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Infusion of diverse types of bone marrow cells, as a source of endothelial progenitor cells (EPCs), into the ischemic myocardium is emerging as a promising therapy for coronary ischemia, probably mediated by the formation of new blood vessels. Studies have shown that while the procedure is safe and feasible, efficacy results are contentious. The investigators in the present preclinical translation study hypothesized that the infusion of a combination cell product consisting of EPCs and other cell types, such as mesenchymal stem cells, promotes the formation of more stable and mature blood vessels resulting in improved clinical outcomes. The safety and feasibility of the intracoronary infusion of such a cell combination was assessed in a canine model.
A mixture of canine autologous mononuclear cells (as the source of EPCs) and ex vivo-expanded bone marrow-derived mesenchymal stem cells or a placebo solution were intracoronarily infused into healthy dogs. Follow-up after cell/placebo infusion included an electrocardiogram, serum cardiac enzyme testing, a transthoracic echocardiography and a histopathological heart examination.
On follow-up at all time points after infusion, no significant changes or abnormalities in vital signs, electrocardiogram, transthoracic echocardiography and heart histology were detected.
From a clinical perspective, the safety and feasibility of the protocol used in the present animal study demonstrated clinical relevance and provided direct evidence supporting the intracoronary infusion of combination stem/progenitor cell products.
In past years, several clinical studies (1–4) have been initiated to determine the safety, feasibility and effectiveness of the use of bone marrow (BM)-derived stem/progenitor cells for the treatment of myocardial infarction. The common rationale among these studies has been based on the concept that the infusion of a source of endothelial progenitor cells (EPCs) may enhance angiogenesis and promote myocardial repair. However, the formation of new and mature blood vessels requires, in addition to EPCs, the coordinated participation of other components such as mural cells (eg, vascular smooth cells, pericytes), local/distant growth factors, chemokines and extracellular matrix molecules (5–8).
We hypothesized that the use of a combination, instead of a single cellular product, consisting of a mixture of autologous BM-derived mononuclear cells (BM-MNCs, as a source of EPCs) and ex vivo-expanded mesenchymal stem cells (BM-MSCs, as a source of pericyte progenitors and angiogenic factors) (9,10) represented a potent cellular and molecular mechanism for the formation of blood vessels. To our knowledge, such a therapeutic approach has not been assessed in myocardial infarction patients.
An important issue in cell therapy is to demonstrate that the route of infusion of a particular cell product is feasible and safe. The present study was designed and performed to explore the safety and feasibility of the intracoronary infusion of a combination of canine autologous BM-MNCs and ex vivo-expanded BM-MSCs into normal dogs.
Studies were performed using healthy male dogs (n=9; two to four years of age, weight 17 kg to 20 kg, average heart weight 0.14 kg), in accordance with the Guide for the Care and Use of Laboratory Animals (www.nap.edu/readingroom/books/labrats/), approved by the Facultad de Medicina Veterinaria, Universidad de Chile, Santiago, Chile.
The preparation and ex vivo expansion of canine BM-MSCs was performed by following the procedures described for human BM-MSCs (11). Briefly, a bone marrow aspiration (10 mL to 15 mL) was performed at the wing of the ilium and sent to the GTP facility for cell processing. Mononuclear cells, isolated by density gradient centrifugation (Histopaque-1077, Sigma-Aldrich, USA) were suspended in culture medium (alpha-minimum essential medium [MEM]) containing 10% fetal bovine serum), seeded at a concentration of 1×106 cells/cm2 and incubated at 37°C with 5% CO2. One week later, when the monolayer of adherent cells had reached confluence, cells were trypsinized (0.25% trypsin), washed, resuspended in culture medium and expanded by successive subcultures. Expanded BM-MSCs were suspended in infusion medium (alpha-MEM containing 5% dog serum). For preparation of BM-MNCs, a secondary bone marrow aspirate was obtained on the day of the infusion and cells were processed by density gradient centrifugation as described above. The resulting fraction of BM-MNCs was suspended in infusion medium.
