In situ labeling of NPCs with iron oxides first involved injecting very small ∼20 nm particles into the carotid artery, then disrupting the blood-brain barrier (BBB) with mannitol to allow the particles to enter the brain and label NPC (Neuwelt et al., 1994). Shapiro et al. (2006a) developed the technique of injecting MPIOs in a volume of 10–50 μL of stock particles (1.63 μm diameter particles, ∼3 μg Fe/μL) into the anterior horn of the lateral ventricle to label NPCs and track them over time in their migration through the RMS to the OB. This foundational work has been the basis for endogenous NPC labeling tracking studies. Figure 2 shows an example of migrating cells in the RMS and OB after injection of MPIO particles into the SVZ. The literature since has used several different doses and coordinates (Table 1). Granot et al. (2011) attempted to further optimize in situ labeling and found that a more rostral injection site and a smaller dose of 20 μL of MPIOs produced the best labeling of NPCs in the RMS, however, in a later publication the same authors used different, more rostral coordinates (Granot and Shapiro, 2014).

Smaller particles were less effective for monitoring migration in the RMS and OB. The clinically approved SPIO called Endorem has a particle size of 120–180 nm, and these particles were evaluated for NPC labeling after injection into the ventricles of healthy rats either with, or without, co-injection of protamine sulfate at a dose of ∼7 μg Fe/μL in a volume of 2.5 μL (Panizzo et al., 2009). The protamine sulfate-Endorem complexes labeled the NPC in situ while the uncomplexed Endorem did not, and in fact, was shown to spread throughout the ventricles after injection. Migrating cells labeled with these smaller Endorem particles after co-injection with protamine sulfate were visible in the RMS, but not strongly, when imaged in situ at 2.35T, and were not visible in the OB. Ex vivo imaging at 9.4T was needed to visualize NPC in the OB after labeling with Endorem particles (Panizzo et al., 2009).

Transfection agents have also been used to assist in situ cell labeling with MPIOs. In healthy mice, co-injection of 1.5 μL of a mixture of MPIOs and PLL (∼0.67 μg Fe/μL) provided better labeling than an injection of MPIOs and saline in the same volume. Use of the transfection reagent led to good labeling that enabled visualization of cells in the RMS and there seemed to be minimal effects of the labeling on NPC proliferation and no significant inflammation. A higher dose (10 μL, ∼3 μg Fe/μL) of MPIOs did reduce NPC proliferation (Vreys et al., 2010; Guglielmetti et al., 2014).

When MPIOs or SPIOs are injected into the lateral ventricle, uptake of the label is not restricted only to NPCs – scavenger cells and other non-migrating cells can also internalize the particles along the ventricles and choroid plexus. Migrating cells in the RMS can also be ‘pruned’ (Brunjes and Armstrong, 1996; Winner et al., 2002) and the label may be taken up by microglia during this process. This could lead to signals in the MRI image that are not associated with NPCs. The extent and significance of these nonspecific signals has been addressed in a number of studies. Sumner et al. (2009) used flow cytometry to determine the relative proportion of labeled cell types in excised and digested RMS and OB. At 2 weeks post-injection (50 μL, 3 μg Fe/μL) they observed that ∼40% of the labeled cells were astrocytes, ∼30% oligodendrocytes, ∼30% neurons, with ∼5% microglia in these excised samples of the RMS and SVZ. In the OB, the fraction of astrocytes remained the same, but the proportion of oligodendrocytes increased slightly and neurons decreased to 10% of labeled cells, with a greater percent of microglia (over 10%; Sumner et al., 2009). In contrast, another study in mice using histological staining found that 2 days after labeling with 1.63 μm particles (50 nL, 3 μg Fe/μL), the label was predominantly confined to migrating NPCs (∼60%) and astrocytes (∼25%), but after 21 days, up to 35% of labeled cells were microglia instead of migrating cells, with NPCs remaining at ∼50% and astrocytes ∼25%; there was no difference between the labeled populations in the RMS and the OB (Nieman et al., 2010). This suggests that as the experiment progresses, the label is less likely to be inside migrating cells derived from NPCs. One suggestion is that labeled microglia can be discriminated from labeled NPCs because the microglia will be stationary, while the NPCs will be moving (Shapiro et al., 2006a), though this experiment would be time consuming, requiring multiple scans.

