Jennifer Lyons

Preclinical derivation and imaging of autologously transplanted canine induced pluripotent stem cells

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell–Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

Rationale: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. Objective: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. Methods and Results: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. Conclusions: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

Microfluidic Single Cell Analysis Show Porcine Induced Pluripotent Stem Cell–Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

Rationale: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. Objective: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. Methods and Results: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. Conclusions: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

Fractional Flow Reserve Assessment of Left Main Stenosis in the Presence of Downstream Coronary Stenoses

Background—Several studies have shown that fractional flow reserve (FFR) measurement can aid in the assessment of left main coronary stenosis. However, the impact of downstream epicardial stenosis on left main FFR assessment with the pressure wire in the nonstenosed downstream vessel remains unknown. Methods and Results—Variable stenoses were created in the left main coronary arteries and downstream epicardial vessels in 6 anaesthetized male sheep using balloon catheters. A total of 220 pairs of FFR assessments of the left main stenosis were obtained, before and after creation of a stenosis in a downstream epicardial vessel, by having a pressure-sensor wire in the other nonstenosed downstream vessel. The apparent left main FFR in the presence of downstream stenosis (FFRapp) was significantly higher compared with the true FFR in the absence of downstream stenosis (FFRtrue; 0.80±0.05 versus 0.76±0.05; estimate of the mean difference, 0.035; P<0.001). The difference between FFRtrue and FFRapp correlated with composite FFR of the left main plus stenosed artery (r=−0.31; P<0.001) indicating that this difference was greater with increasing epicardial stenosis severity. Among measurements with FFRapp >0.80, 9% were associated with an FFRtrue of <0.75. In all instances, the epicardial lesion was in the proximal portion of the stenosed vessel, and the epicardial FFR (combined FFR of the left main and downstream stenosed vessel) was ⩽0.50. Conclusions—A clinically relevant effect on the FFR assessment of left main disease with the pressure wire in a nonstenosed downstream vessel occurs only when the stenosis in the other vessel is proximal and very severe.

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.

Reporter Gene Imaging Following Percutaneous Delivery in Swine: Moving Toward Clinical Applications

To the Editor: Noninvasive monitoring of cardiac gene therapy is critical to fully understand the biology of gene therapy in living subjects. We and others have monitored reporter gene expression in the myocardium of small ([1][1]) and large ([2][2]) animals (reviewed in reference [3][3]). However,

Development of animal model for calcified chronic total occlusion

Chronic total coronary occlusion (CTO) remains a major problem for percutaneous revascularization, with relatively low primary success rates and a high incidence of restenosis and reocclusion compared with those of subtotal stenoses. No reproducible animal model simulating human CTOs has previously been developed. We hypothesized that an apatite‐coated bioabsorbable polymer sponge could be implanted to produce calcified CTO lesions in animal coronary arteries/peripheral arteries. A total of 10 swine and six rabbits were used for this study. The apatite‐coated bioabsorbable polymer sponges were implanted into a preselected segment of coronary and peripheral arteries. Four weeks after implantation, both angiography and histopathology were performed to document the presence or absence of CTO lesions. We could reproducibly develop CTO lesions in animal coronary/peripheral arteries that mimic human CTO lesions. These lesions were found to have microvascular channels and microcalcification similar to those of human older CTO lesions and demonstrate the development of adventitial arterioles, a consistent finding in human CTO. This CTO model might provide a platform for evaluating future CTO technologies as well as contributing to a better understanding of CTOs in both educational and practical terms.

Novel percutaneous adventitial drug delivery system for regional vascular treatment

A novel intracoronary microsyringe system (MicroSyringe) was developed for regulated drug injection into the adventitial space. In this report, the feasibility, safety, and distribution pattern of vascular treatment with this modality were tested in 17 swine by delivering Oregon green‐labeled paclitaxel (OGP) and tacrolimus. Coronaries were harvested 0.5–96 hr postinjection and analyzed for drug by fluorescence histochemistry (OGP) and liquid chromatography‐mass spectrometry (tacrolimus). Histopathological analysis was subsequently performed. The microsyringe deliveries were performed safely in all cases. In the OGP‐injected group, within 2 hr postprocedure there was intense staining of the adventitia, media, and endothelium around the injection site, and by 23 hr staining extended distally by 27.5 mm. With tacrolimus, similar longitudinal drug distribution was seen; furthermore, by 48 hr there was detectable drug over 40 mm proximal and distal to the injection site. Significant levels of tacrolimus were detectable in coronaries at 96 hr. Percutaneous adventitial delivery is safe, feasible, and provides consistent dosing for complete treatment of a vascular territory. Catheter Cardiovasc Interv 2004;63:222–230.

