed that release of cytokines that exert trophic effects on host cardiac cells and induce neovascularization may be of equal or greater importance.7,15 In the present study, we examined the effects of allogeneic bone marrow–derived adherent stem cells (pMultistem) transplanted into the BZ of acutely infarcted swine hearts on BZ and remote zone structure (LV chamber size and infarct size), contractile performance, and energetic status (PCr/ATP ratio) using 31P magnetic resonance spectroscopy (MRS). pMultistem cells were expanded by Athersys, Inc (Cleveland, Ohio) from a cell line of multipotent adult progenitor cells generated from a young pig (45 days of age)16 using clinically suitable expansion methods. pMultistem cells were transduced with a β-galactosidase–expressing retrovirus, expanded to generate a master cell bank and working cell bank, which were cryopreserved. pMultistem cells can differentiate into smooth muscle cells and endothelial cells. We hypothesized that the primary beneficial effects of cell transplantation occur in the infarct zone (IZ) and BZ of infarcted hearts and that preservation of structure and function is primarily the result of limitation of BZ deterioration. We reasoned that grafting of pMultistem cells into the BZ of acutely infarcted hearts would attenuate BZ contractile and energetic deterioration and thereby limit overall LV chamber dilation. To determine whether cell transplantation could augment oxygen availability in the BZ, myocardial deoxymyoglobin (Mb-δ) was assessed with 1H MRS.17,18 The results were compared with nontransplanted infarcted hearts. We also evaluated the effects of immunosuppression with cyclosporine on the pMultistem engraftment rate.
pMultistem cells were derived from 1 of the male swine multipotent adult progenitor cell stocks generated at the University of Minnesota.16 Details on the generation of the pMultistem cells, their banking and cryopreservation, and in vitro experiments characterizing the phenotype and differentiation potential are described in the online Data Supplement.
Creation of Infarct by Coronary Artery Ligation
The investigation conformed to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). Details of the animal model of postinfarction LV remodeling have been described previously.1,17 Briefly, young Yorkshire female swine (45 days old, ≈10 kg; Manthei Hog Farm, Elk River, Minn) were anesthetized with pentobarbital (30 mg/kg IV), intubated, and ventilated with a respirator with supplemental oxygen. A left thoracotomy was performed, and 0.5 cm of the left anterior descending coronary artery (LAD) distal to the second diagonal branch was dissected free and permanently occluded with a ligature, which usually results in ≈10% LV mass damage. Lidocaine (1 mg · kg−1 · min−1 IV for 70 minutes, 2 mg/kg IV bolus before LAD occlusion) and nitroglycerine (0.5 μg · kg−1 · min−1 IV for 70 minutes starting 10 minutes before LAD occlusion) were given before and during LAD ligation to decrease the arrhythmia. Animals were observed in the open-chest state for 60 minutes. If ventricular fibrillation occurred, electrical defibrillation was performed immediately and was usually successful. Sixty minutes after LAD ligation, the surviving pigs were randomized to ligation only (n=7), MI plus cell transplantation group (n=7), and MI plus cell transplantation plus cyclosporine A (CsA) group (n=6). The ischemic myocardial region became cyanotic in response to the LAD occlusion. pMultistem cells were injected directly into 5 regions of the peri-infarct BZ (10 million cells per location; 50 million total diluted in 2 mL of saline). Injection sites were marked with a stitch to allow identification of the areas for histological studies. Animals in the control group (MI) received 2 mL of saline in 5 injections at similar BZ injection sites. The chest was then closed. Animals received standard postoperative care, including analgesia, until they ate normally and became active. pMultistem-treated animals were randomized to receive or not receive CsA (15 mg · kg−1 · d−1 mixed with food). Animals returned to the laboratory 3.5 and 4 weeks later for magnetic resonance imaging (MRI) and 31P MRS studies, respectively.
Tagged and Cine MRI Protocol
MRI was performed ≈25 days after surgery on a 1.5-T clinical scanner (Siemens Sontata, Siemens Medical Systems, Islen, NJ) with a phased-array, 4-channel surface coil and ECG gating. Details on methods of MRI and MRS are described in the Data Supplement.
