Peterson, Karen M.

Increased oxidative stress has been suggested to play an important role in the biology of mesenchymal stem cells (MSCs). However, little is known of the effect of increased oxidative stress in-vivo, partly due to the lack of appropriate imaging technologies for monitoring of stem cell biology in the living subject. Recent developments in molecular imaging permit, for the first time, the longitudinal monitoring of stem cell biology in vivo. We hypothesize that changes in oxidative stress can be monitored using a reporter gene strategy (molecular imaging). Methods: We have developed a novel oxidative stress sensor, using a two-step transcriptional amplification strategy to enhance the expression of firefly luciferase (Fluc), driven by the pro-oxidant enzyme NAD(P)H promoter, and used to monitor oxidative stress in rMSCs. rMSCs was induced by hypoxia (1% O2 for 24 hours) and serum deprivation (SD) and confirmed using a dichlorofluorescein colorimetric assay (measures the production of hydrogen peroxide-H2O2-) and NAD(P)H protein expression. Results: Both serum deprivation and hypoxia led to a pro-oxidant state (Fig. A–B ). Most importantly, increased oxidative stress led to increased activity of the novel reporter gene sensor (Fig. C ) which was decreased in the presence of antioxidants (superoxide dismuatse mimetic Tempol, 5mM, SD+T, Fig D ). Results were reproduced in a different cell type (rat cardiomyoblast, data not shown). Conclusion: Changes in oxidative stress in MSCs can be monitored using molecular imaging. Reporter Gene-based Molecular Imaging, a strategy that is clinically applicable, provides the opportunity to monitor stem cell survival in the living subject.

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.

Background Stem cells undergo significant death after transplantation, but the mechanisms underlying this deleterious response remain unclear. Increased oxidative stress has been shown to be present in states of hypoxia or cellular stress. Thus, we hypothesize that oxidative stress blockade would ameliorate the increased cell death when exposed to hypoxia. Methods Cardiac stem cells were exposed to hypoxic conditions in cell culture (1% O 2 for 24 hours). Oxidative status (by measuring reactive oxygen species‐ROS‐) and cell survival (by the MTT assay) were measured. In a separate group of cells, oxidative stress was blocked using a superoxide dismutase mimetic (Tempol, 1‐10µm/L). Results Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subfraction 47 phox ) and in the amount of ROS (increased in the conversion of dihydroethidium ‐DHE‐ and dichlorofluorescein ‐DCF‐) what led to a decrease in stem cell viability. Pre‐conditioning of stem cells with antioxidants (Tempol) decreased the amount of cellular ROS and improved stem cell survival. The increased expression of NAD(P)H p47 phox seen in hypoxia was unaltered by oxidative stress blockade. Conclusion Increased oxidative stress plays a role in the early cell death after hypoxia. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.

Introduction: Myocardial infarction is an inflammatory condition that leads to scar deposition and cardiac remodeling. Stem cells (SC) are an alternative to prevent cardiac remodeling, but have poor survival after transplanted to an pro-inflammatory ischemic myocardium. Polymeric biocompatible scaffolds are biomaterials that can improve SC retention and survival, with the goal to improve regional cardiac function. Methods: We electrospun fibers of a) uniaxial polycaprolactone (PCL) and b) coaxial PCL (core) and gelatin methacrylate (sheath), an anti-inflammatory substance. We first tested the SC retention of each scaffold. Then, adipose derived mesenchymal SCs (MSCs) were co-cultured with M1 macrophages (pro-inflammatory) with and without scaffold and we measure the expression of inflammatory-related genes (RT-PCR). Lastly, PCL+GelMA scaffolds with and without MSCs (3.5x10 3 MSCs) were implanted after myocardial ischemia/reperfusion (IR) and followed for 2 weeks. Results: The average diameter of PCL and PCL+GelMA fibers were 730±40nm and 1110±50nm, respectively. PCL+GelMA scaffolds increased MSCs adhesion compared to PCL (absorbance: 0.09±.03 for PCL and 0.24±0.02 for coaxial, p<0.05). Figure A shows that MSCs in presence of M1 macrophages had a significant increase in inflammatory genes, which was significantly ameliorated in the presence of GelMA. In preliminary animal studies (n=1-4), MSCs delivered in a PCL+GelMA scaffold had partial preservation of regional function parameteres compared to IR only or IR+PCL+GelMA (no MSCs) (Figure B). Conclusion: Coaxially PCL+gelMA scaffolds attenuate the pro-inflammatory milieu of MSCs under ischemic-like conditions, and may improve regional cardiac remodeling.

