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Cellular Plasticity in the Cardiovascular System
Cardiovascular Division, Departments of 1Medicine and 2Radiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Submitted 30 March 2004 ; accepted in final form 30 March 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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cellular therapy; heart failure; hypertrophy; infarction; neovascularization
In mice, bone marrow mesenchymal stem cells (MSCs) transplanted into the myocardium differentiated into myocytes, endothelial cells, and smooth muscle cells (13). Moreover, neovascularization and increases in functional LV mass occurred (13, 21). The therapeutic potential of cell transplantation in cardiovascular disease is significant, but investigators have not reached a consensus on the best cell type or the best delivery method. As an example of the former point, the use of allogeneic cell sources has the potential for side effects due to the requisite immunosuppression. As an example in the latter point, delivery methods typically described in the literature consist of direct intramyocardial injection (13) or LV injection (21) with low homing efficiency into the myocardium. We have addressed both points by creating a novel fibrin biopolymer patch seeded with autologous bone marrow stem cells that can be surgically implanted directly onto areas of MI. We hypothesized that holding the cells adjacent to damaged myocardium would enhance their reception of soluble ischemic signals and subsequent homing into damaged myocardium.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Adenovirus Vectors
Replication-deficient recombinant adenoviruses carrying the 
-galactosidase reporter gene lacZ under the control of Rous sarcoma virus long-terminal repeat (RSV-LTR) promoters were purchased from the University of Iowa Gene Vector Core (Dr. Richard Anderson).
Isolation of Swine Mesenchymal Stem Cells
MSCs from bone marrow were isolated by gradient density centrifugation (14). Bone marrow was aspirated from the sternum of healthy Yorkshire pigs into a syringe containing 6,000 units of heparin and diluted with Dulbecco's PBS in a ratio of one to one. The marrow sample was carefully layered onto the Ficoll-Paque-1077 (Sigma) in a 50-ml conical tube and centrifuged at 400 g for 30 min at room temperature. The mononuclear cells were collected from the interface, washed with 2–3 volumes of Dulbecco's PBS, and collected by centrifugation at 1,000 rpm. The cells were resuspended and seeded at a density of 200,000 cells/cm2 in T-150 flask coated with 10 ng/ml fibronectin and cultured in medium consisting of 60% low-glucose DMEM (GIBCO-BRL), 40% MCDB-201 (Sigma), 1x insulin transferrin selenium, 1x linoleic acid-bovine serum albumin (LA-BSA), 0.05 µM dexamethasone (Sigma), 0.1 mM ascorbic acid 2-phosphate, 2% FCS, 10 ng/ml PDGF, 10 ng/ml EGF, 100 U/ml penicillin, and 10 µg/ml streptomycin. After 3 days, nonadherent cells were removed by replacing the medium. The attached cells grew and developed colonies in 
5–7 days. After 10 days, the primary cultures of MSC reached nearly 90% confluence; cells were subcultured by incubation with trypsin. The first-passage cells were plated at 4,000–5,000 cells/cm2 and further cultured 2 days for the transduction with AdRSV-LacZ.
Cell Preparation for Transplantation
First-passage swine MSCs were plated at 5,000/cm2 in modified DMEM with 2% FBS. One day later, the cells were washed with serum-free modified DMEM and infected overnight with AdRSV-lacZ at 1,500 multiplicity of infection (MOI). The supernatant was then removed, and the cells were washed with PBS and then cultured with modified DMEM containing 2% FCS. The medium was repeatedly changed over 2 days (
6 changes) to ensure complete removal of viral particles and to allow for the internalization of any particles remaining on the surface. On the day of surgery, the cells were harvested with 0.25% trypsin-EDTA (Invitrogen), washed with PBS, and resuspended in 0.5 ml saline.
