Yoriyasu Suzuki

Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects

Human embryonic stem (hES) cells are pluripotent stem cells capable of self‐renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double‐fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell‐derived endothelial cells [hESC‐ECs]). To compare the two popular imaging modalities, hES cells and hESC‐ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC‐ECs. In particular, hESC‐ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post‐mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC‐ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high‐resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation.

In Vivo Comparison Between Optical Coherence Tomography and Intravascular Ultrasound for Detecting Small Degrees of In-Stent Neointima After Stent Implantation

OBJECTIVES The purpose of this study was to evaluate optical coherence tomography (OCT) for detecting small degrees of in-stent neointima (ISN) after stent implantation compared with intravascular ultrasound (IVUS). BACKGROUND The importance of detecting neointimal coverage of stent struts has grown with the appreciation of the increased risk for late stent thrombosis after drug-eluting stent (DES) implantation. Intravascular ultrasound, the current standard for evaluating the status of DES, lacks the resolution to detect the initial neointimal coverage. Optical coherence tomography has greater resolution but has not yet been compared with IVUS in vivo with histological correlation for validation. METHODS Intravascular ultrasound and OCT were performed with motorized pullback imaging in 6 pigs across 33 stents, 1 month after implantation. Each pig was euthanized, and histological measurements of vessel, stent, and lumen dimensions were performed in 3 sections of each stent. A small degree of ISN was defined as occupying <30% of the stent area measured with histology. The IVUS, OCT, and histological assessment of ISN were compared in matched cross-sections of the stents with a small degree of ISN. RESULTS Eleven stents had a small degree of ISN (average ISN area: 1.26 +/- 0.46 mm(2), and percent area obstruction: 21.4 +/- 5.2%). Compared with histology, the diagnostic accuracy of OCT (area under the receiver operating characteristic curve [AUC] = 0.967, 95% confidence interval [CI] 0.914 to 1.019) was higher than that of IVUS (AUC = 0.781, 95% CI 0.621 to 0.838). CONCLUSIONS Optical coherence tomography detects smaller degrees of ISN more accurately than IVUS and might be a useful method for identifying neointimal coverage of stent struts after DES implantation.

A human ferritin iron oxide nano-composite magnetic resonance contrast agent

Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)‐iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R2 relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn‐iron oxide contrast agent to assess inflammatory disease status such as macrophage‐rich atherosclerotic plaques in vivo. Magn Reson Med 60:1073–1081, 2008.

In vitro comparison of the biological effects of three transfection methods for magnetically labeling mouse embryonic stem cells with ferumoxides

In vivo MRI of stem cells (SCs) is an emerging application to evaluate the role of cell therapy in restoring the injured myocardium. The high spatial and temporal resolution combined with iron‐oxide‐based intracellular labeling techniques will provide a sensitive, noninvasive, dual imaging modality for both cells and myocardium. In order to facilitate this novel imaging approach, much effort has been directed towards developing efficient transfection methods. While techniques utilizing poly‐L‐lysine (PLL), protamine sulfate (PS), and electroporation (ELP) have been proposed, the fundamental biological effects of these methods on mouse embryonic SCs (mESC) have not been investigated systematically. In this study a longitudinal in vitro evaluation of cellular viability, apoptosis, proliferation, and cardiac differentiation of magnetically labeled mESC was conducted. No significant difference was seen in these biological parameters among the three transfection methods. However, cardiac differentiation was most attenuated by ELP, and iron uptake was most effective by PS. Magn Reson Med 57:1173–1179, 2007.

The representative porcine model for human cardiovascular disease.

To improve human health, scientific discoveries must be translated into practical applications. Inherent in the development of these technologies is the role of preclinical testing using animal models. Although significant insight into the molecular and cellular basis has come from small animal models, significant differences exist with regard to cardiovascular characteristics between these models and humans. Therefore, large animal models are essential to develop the discoveries from murine models into clinical therapies and interventions. This paper will provide an overview of the more frequently used large animal models, especially porcine models for preclinical studies.