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

AbstractStem cell−based therapies for various ailments have attracted significant attention for over a decade. However, low retention of transplanted cells at the damaged site has hindered their potential for use in therapy. Tissue engineered grafts with fibrillar structures mimicking the extracellular matrix (ECM) can be potentially used to increase the retention and engraftment of stem cells at the damaged site. Moreover, these grafts may also provide mechanical stability at the damaged site to enhance function and regeneration. Among all the methods to produce fibrillar structures developed in recent years, electrospinning is a simple and versatile method to produce fibrous structures ranging from a few nanometers to micrometers. Coaxial electrospinning enables production of a mechanically stable core with a cell‐binding sheath for enhanced cell adhesion and proliferation. Furthermore, this process provides an alternative to functionalized engineered scaffolds with specific compositions. The present article describes the protocol for developing a polycaprolactone (PCL) core and gelatin/gelatin methacrylate (GelMA) sheath laden with stem cells for various regenerative engineering applications. © 2021 Wiley Periodicals LLC.This article was corrected on 18 July 2022. See the end of the full text for details.Basic Protocol 1: Uniaxial PCL electrospinningBasic Protocol 2: Coaxial electrospinningSupport Protocol 1: Scaffold characterization for Basic Protocols 1 and 2Basic Protocol 3: Cell seeding on uniaxial and coaxial electrospun scaffolds and MTS assaySupport Protocol 2: Preparation of scaffold with cells for scanning electron microscopy

Polycystic kidney disease (PKD) has been linked to abnormal structure/function of ciliary proteins, leading to renal dysfunction. Recently, attention has been focused in the significant vascular abnormalities associated with PKD, but the mechanisms underlying this phenomenon remain elusive. Here, we seek to define the molecular events regulating the angiogenic imbalance observed in PKD. Using micro computed tomography (n=7) and protein expression analysis (n=5), we assessed the vascular density and the angiogenic profile of noncystic organs in a well-established PKD rat model (Polycystic Kidney-PCK rat). Heart and lungs of PCK rats have reduced vascular density and decreased expression of angiogenic factors compared with wild type. Similarly, PCK-vascular smooth muscle cells (VSMCs; n=4) exhibited lower levels of vascular markers. Then, using small interfering RNA (n=4), we determined the role of the ciliary protein fibrocystin in wild type-VSMCs, a critical component/regulator of vascular structure and function. Reduction of fibrocystin in wild type-VSMCs (n=4) led to an abnormal angiogenic potential similar to that observed in PCK-VSMCs. Furthermore, we investigated the involvement of the hedgehog signaling, a pathway closely linked to the primary cilium and associated with vascular development, in PKD. Mechanistically, we demonstrated that impairment of the hedgehog signaling mediates, in part, this abnormal angiogenic phenotype. Lastly, overexpression of Gli1 in PCK-VSMCs (n=4) restored the expression levels of proangiogenic molecules. Our data support a critical role of fibrocystin in the abnormal vascular phenotype of PKD and indicate that a dysregulation of hedgehog may be responsible, at least in part, for these vascular deficiencies.

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 &lt; .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.

Polycystic kidney disease (PKD) is a common cause of end-stage renal failure, for which there is no accepted treatment. Progenitor and stem cells have been shown to restore renal function in a model of renovascular disease, a disease that shares many features with PKD. The objective of this study was to examine the potential of adult stem cells to restore renal structure and function in PKD. Bone marrow-derived mesenchymal stromal cells (MSCs, 2.5 × 10 5 ) were intrarenally infused in 6-week-old PCK rats. At 10 weeks of age, PCK rats had an increase in systolic blood pressure (SBP) versus controls (126.22 ± 2.74 vs. 116.45 ± 3.53 mmHg, p < 0.05) and decreased creatinine clearance (3.76 ± 0.31 vs. 6.10 ± 0.48 μl/min/g, p < 0.01), which were improved in PKD animals that received MSCs (SBP: 114.67 ± 1.34 mmHg, and creatinine clearance: 4.82 ± 0.24 μl/min/g, p = 0.001 and p = 0.003 vs. PKD, respectively). MSCs preserved vascular density and glomeruli diameter, measured using microcomputed tomography. PCK animals had increased urine osmolality (843.9 ± 54.95 vs. 605.6 ± 45.34 mOsm, p < 0.01 vs. control), which was improved after MSC infusion and not different from control (723.75 ± 56.6 mOsm, p = 0.13 vs. control). Furthermore, MSCs reduced fibrosis and preserved the expression of proangiogenic molecules, while cyst size and number were unaltered by MSCs. Delivery of exogenous MSCs improved vascular density and renal function in PCK animals, and the benefit was observed up to 4 weeks after a single infusion. Cell-based therapy constitutes a novel approach in PKD.