Phenotype
CD44, CD45, CD90, myosin heavy chain (MHC)-Class I, MHC-Class II, SWC3A, and SLA-DR were detected by flow cytometry. MSCs (0.5 x 106) were placed in 100 µl BSA-PBS solution for each phenotype test and incubated with 2 µg primary mouse monoclonal antibodies (mAbs) against pig CD44, CD45, CD90, MHC-Class I, MHC-Class II, SWC3A, and SLA-DR for 30 min at 4°C. The second polyclonal antibody IgG against mouse conjugated FITC (1 µg/tube) was added and incubated at 4°C for an additional 30 min in a dark room. Mouse IgG (2 µg) instead of primary mAbs was added to 0.5 x 106 cells for a negative control.
Cardiomyocyte Differentiation of MSCs
The differentiation of myocytes from MSCs was performed as previously described (19). The second passage of porcine bone marrow-derived MSCs was seeded into 35-mm dishes at a density of 20,000 cells/cm2 in DMEM medium containing 10% FCS and 1% antibiotics (100 U/ml penicillin and 10 µg/ml streptomycin). On the second day, the medium was changed, and the cells were exposed to the medium with 20% FCS or treated for 24 h with medium consisting of 20% FCS and 5-azacytidine (6 µM/l). Thereafter, the cells were cultured in medium consisting of 20% FCS and 1% antibiotics, and the medium was changed twice a week for 2 wk until the experiment was terminated. Control cells were cultured in DMEM supplemented with 5% FCS and antibiotics. Myogenesis was determined by RT-PCR and immunostaining. PCR primers and reaction conditions are depicted in Table 1.
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-Galactosidase Reporter Gene in the Patch and in the Periscar Region by PCR
MSCs (5 x 106) at 95% transduction efficiency with Ad5RSV-lacZ vector (Gene Transfer Vector Core, University of Iowa) were suspended in a reconstituted solution of thrombin and fibrinogen to form the patch. The patch containing swine MSCs that express the lacZ reporter gene was applied to the epicardial surface of the expected infarct area with a biological adhesive. At 21 days posttransplantation, the patch and the periscar region of LV were resected for PCR analysis. The DNA template was extracted with a High Purity PCR Template Preparation Kit (Roche Molecular Biochemicals). Advan Taq plus DNA polymerase and 10x Advan Taq plus PCR buffer (Clontech) were used in the reaction system; PCR nucleotide mix (10 mM) was purchased from USB. The forward primer was 5'-CAT-GCC-GAT-TGG-TGG-AAG-TAA-3' complementary to sequences of RSV-LTR to detect RSV promoter in replication-deficient recombinant adenoviruses (Ad5RSV-lacZ); the reverse primer was 5'-AAA-GCG-CCA-TTC-GCC-ATT-3'.
Analyses of Neovascularization: RNA Isolation and cDNA Preparation
Snap-frozen LV specimens were pulverized in liquid nitrogen. Total RNA was isolated with RNeasy columns with RNase-free DNase treatment. Reverse transcription reactions were performed using 1 µg of total RNA as described by the manufacturer (Clontech) using oligo(dT)18 as a primer.
Quantitative Real-Time RT-PCR
Changes in mRNA levels under different experimental conditions were compared by quantitative real-time RT-PCR analysis using the Light Cycler thermocycler (Roche Diagnostics) as previously described (24).
Animal Model
A swine model of postinfarction LV remodeling (10, 26) was used to examine the behavior of autologous swine MSCs, homing into the heart. Briefly, young pigs (45 days) were anesthetized, and a left thoracotomy was performed. The left descending coronary artery distal to the first diagonal segment was dissected free and occluded with a ligature to generate MI. The coronary artery was reopened after 60 min of no-flow ischemia. After systemic hemodynamics had stabilized, the fibrin-swine MSC patch was applied to the LV anterior wall. After the surface was prepared, about 5 ml of fibrin gel mixed with MSCs (107) were applied to the MI area.