Spatially Localized 31P-MRS and 1H-MRS Technique
Spatially localized 31P nuclear magnetic resonance spectroscopy was performed with the rotating frame experiment using adiabotic plane-rotation pulses for phase modulation–imaging selected in vivo spectroscopy/Fourier series window method.17,19–21 There is absolutely no overlap between the 135° voxel (corresponding to the subepicardium) and the 45° voxel (corresponding to the subendocardium).17,19–21 The 31P-MRS and 1H-MRS techniques are described briefly in the Data Supplement.
Surgical Preparation for Open-Chest MRS Study
Detailed surgical preparations for the MRS study have been published previously and are described briefly in the Data Supplement.
Aortic and LV pressures were measured with pressure transducers positioned at the midchest level19–21 and recorded on an 8-channel recorder.
At the completion of the open-chest MRS and hemodynamic measurements, animals were euthanized by an overdose of pentobarbital, and the heart was explanted. The LV was opened at the lateral wall from base to apex, and a photograph was taken for infarct size measurement. Infarct size was expressed as a percent of LV surface area by an image-analysis system (NIH Image J program, available at http://rsb.info.nih.gov/ij).22
Detailed immunohistochemistry methods are described in the Data Supplement.
Hemodynamic data were measured from the strip chart recordings.18–20 Data were analyzed with 1-way ANOVA. A value of P<0.05 was considered significant. When ANOVA demonstrated a significant effect, post hoc analysis was performed with the 2-tailed t test with Bonferroni correction. A least-squares linear regression model was used for fitting the regression lines in the evaluation of the relationships between PCr/ATP and myocardial systolic shortening fraction.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Phenotypic Characteristics of pMultistem
Methods for pMultistem generation, expansion, banking, and quantification are described in detail in the online Data Supplement. Figures 1 and 2⇓ illustrate the results of in vitro differentiation of pMultistem cells to endothelial and smooth muscle cells, respectively. These data demonstrate that with appropriate inducers, pMultistem cells differentiate into smooth muscle–like and endothelium-like cells and are consistent with the in vivo cell fate detected in these experiments.
Animal Model and Anatomic Data
Seven of the 27 pigs with LAD ligation died within the first 60 minutes after coronary occlusion. The surviving 20 pigs were randomized to ligation only (MI; n=7), ligation plus cell transplantation (n=7), and ligation plus cell transplantation plus CsA (n=6). In addition, 8 size-matched normal pigs and 2 ligation-only pigs were subjected to the identical study protocol before randomization. These 10 pigs have been included in the respective study groups. Table 1 summarizes anatomic data from 13 swine with LAD ligation into which pMultistem cells were grafted with or without CsA, compared with 9 swine with LAD ligation with no cells grafted and no CsA treatment, as well as 8 normal pigs. The LV weight to body weight ratio and right ventricular weight to body weight ratio were 32% and 21% greater in hearts after LAD occlusion than in hearts of size-matched normal swine, respectively (P<0.05; Table 1). LAD occlusion resulted in 10% to 13% LV infarct, expressed as the ratio of scar surface area to LV surface area (Table 1). Infarct size, LV weight to body weight ratio, and right ventricular weight to body weight ratio were not significantly affected by pMultistem transplantation in the presence or absence of CsA (Table 1).
Hemodynamics and LV Function
Hemodynamic and LV ejection fraction data are summarized in Table 2. One month after LAD ligation, hemodynamic variables were not significantly different between the hearts with or without pMultistem treatment (Table 2). The cine MRI–measured LV ejection fraction decreased from 55% (normal hearts) to 30% in hearts with postinfarction LV remodeling without pMultistem treatment; LV ejection fraction was significantly greater in animals with pMultistem transplantation irrespective of CsA treatment (P<0.05; Table 2).
LV end-systolic wall thickness and thickening fraction measured by MRI are summarized in Table 3. The LV IZ wall tended to be thinner than other regions of the LV (P<0.01; Table 3), which suggests higher wall stress in this region.
Myocardial High-Energy Phosphate and Pi Levels
Figure 3 illustrates typical transmurally differentiated 31P nuclear magnetic resonance spectra for a normal heart (Figure 3) compared with the BZ of an infarcted heart not treated with pMultistem (Figure 3) and the BZ of a heart with pMultistem transplantation with CsA (Figure 3). The voxel labeled EPI was positioned over the outer edge of the LV wall, whereas the voxel most distant from the coil (labeled ENDO) was positioned over the subendocardium. Myocardial high-energy phosphate and Pi levels are summarized in Table 4. PCr/ATP was most severely decreased in the inner layer of the BZ, being ≈56% of that in normal hearts (Figure 3; Table 4). The BZ PCr/ATP ratio was significantly improved in response to pMultistem transplantation (P=0.03, Table 4). The BZ PCr/ATP ratio was not significantly different in animals that did or did not receive CsA (Table 4). The combined pMultistem-treated hearts showed a significant increase of PCr/ATP across the LV wall compared with the hearts with postinfarction LV remodeling without cell treatment (P<0.05; Table 4).
