Direct Cell Labeling Research Articles

PET Imaging for Monitoring Cellular and Immunotherapy of Cancer.

Cancer immunotherapy, including checkpoint blockade and cellular therapy, has become a cornerstone in cancer treatment. However, understanding the factors driving patient response or resistance to these therapies remains challenging. The dynamic interplay between the immune system and tumors requires new approaches for characterization. Biopsies and blood tests provide valuable information, but their limitations have led to increased interest in positron emission tomography (PET)/computed tomography imaging to complement these strategies. The noninvasive nature of PET imaging makes it ideal for monitoring the dynamic tumor immune microenvironment. This review discusses various PET imaging approaches, including immune cell lineage markers, immune functional markers, immune cell metabolism, direct cell labeling, and reporter genes, highlighting their potential in targeted immunotherapies and cell-based approaches. Although PET imaging has limitations, its integration into diagnostic strategies holds promise for improving patient outcomes and accelerating drug development in cancer immunotherapy.

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections.

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.

A multi-modality imaging strategy to determine the multiple in vivo fates of human umbilical cord mesenchymal stem cells at different periods of acute liver injury treatment.

Human umbilical cord mesenchymal stem cells (HUCMSCs) are applied for disease therapy as a new type of drug in many countries. Their effects are not only presented by live cells, but also apoptotic bodies or cell fragments of dead cells. Therefore, it is meaningful to determine the multiple fates of HUCMSCs in vivo. Although various probes combining different imaging modalities have been developed to label and trace transplanted HUCMSCs in vivo, the status of the cells (live, dead, or apoptotic) was not distinguished, and a thorough understanding of the multiple fates of HUCMSCs after transplantation in vivo is lacking. Therefore, a magnetic resonance (MR)/near infrared fluorescent (NIRF)/bioluminescence (BI) multi-modality imaging strategy was developed. Iron oxide nanoparticles (IONPs) were assembled into 100 nm nanoparticles using epigallocatechin gallate as a chemical linker to increase the MR signal and reduce the exocytosis of IONPs for direct cell labeling and longitudinal MR imaging tracking. Fluorescent probes for apoptosis (DEVD-Cy-OH) were also loaded in the above assemblies to monitor the cell status. Meanwhile, the cell surface was labeled with the fluorescent dye Cy7 via bioorthogonal reactions to visualize the NIRF signal. Luciferase was lentivirally transfected into live cells to generate bioluminescence. Such labeling did not affect either the viability, proliferation, migration, differentiation characteristics of HUCMSCs or their therapeutic effects on acute liver injury mice in vivo. The in vivo fates of HUCMSCs were monitored via MR/NIRF/BI multi-modality imaging in acute liver injury mice. Although MR and Cy7 signals aggregated in injured liver for 7 days, the BI signals persisted for less than 24 hours. There was an increase in DEVD-Cy-OH signals in the injured liver, but they were almost at the basal level. That means that HUCMSCs survive in mice for a short time, and the dead form of HUCMSCs accumulated in a large quantity and sustained for a long time, which might contribute to their therapeutic effect.

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies

Plain Language SummaryWe developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells’ platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband’s or patient’s own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.

Neural-fated self-renewing cells regulated by Sox2 during secondary neurulation in chicken tail bud

In amniotes, unlike primary neurulation in the anterior body, secondary neurulation (SN) proceeds along with axial elongation by the mesenchymal-to-epithelial transition of SN precursors in the tail bud. It has been under debate whether the SN is generated by neuromesodermal common progenitor cells (NMPs) or neural restricted lineage. Our direct cell labeling and serial transplantations identify uni-fated (neural) precursors in the early tail bud. The uni-fated SN precursor territory is further divided into two subpopulations, neural-differentiating and self-renewing cells, which are regulated by high- and low levels of Sox2, respectively. Unexpectedly, uni-fated SN precursors change their fate at later stages to produce both SN and mesoderm. Thus, chicken embryos adopt a previously unappreciated prolonged phase with uni-fated SN stem cells in the early tail bud, which is absent or very limited in mouse embryos.

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs’ presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs’ influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs’ impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs’ influence on treated skeletal myoblasts, not interfering with basic cell functions.

Early segregation of the adrenal cortex and gonad in chicken embryos.

The adrenal gland is an endocrine organ that plays essential roles in stress responses. This organ consists of two types of tissues, adrenomedulla and adrenocortex, deriving from different embryonic origins. Whereas it is well accepted that the adrenomedulla derives from neural crest cells, the origin of the adrenocortex remains elusive. In addition, the adrenocortex and gonads, two major steroid hormone-producing tissues, have been thought to share the same origin, although the experimental evidence is lacking. In this study, to identify the origin of adrenocortex and to compare it to that of gonads, we scrutinized the medial portion of the coelomic epithelium (CE) after the lateral plate mesoderm has split into two CE components with a concomitant opening of the coelomic cavity in between them. We found that early medial CE consists of a two-cell layer-thick band of epithelial-like cells, the outer and inner CEs. The outer CE faces the coelomic cavity, whereas the inner CE is juxtaposed to nascent blood vessels. Combining direct cell labeling with early molecular markers, we found that outer CE was the origin of the gonad but not the adrenocortex. The adrenocortex, instead, appears to derive from inner CE. Thus, the adrenocortical and gonadal progenitors are already segregated from each other when the coelomic cavity has opened. This study provides a new basis for understanding how the adrenal gland forms and how steroid hormone-producing tissues arise during development.