Fibrin Biomatrix
A fibrin biomatrix gel was formed from a fibrinogen solution (2.5 ml) (i.e., a solution containing fibrinogen polypeptides) by combining the fibrinogen solution with a solution containing fibrinogen-converting serine protease thrombin. Without being bound by a particular mechanism, fibrinogen in the fibrinogen solution was converted to fibrin through a proteolytic reaction catalyzed by thrombin. Fibrin monomers then aggregated to form a flexible biomatrix. About 107 MSCs were added to the thrombin solution (2.5 ml). On exposure to a fibrinogen solution, the forming fibrin biomatrix included the MSCs. Fibrinogen solution that contained 100 mg/ml and thrombin solution containing 400 IU/ml were chosen for making the biomatrices, which was suitable for cell survival and function based on the pilot studies. Flexibility can be altered by adding fibrinolytic inhibitors (e.g., tranexamic acid at 9.2% wt/vol or aprotinin at 3,000 KIU/ml, where KIU is kallikrein IU) or anticoagulants (e.g., trisodium citrate at 3–10 mg/ml or glycine at 10–40 mg/ml) to the solutions. In addition, such components can be used to alter the polymerization time associated with biomatrix formation. Typically, the biomatrix reaches a gel-like state within a few seconds of the two solutions mixed by injection to a restricted area and stabilizes into a flexible semisolid state within 1 min. A homemade plastic ring was placed on top of the surface of the MI area to restrict the flow of the solutions while solutions form the fibrin gel within 1 min.
The plastic ring was then removed. The thoracotomy was then repaired and the animal allowed to recover. The times forming the matrix can be altered by using the components listed above or additionally by varying the concentration of fibrinogen, fibrinogen-converting agent or cofactors that assist in fibrinogen conversion (e.g., CaCl2). Biomatrix components fibrinogen and thrombin were from Sigma (St. Louis, MO).
Eighteen to twenty-one days after surgery, the animals were returned to the laboratory for magnetic resonance imaging (MRI) and terminal studies.
MRI
All MRI studies were performed 18 days after coronary artery ligation on a standard Siemens Medical System Vision operating at 1.5 Tesla as previously described (26).
Cell Immunohistochemistry
The cultured MSCs were immunochemically stained. The monoclonal antibodies, including a mouse monoclonal antibody against troponin T, cardiac isoform Ab-1 (clone 13–11, NeoMarkers; Fremont, CA), a mouse monoclonal antibody against phospholamban (MA3-922, Affinity Bioreagents; Golden, CO), a mouse monoclonal antibody against muscle-specific actin that reacts with smooth muscle as well as 
-skeletal, and -cardiac sarcomeric isoforms of actin (clone MSA06; same as HUC1-1, NeoMarkers) were used. The cells were incubated with primary antibodies. After five washes in Tris-buffered saline (TBS) for 5 min each, the biotinylated anti-mouse IgG (Dako) was applied for 30 min at a dilution of 1:400. Visualization was achieved with the streptavidin-biotin horseradish peroxidase detection system.
Tissue Preparation
After the MRI experiments were completed, the animals were anesthetized, and the thoracotomy incision was reopened. The area of the patch was examined for adhesion, fibrosis, and residual remnants of the fibrin matrix. The heart was excised, and integrated specimens including the fibrin patch and the underlying myocardium were collected and either frozen in liquid nitrogen for blotting, PCR, and X-gal staining or fixed in 4% formaldehyde for histological and immunohistochemical analyses. Sections 5 µm in thickness were cut and stained with hematoxylin and eosin (H&E).
