Paulmurugan, Ramasamy

Introduction: Stem cells have poor survival after transplantation, what can limit the use of these therapies. Mitochondrial dysfunction has been recognized as a limiting event that can lead to cell death. Here, we hypothesized that the mitochondrial function of progenitor cells can be monitored in vivo using reporter gene-based molecular imaging. A better understanding of the biology of transplanted stem cells in the ischemic myocardium can lead to optimization of cell therapies. Methods: Using RT-PCR we identified the antioxidant NAD(P)H:Quinone oxidoreductase (NQO1) enzyme as a marker of mitochondrial dysfunction in mouse Mesenchymal Stem Cells (MSCs) that were treated with a mitochondrial stressor, diethyl maleate (DEM). We then constructed a luciferase reporter gene driven by the NQO1 promoter. After in vitro validation, MSCs were transplanted into a mouse model of ischemia/reperfusion (IR, n=4) and mitochondrial function was monitored by bioluminescence imaging. Results: MSCs treated with DEM had an increase in NQO1 reporter gene signal that inversely correlated with the amount of the mitochondrial by-product adenosine tri-phosphate (ATP, r = -0.9643, p = 0.0028; Fig 1A). Mitochondrial dysfunction was preserved by the use of a mitochondria-targeted antioxidant Mito-Tempo (MT), correcting ATP and NQO1 luciferase signal. In vivo studies showed that MSCs yielded higher Firefly luciferase activity in IR mice compared to sham and this signal was normalized when cells were pre-treated with MT (Fig 1B). Data was confirmed by ex vivo measurement of Firefly luciferase activity, which was 4.9-fold higher in the IR group than in shams, after correction for cell number (Fig 1C). Conclusions: We have developed a novel imaging strategy that allows noninvasive assessment of the mitochondrial status of transplanted stem cells in the ischemic myocardium. Knowing when transplanted cells “suffer’ from mitochondrial dysfunction may allow optimization of these therapies.

AbstractCell therapy is being investigated as a powerful intervention to ameliorate the consequences of coronary artery disease. Among the different stem cell options, mesenchymal stem cells (MSCs) are particularly attractive due to their high availability, as well as immune‐privileged status. However, it is still unclear whether mesenchymal stem cells can acquire cardiomyogenic characteristics after they are transplanted to the myocardium. In this article, we outline protocols that illustrate the plasticity of MSCs and their ability to acquire cardiogenic characteristics when they are in an ischemic‐like environment, as typically encountered after transplantation into the ischemic heart. © 2021 Wiley Periodicals LLC.This article was corrected on 26 July 2022. See the end of the full text for details.Basic Protocol 1: Isolation of mesenchymal stem cells (MSCs)Support Protocol 1: Characterization of MSCs by flow cytometryBasic Protocol 2: Isolation of neonatal cardiomyoctes (NCMs)Support Protocol 2: Characterization of NCMsBasic Protocol 3: Cardiogenic plasticity of MSCs under ischemic‐like conditionsSupport Protocol 3: Characterization of the cardiomyogenic potential of MSCs

Introduction: Stem cell (SC) therapy is a potential method of repairing the heart after injury. However, SCs have been shown to have poor survival after transplantation, at least in part, due to a hostile pro-inflammatory ischemic myocardium. Thus, there is significant interest in the identification of strategies that can increase the retention/survival of SCs after transplantation. Here we seek to investigate whether the use of an immunomodulatory hydrogel, containing anti-inflammatory sinapic acid (SA), promotes the survival of SCs after transplantation. Methods: Myocardial infarction was induced in rats followed by the delayed delivery of A) mesenchymal stem cells (MSCs), B) MSCs in a hydrogel without SA or C) MSCs in a hydrogel containing SA (10μM). Delivery of MSCs (7.5 x 10 5 in 30μL) occurred 7 days after MI, representing the subacute stage and to minimize acute inflammation. Survival of MSCs, stably expressing firefly luciferase under the constitutive CMV promoter, was followed longitudinally using bioluminescence imaging. Results: Panels A-C provide representative images of the viability of MSCs in different groups, with the quantification shown in panel D. There were no differences in survival when MSCs were delivered alone (A) or in a hydrogel without SA (B). However, the survival of MSCs was significantly increased when they were transplanted in a hydrogel with the anti-inflammatory SA (C). Conclusions: Here we provide evidence of the potential for an immunomodulatory hydrogel to enhance the viability of MSCs after infarction, which may lead to improvement of cardiac function. Furthermore, use of this hydrogel in combination with stem cells could be a potential therapeutic strategy for ameliorating cardiac repair after significant damage.

Abstract Little is known on the phenotypic characteristics of stem cells (SCs) after they are transplanted to the myocardium, in part due to lack of noninvasive platforms to study SCs directly in the living subject. Reporter gene imaging has played a valuable role in the noninvasive assessment of cell fate in vivo. In this study, we validated a pathway-specific reporter gene that can be used to noninvasively image the phenotype of SCs transplanted to the myocardium. Rat mesenchymal SCs (MSCs) were studied for phenotypic evidence of myogenic characteristics under in vitro conditions. After markers of myogenic characteristics were identified, we constructed a reporter gene sensor, comprising the firefly luciferase (Fluc) reporter gene driven by the troponin T (TnT) promoter (cardio MSCs had threefold expression in polymerase chain reaction compared to control MSCs) using a two-step signal amplification strategy. MSCs transfected with TnT-Fluc were studied and validated under in vitro conditions, showing a strong signal after MSCs acquired myogenic characteristics. Lastly, we observed that cardio MSCs had higher expression of the reporter sensor compared to control cells (0.005 ± 0.0005 vs 0.0025 ± 0.0008 Tnt-Fluc/ubiquitin-Fluc, P < .05), and that this novel sensor can detect the change in the phenotype of MSCs directly in the living subject. Pathway-specific reporter gene imaging allows assessment of changes in the phenotype of MSCs after delivery to the ischemic myocardium, providing important information on the phenotype of these cells. Imaging sensors like the one described here are critical to better understanding of the changes that SCs undergo after transplantation.