Direct Labeling

The most effective tracer of cell morphology is a fluorescent probe, which has the capacity for localized introduction into a cell or organelle, as well as long-term retention within that structure, and is biologically inert and non-toxic. Fluorescent tracers can be used to investigate the movements of labeled cells in culture, tissues or intact organisms. There are four major different types of fluorescent probes for stem cell tracking.

• •
  

  Membrane tracers : DiI, DiO, DiD, DiR, DiA and their analogs. Di dyes can be introduced into membranes by direct application of dye onto the cell by loading from culture media . Lateral diffusion of the dye within the membrane eventually stains the entire cell. These probes are widely used for stem cell tracking and long-term assays of cell–cell association . DiI dye has been applied in EPC labeling and tracking in the kidney. EPCs were incubated with CM-DiI for 30 min, then transplanted into the stenotic kidney of pigs with unilateral renal artery stenosis. Red fluorescence EPCs could be visualized in kidney sections as late as 4 weeks after transplantation ( Fig. 17.2 ). Furthermore, combining Di dye with other immunohistological staining, it was possible to track stem cell change in phenotype (transdifferentiation). For example, a histological section from a kidney injected with Di dye-labeled EPCs (fluorescent red) was stained with an antibody against the endothelial marker CD31 (labeled fluorescent green). The cells showing both red and green fluorescence (i.e. yellow) were mainly confined to perivascular capillary structures, indicating the ability of EPCs to transdifferentiate into mature vascular endothelial cells ( Fig. 17.2 ).

  
  

  
  

  
  

  

  
  

  
  

  Top: Representative images of endothelial progenitor cells (EPCs) labeled with fluorescence dye (CM-DiI, red, left) and yellow–green (YG) microspheres (right) in the kidney 4 weeks after transplantation. Bottom: Representative merged images of CM-DiI-labeled EPCs and the same kidney immunostained for the endothelial marker CD31, indicating transdifferentiation of EPCs to vascular endothelial cells. Please see color plate at the end of the book.

  
  
  
  
• •
  

  Fluorescent microspheres . Those microspheres contain approximately 10 ² to 10 ¹⁰ fluorescent dyes per bead and are the most intensely fluorescent tracers available . Furthermore, they are often biologically inert and physically durable, which makes fluorescent beads particularly useful as long-term markers for transplantation studies. Submicrometer microspheres can be taken up by phagocytosis. This feature enabled EPCs to be labeled by incubation with microspheres in the culture medium, and then EPCs (yellow fluorescence) were tracked in the kidney 4 weeks after injection ( Fig. 17.2 ).

  
  
• •
  

  Cell-permeant cytoplasmic labels (thiol-reactive CellTracker probes) yield fluorescent products that are retained in many live cells through several generations and are not transferred to adjacent cells, except possibly by transport through gap junctions. These probes constitute excellent long-term tracers for transplanted cells or tissues .

  
  
• •
  

  Fluorescent dextran conjugates . Dextran conjugates are ideal cell lineage tracers because they are relatively inert, exhibit low toxicity and are retained in cells for long periods . However, they are membrane-impermeant probes, and usually need to be loaded into cells by invasive techniques such as microinjection, whole-cell patch clamping or scrape loading. This may limit the application of dextran conjugates in stem cell tracking.

Histological techniques are also used to track iron-labeled stem cells in the kidney in conjunction with MRI studies. Prussia blue staining is specific for iron, and comparing MRI images with histological sections is useful to confirm and determine the accuracy of MRI tracking.

Indirect Labeling

For histology, cells can be labeled with a fluorescent reporter gene, and then when the tissue is excised, the labeled cells that remained in the tissue can be easily identified using histological methods . Similar to in vivo conditions, different reporter genes emit light in different spectra permitting detection of corresponding fluorophores in the sample. These applications play a very important role in the study of stem cell biology in cell cultures and ex vivo. Furthermore, owing to their light emission and detection characteristics, they are commonly used as a basic strategy when designing novel reporter gene constructs. A good example for reporter genes is green fluorescent protein (GFP) . Stem cells isolated from GFP transgenic animals can be allogenically transplanted into the host body; those cells consistently express GFP and can be visualized using fluorescence microscopy.

In summary, although histology is a traditional ex vivo imaging technique, which can only provide information obtained by invasive techniques, it is still a basic and powerful tool that allows accurate cell tracking, and provides cell fusion information and cell–tissue interaction, especially in animal studies.

Direct Labeling

This method employs an incident of light that excites a fluorophore and emitted light is captured by a CCD camera. So far, no single ideal optical contrast agent is available. In general, all OI contrast agents need to be biocompatible, possess a tolerable toxicity profile, be small in size and exude a bright signal.

• •
  

  Fluorescent dyes . These fluorochromes are continuously being designed (hundreds are commercially available) and include DiI, DiO, DiD and CM-DiI. They have proven effective for in vitro cell labeling (as discussed in the last section) and in vivo cell tracking with OI.