Histology and Microscopy to Evaluate Myocyte Differentiation
For immunohistochemical staining to evaluate myocyte differentiation, tissues were harvested, cryoprotected in cold 2-methylbutane for 1 h, and then embedded in Tissue-Tek OCT (Fisher Scientific). Cryostat sections (10 µm) were obtained for X-gal staining and immunohistochemistry study.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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To determine the phenotypic nature of swine MSCs, the surface antigens CD44, CD45, CD90, MHC-Class I, MHC-Class II, SWC3A, and SLA-DR were examined by flow cytometry using about 1.5 x 105 cells per 100 µl labeled with primary mAbs (2 µg each) against pig CD44, CD45, CD90, MHC-Class I, MHC-Class II, SWC3A, and SLA-DR. Cells were incubated at 4°C for 30 min and washed; the second polyclonal antibody-FITC conjugated against mouse IgG (1 µg/tube) was added and incubated at 4°C for an additional 30 min. The mouse isotype IgG (2 µg) was added to 1.5 x 105 cells instead of primary mouse antibodies as a negative control. The results are illustrated in Fig. 1. The thick lines indicate phenotype profiles of swine MSC; the thin line represents the isotype control IgG-staining profile. The phenotype profiles of swine MSC were shown to be negative for CD45, MHC-Class II, and SLA-DR and positive for CD90, CD44, SWC3A, and MHC-Class I (Fig. 1).
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To demonstrate the potential of swine MSC differentiation into cardiomyocytes in vitro, cells were cultured in 20% FCS and 5-azacytidine medium. After 14 days, early and late gene markers of myocyte differentiation were identified by immunohistochemistry and RT-PCR (Fig. 2A). Figure 2A illustrates the RT-PCR experiments from adult cardiac myocytes (left), swine MSC (middle), and myogenic differentiation cells on day 14 (right). Results showed that swine MSC expressed a number of muscle-specific protein genes, including phospholamban and -muscle-specific actin (Fig. 2A). After myogenic differentiation by using 5-azacytidine as an inducer, desmin changed from negative to positive, and phospholamban expression was significantly increased (Fig. 2A). Figure 2, B and C, illustrates immunostaining with troponin T antibody that clearly showed longitudinal fibers in 20% FCS and 5-azacytidine-treated cells (Fig. 2C, day 14), which was not seen in control cells (Fig. 2B).
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To track and identify swine MSC in vivo, the cells were transduced by the recombinant AdRSV-lacZ adenovirus at different concentrations. The maximum number of cells expressing -galactosidase was achieved at 1,500–2,000 MOI in the absence of an effect on growth and differentiation (Fig. 2, D–F). Consequently, this concentration was selected to use for labeling cells for patch transplantation experiments.
To examine transduced swine MSC behavior and proliferation potential within the fibrin matrix in vitro, equal volumes (2.5 ml) of the reconstituted fibrinogen and thrombin solution containing 2 x 106 cells were mixed and pooled into a 25-mm Falcon culture dish using two syringes that hold fibrinogen and thrombin solutions, respectively. The mixture of two solutions that entrapped MSCs was gelled within a few seconds and then overlaid with 3 ml of the stem cell medium; cultures were incubated at 37°C in 5% CO2-95% air, and the medium was changed every 2 days. Using an inverted contrast microscope, we found transduced MSC entrapped in the fibrin gel that started to migrate from the fibrin matrix into the stem cell culture medium (Fig. 2F).
Swine MSC Patch Transplantation
We tested the hypothesis that transduced swine MSC delivered by a fibrin patch would respond to the ischemic stimuli to survive, migrate, and home into the infarcted myocardium, thereby improving heart function. In vivo studies were performed in three groups of animals: transplantation group (Tx, n = 8), patch without stem cell group (P, n = 6), and a group with left anterior descending coronary artery ligation alone (60 min) (L, n = 6). A fibrin-MSC patch was applied on the surface of the myocardial infarct. Eighteen to twenty-one days after left anterior descending coronary artery ligation, MRI was employed to assess LV function. The MRI data demonstrated that in hearts with postinfarction LV remodeling, the systolic wall thickening fraction was significantly improved in hearts with fibrin-stem cell patch transplantation (Fig. 3).
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-galactosidase). Striation of myocytes can be clearly observed with staining for cardiac-specific troponin T (Fig. 4F). In Fig. 4G, H&E and X-gal costaining are as indicated.