Figure 4 illustrates relationships between BZ myocardial PCr/ATP and tagged MRI measuring LV systolic shortening fraction from the combined IZ and BZ. Regional LV contractile function calculated for segments 1 through 6 represents the anterior papillary, anterior, septal, posterior, posterior papillary, and lateral LV segments. LAD ligation resulted in a thin-walled infarct that mainly involved the anterior wall within segment 2, whereas the septal and anterior papillary segments (segments 1 and 3, respectively) represent the BZ. The remaining segments (4, 5, and 6) represent the remote zone. pMultistem treatment significantly increased IZ LV wall contractile function irrespective of CsA. When each PCr/ATP ratio was plotted against the respective IZ/BZ systolic shortening fraction for each heart, significant correlations were observed (Figure 4). These data suggest that pMultistem transplantation significantly improved IZ contractile performance, which in turn was associated with remarkably improved BZ bioenergetics.
In principle, the deeper voxels (ie, more distant from the outer LV wall) contain contributions from LV cavity blood because of partial volume effects (ie, they can be occupied both by LV wall and LV chamber), recognizable by the presence of 2,3-diphosphoglycerate resonances in the ≈3 ppm region of the spectra.1,23 The presence of both blood and cardiac muscle in the same voxel has the potential to distort ATP levels and PCr/ATP ratios, because blood contains ATP but not PCr. The ATP contribution from blood to the subendocardial spectrum PCr/ATP has been examined previously1,23 and found to be trivial. In the present study, the contribution of blood in the nuclear magnetic resonance region of interest might be greater because of the thinner wall in the peri-infarct area. To assess this possibility, the blood ATP contribution was examined with a phantom filled with fresh heparinized blood using the identical spectrometer setup. Prominent resonance peaks of 2,3-diphosphoglycerate appeared at ≈3 ppm. No ATP resonance was detected. These data demonstrate that the contribution of LV cavity blood ATP to the subendocardial PCr/ATP ratio was negligible.
Myocardial Oxygenation Evaluated by 1H-MRS
No deoxymyoglobin resonance peak was detected either in normal hearts or in the peri-infarct or remote regions of infarcted hearts (spectra not shown). On the basis of the signal to noise ratio of Mb-δ during partial and complete LAD occlusions,16 the resonance peak should be recognized when there is >10% myoglobin desaturation.17 These data indicate that the BZ bioenergetic abnormality in hearts with postinfarction LV remodeling (Figure 3; Table 4) was not the result of persistent myocardial ischemia.
Transplanted pMultistem Engraftment and Differentiation In Vivo
Four weeks after transplantation, 0.35±0.05% of a total 50 million injected LacZ pMultistem cells (CsA: n=6, no CsA: n=7) could be detected. Approximately 70% of these cells were found at areas near the injection sites in the BZ, whereas ≈30% migrated into the IZ. In hearts that received stem cell transplantation, patchy spared myocytes were observed in the IZ (Figure 5A), and this was only observed in the hearts with cell transplantation. Among these cells, a small number (shown by the arrows) were costaining positive for X-Gal and cardiac-specific troponin T (≈2%; Figure 5A). LacZ-labeled pMultistem cells were also detected in coronary vessels, where they expressed von Willebrand factor, which suggests differentiation into endothelial cells (≈3%; Figure 5B).
Measurements of vascular density are illustrated in Figure 6. Transplantation of pMultistem cells was associated with a significant increase of vascular density (per mm2) in both IZ and BZ (P<0.05). The vascular density was not significantly different between hearts with or without cell transplantation in the remote zone (Figure 6).
Myocardial CK Isoform Protein Expression
Figure 7 illustrates Western blots examining CK isoform proteins in the BZ. The protein levels of CK-M and CK-mito were decreased significantly in hearts with MI and recovered significantly in response to cell transplantation (Figure 7). In contrast, CK-B isoform protein expression did not change significantly.
