To our knowledge, this is the first imaging study to evaluate bilabeled hMSCs in an arthritis model over time with OI and MRI. Our major findings are that labeling of hMSCs with ferucarbotran and DiD provides effective cell depiction with OI and MRI in vitro and in vivo. The OI and MRI contrast effect increased nonlinearly with cell concentration. There was no significant difference in the sustained contrast effect on OI or MRI between viable and nonviable hMSCs up to 72 hours postinjection. However, viable and nonviable hMSCs could be differentiated based on their migration/homing pattern in vivo because only viable cells migrated to the ipsilateral arthritic ankle joints as demonstrated with OI and confirmed with histology.
The domain of exogenous bifunctional labeling of stem cells for in vivo tracking remains very limited.
There are three approaches to bifunctional cell labeling for cell depiction with OI and MRI:
- Several investigators have attached a fluorescent dye to an MRI contrast agent, thereby creating one contrast agent molecule that can be depicted with both OI and MRI. Examples of such bifunctional contrast agents, being investigated in vitro, include gadolinium-based DO3A-ethylamine–derived agents,23 gadofluorine M attached to a Cy dye,24 arginyl peptides cross-linked to nanoparticles and attached to Cy5.5, and lanthanide chelators (diethylenetriaminepentaacetic acid [DTPA] and tetraazacyclododecane-tetraacetic acid [DOTA]) bound to gadolinium and covalently attached to fluorescent and multifunctional nanoprobes.25 Bifunctional probes that have been used for in vivo cell tracking studies include gadophrin-2, gadofluorine-Cy3, and nanoparticles with attached fluorochromes.26 The disadvantage of these probes is that their optimal concentration for depiction with MRI is usually much higher compared to their optimal concentration for OI. Thus, internalized concentrations of these agents for cell labeling purposes usually represent a suboptimal compromise between the desired concentrations for the two imaging modalities.
- Other investigators have introduced MRI contrast agents into genetically modified cells with intrinsic fluorescence (eg, green fluorescent protein [GFP] expression). This method was mainly used to correlate MRI data of transplanted cells with direct depiction of the same cells on histopathologic specimens.27,28 The disadvantage of this technique is in the translation for clinical application as the cells are genetically engineered. Further, OI studies using GFP-expressing cells have poor tissue penetration owing to the increased excitation and emission maxima compared to NIR labels.29
- Yet other investigators achieved bifunctional cell labeling by the internalization of separate fluorescent and MRI contrast agents within their target cells. This approach has the advantage that labeling can be optimized for both OI and MRI. This approach has been applied for in vivo cell tracking with MRI and postmortem correlation with fluorescent microscopy.13,30–32 The approach has been used for neural stem cells (NSCs) labeled with PKH26 (fluorescent dye) and SPIO particles,13 NSC labeled with gadolinium rhodamine dextran,30,32 and MSCs labeled with magnetic silica nanoparticles.31
To date, no studies have used bifunctional fluorescent iron oxide labels for in vivo dual-modality imaging of MSCs, allowing us to establish proof of concept. This study was the first to demonstrate the detection and stable signal of bilabeled hMSCs with OI and MRI over several days. Synergistic imaging combined the single-cell sensitivity of OI, resulting in the detection of viable bilabeled hMSC migration to the right ankle joint. The high spatial and anatomic resolution of MRI allowed us to visualize the intra-articular hMSC distribution.
We chose ferucarbotran, a clinically applicable iron oxide–based contrast agent (ie, approved for clinical use in Europe and Japan), because its size and physiochemical surface properties allow for efficient internalization into stem cells by simple incubation without transfection agents. It is compartmentalized within secondary lysosomes, where it is slowly metabolized over time.33,34
Other SPIO particles would provide the same sensitivity for cell detection with MRI but require more complicated labeling procedures for stem cells. Another alternative would be gadolinium chelates, which have the advantage of providing T1
contrast (positive contrast) depending on the environment and image weighting27
instead of the described decrease in T2
signal seen with iron oxides that could be confused with bleeding or air. However, the sensitivity of gadolinium chelates is several magnitudes lower than that of iron oxides.
We chose DiD because it is an NIR probe and effectively labels cells by simple incubation. It localizes to a different cell compartment, the cell membrane, than ferucarbotran, thereby minimizing interferences between the two labels. In addition, DiD has demonstrated low cytotoxicity, high labeling efficiency, and high resistance to intracellular transfer.14
Research on exogenous NIR fluorescent probes is extremely broad. Numerous alternative fluorescent agents can be used for double labeling; however, cyanine dyes such as DiD represent the most prominent class and have proven effective for in vivo cell tracking with OI.29,35,36
The effect of OI and MRI contrast agents on cell properties and environment is imperative for the progression of preclinical research but remains to be entirely addressed.27
Potential impairments in the viability of labeled stem cells compared to nonlabeled controls have been reported to be dependent on the type of cells being investigated, the type of contrast agent, and the contrast agent dose, concentration, and incubation time.29
Iron oxide particles are biocompatible, and the viability of iron oxide–labeled MSCs appears to be most related to the internalized iron load. When labeled at less than 10 pg/cell, iron oxides appear to be slowly incorporated into the regular iron metabolism and do not change the physiology of the cells.15,37–42
Likewise, cyanine dyes could cause a dose-dependent toxic effect to the nucleus.43
The ferucarbotran and DiD concentrations in our MSCs did not show any effect on cell viability, as determined by the relatively simple trypan blue exclusion test.