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Twelve biopsy specimens (1 mm3 each) from necrotic and border zones were taken from each LV and placed into T25 flasks for culture using stem cell culture medium. The total number of stem cells that migrated into the ischemic myocardium was obtained relative to the volume of the biopsy specimen (weight x 1.06 as the density of myocardium); data represent the mean of eight counts of specimens from each heart. The identical specimen collection and culture procedure were also performed in areas remote from the infarct in hearts in P and L groups. No blue cells were found from the cultured specimen of the P or L hearts. The homing efficiency (%) was calculated as efficiency % = 100 x total number of cells migrated into the LV per number of stem cells transplanted. Blue areas were carefully cut and weighed. Counting the blue cells in the infarcted area (including the periscar zone) indicated that 10% of transplanted cells migrated to the myocardial infarct.
Pathological Evaluation
Figure 4, H and I, illustrates typical light microscope evaluation of pathological sections of the periscar areas of a transplanted heart. The spindle-shaped autologous blue MSCs were found in the necrotic areas (Fig. 4I, bottom right panel). In addition, blue cells migrated into the periscar area where there were spared myocytes (Fig. 4I, top right panel). These cells assumed a rod shape, suggesting differentiation into myocytes. Furthermore, these rod-shaped blue cells were oriented in the same direction as the native cardiac myocytes (Fig. 4I, left panel). To prove these rod-shaped blue cells were nascent myocytes, double staining for cardiac-specific proteins was performed (Fig. 4, J–Q). Myogenic differentiation is evidenced by -galactosidase colocalized with -actin (Fig. 4, J and K), desmin (Fig. 4, L and M), phospholamban (Fig. 4, N and O), and cardiac-specific troponin T (Fig. 4, P and Q). Figure 4, J, L, N, and P, and Fig. 4, K, M, O, and Q, show cross-sectional and longitudinal sections, respectively. These data demonstrate that these blue rod-shaped cells derived from exogenous MSCs were cardiomyocytes. Taken together, these data indicate that stem cell differentiation is critically dependent on the microenvironment in which the stem cell resides. Only the stem cells that had migrated into the vicinity of native cardiomyocytes showed evidence for differentiation into new myocytes.
The MRI cine data demonstrate such a remarkable improvement of LV systolic contractile performance in the MI area of hearts that received patch-based stem cell transplantation (Fig. 3) that, with only a few blue myocytes identified, it indicates that mechanisms other than the regenerating contractile mass may also contribute to the LV function improvement.
MSC Patch Transplantation Promotes Neovascuarization
Figure 5A shows a specimen taken from the periscar area, including the interface between the patch and spared myocardium. The pale white area was from the patch, and the brownish-red area was from the spared myocardium. The arrow points to a vessel, which proved (H&E staining) to be an artery (Fig. 5A, top and bottom panels). The arrowhead in the left panel points to the patch area, which demonstrated (H&E staining) active neovascularization processes (Fig. 5B). The arrow points to a new vessel. H&E staining and high-power evaluation of the arrow-pointing area demonstrate the vesicular structure (Fig. 5A, right). These data indicate very active vessel formation in the scaffold of the patch area (Fig. 5, A and B). Immunofluorescent staining utilizing von Willebrand Factor (vWF) and caveolin-1 antibodies revealed a significant increase in neovascularization, because many vWF-containing angioblasts and capillaries were observed in the patch area of Tx hearts (Fig. 5, C–K, and Fig. 6, A–C). The histological data revealed a significant increase of neovascularization as evidenced by microvascularity, cellularity, and numbers of vWF-containing angioblasts and capillaries in the myocardial tissue underlying the patch: mean numbers of vWF+ capillary per high-power field are 58 ± 6 and 31 ± 5 (P < 0.001) for Tx and P hearts, respectively. There is no significant difference in capillary density in areas remote from the LV scar among the three groups.