In the present study, injection of banked cryopreserved allogeneic pMultistem cells at the time of acute coronary occlusion attenuated the structural, contractile, and energetic abnormalities that occurred in the IZ and peri-infarct BZ at 4 weeks after occlusion, with resultant preservation of global LV function. The beneficial effects of cell transplantation occurred despite the fact that (1) cell transplantation did not reduce infarct size, (2) the engraftment rate 4 weeks after transplantation was very low and mainly confined to the BZ and IZ, and (3) differentiation of pMultistem cells into cardiomyocytes and endothelial cells was minimal. The results support the hypothesis that protection is mediated by paracrine effects of pMultistem on IZ and BZ cardiomyocytes and neovascularization. Interestingly, immunosuppression with CsA did not increase the engraftment rate of the allogeneic pMultistem cells.
Mechanism of Postinfarction LV Remodeling
LV remodeling after MI has been attributed to several factors, including neurohormonal activation and increased systolic wall stress in the BZ, which is mechanically tethered to the dysfunctional region of infarcted myocardium.1–3 Increased biomechanical strain has been found to trigger stretch-activated signaling pathways that lead to cardiomyocyte hypertrophy (with associated changes in gene expression patterns) and apoptosis.1 We recently reported that high-energy phosphate levels, PCr/ATP values, and the expression of several proteins crucial to oxidative ATP production are significantly decreased in the preserved myocardium of infarcted swine hearts and that these abnormalities are most severe in the BZ (especially the BZ subendocardium).1 The profound depression of high-energy phosphate levels and PCr/ATP in the BZ myocardium was in marked contrast to remote-zone bioenergetic characteristics, which remained relatively normal.1,17,27,28 These data are compatible with the concept that impaired energetic capacity in the BZ contributed to contractile dysfunction.
Mechanisms of pMultistem Effects on IZ Contractile Function and BZ Energetics
There are several possible mechanisms by which cell engraftment might protect myocardial energetics. First, the engrafted cells, via differentiation, could make direct contributions to IZ and BZ cardiomyocyte regeneration and/or neovascularization. This possibility is contradicted by the small fraction of the grafted cell population detected 4 weeks after transplantation and the even smaller number of engrafted cells that acquired cardiomyocyte or endothelial cell features. Second, recruitment of endogenous cardiac progenitors to the injury site might provide a protective effect.9,22 Third, engrafted cells could influence IZ and BZ structure and function by exerting trophic effects to enhance native cardiomyocyte survival and function and promote neovascularization,29,30 which is likely the mechanism underlying the observed effects on BZ energetics and function. Gnecchi et al29 recently reported that injection of concentrated conditioned medium harvested from cultured Akt-overexpressing mesenchymal stem cells into infarcted heart was highly protective, decreasing infarct size and improving contractile function in a rat ischemia-reperfusion model. That study demonstrated high concentrations of a number of cytokines and growth factors in the concentrated culture medium, namely, vascular endothelial growth factor (VEGF), insulin-like growth factor, hepatocyte growth factor, and fibroblast growth factor.29
The IZ and BZ of pMultistem-treated hearts demonstrated significant neovascularization compared with the untreated hearts (Figure 6). This could enhance delivery of oxygen and carbon substrate to the BZ and might thereby facilitate ATP production.15 The lack of myoglobin desaturation in the BZ argues against limitation of oxygen and carbon substrate delivery to the BZ, and previous reports31,32 have suggested that BZ perfusion is not compromised in single-vessel coronary artery ligation models. However, the myoglobin saturation data indicate only that ischemia was not present during basal conditions, so the possibility of ischemia during increased periods of increased cardiac work is not excluded, especially because oxygen demands are likely increased in the BZ due to the increased systolic wall stress. Increased BZ vascularity15 could potentially prevent recurrent episodes of demand-induced ischemia.
