Traditionally, the transplanted stem cells are labeled in vitro by various techniques and then visualized by immunohistochemistry after tissue extraction. Such invasive techniques have limited application. There is a constant demand for a better and more effective noninvasive technique for long-term stem cell imaging and tracking of transplanted stem cells. This technique should monitor survival, migration, and differentiation. Considerable research has been conducted with various types of nanoparticles, including magnetic nanoparticles, silica nanoparticles, quantum dots, and gold nanoparticles as vehicles for stem cell delivery [17]. Various types of nanoparticles show variable effects on stem cell viability, proliferation, differentiation, cytotoxicity, and ability to track the stem cells. Hence, each technique has its own advantages and limitations.

SPIO nanoparticles have been utilized to label stem cells, as previously mentioned. Magnetic nanoparticle-labeled stem cells can then be visualized noninvasively by utilizing MRI. These iron oxide nanoparticles are often coated with polymeric materials, such as dextran or carboxydextran, to prevent aggregation and to enhance solubility [18]. SPIO nanoparticles are typically internalized into the stem cells during in vitro processing, with or without the use of transfection agents [19]. These nanoparticles can be derivatized with internalizing peptides, such as the activating transcriptional activator peptide. SPIO can further be labeled for tracking. It can be isolated by conjugation with other probes, such as fluorescent molecules. Such modification instances allow for better tracking, as well as sorting and purification of stem cells [20]. Jendelova et al. utilized MRI for the tracking of transplanted bone marrow and embryonic stem cells, labeled by iron oxide nanoparticles in rat brain and spinal cord. To differentiate between the two stem cell populations, the authors labeled the rat bone marrow stromal cells with bromodeoxyuridine (BrdU) and transfected embryonic stem cells with promoter-driven enhanced green fluorescent protein (pEGFP-C1) to introduce the expression of green fluorescence protein. The authors were able to demonstrate that grafted mesenchymal and embryonic stem cells labeled with iron oxide nanoparticles migrate into the injured CNS and could be tracked noninvasively for 50 days. This migration was further confirmed by histological staining with fluorophores [21]. Jin et al. utilized a similar approach for tracking the survival, migration, and differentiation of magnetically labeled seed cells-bone marrow- derived mesenchymal stem cells. These cells were injected into the intra-articular space of knee joints in rabbits. These colabeled stem cells with SPIO and BrdU were then utilized in combination with Prussian blue staining and transmission electron microscopy to observe the intracellular iron. Such images were able to track the cells noninvasively for 12 weeks. Again combining with immunohistochemical staining these processes were able to confirm the tracking of the stem cells reaching the defect site [22].

Cell tracking with iron oxide nanoparticles has been well established in MRI. However, in experimental animal models, the readout can often be unreliable as the intrinsic iron signal derived from erythrocytes sometimes masks the labeled cells. Interference can also be observed because ofpostoperative local signal voids such as metal, hemorrhage, or air. These shortcomings in this technique are often overcome by gadolinium-based contrast agents, which have an advantage over regular iron oxide—based SIPO. Such nanoparticles provide a positive contrast on T1-weighted images, which are less prone to interference. Shen et al. utilized MRI of mesenchymal stem cells labeled with dual agents (magnetic and fluorescence) in rat spinal cord injury. In this study stem cell from the marrow were paramagnetically and fluorescently labeled with a complex of gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) with rhodamine- conjugated polyethylenimine (PEI)-FluoR. The rats implanted with labeled stem cells were successfully observed and tracked by serial MRI. The images are correlated with fluorescent microscopy. The rats treated with mesenchymal stem cells achieved significantly higher Basso—Beattie—Bresnahan locomotors test scores than controls [23]. Similarly, Liu et al. evaluated cell tracking effects of transplanted mesenchymal stem cells with jetPEI/Gd-DTPA complexes in animal models of hemorrhagic spinal cord injury. In their study, first the mesenchymal stem cells were labeled with jetPEI/Gd-DTPA particles and examined for transfection efficiency by MRI in vitro. Differentiation assays were also carried out to confirm that the gedunin labeling did not alter the differentiation ability of the stem cells. The labeled cells were then transplanted into rat spinal cord injury and monitored by MRI in vivo and confirmed with fluorescence images. The results also confirmed the applicability of gedunin-based contrast agents in stem cell tracking [24].