Liposome-Indocyanine Green Nanoprobes for Optical Labeling and Tracking of Human Mesenchymal Stem Cells Post-Transplantation In Vivo.

Direct labeling of human mesenchymal stem cells (hMSC) prior to transplantation provides a means to track cells after administration and it is a powerful tool for the assessment of new cell-based therapies. Biocompatible nanoprobes consisting of liposome-indocyanine green hybrid vesicles (liposome-ICG) are used to safely label hMSC. Labeled hMSC recapitulating a 3D cellular environment is transplanted as spheroids subcutaneously and intracranially in athymic nude mice. Cells emit a strong NIR signal used for tracking post-transplantation with the IVIS imaging system up to 2 weeks (subcutaneous) and 1 week (intracranial). The transplanted stem cells are imaged in situ after engraftment deep in the brain up to 1 week in living animals using optical imaging techniques and without the need to genetically modify the cells. This method is proposed for efficient, nontoxic direct cell labeling for the preclinical assessment of cell-based therapies and the design of clinical trials, and potentially for localization of the cell engraftment after transplantation into patients.

Direct Cell Labeling to Image Transplanted Stem Cells in Real Time Using a Dual-Contrast MRI Technique.

Exogenous direct cell labeling with superparamagnetic iron oxide nanoparticles (SPIONs) is currently the most employed cell-labeling technique for tracking transplanted cells using magnetic resonance imaging (MRI). Although SPION-based cell labeling is effective for monitoring cell delivery and migration, monitoring cell survival is still a challenge. This unit describes an MRI technique that permits detection of the delivery, migration, and death of transplanted cells. This dual-contrast technique involves labeling cells with two different classes of MRI contrast agents, possessing different diffusion coefficients: SPIONs (T2 /T2* contrast agents, with lower diffusion coefficients) and gadolinium chelates (T1 contrast agents, with higher diffusion coefficients). In live cells, where both agents are in close proximity, the T2 /T2* contrast predominates and the T1 contrast is quenched. In dead cells, where the cell membrane is breached, gadolinium chelates diffuse from the SPIONs and generate a signature T1 contrast enhancement in the vicinity of dead cells. © 2017 by John Wiley & Sons, Inc.

A CD45‐based barcoding approach to multiplex mass‐cytometry (CyTOF)

CyTOF enables the study of the immune system with a complexity, depth, and multidimensionality never achieved before. However, the full potential of using CyTOF can be limited by scarce cell samples. Barcoding strategies developed based on direct labeling of cells using maleimido-monoamide-DOTA (m-DOTA) provide a very useful tool. However, using m-DOTA has some inherent problems, mainly associated with signal intensity. This may be a source of uncertainty when samples are multiplexed. As an alternative or complementary approach to m-DOTA, conjugating an antibody, specific for a membrane protein present on most immune cells, with different isotopes could address the issues of stability and signal intensity needed for effective barcoding. We chose for this purpose CD45, and designed experiments to address different types of cultures and the ability to detect extra- and intra-cellular targets. We show here that our approach provides an useful alternative to m-DOTA in terms of sensitivity, specificity, flexibility, and user-friendliness. Our manuscript provides details to effectively barcode immune cells, overcoming limitations in current technology and enabling the use of CyTOF with scarce samples (for instance precious clinical samples). © 2015 The Authors. Published by Wiley Periodicals, Inc.

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

Nanoparticles for cell labeling.

Cell based therapeutics are emerging as powerful regimens. To better understand the migration and proliferation mechanisms of implanted cells, a means to track cells in living subjects is essential, and to achieve that, a number of cell labeling techniques have been developed. Nanoparticles, with their superior physical properties, have become the materials of choice in many investigations along this line. Owing to inherent magnetic, optical or acoustic attributes, these nanoparticles can be detected by corresponding imaging modalities in living subjects at a high spatial and temporal resolution. These features allow implanted cells to be separated from host cells; and have advantages over traditional histological methods, as they permit non-invasive, real-time tracking in vivo. This review attempts to give a summary of progress in using nanotechnology to monitor cell trafficking. We will focus on direct cell labeling techniques, in which cells ingest nanoparticles that bear traceable signals, such as iron oxide or quantum dots. Ferritin and MagA reporter genes that can package endogenous iron or iron supplement into iron oxide nanoparticles will also be discussed.