For preparation of the combination cell product for infusion, proper aliquots of each cell type (2.5×106 BM-MNCs and 2.5×106 BM-MSCs) were mixed, filtered through a 100 μm cell strainer and centrifuged. The resulting cell pellet was resuspended in 2 mL of infusion medium and transferred to a 3 mL infusion syringe. Therefore, cell-infused dogs received a total of 5×106 cells (approximately 35×106 cells/kg heart weight) and placebo-infused animals received 2 mL of infusion medium.
Aliquots of the cell product for infusion were taken to assess cell viability (trypan blue exclusion test), sterility (Gram-staining) and expression of specific antigens by flow cytometry. Mesenchymal (CD73, alpha-smooth muscle actin and vimentin) and myeloid (CD45 and CD34) antigens were evaluated (11,12).
For the intracoronary infusion of the cell or placebo product, six and three dogs, respectively, were anesthetized. The carotid artery was isolated and canulated with a 6 Fr sheath. A 6 Fr Judkins 3.5 catheter (Vista Brite Tip IG, Cordis Corporation, USA) was then advanced into the ascending aorta and the left main coronary artery was engaged. A 0.014-inch balance middleweight guide wire (Guidant Corporation, USA) was used to navigate into the left circumflex artery. A Voyager (Guidant Corporation, USA) ‘over the wire’ dilation catheter was advanced into the circumflex coronary artery, followed by the removal of the guide wire. The balloon catheter was inflated (1 atm to 4 atm for 3 min) and, subsequently, either the cell product or the infusion medium (placebo) was administered through the main catheter lumen. Once the infusion was completed, an angiogram was performed to assess coronary flow.
Vital signs (blood pressure, heart rate, temperature and O2 saturation) as well as laboratory tests (blood count, blood electrolytes, glucose, creatinine, troponin I and creatinphosphokinase) were obtained immediately before (baseline) and at one, seven and 26±4 days after cell/placebo infusion. Cardiac noninvasive monitoring, including lead II electrocardiograms (ECGs) and transthoracic echocardiograms were recorded before and at various time intervals after cell infusion.
Transthoracic echocardiography was performed under sedation using a 3.5 MHz probe connected to a Vivid 7 Ultrasound System (GE Medical Systems, Norway). Examinations were performed immediately before infusion and at days 1, 7 as well as the day of sacrifice. Two parasternal left ventricular (LV) long-axis views (including the mitral and aortic valves, and the apex) and three parasternal short-axis views (base, papillary muscles and apex) were recorded. LV end-diastolic and LV end-systolic volumes were calculated (Teichholz’s method) and used to calculate the ejection fraction.
At 26±4 days after cell or placebo infusion, the animals were sacrificed. The hearts were exposed by median sternotomy and quickly removed. After gross examination, the hearts were fixed in 10% formaldehyde for 48 h and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin for qualitative histopathological analysis specifically targeted to assess for scar tissue, inflammation and/or infarction lesions.
As revealed by phase-contrast microscopy, primary cultures of canine BM-MSCs consisted of adherent colonies of bipolar fibroblast-like cells (Figure 1A). After ex vivo expansion, MSCs (diameter 20 μm, Figure 1B) were grown as a homogenous population (Figure 1C) reaching a confluent growth-arrested condition after six to seven days (Figure 1D). Ex vivo-expanded canine MSCs express the mesenchymal markers CD73, alpha-smooth muscle actin and vimentin (Figures 1E to to1G),1G), but not the myeloid markers CD34 and CD45, which were expressed by MNCs (data not shown).
Before and after bone marrow aspiration, and during cell infusion, blood pressure and heart rate were continuously monitored, with no abnormalities detected. During cell infusion, continuous ECG tracings revealed minor changes consistent with transient ST-segment elevation and occasional premature ventricular contractions. During the first 24 h, continuous monitoring revealed frequent premature ventricular contractions, junctional rhythms and one case of slow nonsustained ventricular tachycardia, which resolved spontaneously.
At day 1 after cell infusion, ECG tracings revealed minor ST-T abnormalities but no evidence of injury or infarction. During the first week after cell infusion, only nonspecific ST-T abnormalities were noted. Monitoring at various time points after cell infusion revealed no difference from baseline tracings (a representative tracing is shown in Figure 2).
