Induction of myocardial infarction
All animal studies were approved by the Johns Hopkins University Institutional Animal Care and Use Committee and comply with the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 80-23, revised 1985). Female Göttingen minipigs were purchased from Marshall BioResources (North Rose, NY).
Myocardial infarction was induced by temporary balloon occlusion of the left anterior descending coronary artery (LAD) with an inflated angioplasty balloon as previously described in detail.19
After 120 min occlusion was terminated by balloon deflation and removal, and reperfusion was established.
Bone-marrow harvest, mesenchymal stem cell isolation, and characterization
Porcine BM cells were aspirated from the iliac crest of each animal. The mononuclear cells (MNCs) were then isolated on a Ficoll gradient. The low-density fraction, which contained the MNCs, was collected and after washing placed in T162 cm2 costar tissue culture flasks at 1–5 × 106 cells per mL of alpha MEM media containing 20% fetal calf serum. The cells were incubated at 5% CO2, 37°C, and the media changed weekly. Adherent cells were identified by microscopic evaluation at Day 3 and the non-adherent cells discarded with the media with each media change. The MSC became confluent after ~2 weeks of culture and were passaged using trypsin–EDTA for cell mobilization. Cultures were expanded with each passage of the MSC until sufficient numbers of MSC were obtained. A total of four to seven passages were required.
Phenotypic characterization of the MSCs was achieved with porcine specific antibodies or human antibodies that are cross-reactive with porcine antigens. The porcine MSCs were CD45− and CD90+, consistent with human MSCs. Porcine MSCs formed CFU-F colonies similar in size, appearance, and frequency to human BM-derived MSCs. Cytogenic abnormalities were highly unlikely given the normal cell appearance and growth rate, as previously described.20
The cells were then frozen in liquid nitrogen until needed. Prior to injection, the cells were thawed rapidly, washed to remove dimethylsulfoxide, and then re-suspended in PBS plus 1% human serum albumin to the required cell dose.
Fifteen pigs were used in this randomized, blinded, placebo-controlled pre-clinical study. Animals were allowed to recover from MI and given sufficient time for formation of a transmural MI, 111 ± 4 days (3.7 ± 0.1 months). Pigs were randomized to one of the following three groups at an age 549 ± 22 days (18.3 ± 0.7 months, Table
). All animals received 15–25 needle injections, and were allocated to:
- High MSC dose (n = 6, 200 × 106 MSCs),
- Low MSC dose (n = 3, 20 × 106 MSCs), and
- Placebo [n = 6, phosphate buffered saline (PBS)].
Initially, nine animals were randomized equally to the placebo, low dose, and high dose MSC groups. After an interim analysis (3 placebo, 2 low dose, 4 high dose), the six remaining animals were randomized continuing the 2:1 ratio between high and low dose and placebo. The results of these studies supported Food and Drug Administration approval for The PROMETHEUS Study (Prospective Randomized Study of Mesenchymal Stem Cell Therapy in Patients Undergoing Cardiac Surgery, clinicaltrials.gov NCT00587990, 362).
An anterior thoracotomy at the fifth intercostal space was performed. After pericardial excision, the heart was freely rotated superiorly to gain access to the apex. The apical infarction zone was visualized, and a moist gauze pack was placed on the underside of the heart to allow for exposure. The autologous BM-derived MSCs or placebo volumes were injected in 0.25 mL volumes with a 1 mL syringe equipped with a 29-gauge needle. The injections were administered epicardially into and surrounding akinetic or severely hypokinetic areas, as determined by direct visualization. A total of 15–25 injections were administered. After injections, careful haemostasis was achieved with 4-0 pledgeted prolene sutures, as necessary, and the left chest was copiously irrigated. A 20-French chest tube was inserted through a small incision just anterior to the thoracotomy and placed posteriorly in the chest. Prior to closure, intrapleural bupivicane was injected for additional peri-operative analgesia. The surgical team remained blinded to the content of the aliquots throughout the procedure and the post-operative care.
Magnetic resonance imaging
Global and regional function
Magnetic resonance images were acquired using a 1.5 T MR scanner (CV/i, GE Medical Systems, Waukesha, Wisconsin) at baseline, following MI, prior to injection and 12 weeks after cell delivery. Global and LV function was assessed using a steady-state free precession pulse sequence.21–23
Six to eight contiguous short-axis slices were prescribed to cover the entire heart from base to apex. Image parameters were the following: TR/TE = 4.2 ms and 1.9 ms; Flip angle = 45°; 256 × 160 matrix; 8 mm slice thickness/no gap; 125 kHz; 28 cm FOV and 1 NSA.