Migration of free particles could also be a problem and is not easily differentiated from labeled cells by MRI. Sumner et al. (2009) found that MPIOs did not migrate spontaneously along the RMS but needed to be carried by a migrating cell. In another experiment, cortical stroke was induced in rats by the injection of ET1, followed by injection of 20 μL of MPIO (0.86 μm, 2 μg Fe/μL). Signal voids were seen at the site of the stroke; however, they were not caused by labeled NPCs, but were due to migration of free particles that were carried by the CSF through the corpus callosum toward the lesion. In this model, the ET1 also acts to disrupt the blood–brain barrier and allow the influx of particles into the brain, which is not a concern in healthy animals, but this does indicate that the location of the lesion must be chosen carefully to avoid the migration of free particles (Granot and Shapiro, 2014). In another study, when magnetoliposomes were injected into the RMS of mice (1.5 μL at 0.67 μg Fe/μL), free magnetoliposomes were seen in white matter tracts and in the RMS but were rarely seen encapsulated in migrating cells (Vreys et al., 2011). They also observed that there was less free motion of cationic magnetoliposomes than anionic ones, so the authors concluded that it may be possible to use larger cationic magnetoliposomes to label NPCs without the risk of migration of free particles (Vreys et al., 2011).

Elvira et al. (2012) made targeted magnetic glyconanoparticles (4 nm diameter) conjugated to Nilo2, an antibody directed against NPCs. These particles were injected into the contralateral ventricle of mice with astrocytoma (1 μL of particles at 0.1 μg Fe/μL). One day after injection, contrast was seen in the tumors as the NPCs at the site of the particle injection migrated to the tumor; indeed, most of the neuroblasts at the tumor were labeled. When a non-targeted particle was injected, there was no accumulation of contrast at the tumor, although NPCs were present (Figure 3). Another study used SPIOs conjugated to CD15 to label in situ NPCs (Zhong et al., 2015), increasing the labeling efficiency while decreasing distortions caused by direct MPIO injection. The authors go on to demonstrate the effectiveness of the technique using a stroke model (Zhang et al., 2016). These studies show the potential of using small particles for MR tracking of NPCs in the situation where large numbers of labeled cells are expected to be localized in the brain. It is anticipated that the use of smaller magnetic particles would not have allowed for single-cell detection if the labeled cells were sparsely distributed.

The contrast due to iron labeled cells is visually apparent for qualitative analysis, although there can be some ambiguity with respect to other hypointense structures in the brain; these depend on the acquisition parameters. Some more complex image analysis methods have also been used to quantify the signal or to discern more subtle or time-dependent effects. Sumner et al. (2009) plotted signal over a line segment to show that the signal in the RMS dropped 60% from the background when there were migrating labeled cells present, while similar studies in phantoms from the same group found that a single cell labeled with MPIOs would cause a 30% signal drop (Shapiro et al., 2005). Subsequently, Granot et al. (2011) registered images of rat brains pre- and post-MPIO injection and looked for regions with a signal decrease of >30% to quantify the migration of cells in the OB. Using this method, they were able to quantify the volume of the OB that was occupied by signal from migrating cells. This signal appeared 2–3 days after MPIO injection, increased linearly until day 7, and then plateaued (Granot et al., 2011). Nieman et al. (2010) used image registration and intensity-based analysis to identify labeled cells and determine their speed of migration. They estimated that labeled NPCs move at 100–120 μm/h in the RMS and 50 μm/h in the OB (Nieman et al., 2010; Pothayee et al., 2017; Shuboni-Mulligan et al., 2018), slightly slower than the speeds of 150–700 μm/h that were measured in mice with tumors (Elvira et al., 2012). These migration rates are not far from those determined in studies of neurogenesis that employ immunohistochemical methods of detection, which determined a migration rate of 70–80 μm/h in naïve rodents (Nam et al., 2007). Recently, the technique demonstrated that both environmental stimuli, olfactory activity (Pothayee et al., 2017) and aging (Shuboni-Mulligan et al., 2018) impacted migration rates in vivo. Understanding the dramatic decrease in NPC migration as animals aged is critical, as many diseases that recruit NPC are more prevalent in older individuals (Wang et al., 2013).

