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a Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, Shanghai, China
b Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio
c National Heart Center, Singapore
d Department of Surgery, National University of Singapore, Singapore
e Gleneagles JPMC Cardiac Center, Brunei Darussalam
Accepted for publication August 14, 2008.
* Address correspondence to Dr Guo, Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Rd, Shanghai City, 200032, China (Email: drguocf{at}yahoo.com; sursimkw{at}nus.edu.sg). A/P Eugene K. W. Sim, Department of Surgery, National University of Singapore, Singapore 117597 (Email: drguocf{at}yahoo.com; sursimkw{at}nus.edu.sg).
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Abstract |
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Methods: One week after myocardial infarction, 128 animals were divided into four groups: group 1 (n = 24, receiving medium only), group 2 (n = 24, receiving medium and cyclosporine), group 3 (n = 40, autologous skeletal myoblast transplantation), and group 4 (n = 40, allogeneic skeletal myoblast transplantation with cyclosporine treatment). Rats were euthanized 10 minutes, 1 day, and 4, 7, and 28 days later. Host immune cell kinetics were assessed by immunohistochemical studies for macrophages, and CD4+ and CD8+ lymphocytes. Donor skeletal myoblast survival was confirmed by tracking prelabeled signals, and quantified by β-gal assay. Heart function was evaluated by echocardiography.
Results: A transient immune cell infiltration was demonstrated in group 3, with macrophage infiltration on day 1 and day 4, CD8+ cell infiltration on day 4 and day 7, and CD4+ cell infiltration on day 4. In group 4, immunocyte infiltration was slightly more severe than that in group 3. Automyoblasts and allomyoblasts showed no significant difference of survival from day 1 to day 7 (p > 0.10); however, on day 28, automyoblasts showed better survival than allomyoblasts (p < 0.05). Transplantation of allomyoblasts increased systolic heart function and limited heart dilation after myocardial injury to a similar degree as automyoblasts (p > 0.10).
Conclusions: The use of allomyoblasts is feasible and effective for cardiac repair with immunosuppressive treatment as compared with automyoblasts.
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Introduction |
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In the present study, we transplanted an equal number of SkMs from autologous or allogeneic origin into infarcted myocardium, and therefore provided direct comparison between automyoblasts and allomyoblasts after engraftment with respect to host immune cell dynamics, SkM survival profile, and SkM graft efficacy for cardiac repair. This study would give us a better understanding of the early cellular behavior of automyoblasts and allomyoblasts and allows us to assess the feasibility of allogeneic myoblast transplantation.
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Material and Methods |
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Isolation and Expansion of SkMs
Isolation of SkMs was performed from Wistar rats, as described previously [7]. Forty-eight hours before muscle harvest, 0.5 mL bupivacaine (0.5%; Astra, North Ryde, Australia) was injected into the tibialis anterior muscles of rat hind limbs to achieve a better cell yield. Skeletal muscle (1 g) was harvested, minced, and enzymatically dissociated with collagenase IA (2 mg/mL; Sigma, St. Louis, MO) for 1 hour, dispase (2.4 U/mL; Invitrogen, Carlsbad, CA) for 1 hour, and trypsin-ethylenediamine tetraacetic acid (0.25%; Sigma) for 30 minutes. The muscle cell extract was preplated three times at regular time intervals to increase the purity of the purified SkMs. The cells were cultured in patented Super Medium (Cell Transplants, Singapore) for 4 days after initial seeding, and then fed with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.1 mg/mL L-glutamine until they achieved the required number. The purity of SkMs was assessed by means of immunostaining and flow cytometric analysis for desmin expression with desmin-specific antibody (1:50, Sigma).
Preparation of SkMs Before Transplantation
For post-transplantation identification, SkMs were double-labeled with lac-z reporter gene and 4', 6'-diamidino-2-phenylindole hydrochloride (DAPI [Sigma]). The SkMs were grown to achieve 60% confluence. The cells were first incubated in 15 mL transduction medium containing 1 x 106 particles/mL retroviral vector carrying lac-z reporter gene. Eight hours later, the viral infection medium was replaced with culture medium for 24 hours. The transduction procedure was repeated three times. Transduction efficiency was assessed by means of histochemical staining for lac-z expression.
Before transplantation, SkMs were also incubated in DAPI culture medium (20 µg/100 mL) for 24 hours. Incorporation efficiency of DAPI was calculated by dividing the number of blue fluorescent nuclei counted by means of fluorescence microscopic analysis by the total number of cells counted by means of phase-contrast microscopic analysis. Before cell transplantation, the viability of SkMs was assessed using trypan blue (0.4%, GIBCO, Gaithersburg, MD) exclusion method.