To assess regional cardiac function, tagged MRI images were acquired with an ECG-gated, segmented K-space, fast gradient recalled echo pulse sequence with spatial modulation of magnetization to generate a grid tag pattern. Images were obtained at the same location as the cine-MRI images, and image parameters were as follows: TR/TE = 6.7 and 3.2 ms; flip angle = 12°; 250 × 160 matrix; views per second: 4; 8 mm thickness/no gap; 31.25 kHz; 28 cm FOV; 1 NSA and six pixels tagging space.
Myocardial perfusion imaging and delayed enhancement
First-pass perfusion imaging was performed continuously for ~1.5 min at rest immediately after an intravenous bolus injection of Gd-DTPA (0.1 mmol/kg, 5 mL/s; Magnevist, Berlex, Wayne, NJ) with an ECG-gated interleaved saturation recovery gradient echo planar imaging pulse sequence (EFGRET-ET). An entire short-axis stack was acquired every 2–4 heartbeats. Imaging parameters were as follows: TR/TE = 7.2 and 1.8 ms; flip angle = 20°; 128 × 128 matrix; 8 mm slice thickness/no gap; bandwidth 125 kHz; 28-com FOV; and 0.5–1 NSA.
After completion of first-pass image acquisition a second bolus of Gd-DTPA (0.1 mmol/kg) was injected. Prior to the second bolus of Gd-DTPA, infusion of adenosine was used to simulate a stress response. Delayed enhancement (DE) images were acquired 15 min later with an ECG-gated, breath-hold, interleaved, inversion recovery, fast gradient echo pulse sequence. DE-MRI images were acquired at the same location as the short-axis cine images. Imaging parameters were TR/TE/inversion time (TI) = 7.3, 3.3, and ~200 ms; flip angle = 25°; 256 × 196 matrix; 8 mm slice thickness/no gap; 31.2 kHz; 28 cm FOV; and 2 NSA. Inversion recovery time was adjusted as need to null the normal myocardium.9
Magnetic resonance imaging analysis
Cine and DE MR images were analysed for determination of global function, infarct quantization, and remodelling parameters using a custom research software package (Segment; http://segment.heiberg.se
) as previously described.24
Tagged and perfusion MRI images were analysed using a commercially available software package (PLUS, Diagnosoft Inc., Baltimore, MD). The four most apical tagged slices were selected and overlaid with the corresponding DE slices. Meshes were constructed with a total of 72 evenly distributed regions (24 endocardial, 24 mid-wall, and 24 epicardial) incorporating the entire myocardium. The meshes were designed such that the edges of the infarct scar corresponded with the edges of mesh regions. The border zone was defined as the left and right most infarct containing mesh regions, and the infarct zone encompassed all regions in between. The same slices, mesh, and defined regions were used at all time points on an individual pig basis. The values calculated were averaged between all like defined regions and across all slices.
Laboratory measurements and cytokine analysis
Blood chemistry and white blood counts were obtained through a clinical laboratory immediately following each blood draw. Blood for cytokine analysis was also collected at this time, centrifuged at 2000g for 15 min at 4°C, serum recovered, aliquoted, and stored in a −70 freezer until analysed. Porcine-specific serum levels of TGF-beta and TNF-alpha were determined using commercially available enzyme-linked immunosorbent assay (EIA) kits (R&D Systems, Minneapolis, MN). Serum samples were tested in duplicate according to the manufacturer's instructions. The mean minimum detectable dose for each cytokine measured is TGF-β = 4.6 pg/mL and TNF-α = 3.7 pg/mL.
All measurements are expressed as mean ± standard error. The Mann–Whitney test and t-tests were performed, as appropriate, to test for significance between variables using commercially available software (Stata, StataCorp LP, College Station, TX). P < 0.05 was defined as statistically significant. The Kruskal–Wallis test was used to compare age, bodyweight, and follow-up time (Table ). The impact of cell therapy on TGFb levels were evaluated using repeated measures ANOVA. Comparisons between placebo and the two cell dose groups used two-way ANOVA. Post hoc analysis was performed with the Mann–Whitney test.