**18F-FDG PET-Based Imaging of Myocardial Inflammation Predicts a Functional Outcome Following Transplantation of mESC-Derived Cardiac Induced Cells in a Mouse Model of Myocardial Infarction**

**Praveen Vasudevan 1,2, Ralf Gaebel 1,2, Piet Doering 3,4, Paula Mueller 1,2, Heiko Lemcke 1,2, Jan Stenzel 5, Tobias Lindner 5, Jens Kurth 3, Gustav Steinho**ff **1,2, Brigitte Vollmar 5, Bernd Joachim Krause 3, Hueseyin Ince 6, Robert David 1,2 and Cajetan Immanuel Lang 6,\***


Received: 21 October 2019; Accepted: 9 December 2019; Published: 11 December 2019

**Abstract:** Cellular inflammation following acute myocardial infarction has gained increasing importance as a target mechanism for therapeutic approaches. We sought to investigate the effect of syngeneic cardiac induced cells (CiC) on myocardial inflammation using 18F-FDG PET (Positron emission tomography)-based imaging and the resulting effect on cardiac pump function using cardiac magnetic resonance (CMR) imaging in a mouse model of myocardial infarction. Mice underwent permanent left anterior descending coronary artery (LAD) ligation inducing an acute inflammatory response. The therapy group received an intramyocardial injection of 10<sup>6</sup> CiC into the border zone of the infarction. Five days after myocardial infarction, 18F-FDG PET was performed under anaesthesia with ketamine and xylazine (KX) to image the inflammatory response in the heart. Flow cytometry of the mononuclear cells in the heart was performed to analyze the inflammatory response. The effect of CiC therapy on cardiac function was determined after three weeks by CMR. The 18F-FDG PET imaging of the heart five days after myocardial infarction (MI) revealed high focal tracer accumulation in the border zone of the infarcted myocardium, whereas no difference was observed in the tracer uptake between infarct and remote myocardium. The CiC transplantation induced a shift in 18F-FDG uptake pattern, leading to significantly higher 18F-FDG uptake in the whole heart, as well as the remote area of the heart. Correspondingly, high numbers of CD11<sup>+</sup> cells could be measured by flow cytometry in this region. The CiC transplantation significantly improved the left ventricular ejection function (LVEF) three weeks after myocardial infarction. The CiC transplantation after myocardial infarction leads to an improvement in pump function through modulation of the cellular inflammatory response five days after myocardial infarction. By combining CiC transplantation and

the cardiac glucose uptake suppression protocol with KX in a mouse model, we show for the first time, that imaging of cellular inflammation after myocardial infarction using 18F-FDG PET can be used as an early prognostic tool for assessing the efficacy of cardiac stem cell therapies.

**Keywords:** inflammation; 18F-FDG PET; cardiomyocytes; cardiac induced cells; cardiac function; non-invasive imaging

#### **1. Introduction**

Inflammatory activity of the innate immune system following myocardial infarction substantially influences remodeling after myocardial infarction (MI) and the evolution of heart failure [1]. Therefore, anti-inflammatory therapies have been under intense investigation since the early 1970s. Yet, none of the clinical trials testing pharmacological strategies aiming at unspecific reduction of myocardial inflammation has proven successful [2]. The main reason for these disappointing findings is the complexity of the well-orchestrated activity of different leukocyte populations. The amplitude and duration of inflammation and the timely and spatial resolution within the heart define the quality of the scar following MI and the amount of tissue loss rather than the mere extent of inflammation [1]. Hence, the concept of suppressing inflammation has changed to a further elaborated concept of "modulating cellular inflammation". The balance of pro- and anti-inflammatory immune cells and their recruiting mechanisms are discussed as both therapeutic and prognostic targets [1]. Further understanding of this intricate cellular immune response to myocardial ischemia in a translational setting requires non-invasive molecular imaging tools. The 18F-FDG PET (Positron emission tomography) using specific protocols for suppressing glucose uptake in cardiomyocytes has recently been introduced to detect cellular inflammation following MI in both patients and C57BL/6 mice [1,3–5]

Protocols to suppress physiological myocardial 18F-FDG uptake require low-carbohydrate diet the day before imaging followed by a 12 h fasting period and intravenous application of heparin before the actual scan [3]. In mice, myocardial glucose metabolism can be effectively suppressed by replacing the commonly used anaesthetic isofluorane for a ketamine/xylazine-based protocol [1].