Initial Success Rate of Percutaneous Coronary Intervention for Chronic Total Occlusion in a Native Coronary Artery Is Decreased in Patients Who Underwent Previous Coronary Artery Bypass Graft Surgery

OBJECTIVES This study sought to compare the initial success rate of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) in a native coronary artery (NCA) in patients with and without previous coronary artery bypass grafting (CABG) and to assess predictive factors. BACKGROUND Landmark novel wiring techniques for CTO-PCI have contributed to improvement in the initial success of CTO-PCI. However, challenges persist in CTO-PCI in NCA in pCABG patients. METHODS Patients who underwent CTO-PCI in an NCA were selected and classified into 2 groups: pCABG (206 PCIs in 153 patients) and nCABG (1,431 PCIs in 1,139 patients). RESULTS CTO was located more often in the left anterior descending artery (p = 0.0003), and severe calcified lesions were observed more frequently in the pCABG group (p < 0.0001). Although the retrograde attempt was tried more frequently in the pCABG group, the CTO-PCI success rate was significantly lower in the pCABG patients than in the nCABG patients (71% vs. 83%). Longer procedural time and greater radiation exposure were needed in the pCABG patients. Logistic regression analysis among the pCABG patients revealed that intravascular ultrasound use and parallel wiring were positive factors, and lesion tortuosity was a negative factor. CONCLUSIONS The initial success rate of CTO-PCI of an NCA in the pCABG group was significantly decreased compared with that in the nCABG group. Anatomic complexity and unstable hemodynamic state were unfavorable conditions. This study reveals that the issues to be overcome are lying with CTO revascularization in an NCA in pCABG patients.

In vivo porcine model of reperfused myocardial infarction: In situ double staining to measure precise infarct area/area at risk

Objectives: The aim of this study is to evaluate a catheter‐based porcine model for reperfused myocardial infarction and investigate the appropriate location and duration of the occlusion. Material and Methods: A balloon catheter was placed in the left descending coronary artery (LAD) in 78 swine, and used to occlude the LAD. To evaluate this model, left ventricular ejection fraction (LVEF), infarct size, incidence of ventricular fibrillation (VF), and mortality was compared among three groups: 60‐min proximal LAD occlusion (60P), 60‐min mid LAD occlusion (60M), and 30‐min proximal LAD occlusion (30P). Results: In 72 of the 78 pigs, the procedures were successfully completed. Both mortality and incidence of VF were highest in the 60P group (66.7% and 91.7%, respectively). Myocardial infarction was successfully induced in all 72 animals and in situ double‐staining with Evans blue dye and 2,3,5‐triphenyltetrazolium chloride was performed to delineate area at risk for ischemia and infarcted myocardium. There was no difference in infarct size, expressed as a percentage of the area at risk, between the 60P and 60M groups (49.5% ± 3.9% vs. 45.4% ± 13.3%, respectively). Serial changes in LVEF of the 60M group demonstrated that until 14 days after reperfusion, LVEF improved naturally over time (36.4% ± 6.6% at 24 hr, and 47.3% ± 10.1% at 14 days). Conclusion: This model and methodology could provide a reproducible and consistent infarct size. The current study demonstrated that 60‐min mid LAD occlusion can be the most feasible to serve as a porcine reperfused myocardial infarction model.

In vivo serial evaluation of superparamagnetic iron-oxide labeled stem cells by off-resonance positive contrast.