pMultistem Paracrine Effects on BZ and IZ Cardiomyocytes
As previously discussed, there is strong evidence that stretch can activate signaling pathways that can trigger pathological hypertrophy and apoptosis of cardiomyocytes.33 The resultant alterations can include genes involved in carbon substrate metabolism and ATP generation.1 There is similarly strong evidence that other marrow-derived (or other tissue-derived) progenitor cells secrete cytokines involved in stimulation of cell growth and suppression of apoptosis.7,15,29 We have shown that transplantation of mesenchymal stem cells overexpressing VEGF into swine myocardium at the time of imposition of an acute pressure overload (ascending aortic banding) limited the development of LV hypertrophy, contractile dysfunction, and bioenergetic abnormalities.15 Transplantation of cells not overexpressing VEGF produced significant but smaller beneficial effects.15 In an experiment in which mesenchymal stem cells overexpressing VEGF were co-cultured with neonatal myocytes, the VEGF-modified mesenchymal stem cells inhibited myocyte apoptosis.15 Similarly, Dzau and colleagues have shown that paracrine factors released by mesenchymal stem cells induce neovascularization and protect surviving cardiomyocytes in infarcted mouse hearts.7,29 In other studies, we have shown that mouse multipotent adult progenitor cells secrete a number of cytokines and chemokines, including VEGF, platelet-derived growth factor, and monocyte chemoattractant protein-1.34 Taken together, these data suggest that pMultistem, like mouse multipotent adult progenitor cells, may secrete soluble factors that exert paracrine effects that inhibit activation of pathological signaling pathways induced by the increased mechanical and oxidative stresses in the infarct BZ, and that limiting the activation of these pathways may restrict the development of gene expression patterns that are associated with contractile and energetic dysfunction. The resultant preservation of cardiomyocyte contractile and energy-generating systems could preserve BZ function and thereby limit global LV remodeling. It is likely that these paracrine effects are crucial to the protective effects of cell transplantation on native cardiomyocytes in the acutely infarcted heart.
BZ Myocardial CK Isoform Protein Expression
The BZ bioenergetic improvement produced by cell transplantation was accompanied by increased expression of CK-M and CK-mito isoform proteins but unchanged CK-B isoform (Figure 7), which occurred independent of changes in mitochondrial density, which suggests again that alterations in protein expression secondary to mechanical or oxidative stress were prevented by a paracrine effect of stem cell transplantation. These observations also indicate an urgent need for studies of BZ signaling pathways that are activated and/or inhibited by cell transplantation.
Does Immune Rejection Contribute to the Low pMultistem Engraftment Rate?
In the present study, immunosuppression with CsA did not increase the rate of engraftment of pMultistem. This suggests that either intrinsic rejection processes are limited over the 4-week observation period or that, as suggested by Thum et al,35 apoptosis of transplanted cells induces immunosuppression. Other possible mechanisms for the low engraftment rate include early loss of pMultistem from the injection sites and apoptotic loss of cells over time. The latter is supported by our recent observation in a murine infarct model that the number of engrafted cells fell sequentially over a 4-week period after transplantation.22 It remains to be determined whether higher engraftment rates would be associated with greater protective effects.
Transplantation of pMultistem at the time of coronary artery ligation resulted in improved IZ contractile function and prevented BZ bioenergetic deterioration. It is likely that the LV chamber response to cell transplantation resulted from the beneficial effects of sparing myocytes and increasing revascularization in both IZ and BZ. A direct structural contribution of the engrafted cells to cardiomyocyte regeneration or neovascularization appears unlikely. It remains to be determined whether the primary benefits of pMultistem transplantation are a consequence of (1) increased blood flow reserve resulting from increased BZ capillarity, (2) paracrine effects emanating from pMultistem, or (3) a combination of these effects. The results imply that progenitor cell transplantation can exert beneficial effects at the time of an acute myocardial infarct.
Sources of Funding
This work was supported by US Public Health Service grants HL50470, HL61353, HL 67828, HL71970, and HL21872.
P. Suntharalingam, S. Boozer, Dr Mhashilkar, and Dr Deans were employees of Athersys, Inc. None of these 4 coauthors were involved in data collection or analysis. Dr Verfaillie has received research funding from Athersys, Inc, for work with multipotent adult progenitor cells unrelated to the studies in this report. The remaining authors report no conflicts.
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Cell transplantation into border-zone myocardium at the time of percutaneous intervention for acute myocardial infarction is a potentially feasible therapeutic strategy, because banked frozen bone marrow–derived stem cells (pMultistem) can be made readily available, and the methodology for percutaneous cell injection already exists. It must first be shown, however, that the observed short-term therapeutic effects translate into long-term benefit. It is possible that strategies to enhance the pMultistem engraftment rate or in vivo proliferation and differentiation could also contribute to the long-term efficacy of this therapeutic approach.
↵*The first 3 authors contributed equally to this work.
The online-only Data Supplement, consisting of Methods and a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.659730/DC1.