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J Thorac Cardiovasc Surg 2003;126:114-122
© 2003 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Division of Cardiothoracic Surgery, Montreal General Hospital, McGill University Health Center, Montreal, Quebec, Canada
b Department of Cardiovascular Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Read at the Eighty-second Annual Meeting of The American Association for Thoracic Surgery, Washington, DC, May 5-8, 2002.
Received for publication March 29, 2002; revisions received November 19, 2002; accepted for publication December 4, 2002.
* Address for reprints: Ray C.-J. Chiu, MD, PhD, Division of Cardiothoracic Surgery, The Montreal General Hospital, MUHC, 1650 Cedar Ave, Suite C9-169, Montreal, Quebec H3G 1A4, Canada
rchiu{at}po-box.mcgill.ca
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Abstract |
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METHODS: Isolated bone marrow stromal cells from the isogenic donor rats were transfected with LacZ reporter gene for cell labeling. To induce cardiomyogenic differentiation, the bone marrow stromal cells were treated with 5-azacytidine before implantation. Two weeks after left coronary ligation, these cells (1 x 106 in 150 µL) were infused into the briefly distally occluded ascending aorta of the recipient rats (n = 15) to simulate direct coronary infusion clinically. Control animals were infused with cell-free medium (n = 14). Cardiac function was evaluated by echocardiography at preimplantation and 4 and 8 weeks postimplantation. The hearts were then immunohistochemically studied to identify phenotypic changes of implanted bone marrow stromal cells.
RESULTS: Immediately after cell infusion, the bone marrow stromal cells were trapped within coronary vessels in both infarcted and noninfarcted areas. However, after 8 weeks, most of the cells were identified in the scar and periscar tissue, expressing sarcomeric myosin heavy chain and cardiomyocyte-specific protein troponin I-C. Some bone marrow stromal cells were found to be connected to the adjacent host cardiomyocytes with gap junction. Two-way repeated-measures analysis of variance revealed significant improvement in fractional shortening and end-diastolic and end-systolic diameter of the left ventricle (P = .0465, .002, .0004, respectively) in the bone marrow stromal cell group.
CONCLUSIONS: Although bone marrow stromal cells had been reported to improve cardiac function when injected directly into the myocardial scar, this study demonstrated for the first time that bone marrow stromal cells can be delivered via the coronary artery, as they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.
With the recent advent in stem cell biology, it has been shown that an adherent population of bone marrow cells in culture that can be expanded in vitro, known as "marrow stromal cells" (MSCs), contain adult stem cells that can give rise to various mesenchymal and nonmesenchymal cell types.1-4 Since Makino and colleague5 demonstrated that cardiomyogenic differentiation of MSCs in vitro in 1999, several in vivo studies including those of ours have confirmed this capability of the MSCs.6-9 The advantages of using MSCs as donor cells for cellular cardiomyoplasty are that MSCs can be easily harvested from patients’ own bone marrow by aspiration, can be expanded vastly in culture to provide adequate numbers, and can then be autoimplanted without encountering immunorejection. A clinical trial for the autologous transplantation of bone marrow cells into an unrevascularizable area of the ischemic hearts to induce angiogenesis has already been reported.10
Cells can be implanted into the myocardium in a number of ways. To date, most of the experimental studies reported have employed direct injection of various types of donor cells into the myocardium.6,7,9,11-14 The advantage of this procedure is that it enables one to transplant cells selectively into specific areas of the myocardium. However, such an approach is rather invasive and in particular not suitable for patients with diffuse cardiomyopathy. In a recent study, we confirmed the feasibility of delivering MSCs via the coronary artery.8 As the MSCs were found to be capable of migrating to an injured area after transplantation,15,16 we hypothesized that MSCs, when infused into the coronary artery, could engraft into an infarcted myocardium and participate in myocardial regeneration and perhaps also angiogenesis,10 resulting in an improved cardiac function. In this study, we used a rodent model proximally injecting MSCs into the briefly occluded ascending aorta, to simulate clinical coronary delivery of these cells such as by selective coronary artery catheterization, or by aortic root injection during cardiopulmonary bypass.