Quantum dots are another type ofnanoparticles that have been extensively applied in cellular imaging and tracking due to their unique spectral, physical, and chemical properties. These advantages allow for concurrent monitoring of several intercellular and intracellular interactions in live cells over short and long periods. Quantum dots delivered to cytoplasm result in intense and stable fluorescence that can be easily tracked and imaged. Furthermore, these images are not known to alter the differentiation potential of stem cells, as demonstrated in rat pancreatic stem cells [25]. Quantum dots have also been applied in vivo to view mouse embryonic stem cells labeled with Qtracker through multiplex imaging [26]. Lin et al. reported murine embryonic stem cells that were labeled with six different quantum dots. None affected the viability, proliferation, and differentiation of the stem cells adversely. The authors were able to view cells that were injected subcutaneously onto the backs of athymic nude mice with good contrast. Although it was not suitable for deep tissue imaging, it could be utilized as a proof-of-concept study for viewing labeled stem cells in vivo [26].

Quantum dots also have been bioconjugated to enhance their internalization, targeting, as well as tracking. Barnett et al. reported a conjugate of high quantum efficiency photostable and multispectral quantum dot nanocrystals with fluorescent tracer, 1,10-dioctadecyl- 3,3,3/,3/-tetramethylindocarbo cyanine perchlorate-labeled acetylated LDL. The nanocrystals can be applied for long-term tracking of endothelial progenitor cell subpopulations in ocular angiogenesis with improved signal-to-noise ratio [27]. Shah et al. further utilized quantum dot bioconjugates for labeling and imaging of human mesenchymal stem cells during proliferation and osteogenic differentiation. In this study, the researchers utilized a specialized peptide CGGGRGD, which was cross-linked onto the CdSe—ZnS quantum dots coated with carboxyl groups utilizing amine groups. The peptide causes the quantum dots to bind with selected integrins on the membrane of human mesenchymal stem cells, which allowed for long-term labeling [28]. Quantum dots—based multifunctional nanoparticles were also developed by Li et al. for effective differentiation and long-term tracking of human mesenchymal stem cells. These investigators modified the quantum dots with b-cyclodextrin and CKKRGD peptide (Cys-Lys-Lys-Arg-Gly-Asp), which resulted in enhanced cellular uptake of nanoparticles as well as siRNA, which regulates differentiation. Such combination provides a powerful tool to simultaneously enhance differentiation and long-term tracking of stem cells, both in vitro and in vivo [29].

Due to the inert and biocompatible characteristics of gold, nanotracers or gold nanoparticles have also been prepared as labeling and tracking agents for stem cells. The inherent properties of gold make it nonreactive to any biological material. Studies have shown that gold nanoparticles do not interfere with the viability or cellular functioning of stem cells, making them useful candidates for long-term imaging and tracking of stem cells in vivo [30]. Nam et al. utilized gold nanotracers for in vivo ultrasound and photoacoustic monitoring of mesenchymal stem cells. The gold nanotracer-labeled mesenchymal stem cells injected intramuscularly in the lower limb of the Lewis rat were detected with ultrasound-guided photoacoustic imaging. High detection sensitivity, spatial resolution, and greater penetration depth render it as useful techniques [31].