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Utilizing real-time quantitative PCR, we found a significant increase in vWF mRNA expression in the patch area of Tx hearts compared with P hearts (Fig. 6D). No significant difference in capillary numbers and vWF mRNA expression was observed in areas remote from the LV scar among the three groups (Fig. 6D). Taken together, these data suggest that increased neovascularization in response to the cellular therapy also contributes importantly to sparing of the periscar myocardium by supplying oxygen and nutrients to newly derived myocytes from MSC and consequently improving LV contractile performance.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The characteristics of bone marrow MSCs were established by Pittinger et al. (14). These investigators included 10% FBS in the culture medium and emphasized that the selection of the specific FBS lot number was important for the success of MSC selection (14). It is likely that the high concentration of serum in the culture medium contained some unknown growth factors that made these selection experiments successful. In the present study, the use of a culture medium with a low serum concentration (FBS, 2%), but added growth factors (PDGF, epidermal growth factor), resulted in 100% successful MSC selection from each individual marrow aspirate. These data demonstrate that a growth factor-enriched, low-serum culture medium is effective for the selection of MSCs.
Stem Cell-Fibrin Patch
We aimed to create an environment in which the autologous MSCs would be in direct contact with the ischemic myocardium to observe whether the MSCs would migrate toward the ischemic tissue. The fibrin patch is formed when the solutions of fibrinogen and thrombin are combined and the MSCs are entrapped in the resultant matrix. This patch method could potentially be employed with a minimally invasive technique using endoscopic access to the pericardial sac. Using this technique, we observed MSCs appearing to move out of the fibrin matrix (Fig. 4, D and E) and home into the ischemic myocardium (Fig. 4) with high efficiency (10%).
MSC Differentiation
It has been customarily believed that myocytes do not regenerate after birth. However, the recent finding that stem cells with transdifferentiation potential exist in postnatal tissues opens the possibility of using stem cells to treat MI and heart failure secondary to discrete LV injury (3, 5–6, 13–16, 18, 21). With the use of a porcine model of postinfarction LV remodeling and immunohistochemical methods, the present study demonstrated that autologous MSCs homed into the infarcted region and differentiated into myocytes as evidenced by cardiac-specific troponin T staining (Fig. 4, J–K). This is in agreement with recent reports that transplanted allogenic bone marrow stem cells were able to differentiate into myocytes in periscar areas (4, 13). There is concern that the blue cells might represent host cells expressing some level of endogenous -galactosidase. To exclude this confounding factor, we designed a primer and performed PCR to determine whether these blue cells do express the Ad5RSV-lacZ gene. Indeed, PCR amplification of specimens from the blue area gave the Ad5RSV vector and lacZ-combined DNA sequence that was transduced by the adenovirus (Fig. 4C). This is the first evidence at the gene level of transplanted autologous MSC surviving in the postischemic myocardium. These data demonstrate that X-gal-positive myocytes are derived from transplanted stem cells and are not endogenous cells expressing -galactosidase. The blue myocyte-like cells were smaller than normal myocytes (60 µm in length, Fig. 4, F and G) and were observed in the periscar areas where they were surrounded by native myocytes (Fig. 4, J–Q). In scar areas, the abundant blue cells retained the stem cell appearance with a large nuclear-to-cytoplasmic ratio (Fig. 4I, right bottom). These data suggest that stem cell differentiation critically depends on the surrounding microenvironment.
LV Function
Employing fetal myocyte transplantation, Rubart et al. (17) demonstrated that donor myocytes can electrically and mechanically synchronize to the host myocardium and improve LV contractile function. The exact mechanism of cardiac functional improvement in response to the cellular therapy remains unclear. The following four lines of mechanisms can be considered.
Regenerating cardiomyocytes.