MRI is emerging as a diagnostic modality to track iron‐oxide‐labeled stem cells. This study investigates whether an off‐resonance (OR) pulse sequence designed to generate positive contrast at 1.5T can assess the location, quantity, and viability of delivered stem cells in vivo. Using mouse embryonic stem cell transfected with luciferase reporter gene (luc‐mESC), multimodality validation of OR signal was conducted to determine whether engraftment parameters of superparamagnetic iron‐oxide labeled luc‐mESC (SPIO‐luc‐mESC) could be determined after cell transplantation into the mouse hindlimb. A significant increase in signal‐ and contrast‐to‐noise of the SPIO‐luc‐mESC was achieved with the OR technique when compared to a gradient recalled echo (GRE) sequence. A significant correlation between the quantity of SPIO‐luc‐mESC and OR signal was observed immediately after transplantation (R2 = 0.74, P < 0.05). The assessment of transplanted cell viability by bioluminescence imaging (BLI) showed a significant increase of luciferase activities by day 16, while the MRI signal showed no difference. No significant correlation between BLI and MRI signals of cell viability was observed. In conclusion, using an OR sequence the precise localization and quantitation of SPIO‐labeled stem cells in both space and time were possible. However, the OR sequence did not allow evaluation of cell viability. Magn Reson Med 60:1269–1275, 2008.

High‐contrast in vivo visualization of microvessels using novel FeCo/GC magnetic nanocrystals

FeCo‐graphitic carbon shell nanocrystals are a novel MRI contrast agent with unprecedented high per‐metal‐atom‐basis relaxivity (r1 = 97 mM‐1 sec‐1, r2 = 400 mM‐1 sec‐1) and multifunctional capabilities. While the conventional gadolinium‐based contrast‐enhanced angiographic magnetic MRI has proven useful for diagnosis of vascular diseases, its short circulation time and relatively low sensitivity render high‐resolution MRI of morphologically small vascular structures such as those involved in collateral, arteriogenic, and angiogenic vessel formation challenging. Here, by combining FeCo‐graphitic carbon shell nanocrystals with high‐resolution MRI technique, we demonstrate that such microvessels down to ∼100 μm can be monitored in high contrast and noninvasively using a conventional 1.5‐T clinical MRI system, achieving a diagnostic imaging standard approximating that of the more invasive X‐ray angiography. Preliminary in vitro and in vivo toxicity study results also show no sign of toxicity. Magn Reson Med, 2009.

Multimodality evaluation of the viability of stem cells delivered into different zones of myocardial infarction.

Background—We tested the hypothesis that multimodality imaging of mouse embryonic stem cells (mESCs) provides accurate assessment of cellular location, viability, and restorative potential after transplantation into different zones of myocardial infarction. Methods and Results—Mice underwent left anterior descending artery ligation followed by transplantation of dual-labeled mESCs with superparamagnetic iron oxide and luciferase via direct injection into 3 different zones of myocardial infarction: intra-infarction, peri-infarction, and normal (remote). One day after transplantation, magnetic resonance imaging enabled assessment of the precise anatomic locations of mESCs. Bioluminescence imaging allowed longitudinal analysis of cell viability through detection of luciferase activity. Subsequent evaluation of myocardial regeneration and functional restoration was performed by echocardiography and pressure–volume loop analysis. Using 16-segment analysis, we demonstrated precise localization of dual-labeled mESCs. A strong correlation between histology and magnetic resonance imaging was established (r=0.962, P=0.002). Bioluminescent imaging data demonstrated that cell viability in the remote group was significantly higher than in other groups. Echocardiography and pressure–volume loop analysis revealed improved functional restoration in animals treated with mESCs, although myocardial regeneration was not observed. Conclusions—Multimodality evaluation of mESC engraftment in the heterogeneous tissue of myocardial infarction is possible. Magnetic resonance imaging demonstrated accurate anatomic localization of dual-labeled mESCs. Bioluminescent imaging enabled assessment of variable viability of mESCs transplanted into the infarcted myocardium. Echocardiography and pressure–volume loop analysis validated the restorative potential of mESCs. Although mESCs transplanted into the remote zone demonstrated the highest viability, precise delivery of mESCs into the peri-infarction region might be equally critical in restoring the injured myocardium.