Histochemistry for Lac-z Expression
The lac-z–transduced SkMs were fixed with 0.5% glutareldehyde for 15 minutes at room temperature. After rinsing, the cells were incubated overnight at 37°C with 40 mg/mL 5-bromo-4-chloro-3indoyl-β-D-galactosidase (Bio-Rad, Hercules, CA) prepared in 1 x PBS buffer containing 30 mmol/L potassium ferricyanide, 30 mmol/L potassium ferrocyanide, and 2 mmol/L magnesium chloride. Next, the cells were observed with a light microscope (Olympus, Tokyo, Japan) for bluish nuclei. The labeling efficiency was calculated by dividing the number of bluish-nuclei cells counted by means of light microscopic analysis by the total number of cells counted by means of phase-contrast microscopic analysis.
Myocardial Infarction Model and Experimental Groups
A rat model of myocardial infarction was developed in Wistar rats. The animals were anesthetized with ketamine (50 mg/kg administered introperitoneally) and xylazine (10 mg/kg administered introperitoneally), and mechanically ventilated at a tidal volume of 5 mL (75 cycles/min). The infarction was created by means of permanent ligation of the left anterior descending coronary artery with a 6-0 polypropylene snare.
One week later, 128 animals with myocardial infarction were divided into 4 groups: group 1 (n = 24, receiving medium only), group 2 (n = 24, receiving medium and cyclosporine), group 3 (n = 40, autologous SkM transplantation), and group 4 (n = 40, allogeneic SkM transplantation with cyclosporine treatment). The hearts were injected with a total of 150 µL DMEM without cells or containing 3 x 106 SkMs at five different sites in and around the infarcted area. In group 2 and group 4, cyclosporine (10 mg/kg per day administered introperitoneally) was administered 5 days before cell transplantation and continuing to euthanasia.
The animals were euthanized at 10 minutes, 1 day, and 4, 7, and 28 days after cell transplantation. In control group 1 and group 2, there were 4 rats at each time point except 8 rats on day 28. The hearts were frozen in liquid nitrogen cooled isopentane only for histochemical studies (no heart was used for β-gal assay, because in our pilot study, no signal was detected). In group 3 and group 4, there were 8 rats at each time point. Four hearts were used for histochemical study, and the whole left ventricular free wall of the remaining 4 were homogenized for β-gal assay analysis.
Myoblast Survival by β-gal Assay
In group 3 and group 4, assay for β-gal activity was performed to evaluate the number of transplanted SkMs existing in the myocardium. Cultured SkMs were used as standard. Briefly, SkMs were trypsinized and counted. Such numbers (3 x 106, 1 x 106, 5 x 105, 2.5 x 105, 1 x 105, 5 x 104) of SkMs were homogenized in 0.25 mol/L Tris-HCl (pH 7.8). After SkM transplantation, the hearts (n = 4 at each point) were harvested at different timepoints and then homogenized. The homogenates (including those from serial dilated SkMs and from heart tissues) were centrifuged at 3,500g for 5 minutes and then 12,000g for a further 5 minutes. The supernatant (10 µL) was mixed with 90 µL of 4 mg/mL 5-bromo-4-chloro-3indoyl-β-D-galactosidase (X-gal; Bio-Rad) prepared in 0.1 mol/L sodium phosphate buffer containing 3 mmol/L potassium ferricyanide, 3 mmol/L potassium ferrocyanide, and 0.2 mmol/L magnesium chloride. The mixture was incubated at 37°C for 30 minutes, and the reaction was stopped by adding 500 µL of 1 mol/L Na2CO3. Optical density was read on a spectrophotometer at a wavelength of 420 nm. The SkM numbers after cell transplantation into myocardium were calculated using the standard curves formed by serial dilated SkMs.
Immunohistochemical Studies
Sections of 6 µm were cut from frozen slices and observed under a microscope for DAPI florescence (blue). The tissue sections positive for DAPI were processed for lac-z staining and further stainings. Host immune responses was monitored by immunostaining for the infiltration of CD4+ and CD8+ lymphocytes (1/50, 1 hour, 37°C; BD Pharmingen, San Diego, CA) and macrophages (1/20, 1 hour, 37°C; Serotec, Oxford, UK) using their specific antibodies, and then followed by the rhodamine conjugated secondary antibody (1/200, 1 hour, room temperature; Chemicon, Temecula, CA). The numbers of the infiltrating macrophages and CD4+ and CD8+ lymphocytes were counted by means of a microscope. In the process of counting cell numbers, five sections (positive for DAPI) were chosen in each rat, three sections in the middle of infarction and two sections at remote regions. For each section, three fields were chosen under microscopy of x400 magnification.