Dual Manganese-Enhanced and Delayed Gadolinium-Enhanced MRI Detects Myocardial Border Zone Injury in a Pig Ischemia- Reperfusion Model

Background— Gadolinium (Gd)-based delayed-enhancement MRI (DEMRI) identifies nonviable myocardium but is nonspecific and may overestimate nonviable territory. Manganese (Mn2+)-enhanced MRI (MEMRI) denotes specific Mn2+ uptake into viable cardiomyocytes. We performed a dual-contrast myocardial assessment in a porcine ischemia-reperfusion (IR) model to test the hypothesis that combined DEMRI and MEMRI identifies viable infarct border zone (BZ) myocardium in vivo. Methods and Results— Sixty-minute left anterior descending coronary artery IR injury was induced in 13 adult swine. Twenty-one days post-IR, 3-T cardiac MRI was performed. MEMRI was obtained after injection of 0.7 mL/kg Mn2+ contrast agent. DEMRI was then acquired after injection of 0.2 mmol/kg Gd. Left ventricular (LV) mass, infarct, and function were analyzed. Subtraction of MEMRI defect from DEMRI signal identified injured BZ myocardium. Explanted hearts were analyzed by 2,3,5-triphenyltetrazolium chloride stain and tissue electron microscopy to compare infarct, BZ, and remote myocardium. Average LV ejection fraction was reduced (30±7%). MEMRI and DEMRI infarct volumes correlated with 2,3,5-triphenyltetrazolium chloride stain analysis (MEMRI, r=0.78; DEMRI, r=0.75; P<0.004). MEMRI infarct volume percentage was significantly lower than that of DEMRI (14±4% versus 23±4%; P<0.05). BZ MEMRI signal-to-noise ratio (SNR) was intermediate to remote and core infarct SNR (7.5±2.8 versus 13.2±3.4 and 2.9±1.6; P<0.0001), and DEMRI BZ SNR tended to be intermediate to remote and core infarct SNR (8.4±5.4 versus 3.3±0.6 and 14.3±6.6; P>0.05). Tissue electron microscopy analysis exhibited preserved cell structure in BZ cardiomyocytes despite transmural DEMRI enhancement. Conclusions— The dual-contrast MEMRI-DEMRI detects BZ viability within DEMRI infarct zones. This approach may identify injured, at-risk myocardium in ischemic cardiomyopathy.

Long-Term Histopathologic and IVUS Evaluations of a Novel Coiled Sheet Stent in Porcine Carotid Arteries

Carotid angioplasty with stent placement has been proposed as an alternative method for revascularization of carotid artery stenosis. A novel stent with a laser-cut, rolled sheet of Nitinol (EndoTex Interventional Systems, Inc., Cupertino, CA) has been developed to customize treatment of stenotic lesions in carotid arteries utilizing a single stent, designed to adapt to multiple diameters and to tapered or nontapered configurations. The purpose of this study is to evaluate the conformability and vascular response to a novel stent in a chronic porcine carotid model using serial three-dimensional intravascular ultrasound (IVUS) analysis as well as histological examination. Ten Yucatan pigs underwent stent implantation in both normal carotid arteries with adjunctive balloon angioplasty. Three-dimensional IVUS analysis was performed before stent implantation, after adjunctive balloon angioplasty, and at follow-up [1 month (n = 6), 3 months (n = 6), or 6 months (n = 8)]. Histological examination (injury score, percent plaque obstruction, and qualitative analysis) was also performed. All stents were successfully deployed and well apposed in different sized vessels (lumen area range: 19–30 mm2). Volumetric IVUS analysis showed no significant difference between the lumen areas before stent implantation and after adjunctive balloon angioplasty and no stent area change at each follow-up point compared to immediately postprocedure. Histological examination revealed minimal injury and neointimal hyperplasia at each follow-up point. In the chronic porcine carotid model, the novel stent system demonstrated good conformability, resulting in minimal vessel injury and neointimal formation.