Choi et al. utilized a polydopamine-coated gold core-shell nanoprobe for longterm intracellular detection of differentiation in human mesenchymal stem cells by targeting specific microRNA (miRNA). The polydopamine shell was utilized to immobilize the fluorescently labeled hairpin DNA strands on the nanoparticles, which were capable of targeting specific miRNA (miR-29b and miR-31). The gold core and polydopamine shell were able to quench the fluorescence of the immobilized hairpin DNA. Once internalized, the nanoprobes target the miRNA that are present only within the differentiating stem cells. Interaction of the hairpin DNA with miR- NAs within the cells leads to dissociation of the miRNA from the gold-polydopamine nanoparticles and thus enables recovery of the fluorescence signal. This technique allows for identification, isolation, and tracking of specific types of stem cells within a diverse population [32]. In a different study, a dual gold nanoparticle system for mesenchymal stem cell tracking was developed. This construct is capable of monitoring both delivered stem cells and infiltrating macrophages by photoacoustic imaging. Macrophages play an important role in wound healing and vascular regeneration. These cells also interact with stem cells, due to phagocytosis. The dual gold nanoparticle system enables monitoring of macrophage infiltration and endocytosis of stem cells. Two separate contrast agent nanoparticles were needed. This technique is based on transfer of contrast agents from stem cells to macrophages as a result of internalization. Gold nanorods (absorbance in the near-infrared region) labeled with mesenchymal stem cells and gold nanospheres (absorbance in the visible light region) were applied to label macrophages. Nanosphere endocytosis leads to peak broadening due to plasmon coupling, thus allowing two different cell types as well as interaction monitoring [33].

Silica nanoparticles were also studied for stem cell labeling with multiple imaging techniques. Photostable cyanine dye—loaded fluorescent silica nanoparticles can improve optical imaging ofhuman mesenchymal stem cells, which aids in the direct discrimination between live and early-stage apoptotic cells. With this nanoparticle technology, the labeled cells can be visualized by flow cytometry, confocal and transmission electron microscopy. Dye-loaded silica nanoparticles also known as IRIS Dots are capable of discriminating between live and early-stage apoptotic stem cells through a distinct external cell surface distribution [34]. Engineered silica nanoparticles, due to resistance to degradation and ease of functionalization, have been utilized as contrast agents for labeling stem cells in multiple disease conditions. Gallina et al. developed fluorescent, amorphous 50-nm silica nanoparticles. This group examined the effects on viability and function ofhuman bone marrow—derived mesenchymal stem cells in a beating heart model. The bright fluorescence emitted by the internalized nanoparticles made them optimal candidates for stem cell tracking inside heart tissue [35]. Fig. 5.2 shows improved results of stem cell therapy by guiding this growth in the right direction through nanoscale scaffolds.

Emerging Nanotechnology for Stem CellTherapy 93

Stem cells can be used to regrow damaged tissue in many areas of the body. Nanoscale scaffolds can improve results of stem cell therapy by guiding this growth in the right direction. (Image credit

Figure 5.2 Stem cells can be used to regrow damaged tissue in many areas of the body. Nanoscale scaffolds can improve results of stem cell therapy by guiding this growth in the right direction. (Image credit: National Eye Institute.)

Silica nanoparticles can be combined with multiple technologies to form multifunctional tracking agents. Jokerst et al. developed a multimodal, silica-based nanoparticlulate system that can serve multiple functions, such as cell sorting through fluorescence, guided cell implantation in real time with ultrasound, and high-resolution long-term monitoring by MRI. This technology can be very useful for improving stem cell therapy by improving delivery and preventing death of implanted stem cells in ischemia, inflammation, or immune response [36]. Cobalt zinc ferrite nanoparticles encapsulated by amorphous silica have been developed as a contrast agent. It can also act as magnetic label for tracking transplanted stem cells within an organism using MRI [37]. Similarly fluorescein isothiocyanate (FITC)-incorporated silica-coated magnetic nanoparticles have been developed for specific labeling of neurogenic, endothelial, and myogenic differentiated cells derived from human amniotic fluid stem cells [38]. The multiple mechanisms for tracking the cells in vivo provide a more precise and confirmatory scientific technique allowing for more accurate determination of stem cell health and activity.

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