However, to our knowledge, no studies have used these protocols for measuring therapeutic effects of therapies aiming at improving myocardial healing following MI. Therefore, we implanted syngeneic cardiac induced cells committed to the cardiac lineage in order to improve post-MI cardiac function in 129Sv mice as measured my cardiac magnetic resonance (CMR). Cellular inflammation was detected by 18F-FDG PET in vivo and by post-mortem flow cytometry in both infarcted and remote myocardium.

We hypothesize that PET-based imaging and quantification of cellular inflammation can be used as a molecular imaging tool for both quantification and spatial distribution of monocytes and as an early prognostic tool to predict the effect of cardiac stem cell therapies modulating post-MI inflammation.

#### **2. Materials and Methods**

#### *2.1. Stem Cell Culture and Cardiovascular Di*ff*erentiation*

W4 murine embryonic stem cells (mESCs), originally isolated from the 129S6 mouse strain [6], were grown according to standard protocols as described previously [7]. In brief, cells were cultured in DMEM supplemented with 15% FBS Superior (Biochrom AG, Berlin, Germany), 1% Cell Shield® (Minerva Biolabs GmbH, Berlin, Germany), 100 μM non-essential amino acids, 1000 U/mL leukemia inhibitory factor (Phoenix Europe GmbH, Mannheim, Germany) and 100 μM β-mercaptoethanol (Sigma-Aldrich GmbH, Steinheim, Germany) at 37 ◦C, 5% CO2, and 20% O2. For cardiovascular differentiation we used cardiogenic differentiation medium, containing IBM (Iscove's Basal Medium, Biochrom AG) supplemented with 10% FBS Superior, 1% Cell Shield®, 100 μM non-essential amino

acids, 450 μm 1-thioglycerol (Sigma-Aldrich GmbH), and 213 μg/mL ascorbic acid (Sigma-Aldrich GmbH), as described previously [7,8]. Cardiovascular differentiation was initiated by hanging-drop culture for two days at 37 ◦C, 5% CO2, and 20% O2. 400 cells per drop were plated on the cover of a square petri dish and grown for 2 days to start formation of embryoid bodies (EB). Afterwards, EB were grown additional four days in suspension culture [9] and then harvested for transplantation and PCR analysis. These cells will be referred to as cardiac induced cells (CiC). They were cultured till day 30 for beating foci analysis.

#### *2.2. Animal Model*

The present study was approved by the federal animal care committee of the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern (LALLF, Germany) (registration no. LALLF M-V/TSD/7221.3-1.1-054/15). The 129S6/SvEvTac were bred in the animal facility of the Rostock University Medical Center and maintained in specified pathogen-free conditions. The mice had access to water and standard laboratory chow ad libitum and received humane care according to the German legislation on protection of animals and the Guide for the Care and Use of Laboratory Animals (NIH publication 86–23, revised 1985), and all efforts were made to minimize suffering. Mice were anaesthetized with pentobarbital (50 mg/kg, intraperitoneal). Following thoracotomy, the left anterior descending coronary artery (LAD) was ligated to induce acute myocardial infarction. The MI group received an intramyocardial injection of 10 μL PBS mixed with 10 μL Growth Factor Reduced Matrigel™ Matrix (Corning, Berlin, Germany). The MI induction plus cell transplantation (MIC) group received a suspension of 1 <sup>×</sup> 10<sup>6</sup> syngeneic in PBS (10μL) mixed with 10 <sup>μ</sup>L Growth Factor Reduced Matrigel™ Matrix. Injections of 4 × 5 μL were given along the infarct border. The injection site was controlled visually at the time of transplantation [10].

Healthy mice (n = 33) were divided into 7 groups for 18F-FDG PET imaging 5 days following MI using different protocols for anaesthesia:


Cardiac function was assessed in separate groups three weeks following MI by cardiac MRI:


Flow cytometric analysis was performed in separate groups (MI, n = 5 and MIC, n = 5).