  
  
• •
  

  Targeted probes . Colloidal quantum dots (QDs) have a narrow emission and continuous broad absorption spectrum (i.e. broad excitation spectrum), which allows fluorescent excitation by any wavelength below the emission maximum.

Indirect Labeling

One of the main advantages of fluorescent reporter genes is that different fluorophores (e.g. red or green fluorescent proteins) can be used to label different cell populations, permitting their concomitant imaging .

• •
  

  Bioluminescence . Bioluminescent imaging typically involves the luciferase (luc) gene and based on luc-mediated oxidation of D-luciferin (with emission spectra: 400–620 nm). A chemical process following intravenous administration of luciferin generates light emission. Photon generation takes place exclusively at the site of luciferase expression; therefore, the target-to-background signal ratio is extremely high, thus allowing for detection at lower wavelengths with a maximal penetration depth of 3 cm.

  
  
• •
  

  Fluorescent proteins . The first representative of this class of proteins was GFP, which has been commonly used for cell tracking with OI in small animal models.

Perhaps the most frequent application of fluorescent reporter genes in cell imaging is for ex vivo analysis, where they can be used for cell sorting. For example, not all stem cells transduced with a bioluminescent or PET reporter gene incorporate the gene of interest into their genome. A common method of sorting and selecting the cells that did incorporate the gene of interest takes advantage of fluorescent reporter gene cell markers (e.g. Gfp, Rfp) .

Application of Optical Imaging in the Kidney

Few studies have applied OI to track stem cells in the kidney. Tögel et al. tested bioluminescence to track MSCs in a mice acute kidney injury (AKI) model using the OI technique. Bone marrow-derived MSCs were infected with luciferase/neomycin phosphotransferase. The transinfection had no impact on MSC biology, as tested by a multidifferentiation assay. Transinfected MSCs (1 × 10 ⁵ ) were injected into mice through the jugular vein or carotid artery. Immediately or 24 h after injection, animals were imaged using the Xenogen IVIS 100 system. MSCs showed distinct accumulation in the kidney only in AKI mice, mainly after the cells had been administered by intra-arterial injection. Importantly, intravenous injection of MSCs resulted in substantial accumulation in the lung ( Fig. 17.3 ), so that fewer cells reached the kidney. This study demonstrated that bioluminescence is sensitive for in vivo MSC tracking, and is suitable for non-invasive kidney localization of injected MSCs in small animals.

Optical imaging of mesenchymal stem cells (MSCs) in the kidney. (A) Immediately after intra-arterial infusion, animals with acute kidney injury (AKI) showed distinct accumulation of cells in kidneys (as shown by green/red areas). (B) Normal animals show diffuse whole-body distribution with eventual accumulation in the lungs in some animals. Bottom: Intravenous injection in a normal animal (first on the left) showed accumulation of MSCs in the lungs. In the middle panels three different AKI animals showed accumulation of cells in the lungs immediately after injection, and only one animal with AKI showed retroperitoneal uptake indicating cell localization in the kidneys.

[Adapted from Tögel et al., 2008 with permission.] Please see color plate at the end of the book .

Limitations

The fluorescence direct imaging and reporter gene approaches have limited tissue penetration (around 2 mm), restricting the use of these techniques to superficial tissues in small animals (e.g. mice) . Furthermore, most common fluorescence imaging devices do not have tomographic capabilities, which further limits the identification of deeper organs. Several studies are underway to attempt to provide tomographic fluorescence imaging .

There are also some other drawbacks to the use of fluorescent reporter genes. Similar to other imaging modalities, the detected signal constitutes only a small fraction of the emitted signal. Significant efforts are underway to use multiple cameras, which may enable detection of a higher fraction of the emitted signal . Novel developments, such as time-domain imaging , incorporate the time domain in the analysis, and have the potential to provide depth information of the fluorescent signal. However, such strategies are under development and not ready to be routinely applied by mainstream imagers. In addition, fluorescence techniques are associated with background autofluorescence, which necessitates a stronger signal to overcome the noise in the data. The presence of autofluorescence could be a significant disadvantage, especially when the emitted signal is not high, leading to a low signal-to-noise ratio.

Direct Labeling

To visualize cells against the background of host cells and to increase the likelihood and sensitivity of their detection with MRI, cells need to be labeled with specific MRI contrast agents. Four types of agents are currently available for MRI cell imaging, and selection of a specific agent depends on the purpose of imaging.

• •
  

  Iron oxide-based contrast agents. Superparamagnetic iron oxide particles (SPIOs) are the most commonly used strategy in MRI cell labeling. These highly magnetic particles induce changes in T2 relaxivity, which makes them detectable in vivo . In a direct labeling strategy, stem cells are prelabeled with SPIOs, before transplantation and imaging, so that the signal originating from the SPIOs is considered to reflect number of cells retained in the tissue.

