Jaehoon Chung

Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells

OBJECTIVES The goal of this study is to characterize resident cardiac stem cells (CSCs) and investigate their therapeutic efficacy in myocardial infarction by molecular imaging methods. BACKGROUND CSCs have been isolated and characterized in vitro. These cells offer a provocative method to regenerate the damaged myocardium. However, the survival kinetics and function of transplanted CSCs have not been fully elucidated. METHODS CSCs were isolated from L2G85 transgenic mice (FVB strain background) that constitutively express both firefly luciferase and enhanced green fluorescence protein reporter gene. CSCs were characterized in vitro and transplanted in vivo into murine infarction models. Multimodality noninvasive imaging techniques were used to assess CSC survival and therapeutic efficacy for restoration of cardiac function. RESULTS CSCs can be isolated from L2G85 mice, and fluorescence-activated cell sorting analysis showed expression of resident CSC markers (Sca-1, c-Kit) and mesenchymal stem cell markers (CD90, CD106). Afterwards, 5 x 10(5) CSCs (n = 30) or phosphate-buffered saline control (n = 15) was injected into the hearts of syngeneic FVB mice undergoing left anterior descending artery ligation. Bioluminescence imaging showed poor donor cell survival by week 8. Echocardiogram, invasive hemodynamic pressure-volume analysis, positron emission tomography imaging with fluorine-18-fluorodeoxyglucose, and cardiac magnetic resonance imaging demonstrated no significant difference in cardiac contractility and viability between the CSC and control group. Finally, postmortem analysis confirmed transplanted CSCs integrated with host cardiomyocytes by immunohistology. CONCLUSIONS In a mouse myocardial infarction model, Sca-1-positive CSCs provide no long-term engraftment and benefit to cardiac function as determined by multimodality imaging.

αB-Crystallin Improves Murine Cardiac Function and Attenuates Apoptosis in Human Endothelial Cells Exposed to Ischemia-Reperfusion

BACKGROUND This study investigates the protective effect of exogenous αB-crystallin (CryAB) on myocardial function after ischemia-reperfusion injury. METHODS Mice underwent temporary left anterior descending artery occlusion for 30 minutes. Either CryAB (50 μg) or phosphate-buffered saline (100 μL [n=6, each group]) were injected in the intramyocardial medial and lateral perinfarct zone 15 minutes before reperfusion. Intraperitoneal injections were administered every other day. Left ventricular ejection fraction was evaluated on postoperative day 40 with magnetic resonance imaging. To investigate the effect of CryAB on apoptosis after hypoxia/reoxygenation in vitro, murine atrial cardiomyocytes (HL-1 cells) or human microvascular endothelial cells (HMEC-1) were incubated with either 50 μg CryAB (500 μg /10 mL) or phosphate-buffered saline in a hypoxia chamber for 6, 12, and 24 hours, followed by 30 minutes of reoxygenation at room air. Apoptosis was then assessed by western blot (Bcl-2, free bax, cleaved caspases-3, 9, PARP) and enzyme-linked immunosorbent assay analyses (cytoplasmic histone-associated DNA fragments and caspase-3 activity). RESULTS On postoperative day 40, CryAB-treated mice had a 1.8-fold increase in left ventricular ejection fraction versus control mice (27%±6% versus 15%±4% SD, p<0.005). In vitro, (1) the HL-1 cells showed no significant difference in apoptotic protein expression, cytoplasmic histone-associated DNA fragments, or caspase-3 activity; (2) the HMEC-1 cells had increased but not significant apoptotic protein expression with, however, a significant decrease in cytoplasmic histone-associated DNA fragments (1.5-fold, p<0.01) and caspase-3 activity (2.7-fold, p<0.005). CONCLUSIONS Exogenous CryAB administration significantly improves cardiac function after ischemia-reperfusion injury, in vivo. The protective anti-apoptotic affects of CryAB may target the endothelial cell.

A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.

Cell death by apoptosis is critical in myocardial diseases, and noninvasive detection of early, reversible apoptosis might be useful clinically. Exogenous Annexin‐V (ANX) protein binds membrane phosphatidylserine, which is externalized in early apoptosis. A molecular MRI probe was constructed with superparamagnetic iron oxide (SPIO) conjugated to recombinant human ANX (ANX‐SPIO). Apoptosis was induced with doxorubicin, a cardiotoxic cancer drug, in culture in neonatal rat ventricular myocytes, cardiac fibroblasts, and mesenchymal stem cells, and in vivo in the mouse heart. ANX‐SPIO was validated using T2*‐weighted 3T MRI. ANX‐SPIO produced T2* signal loss, reflecting iron content, that correlated highly with independent apoptosis markers; bound with high affinity to apoptotic myocytes by competition assay (Ki 69 nM); detected apoptosis in culture much earlier than did TUNEL stain; and revealed fibroblast resistance to apoptosis. With apoptosis in vivo, ANX‐SPIO produced diffuse myocardial T2* signal loss that correlated with increased iron stain and caspase activity. Treatment with an alpha‐1‐adrenergic agonist in vivo reversed apoptosis and eliminated the ANX‐SPIO MRI signal. It is concluded that cardiac MRI of ANX‐SPIO detects early, nonischemic cardiac apoptosis in culture and in vivo, and can identify reversibly injured cardiac cells in diseased hearts, when treatment is still possible. Magn Reson Med, 2011.