Recent studies reported different MRI signal intensities in vitro of viable versus nonviable iron oxide–labeled MSC whereby the intracellular iron oxides in viable hMSCs provided a significantly decreased T2
signal compared to released iron oxides from MSCs that had undergone apoptosis.15
With this study, we evaluated if the same effect could be observed in vivo. Our results showed that the cell’s functional fate could not be characterized with OI or MRI based on the signal characteristics in vivo. An explanation for the observed difference is that cell death can occur via (1) cell necrosis, which is associated with cell lysis and release of iron oxides, or (2) apoptosis, which is associated with fragmentation but not lysis (ie, no release of iron oxides). The in vitro studies were done using a model of cell necrosis, whereas our experiments used a model of cell apoptosis.15
Given that iron oxides are not released in the latter, their T2
effect is expected to change less than in the necrosis model. The heterogeneous distribution of iron oxide–labeled MSCs within inflamed joints of variable volume and variable effusion may create additional confounding factors, which may mask subtle differences in T2
effects between iron oxide–labeled viable and apoptotic cells. However, we were able to differentiate viable and nonviable cells based on their physiology in vivo: viable cells migrated to adjacent joints, whereas apoptotic cells did not. The identification of apoptotic and dead stem cells with noninvasive diagnostic studies would have important implications for improvement and management of stem cell transplants. Thus, other strategies to identify dead stem cells are being explored, such as intrinsic contrast reporters of gene expression and smart probes that “turn on” with expression of a particular enzyme associated with cell death.39
However, modification of a cell’s genetic profile through the introduction of transgenes is an inherent risk and would not be a clinically feasible option. Thus, further studies are needed to define the normal physiology of stem cell homing and engraftment as well as clinically applicable diagnostic techniques to detect deviations from this “normal” process.
The labeling techniques that are used with OI and MRI were founded on radionuclide methods (eg, 111
In-tropolonate, and 99m
Radionuclide labeling can be done by either simple incubation or transfection depending on the desired probe. Various agents, including 99m
Ga, and 111
In, have been used to successfully label and track cells for the evaluation of RA. The advantages of radiotracers include sensitive tissue penetration, easy signal quantification, high spatial resolution, and rapidity. It is the only imaging modality that has been used to track cells in humans. However, the equipment is complex and expensive, the probes are radioactive and can only be produced at certain times, and their half-life is less than that defined for OI and MRI.8
Nevertheless, novel agents such as 99m
Tc-HMPAO have successfully been used to track cells in arthritic models and offer distinct improvements, including relatively low cost, ease of use, low radiation burden, general accessibility, and high sensitivity.46
Of note, no nuclear medicine studies have characterized stem cell viability before and after exposure to radiotracers.
We are aware of several limitations in our work. Our model of immune-mediated arthritis was transient and evolved over time, with the disease most severe at 7 days postinduction (as defined by daily quantitative joint measurements), and slowly resolved thereafter, thus precluding extended observations. The chosen disease model was to establish an effective method and proof of concept; however, the evaluation of normal controls would have provided additional information. The number of rats investigated was limited to the minimal defined number according to power calculations. Known limitations exist with OI-based cell tracking, including limited depth of penetration, limited quantification, and poor spatial resolution owing to scatter.47
The bifunctional label requires two steps of incubation with contrast agents. Although both labeling techniques take place by simple incubation, their combination results in increased cell loss compared to a single labeling procedure. The internalized contrast agents undergo a dilution effect with proliferation, thus leading to an inherent decrease in signal over time. However, our studies show that we obtained significant OI and MRI signal above-baseline levels for several days. Of note, a recent study from our group demonstrated that ferucarbotran-DiD labeling of hMSCs does not interfere with morphologic differentiation into chondrocytes, but bilabeled cells do exhibit significantly less glycosaminoglycans (GAG) production compared to unlabeled cells.48
No other studies have investigated the differentiation of bilabeled cells in vitro or in vivo. The majority of reports on the differentiation capacity of contrast agent–labeled stem cells focus on MRI contrast agents and have used iron oxide–based particles over gadolinium-based chelates.38,42
Thus, further studies have to evaluate the differentiation potential of bifunctional labels. Finally, additional histopathologic correlation demonstrating colocalization of the CD44 marker with the bilabel (ferucarbotran and DiD) would have further strengthened our results. Nevertheless, prior experiments have shown that DiD is not released from the hMSCs in vivo up to 72 hours postinjection and that free DiD provides a very low fluorescent signal with OI in vivo and is rapidly eliminated via renal excretion.12
Double labeling of cells for visualization with OI and MRI has potential clinical applications. MRI is established as the imaging modality of choice for musculoskeletal disease but is not sensitive or specific in the diagnosis or therapeutic monitoring of arthritis. OI, a newer imaging modality, is gradually being used in the clinical setting and is feasible for imaging small joints transcutaneously and larger joints intra-articularly. Thus double labeling of cells can be seen as complementary and an extension of current clinical practice.7,8
Specifically, double labeling would allow a clinician to monitor the effectiveness of transplantation through long-term MRI follow-up studies of cells and consequently assist in determining the therapeutic benefit and prognosis. This would also provide earlier insight into the need to make appropriate changes if the therapy is not effective. OI could be used for visualization of cells during transplantation (eg, with arthroscopy) and as a rapid and inexpensive means of documenting cell location posttransplantation. Further, preclinical double labeling studies also have indirect benefits to clinical practice in that animal models allow us to perform studies that are difficult, if not impossible, in humans. They also allow us to facilitate imaging technology improvements that can be eventually applied to human beings.29,45