In the present study, we used MRI to evaluate LV contractile performance and observed a significant improvement of LV systolic thickening fraction in hearts that received MSC transplantation (Fig. 3). The mechanism of this beneficial effect of cellular therapy is not known (2, 7–9, 11, 22). The "newly formed myocytes" from adult stem cells could eventually be incorporated into the myocardial syncytium to contract synchronously with the host myocytes. The frequency of myocyte differentiation from transplanted stem cells has been reported in the range of 0–0.02% (4, 12). This myocyte differentiation frequency will have to be increased substantially to improve contractile performance. In the present study, the fibrin patch scaffold that entrapped millions of autologous stem cells was placed on top of the myocardial necrosis. Counting the blue cells in the infarcted area (including the periscar zone) indicated that 10% of transplanted cells migrated into the MI. It is very tempting to speculate that these 10% of stem cells residing in the myocardium continue to divide and differentiate, which results in a better contractile performance.
Increase of neovascularization. It is also possible that the transplanted cells stimulate angiogenesis and therefore spare the ischemic-threatened myocardium at the border of the infarct zone. The present study demonstrates a significant increase of neovascularization in the Tx group of hearts (Figs. 5 and 6), which might facilitate the improved function possibly by improving perfusion of threatened myocardium. This robust neovascularization in the patch area (Figs. 5 and 6) possibly involves both angiogenesis and vasculogenesis.
A trophic effect. It is also possible that improved LV contractile performance is caused by the cytokines released from the engrafted MSCs that improve the spared myocytes performance: a trophic effect.
LV scar compliance. The factors of engrafted cells, increased neovascularization, or cytokines may together or individually result in change of the LV scar compliance and consequently improve the LV systolic contractile performance. The exact mechanisms of improved LV function in response to patch-based stem cell transplantation remain to be elucidated.
In the present study, we used an "open artery" model of postinfarction LV remodeling analogous to the clinical situation of MI followed by therapeutic reperfusion. During the first 2 wk after reperfusion, the necrotic myocardial environment would not seem to be a hospitable homing target for foreign stem cells because the inflammatory response might preclude the stem cells from thriving in the necrotic area. It was consequently surprising to find the opposite, namely, that blue spindle-shaped stem cells were mainly found in the necrotic area as shown in Fig. 4, H and I. The cells migrated into the periscar area where they differentiated into myocyte-like cells. The majority of stem cells migrated into the necrotic area (Fig. 4, A and H), suggesting that signals emanating from the necrotic myocardium directed the stem cells to the injured region (Fig. 4C). These data also suggest that autologous bone marrow stem cells might be protected from the host immune response.
Chemoattracts mesenchymal stem cells to cardiac homing. We have carried out experiments using a similar patch-based stem cell delivery to normal myocardium. The results demonstrate that few stem cells engrafted to normal myocardium. These data suggest that ischemia is necessary for the stem cell engraftment. However, other disease signals may also stimulate stem cell cardiac homing.
Limitations. The fusion of MSC and myocytes could potentially result in cells having the appearance of blue myocytes. However, stem cell fusion with other cells rarely occurs (0.0001–0.000002%) in culture (20, 25). In the present in vivo study, we cannot either exclude or prove that the lacZ-positive myocytes were the result of fusion between MSC with myocytes. Further studies will be required to prove beyond any doubt that the myocytes are not the result of cell fusion.
It should be noted that incorporation of differentiated "myocytes" into the LV wall could result in electrical instability and generation of arrhythmias. We did not find evidence for arrhythmias or excess sudden death in the animals receiving MSC transplantation.
In conclusion, the data in the present study demonstrate that a patch-based delivery of autologous MSC onto myocardial infarcts can improve the LV contractile performance, prevent LV aneurysm formation, and prevent the transition to heart failure. This beneficial effect was associated with the combination of stem cell differentiation and a robust increase of neovascularization in response to stem cell transplantation. This procedure may have potential as a therapeutic modality for treatment of MI.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* J. Liu and Q. Hu contributed equally to this work. 
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