Rodriguez‐Porcel, Martin

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

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.

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.

Regenerative medicine with the use of stem cells has appeared as a potential therapeutic alternative for many disease states. Despite initial enthusiasm, there has been relatively slow transition to clinical trials. In large part, numerous questions remain regarding the viability, biology and efficacy of transplanted stem cells in the living subject. The critical issues highlighted the importance of developing tools to assess these questions. Advances in molecular biology and imaging have allowed the successful non-invasive monitoring of transplanted stem cells in the living subject. Over the years these methodologies have been updated to assess not only the viability but also the biology of transplanted stem cells. In this review, different imaging strategies to study the viability and biology of transplanted stem cells are presented. Use of these strategies will be critical as the different regenerative therapies are being tested for clinical use.

To examine whether changes in renal perfusion pressure (RPP) within the range of autoregulation induce detectable changes in tubular dynamics in an entire nephron population of the intact kidney, we measured, using electron beam computed tomography (EBCT), transit times (TT, s) and intratubular concentration (%) of filterable contrast media in various nephron segments simultaneously with renal regional perfusion. In seven dogs ( group A) this was performed at the upper and lower limits of autoregulation (RPP = 130 and 95 mmHg, respectively) while group B( n = 5) served as control. In group A alone, a decrease in RPP led to an increase in TT by 40%, 68%, and 32% in the proximal tubules, ascending limb of Henle’s loop, and distal tubules, respectively, in association with an increase in intratubular concentration (+50%, 80%, and 42%, respectively). Papillary perfusion decreased, whereas perfusion of the adjacent, outlying inner medulla increased. The decrease in papillary perfusion correlated positively with the concurrent change in sodium excretion ( R = 0.81). This study demonstrates that changes in RPP within the autoregulatory range elicit changes of tubular sodium reabsorption mainly in proximal, distal, and ascending tubules, in which most of the nephrons participate. These tubular changes are associated with an alteration of perfusion circumscribed to two areas of the inner renal medulla.