As shown in Table 1, the median levels of troponin I slightly increased beyond the cut-off level (0.15 ng/mL) at day 1 in animals infused with the cell product. Plasma creatinphosphokinase activity followed a similar trend, with a moderate increase at day 1 after infusion (Table 1). Troponin I and creatinphosphokinase activity returned to baseline levels on day 7. In animals receiving the placebo infusion, changes in the activity of both cardiac serum markers followed a similar pattern.
At different follow-up times, no changes in LV end-diastolic dimension and LV end-systolic dimension volumes were detected in either cell- or placebo-infused animals (data not shown). Likewise, LV ejection fraction was stable during the entire study period (Table 2). Furthermore, no regional wall motion abnormalities were detected using tissue velocity imaging and tissue synchronization imaging.
After gross examination and histopathological analysis performed 26±4 days after cell or placebo infusion, the hearts of all dogs, except one, demonstrated normal myocardial appearance without evidence of inflammation or fibroplasia suggestive of infarction (Figure 3A). The heart of one cell-infused dog showed discrete inflammatory changes, with mild mononuclear infiltration. Distinctive features suggestive of myocardial infarction similar to a circumscribed area of ischemic/necrotic tissue or moderate inflammation were not observed (Figure 3B).
In the present study, we evaluated the safety and feasibility of an intracoronary infusion of a combination cell product consisting of autologous BM-MNCs and BM-MSCs into healthy dogs. After one-month of follow-up, results indicated that the use of a balloon catheter similar to catheters used for coronary angioplasty in humans proved to be feasible in dogs. Although a small balloon was used to select the circumflex artery, the guiding catheter was placed in the left main coronary artery; the above manoeuvre created a transient myocardial ischemia because the diameter of the catheter in relatively small coronary arteries compromises coronary flow. As a consequence, a mild elevation in myocardial markers and ECG parameters was detected 24 h later. However, these changes – observed in both cell- and placebo-infused animals – returned to baseline values after seven days (Table 1 and Figure 2); in every dog, including those that had a transient increase in cardiac markers, functional assessment by echocardiography revealed no changes in global and regional systolic functions after cell infusion (Table 2); and histopathological examination performed after 26±4 days of cell/placebo infusion, confirmed no evidence of myocardial infarction in the cardiac tissue (Figure 3A). Histopathological examination of the heart of one cell-infused dog revealed the presence of a lymphocyte infiltrate suggestive of an inflammatory process such as myocarditis (Figure 3B).
Accordingly, the results of the current preclinical study demonstrated that the intracoronary infusion of a combination cell product into normal dogs is safe and feasible at the cell dose used (5×106 cells – corresponding to approximately 35×106 cells/kg dog heart weight).
To translate the safety and feasibility outcomes of the present animal study into a clinical setting, the following should be considered: if a combination cell product is to be used, patients can be intracoronarily infused with approximately 70×106 cells (assuming a human/dog heart body weight ratio of 2). This cell number, which is within the range currently used in clinical trials to intracoronarily infuse a single cell product (13), assures that a relatively high percentage of infused stem/progenitor cells may reach, home and persist in the cardiac tissue (14); the total elapsed time to prepare the cell infusion product (including the ex vivo expansion of BM-MSCs) is approximately two weeks, which seems to be acceptable, especially when compared with the time needed to complete more conventional therapies.
Clinical studies (1–4) have shown that the intracoronary infusion of a single type of ‘repair’ cell into myocardial infarction patients is safe and feasible. However, efficacy results are contentious (13,15,16). The rationale that supports these studies has been based on the premise that the cellular product (a source of EPCs) will promote tissue revascularization, regeneration and repair of injured and ischemic tissues. Considering that vasculogenesis and angiogenesis are multifactorial processes, we postulate that the use of a combination cell product, including EPCs and other progenitor(s) known to participate in the formation of blood vessels, may represent a more effective therapeutic option for myocardial infarction. Thus, from a clinical perspective, the protocol used in the present animal study demonstrated clinical relevance and provided direct evidence of the safety and feasibility for the intracoronary infusion of a combination stem/progenitor cell product. To our knowledge, only one study (17) has addressed the alternative to use a combination cell product, instead of a single ‘repair’ cell type, in a postinfarcted pig model.
The authors thank Janet Jones PhD for the analysis and preparation of the manuscript.