hiPSC-CMs and cell preparation
The ultra-pure (Purity > 95%) hiPSC-CMs were produced by HELP Therapeutics and cryopreserved in Liquid Nitrogen. The cell preparation for intramyocardial injection has been described in a previous paper11.
Animals and Heart Failure model
Sixteen Rhesus (Macaca mulatta) monkeys, aged 3-5 years and weighting 4.5-6.0 kg, were obtained from Sichuan Green-house Biotech Co., Ltd. The animals were housed in individual cages and acclimatized to the laboratory condition for a period of at least 2 weeks. They received conventional laboratory diet with free access to drinking water as approved by the Laboratory Animal Management Committee. All animal procedures were approved by Institutional Animal Care and Use Committee with code IAC-A2019021-P-01. Rhesus monkeys were divided into two groups: vehicle group (n = 6) and HiCM group (n = 10). All the monkeys subjected to surgical procedure were intubated and ventilated to achieve end tidal CO2 between 35 and 40 mmHg. Standard non-invasive measurements including electrocardiography, cuff blood pressure, pulse oximetry, and capnography were constantly monitored. Intra-arterial and intravenous catheters were used. Anesthesia was induced by intramuscular injection of Atropine Sulfate (0.02~0.05 mg/kg), followed by intramuscular injection with Tiletamine Hydrochloride and Zolazepam Hydrochloride for Injection (Zoletil-50, 0.1~0.15 mL/kg) 30 min later. Sevoflurane (1~3%) inhalation was applied to maintain the anesthetic condition during the surgical procedure. The heart was exposed via a sternotomy using rib shears. The left anterior descending artery (LAD) was ligated between the first and the second diagonal artery for 3 hours. In the vehicle group monkeys, 1.25 mL 5% HSA was injected into the left ventricular wall. In the HiCM group, 1×108 hiPSC-CM resuspension in 1.25 mL 5% HSA was similarly injected. Injections were made in the myocardium with a 22G needle in and around the infarct area. Pericardium and pleura were closed with 4-0 polyethylene sutures after the cardiac hemodynamic became stable. The sternum incision was closed with 10 silk sutures, and the skin incision was closed with silk suture. The endotracheal tube was retracted after the spontaneous breathing was restored.
All animals received immunosuppressive drugs 5 days before surgery: Methylprednisolone (16 mg/day), Tacrolimus (4.5 mg/day), Mycophenolate mofetil Capsules (2.25 mg/day) and Basiliximab (20 mg on the day of HiCM injection and 20 mg at 4 days post HiCM injection).
Two-dimensional echocardiographic measurements (parasternal long- and short-axis, apical 4- and 2-chamber views) were performed in left lateral position for all monkeys using a Vivid I Portable Ultrasound Machine (GE, USA). End-diastolic volume (EDV) and end-systolic volume (ESV) were recorded. EF was calculated as EF = (EDV - ESV) / EDV × 100%.
Two male crab-eating (Macaca fascicularis) monkeys, aged 7~8 years and weighting 5.3-5.7kg, were obtained from Beijing Prima Biotech Inc. The crab-eating (Macaca fascicularis) monkeys were divided into two groups (89Zr labeled hiPSC-CM and 89Zr-oxine). The 89Zr labeled hiPSC-CMs or 89Zr-oxine were resuspended in 5% HSA and injected into the left ventricular wall through open chest surgery.
89Zr labeling of hiPSC-CM
89Zr-oxine complex was synthesized from oxine and 89ZrCl4 produced at the Mitro Biotech (Nanjing, Jiangsu, PRC) as previously reported 29. Human iPSC-CM in phosphate-buffered saline (PBS) were incubated with 89Zr-oxine (50-80 kBq/106 cells) for 30 minutes. Then, labeled 89Zr-HiCM was washed twice with PBS and re-suspended in 5% HSA medium for intramyocardial injection.
Tracking 89Zr-HiCM by clinical PET/CT imaging
PET/ CT imaging was conducted at Mitro Biotech (Nanjing, Jiangsu, PRC) with a Biograph PET/CT (Siemens, Knoxville, TN, USA). For each scan, anesthesia was induced by intramuscular injection with Atropine Sulfate (0.02~0.05 mg/kg), followed by intramuscular injection with Tiletamine Hydrochloride and Zolazepam Hydrochloride for Injection (Zoletil-50, 0.1~0.15 mL/kg) 30 min later. Sevoflurane (1~3%) inhalation was applied to maintain the anesthetic condition for the duration of PET scanning. Body temperature was maintained with circulating water heating pads. Whole-body CT scans were acquired at each time point, for PET attenuation and scatter corrections and region-of-interest (ROI) were delineated. Iterative reconstructions were performed for all PET data using the same set of manufacture-recommended reconstruction parameters. The image analysis software package PMOD (v3.8, PMOD Technologies LLC, Zürich, Switzerland) was used for quantitative analysis of the ROIs. ROIs were manually drawn, referencing both PET and CT data at each time point, including heart, lung, liver, kidney, spleen, testis, muscle, femur bone marrow, tibia bone marrow, and tibia cortical bone. Subsequently, the image-derived biodistributions over the course of the imaging study at designated timepoints were calculated as standardized uptake values (SUVmean) in the above ROIs for all imaging data sets.
Note: MAC (μCi/g) the Mean Activity Concentration in the ROI. ID is the total injected dose on day 0. M is the mass of the monkey.
Post-operative management (1 week after surgery)
For both efficacy and biodistribution experiments, prophylactic medications including Cefoxitin Sodium (50 mg/kg, i.v.), Penicillin sodium (25~75 kIU/kg, i.v.), Fluconazole (50 mg/day), and Cefuroxime Axetil (0.5 g/day) were provided to all monkeys for 1 week. In case of pain, morphine (0.01~0.015 mg/kg, intramuscular injection; Animal care, Dunnington, York, UK) was given as deemed necessary.
Sections from heart tissue samples were cut to 4-6 μm and processed for immunohistochemical examination by a streptavidin-biotin-peroxidase complex method. Tissue sections were placed on 3-amino-propyltrieyhoxysilanecoatede slides, dewaxed, and hydrated. Antigen retrieval was facilitated by heating in citrate buffer (pH 6.0) for 10 min in a microwave oven with a power of 800 watts. The slides were then dipped in freshly prepared absolute methanol containing hydrogen peroxide 3% vol/vol for 15 min to quench endogenous peroxidase activity. Sections were treated with Rabbit anti-cardiac Troponin I antibody (ab52862, Abcam, USA) for 60 min. After washing with PBS, the slides were incubated with rabbit anti-goat immunoglobulin G diluted at 1:300 in PBS for 30 min at room temperature. After washing with PBS, the slides were treated for 5 min at room temperature with 3,3′-diaminobenzidine tetrahydrochloride (DAB) in PBS (0.5 mg DAB/mL) containing hydrogen peroxide 30% vol/vol. Sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted before imaging.
Data analysis was performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). Data are shown as means with standard deviations. Multiple t tests were conducted to evaluate the significancy: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.