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Materials and methods |
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Isolation and cell labeling
Isolation and primary culture of MSCs from the femoral and tibial bone of donor rats were performed as previously described in detail.7,8 A replication defective retrovirus containing the reporter (LacZ) gene that encodes for the bacterial ß-galactosidase enzyme were produced by LacZ-GP+AM12 amphotropic retrovirus producer cells originally obtained from Dr Jacques Galipeau’s Laboratory (McGill University, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada).17 The second-passaged MSC growth medium was replaced with the supernatant from the LacZ-GP+AM12 cells as described in detail before.8
MSC treatment with 5-aza-2'-deoxycytidine (5-azacytidine)
MSCs that were harvested and labeled were treated for 24 hours with 5-azacytidine [0.3 µM solution in 10% Dulbecco’s modified Eagle’s medium (DMEM)] within the culture dish. The cells were incubated overnight in a 37°C incubator with a humidified atmosphere of 5% CO2. The following day, the supernatant was removed and replaced with 10% DMEM solution for 24 hours. The cells were treated once more with 5-azacytidine for 24 hours and the growth medium replaced. MSCs treated with 5-azacytidine were infused into the coronary artery of recipient animals 24 hours after the treatment.
Myocardial infarction and infusion of mSCs
Forty-one rats underwent ligation of the left coronary artery. Rats were anesthetized with isoflurane (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and intubated and mechanically ventilated at 80 breaths/min. The heart was exposed via a left thoracotomy incision. The left coronary artery was ligated proximally with a 6-0 polypropylene suture.8 Two weeks after the coronary ligation, survived rats (n = 29) were divided randomly into two groups; rats infused with MSCs (MSC group: n = 15) and rats infused with culture medium (control group: n = 14). Anesthesia was induced and maintained with isoflurane. The ascending aorta was exposed through upper median sternotomy and looped with a suture after dissection. Under direct vision, transfected MSCs suspension (1 x 106 cells in 150 µL of DMEM) was then injected into the root of the ascending aorta, while distally occluding the aorta briefly for less than 3 seconds using the looped suture. A 28-G needle on an insulin syringe was used for the cell injection.8 Equivalent volume of serum-free culture medium was injected into the control animals using the same procedure. Two animals from the MSC group were sacrificed at 10 minutes after the injection to observe initial cell distribution.
Echocardiography
Transthoracic echocardiography was performed on all animals at 2 weeks after coronary ligation before infusion of MSCs as baseline and at 4 and 8 weeks after injection of MSCs or cell-free medium. Echocardiographies were performed with a commercially available echocardiography system (SONO 5500, Hewlett-Packard) equipped with 7.5-MHz transducer (Hewlett-Packard), and interpreted in a blinded fashion. Under anesthesia the heart was first imaged in the 2-dimensional mode in the parasternal long-axis view of the left ventricle (LV). Measurements of maximal LV long-axis lengths (L) and endocardial area tracings (a), using the leading edge method, were carried out.18 LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were calculated by the single-plane area-length method19: V = 8 x a2/(3 x 
x L). LV ejection fraction (EF) was then inferred: EF = [(LVEDV - LVESV)/LVEDV]. M-mode images were obtained at the level of papillary muscles of the mitral valves. End-diastolic and end-systolic diameter of the LV (LVEDd and LVEDs) were determined. Fractional shortening (FS) was then calculated as [(LVEDd - LVEDs)/LVEDd].
MSCs staining with x-gal for detection of ß-galactosidase activity
Cells were plated in 35-mm dish. They were fixed in 1% glutaraldehyde for 5 minutes at room temperature, then washed with phosphate buffered saline (PBS). Staining solution (500 µL) at pH of 8.0 was added, which contained 1 mg/mL 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-gal), 1 mmol/L ethyleneglycol-bis-(ßaminoethylether)-N,N,N',N'-tetraacetic acid, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6.3H2O, 2 mmol/L magnesium chloride, and 0.01% sodium deoxycholate.8 Then cells were incubated at 37°C while protected from light for 16 hours.