Manganese‐guided cellular MRI of human embryonic stem cell and human bone marrow stromal cell viability

This study investigated the ability of MnCl2 as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T1 and T2 values of MnCl2‐labeled hESC and hBMSC were measured and viability signal of manganese (Mn2+)‐labeled cells was validated. Furthermore, the biological activity of Ca2+‐channels was modulated utilizing both Ca2+‐channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl2‐labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn2+‐guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging. Magn Reson Med, 2009.

In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long‐term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO‐conjugated monoclonal antibody against these antigens (SPIO‐MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO‐MAb. The human and mouse ESCs were transduced with the RG (ESC‐RGs) and transplanted into the peri‐infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO‐MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC‐derived teratoma formation in the injured murine myocardium. Magn Reson Med, 2011.

RevaTen platelet-rich plasma improves cardiac function after myocardial injury.

OBJECTIVE Cell therapy is an exciting area of investigation for repair of injured myocardial tissue. Platelet-rich plasma (PRP) is an autologous fractionation of whole blood containing high concentrations of growth factors including vascular endothelial growth factor and insulin-like growth factor, among many others. PRP has been shown to safely and effectively enhance healing of musculoskeletal tissue primarily by reparative cell signaling. Despite a growing body of evidence on PRPs safety and efficacy, limited studies have been performed using PRP in cardiovascular tissues. Utilizing a murine myocardial permanent ligation and ischemia/reperfusion model, this study sought to determine whether RevaTen PRP (Menlo Park, CA, USA), a proprietary formulation of PRP, improves cardiac function as measured by left ventricular ejection fraction (LVEF). METHODS Via thoracotomy, the left anterior descending arteries (LAD) of 28 mice were occluded by suture either permanently or for 45 min to induce ischemic injury and then reperfused. Mice undergoing permanent ligation had intramyocardial injections of either RevaTen PRP (n=5) or phosphate-buffered saline (PBS; n=4). Magnetic resonance (MR) imaging was performed to calculate LVEF at 7 days. Mice undergoing ischemia and reperfusion had intramyocardial injections of either PRP (n=10) or PBS (n=9) and underwent MR imaging to calculate LVEF at 21 days. Hearts were harvested for histologic examination following imaging. RESULTS Compared with PBS controls, RevaTen PRP-treated animals that underwent LAD ligation had a 38% higher LVEF 7 days after injury (PRP=36.1±6.1%; PBS=26.4±3.6%, P=.027). Compared with PBS controls, PRP-treated animals who underwent ischemia-reperfusion of the LAD had a 28% higher LVEF 21 days after injury (PRP=37.6±4.8%, control=29.3±9.7%, P=.038). Histologic analysis suggested the presence of more scar tissue in the control group compared to the PRP-treated animals. CONCLUSION MR imaging demonstrated a positive effect of RevaTen PRP on left ventricular function in both a ligation and ischemia-reperfusion murine model. Our results suggest RevaTen PRP should be investigated further as a potential point-of-care biologic treatment following myocardial injury.

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.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Theranostic effect of serial manganese‐enhanced magnetic resonance imaging of human embryonic stem cell derived teratoma

Although human embryonic stem cell (hESC) hold therapeutic potential, teratoma formation has deterred clinical translation. Manganese (Mn2+) enters metabolically active cells through voltage‐gated calcium channels and subsequently, induces T1 shortening. We hypothesized that serial manganese‐enhanced MRI would have theranostic effect to assess hESC survival, teratoma formation, and hESC‐derived teratoma reduction through intracellular accumulation of Mn2+. Firefly luciferase transduced hESCs (hESC‐Lucs) were transplanted into severe combined immunodeficient mouse hindlimbs to form teratoma. The chemotherapy group was injected with MnCl2 intraperitoneally three times a week. The control group was given MnCl2 only prior to manganese‐enhanced MRI. Longitudinal evaluation by manganese‐enhanced MRI and bioluminescence imaging was performed. The chemotherapy group showed significant reduction in the teratoma volume and luciferase activity at weeks 6 and 8. Histology revealed increased proportion of dead cells and caspase 3 positive cells in the chemotherapy group. Systemic administration of MnCl2 enabled simultaneous monitoring and elimination of hESC‐derived teratoma cells by higher intracellular accumulation of Mn2+. Magn Reson Med, 2012.