Heart Function Assessment
Heart function was assessed by means of two-dimensional echocardiography at 6 days after infarction as baseline determinations and 4 weeks after SkM transplantation to show the efficacy of SkM transplantation. Two-dimensional (and M-mode) measurements were performed with a 10 MHz linear-array transducer system (General Electric, Milwaukee, WI). Parastenal long-axis views were obtained to view the regional wall motion abnormalities. Short-axis M-mode views were obtained perpendicular to the midventricular level with sweep speeds of 200 mm/s, confirmed by two-dimensional echocardiography. To obtain a measure of heart function, dimensional fractional shortening (FS) and ejection fraction (EF) were computed according to the following formulas: FS = (left ventricular end-diastolic dimension [LVEDD] – left ventricular end-systolic dimension [LVESD]) / LVEDD x 100%; EF = (LVEDD2 – LVESD2) / LVEDD2 x 100%.
Statistical Analysis
The data was analyzed by SPSS 12.0 (SPSS, Chicago, IL). All values were expressed as mean ± SEM. One-way analysis of variance with post-hoc Tukey test was performed to assess the significant difference among multiple groups. The significant difference between two groups was evaluated by Student t test. A p value less than 0.05, was considered statistically significant.
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Results |
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Characterization of SkMs
The purity of SkMs was 74.96% for desmin expression when gated for control at 1.00% by flow cytometric assay (Fig 1A, B). The efficiency of DAPI and lac-z labeling were 100% and 85%, respectively (Fig 1C, D, E). Trypan blue dye exclusion showed more than 95% viability of the cell preparations before transplantation.
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Immune Cell Dynamics After SkM Transplantation
Immunostaining for macrophages, CD4+, and CD8+ cells and their count at different time points are shown in Figure 2. Macrophages were the first cells infiltrating into the donor myoblast pockets (Fig 2A, B). In group 3 (autograft), a transient macrophage infiltration was demonstrated on day 1 and day 4. The macrophage numbers on day 1 and day 4 were 13.2 ± 1.57 and 14.6 ± 1.43, respectively, which showed significant increase as compared with 6.8 ± 0.86 and 7.2 ± 0.85 in control group 1. Then the macrophage number declined to 9.4 ± 1.20 on day 7, and to 8 ± 1.00 on day 28, showing no significant difference as compared with those in control group 1 (p > 0.10). In group 4 (allograft with cyclosporine), a similar macrophage infiltration was observed on day 1 and day 4, with macrophage number was 12.2 ± 1.06 and 15.2 ± 1.39, respectively. Then, on day 7, the macrophage number peaked at 16.8 ± 1.93 and significantly increased as well (control: 7.2 ± 1.11, p < 0.05), whereas on day 28, the macrophage number declined to 7.6 ± 0.81, showing no difference as compared with the control group.
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Figure 2E andF show the CD4+ cell infiltration. In group 3 (autograft), the transient infiltration was demonstrated only on day 4 (6.8 ± 0.97 as compared with 3 ± 0.84, p < 0.05). In group 4, the transient infiltration was on day 4 (6.2 ± 0.49 compared with 3 ± 0.84, p < 0.05) and day 7 (8.8 ± 0.58 compared with 3 ± 0.71, p < 0.05).
Time Course of Auto- and Allo-SkM Survival
Table 1
shows the quantification of SkMs at different time points after cell engraftment. There was no significant difference in actual quantity of SkMs at 10 minutes among groups (1.44 to 1.53 x 106 SkMs were left), and the average SkM number at 10 minutes after cell transplantation was used as baseline. In group 3 and group 4, a remarkably rapid and massive loss of β-gal signal was observed after injection of SkMs. A total of 88.7% and 89.8% of the SkMs were lost in group 3 and group 4, respectively, during the first 24 hours. Subsequently, the number of SkMs showed gradual increase until 4 weeks, which was probably a result of proliferation of surviving SkMs. In group 3 (autograft), the SkM numbers at days 1, 4, 7, and 28 were 11.3% ± 0.9%, 19.1% ± 2.3%, 38.0% ± 4.3%, and 54.8% ± 1.8%, respectively; whereas in group 4 (allograft plus cyclosporine treatment), the SkM numbers at days 1, 4, 7, and 28 were 10.2% ± 1.4%, 17.6% ± 2.2%, 35.2% ± 4.4%, and 49.4% ± 5.1%, respectively. On days 1, 4, and 7, there was no significant difference of the SkM numbers between group 3 and group 4 (p > 0.10); however, on day 28, the SkM survival in allograft (in group 4) showed a significant decrease as compared with that in group 3 (p < 0.05).