  
  
• •
  

  Lanthanide chelates and Mn ²⁺ -based contrast agents. Paramagnetic compounds such as Mn ²⁺ ions or lanthanide chelates usually affect T2 less than SPIOs, but are commonly used for their increase in T1 relaxation, resulting in a hyperintense (bright) contrast in T1-weighted images. Manganese-based contrast agents such as manganese chloride (MnCl ₂ ) or manganese oxide (MnO ₂ ) nanoparticles have also been used for cell labeling. However, their toxicity prevents their widespread application.

  
  
• •
  

  Contrast agents based on heteronuclear MRI. The majority of MRI contrast agents used for clinical and experimental applications is based on modulating the contrast in ¹ H-based MRI . Although relatively sensitive, this presents a major challenge for discriminating labeled cells from the background signal and motivates the search for a less abundant contrast molecule. Most heteronuclear approaches to MRI-based cell imaging have focused on ¹⁹ F-MRI and spectroscopy. One advantage of ¹⁹ F-MRI and spectroscopy is the ability to quantify the signal as long as the signal-to-noise ratio is sufficiently high. Other potential applications of heteronuclear MRI and spectroscopy include ¹³ C and ³¹ P.

  
  
• •
  

  Responsive contrast agents. Contrast agents that change contrast owing to their changed magnetic properties (e.g. relaxation, chemical shift) in response to dynamic changes in physiological, enzymatic and other metabolic properties are frequently referred to as responsive contrast agents . Contrast agents that are (chemically) modified by cellular changes have initially been developed for reporting on the physiological status and metabolic activity of cells. Most of these contrast agents are lanthanide chelates with one or more potential coordination sites for water that can be blocked in the inactive state of the contrast agent. Using these agents enables detection of the interaction between transplanted cells and host organs. A potential limitation is the remaining influence of the “inactive” contrast agent on T1 relaxation owing to the secondary coordination sphere of the water, which further decreases its sensitivity.

Special Cell-labeling Approaches for Magnetic Resonance Imaging

Useful approaches for stable labeling of cells with MRI contrast agents include incorporation of the contrast agent in vitro before transplantation and engraftment into the host, systemic application and subsequent specific internalization of the contrast agent by the targeted cells, receptor-specific binding or internalization of targeted contrast agent to targeted cells, or the generation and accumulation of contrast by genetically modified cells.

• •
  

  In vivo cell labeling. In vivo cell labeling applications include localized injection of iron oxide particles, which allows monitoring of the migration of endogenous stem cells. In a recent study , iron oxide particles were injected in the area of stem cell generation, and labeled cells that translocated along the rostral migratory stream to the target area, assumed to represent stem cells.

  
  
• •
  

  Targeted contrast agents for in vivo cell labeling. To achieve more specific labeling of particular cell types by systemic administration of a contrast agent, targeted agents are the most promising approach. Targeted contrast agents are chemically modified so that they accumulate specifically in certain tissue types, usually owing to the presence of particular cell types. Active accumulation occurs by specific binding or uptake due to biomarkers bound to the contrast agents, such as antibodies or small peptides. Antibodies bound to iron oxide particles or lanthanide chelates accumulate at sites of high expression of the targeted receptor. The most common approach is to use Tat peptides to derivatize either coated nanoparticles or lanthanide chelates. Biological applications of targeted contrast agents include inflammation, angiogenesis, apoptosis, tumors and atherosclerosis. These approaches are often hampered by insufficient accumulation of the contrast agent owing to either low cell densities or low receptor expression on the cell surface.

Indirect Labeling: Magnetic Resonance Reporter Genes

MRI-based reporter genes strategies are based on the production of intracellular metalloproteins . As previously described, iron is a paramagnetic substance that induces changes in relaxivity (i.e. T2 ^∗ effect) that can be detected using specific imaging sequences, and the goal of these strategies is to accumulate large quantities of iron intracellularly for its detection. Two major metalloproteins have been used in MRI reporter gene techniques: ferritin and tyrosinase. Ferritin is a metalloprotein that functions as an iron depot and can contain up to 4000 iron atoms. For example, cells transduced with the ferritin reporter gene were delivered to the brain of mice . After several days, enough iron signal was “collected” inside the transplanted cells (due to the overexpression of ferritin) to enable the non-invasive monitoring of transplanted cells using MRI. In a recent study , the investigators used a replication-defective adenovirus vector to deliver the ferritin transgenes. Following focal inoculation of the vector into the mouse brain, they monitored the reporter activity using in vivo time-lapse MRI. They observed robust contrast in virus-transduced neurons and glia for several weeks ( Fig. 17.4 ). This technology is adaptable to monitor transgene expression in vivo in many tissue types and has numerous biomedical applications, such as visualizing preclinical therapeutic gene delivery.

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related posts:

Role of Macrophages in Renal Injury, Repair and Regeneration Glomerulogenesis and De Novo Nephrogenesis in Medaka Fish: An Evolutionary Approach Use of Genetic Mouse Models to Study Kidney Regeneration Bioartificial Stem Cell Niches: Engineering a Regenerative Microenvironment Potential Risks of Stem Cell Therapies Tissue Engineering in Urology

Stay updated, free articles. Join our Telegram channel

Join