Tissue processing and staining for ß-galactosidase activity
Animals were sacrificed at 1 day after the final echocardiogram (8 weeks after transplantation). As mentioned above, two animals in the MSC group were sacrificed immediately after cell transplantation to confirm the initial distribution of MSCs. Upon sacrifice of animals, hearts were excised. The hearts were rinsed with PBS and then perfusion fixed in 2% paraformaldehyde in PBS. The staining for ß-galactosidase activity was performed as described above, but with the addition of 0.02% of Nonidet P-40 and 0.01% of deoxycholate to the staining solution.8 After X-gal staining at pH of 8.0, the hearts were cut longitudinally and embedded in paraffin.
Histology and immunohistochemistry
Heart sections 5 µm in thickness were processed for H&E, Picrosirius Red, and immunohistochemical stains. Immunohistochemical stains were done for anti-
smooth muscle actin (Sigma Laboratories), endothelial cell marker factor VIII, and cardiomyocyte specific troponin I-C (Santa-Cruz Biotechnology Inc). Stainings were also carried out for sarcomeric myosin heavy-chain molecules with MF 20 (Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences) and connexin 43 (Zymed Laboratories Inc) as previously described.8 Briefly, after deparaffinization, sections were placed in boiled citrate buffer (pH 6.0). After blocking in normal serum, sections were treated with the respective monoclonal antibodies overnight and with secondary antibodies the following day. Diaminobenzidine (DAB) was then used as a chromogen for light microscopy. Counterstaining of sections with hematoxylin and eosin was also performed. Cells derived from MSCs were identified by their blue nuclei (ie, ß-gal labeled) with an Olympus microscopy (BX-FLA, Olympus).
Estimation of the number of ß-gal-positive cells in the heart
We estimated the number of ß-gal-positive cells present in the heart immediately after infusion by means of Weiss’ method,20 which had been validated for the rodent heart model when the cell distribution could be assumed to be homogeneous. Briefly, cell counts were made on 5-µm-thick histological sections and surface area of the myocardium on the section was measured by planimetry. Then cell number in certain tissue volume was referred to the gross myocardium volume, which had been obtained by water displacement technique.
Statistical analysis
All data are expressed as mean ± SEM. Repeated variables were analyzed using a two-way repeated-measures analysis of variance. Differences at each time point were compared by unpaired t test. Statistical analysis was performed using a commercial software (StatView version 5.0.1, SAS Institute Inc). A P value of .05 or less was considered statistically significant.
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Results |
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Histological and immunological assessment of engrafted MSCs
Immediately after infusion of MSCs, ß-gal-positive cells were consistently found in all sections of the heart. They were trapped within the coronary vasculatures in the noninfarcted (Figure 2, A to C)
as well as the infarcted areas (Figure 2, D), mostly within capillaries and some in small arterioles. No MSCs could be seen in the interstitium of the scarred myocardium at this time. Approximately 3.45 x 105 MSCs, that is 34.5% of the cells injected into the ascending aorta, were estimated to have been trapped within coronary capillaries immediately after injection. Among them 73% were seen in the noninfarcted myocardium of the hearts. Most of the MSCs found in the myocardium were located in the outer layer (subepicardial) rather than the inner layer of the ventricular wall.
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-smooth muscle actin (Figure 4, F) or for endothelial cell marker, factor VIII.