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Comment |
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In the clinical setting, human allogeneic SkM transplantation provided an alternative therapy for cardiac repair with many advantages as compared with autografts. The allografts are on-shelf available with virtually unlimited cell availability, which overcomes the time delay of about 3 to 4 weeks in autograft transplantation. Importantly, these allogeneic cells are from the young adults, which might be more regenerative than those from older patients. Another advantage of allomyoblast transplantion is that allomyoblasts are more cost efficient. It is not only a waste of time, but also costs more money for autografting since one isolation procedure of autologous cells only provides the use of one transplantation. Moreover, patients with infectious disease, such as acquired immunodeficiency syndrome, cannot be treated with autografts for fear of contamination of the whole cell culture system.
One contribution of our study is the comparison of the immune cell dynamics after automyoblast and allomyoblast transplantation (with cyclosporine therapy) into infarcted myocardium. In 2003, Pagani and colleagues [3] demonstrated there was little or no evidence of lymphocyte infiltration after autologous myoblast transplantaiton. However, in our present study, we did detect a transient immunocyte infiltration in group 3 (autograft). The discrepancy may lie in the different time spots of observation. In the Pagani study, they observed the infiltration of lymphocytes at an average time of 122 days after myoblast transplantation, whereas we observed the cell infiltration mainly in the acute phase. The immunocyte infiltration might result from the external antigenic molecules expressed after exposure to tissue culture conditions [8] or transfer of genes (lac-z gene in the present study). In the present study, with cyclosporine treatment in allogeneic myoblast transplantation (in group 4), we found a similar or a little bit more severe infiltration of immunocytes as compared with that in group 3. Importantly, the number of infiltrating immunocytes in group 4 was also decreased to a low level after 4 weeks. The declination of the immunocytes at 4 weeks after allomyoblast transplantation may result from the immunosuppression by cyclosporine, the relatively privileged transplantation site of myocardium where presentation and recognition of major histocompatibility complex may not take place because of the lack of a lymphatic drainage system [9], and the nature of the cells with less expression of major histocompatibility complex, which might be due to the differentiation of SkMs or establishment of chimerism [10]. This finding is of great clinical significance because it may bode well for the prospects of using allogeneic SkMs for transplantation in humans.
Our study has also compared the early SkM survival profile after autologous and allogeneic transplanted SkMs. In our study, there was no significant difference in actual quantity of SkMs at 10 minutes among groups (1.44 to 1.53 x 106 SkMs were left). After 10 minutes, the majority of the remaining SkM signals were rapidly lost by day 1, maybe resulting from mechanical leakage and washout [11, 12], cell redistribution to other organs [13], and cell death after transplantation [14–16]. From day 1, a gradual increase in the number of myoblasts was observed until 4 weeks after cell transplantation, resulting from the SkM proliferation outbalancing the gradual loss. Previous studies have shown that SkM proliferation is stimulated by several growth factors, such as fibroblast growth factor 2, epidermal growth factor, insulinlike growth factor 1, and stem cell factor [17]. Because SkM proliferation stops on differentiation, the proliferation might be enhanced by transforming growth factor-β, which represses myoblast differentiation [18]. In addition, some isoforms of platelet-derived growth factor also regulate myoblast proliferation and differentiation [19]. To our knowledge, the mechanism that regulates SkM proliferation remains unclear after transplantation into the myocardium, and might be an interesting field for future investigation. In our study, as compared with automyoblasts, allomyoblasts (with cyclosporine) showed a similar surviving number in the early stage (from day 1 to day 7); however, on day 28, there was a significant difference of the survival numbers between auto-SkMs and allo-SkMs. Previous animal studies have accumulated some evidences of allomyoblast transplantation into myocardium [20, 21], and demonstrated SkM allogeneic acceptance without immunosuppressive as like mesenchymal stem cells [22]. Using cyclosporine as an immunosuppressive, we found the allomyoblast survival could be enhanced by cyclosporine treatment [7]. Furthermore, this present study demonstrated minimal decrease of survival number using allomyoblasts (49.4% ± 3.1% on day 28) for transplantation as compared with automyoblasts (54.8% ± 1.8%). These data strongly suggest the allogeneic myoblast acceptance by the host and feasibility of allomyoblast transplantation.
In our study, allo-SkMs improved systolic heart function and limited the dilation of the left ventricle to a similar degree as compared with auto-SkMs. As yet, the mechanisms responsible for the beneficial effects after myoblast transplantation remain elusive. However, a larger size of skeletal graft would probably lead to greater benefits. A previous study has demonstrated a dose-response effect of SkMs on improvement of heart function [23]. But if the increase of cell survival is not big enough (from 49.4% ± 3.1% in group 4 to 54.8% ± 1.8% in group 3), it might not result in significant improvement in heart function, as shown in our study.
In conclusion, the use of allomyoblasts is feasible and effective for cardiac repair with immunosuppressive treatment.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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