Cardiovascular Molecular Imaging

Series Editor: Zahi Fayad Molecular Imaging of Cardiovascular Disease ATVB In Focus Articles in this series: •Choudhury RP, Fisher EA. Molecular imaging in atherosclerosis, thrombosis, and vascular inflammation. Arterioscler Thromb Vasc Biol . 2009;29:983–991. •Cormode DP, Skajaa T, Fayad ZA, Mulder WJM. Nanotechnology in medical imaging: probe design and applications. Arterioscler Thromb Vasc Biol . 2009;29:992–1000. •Tang TY, Muller KH, Graves MJ, Li ZY, Walsh SR, Young V, Sadat U, Howarth SPS, Gillard JH. Iron oxide particles for atheroma imaging. Arterioscler Thromb Vasc Biol . 2009;29:1001–1008. •Rudd JHF, Hyafil F, Fayad ZA. Inflammation imaging in atherosclerosis. Arterioscler Thromb Vasc Biol . 2009;29:1009–1016. •Jaffer FA, Libby P, Weissleder R. Optical and multimodality molecular imaging: insights into atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29:1017–1024. •Kraitchman DL, Bulte JWM. In vivo imaging of stem cells and beta cells using direct cell labeling and reporter gene methods. Arterioscler Thromb Vasc Biol . 2009;29:1025–1030. •Laufer EM, Winkens MHM, Narula J, Hofstra L. Imaging of cell death in atherosclerosis. Arterioscler Thromb Vasc Biol . 2009;29:1031–1038. Molecular imaging is defined today as the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems, and molecular imaging agents are probes …

In Vivo Imaging of Stem Cells and Beta Cells Using Direct Cell Labeling and Reporter Gene Methods

Cellular transplantation therapy offers a means to stimulate cardiovascular repair either by direct (graft-induced) or indirect (host-induced) tissue regeneration or angiogenesis. Typically, autologous or donor cells of specific subpopulations are expanded exogenously before administration to enrich the cells most likely to participate in tissue repair. In animal models of cardiovascular disease, the fate of these exogenous cells can be determined using histopathology. Recently, methods to label cells with contrast agents or transduce cells with reporter genes to produce imaging beacons has enabled the serial and dynamic assessment of the survival, fate, and engraftment of these cells with noninvasive imaging. Although cell tracking methods for cardiovascular applications have been most studied in stem or progenitor cells, research in tracking of whole islet transplants and particularly insulin producing beta cells has implications to the cardiovascular community attributable to the vascular changes associated with diabetes mellitus. In this review article, we will explore some of the state-of-the art methods for stem, progenitor, and beta cell tracking.

Tracking Stem Cell Therapy in the Myocardium: Applications of Positron Emission Tomography

The introduction of stem cells and/or progenitor cells into damaged myocardium has promising therapeutic potential in ischemic heart diseases and dilated cardiomyopathy. However, understanding the biologic mechanisms and the outcomes of transplanted cells during cardiac regenerative therapy remains mostly limited to histological assessment. Positron emission tomography (PET) is a sensitive molecular imaging modality that can non-invasively assess stem cell retention, survival, and function after transplantation. Two radiolabel approaches have been explored to implement PET: 1) direct cell labeling with a radionuclide; and 2) reporter gene-based cell labeling. Direct cell labeling has previously been used for early tracking of transplanted stem cells into the myocardium in several therapeutic clinical trials. Stem cells can also be labeled after transfection with a reporter gene, which can subsequently be visualized by using a PET reporter probe that binds to the reporter gene, therefore allowing serial in vivo evaluation of cell viability and proliferation in long-term follow-up studies. Recently, some studies successfully used this method to visualize implanted stem cells by PET imaging in animals. With the projected rapid growth of cell therapy for heart disease, PET is expected to play a major role in monitoring relevant changes that occur at every stage in cardiac regenerative therapy. These two cell tracking approaches used for PET imaging are reviewed here and compared against other imaging modalities.

Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells

Embryonic cortical neural stem cells apparently have a transient existence, as they do not persist in the adult cortex. We sought to determine the fate of embryonic cortical stem cells by following Emx1IREScre; LacZ/EGFP double-transgenic murine cells from midgestation into adulthood. Lineage tracing in combination with direct cell labeling and time-lapse video microscopy demonstrated that Emx1-lineage embryonic cortical stem cells migrate ventrally into the striatal germinal zone (GZ) perinatally and intermingle with striatal stem cells. Upon integration into the striatal GZ, cortical stem cells down-regulate Emx1 and up-regulate Dlx2, which is a homeobox gene characteristic of the developing striatum and striatal neural stem cells. This demonstrates the existence of a novel dorsal-to-ventral migration of neural stem cells in the perinatal forebrain.

Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. II. Potential application to studies of lymphocyte migration and maturation

The effect of direct cell labeling with fluorescein or tetramethyl rhodamine isothiocyanate on lymphocyte migration is examined. In vitro conditions of labeling are defined which (1) do not significantly affect immediate or long term viability of lymphocytes (up to 2 weeks after transfer in vivo), (2) do not alter normal lymphocyte migration, (3) do not affect expression or detectability of surface antigens, and (4) permit direct visualization and counter-staining with fluorescent antibody reagents for days after intravenous injection. The potential application of this method to studies of lymphocyte migration and maturation is discussed.