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Discussion |
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Several in vitro and in vivo studies have confirmed the plasticity of MSCs to differentiate into cardiomyocytes.5-9 It has been suggested that to induce MSCs to differentiate into cardiomyocytes, the cells need to be in close contact with the myocardial microenvironment, but within a scar they require in vitro treatment with 5-azacytidine prior to implantation.6,7 Although the exact mechanism of action of 5-azacytidine has not been elucidated, Konieczny and colleagues22 proposed that embryonic cells contain a myogenic determination locus in a methylated state while in a transcriptionally inactive phase, which becomes demethylated and transcriptionally active with 5-azacytidine, causing the cells to differentiate into myogenic cells. Tomita and colleagues have reported that when implanted directly into a scar, only MSCs treated with 5-azacytidine showed positive staining for troponin I and myosin heavy chain, and they contributed to improved cardiac function by preventing scar thinning and chamber dilatation.6 The results of our more recent study comparing 5-azacytidine treated with nontreated MSCs appear consistent with their findings.23 In the current study, we again confirmed the importance of the microenvironment for cardiomyogenic differentiation. The morphological appearance of the MSCs in the center of the scar was different to those in the periscar area, where the cells were surrounded by viable host cardiomyocytes. The latter showed well-organized contractile proteins, with small nuclei to cytoplasm ratio, and connected to adjacent host cardiomyocytes with connexin 43 positive gap junction, although the cells within the scar seemed less differentiated. We speculate that the cells found in the periscar area might have received more adequate in situ signals for cardiomyogenic differentiation than those within the scar. Besides the cardiomyogenic phenotype, we also found some labeled MSCs showing vascular endothelial or smooth muscle cell phenotypes integrated into vascular walls. They were likely involved in the angiogenesis and vasculogenesis in the healing process of myocardial infarction.10
It has been shown that MSCs implantation results in an improved cardiac function.6,9 Even though these studies showed improvement in cardiac function, exact mechanism remained unclear, as there was scanty evidence of gap junction formation between neomyofibers in the scar and the native myocardium outside of the infarct, making synchronous contraction unlikely. It has been speculated that function might be improved by the attenuation of adverse remodeling and enhanced contractility of cardiomyocytes. The improvement in regional function probably resulted from a combination of factors including myogenesis and angiogenesis.6 Clearly further mechanistic studies are warranted.
Limitations in the scope of this study also prevented us from addressing a number of important and relevant issues. Recently there was a major concern about the existence of true "pluripotent adult stem cells," as Terada and coworkers24 and Ying and colleagues25 reported that MSCs could simply fuse with existing differentiated cells, with the appearance that they had differentiated into cells of other phenotypes. However, this notion had been challenged subsequently by a number of authors, such as that from Verfaillie’s group,26 who conclusively demonstrated that a single adult stem cell derived from the bone marrow could be guided to differentiate into various specific phenotype, in the absence of any preexisting differentiated cell to which an adult stem cell could fuse with. Tomita and colleagues27 cocultured MSCs with cardiomyocytes in vitro and reported that the MSCs expressed various phenotype specific proteins, such as troponin I-C, connexin-43, and atrial natriuretic factor, in sequence rather than simultaneously. This observation also supports that MSCs undergo differentiation rather than fusion. This is because in a fusion, the cardiomyocyte that fuses with MSC should already contain all the phenotype-specific proteins, so that it is not possible for these markers to become detectable one by one over several days. We have discussed the phenomenon of such milieu-dependent differentiation in greater detail elsewhere.28
Another important limitation in this study is that we did not attempt to use specific cell markers to characterize and define the preimplant properties of MSCs, as we stated earlier. It should be noted, however, that the clonal definition of MSCs is not fully standardized yet at present, as the investigators still use various different cell markers, cellular culture, and isolation techniques in their studies. Likewise, the specific molecular signals for MSC homing and differentiation remain unknown and will also need to be elucidated in future studies.
Although intracoronary injection of cells provides certain advantages, coronary embolism leading to myocardial infarction is a major concern with this delivery approach.21 In our preliminary study, we bolus injected a greater quantity of cells (eg, 5 x 106) and found higher mortality rate (more than 50%). As soon as the MSCs were infused, hearts became pale and stopped beating due to embolism. Therefore, to prevent embolic myocardial damage, measures to avoid cellular aggregation, as well as slower or multiple infusions of diluted cells, need to be considered. Clinically, this approach may be most suited for patients who have heart failure caused by diffuse cardiomyopathy.
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