Magnetic resonance imaging of endogenous NPCs has also been applied in brain diseases and disorders where there is a need for regeneration of neurons. In neonatal rats with hypoxia-ischemia caused by a ligation for 5 min of the left common carotid artery and then placement into hypoxic chamber (8% O₂ and 92% N₂) for 2 h, MPIOs (10 μL of 0.86 μm particles, 2 μg Fe/μL) were injected into the ventricles to label NPCs. As expected, labeled NPCs migrated through the RMS in healthy rats, but a small portion also migrated toward ischemic regions in the hypoxic-ischemic rats (Yang et al., 2009). The therapeutic effect of the migrating cells was examined using endogenous NPC from the SVZ in a study labeling cells with intraventricular injection of anti-CD15 SPIOs. Proliferation of NPCs in the SVZ was blocked using Ara-A infusion, which ceased migration of cells and prevented the decrease in infarct size over time (Zhang et al., 2016). In another experiment, NPC were labeled with MPIOs (1.63 μm particles, 1.5 μL, 0.67 μg Fe/μL) plus PLL to see if NPC from the SVZ were the source of remyelination following cuprizone treatment in a mouse model of MS. There was no migration of cells from the SVZ to the splenium, so the origin of the new cells was not determined (Guglielmetti et al., 2014).

Magnetic resonance imaging reporter genes can be used to induce cells to produce proteins that create MRI contrast; one example of such a protein is the iron storage protein, ferritin. Cell tracking with ferritin-overexpressing cells would allow for specific imaging of NPCs without confounding images from label uptake by microglia or free particles, and the label would not be lost through cell division. These are two properties that comprise the strengths of reporter genes, however, the limitation is the requirement of genetically engineering the cells. Ferritin consists of a light chain (L) and a heavy chain (H) that form a shell that can be filled with excess intracellular iron. A ferritin transgene (H and L chains) in an adenoviral vector was injected into the striatum of mice and a clear signal loss was seen at the injection site as soon as 5 days post-transplant from transfected glia and neurons (Genove et al., 2005). The specificity and sensitivity of detecting the ferritin gene following transfection via lentiviral and adenoviral vectors has been assessed (Vande Velde et al., 2011). In this study, the control lentivirus, with the gene encoding green fluorescent protein (GFP), caused a hypointense signal in the brain that was equivalent to the contrast induced when the lentivirus containing the gene for ferritin was used; this was due to an inflammatory response (Vande Velde et al., 2011). This inflammatory response was not seen when an adenovirus construct was used in the same study. A further study injected the lentiviral vector with the ferritin gene into the SVZ or striatum of mice. Despite extensive correction of the RF field bias, there was only minimal signal from migrating NPCs, and it required T₂^∗ mapping (Vande Velde et al., 2012). Lentiviral transfection with ferritin of exogenous NPCs could be visualized with MRI and fluorescence imaging when reintroduced intracranially into rats with acute ischemic stroke but also not of the migration of single cells (Zhang et al., 2017).

A different adenovirus encoding the L^∗H chains of ferritin has also been injected into mice and in this study, some astrocytes and neurons were transfected (Iordanova et al., 2010; Iordanova and Ahrens, 2012). Some of the transfected cells in the SVZ were proliferative as indicated by Ki67 staining, but transfection was not specific to proliferative cells. In the OB, transfected cells were visible by histology, but not by MRI (Iordanova and Ahrens, 2012). Sensitivity was quantified as 10⁴ cells per voxel. A follow-up study used the neurotrophic HSV virus, which was thought to target neurons selectively over glia. In this study, the amount of signal correlated with the amount of virus injected, and this study supports the use of the ferritin as a means to quantify gene delivery in nucleic acid based therapies, rather than a method for tracking the migration of individual transfected neurons (Iordanova et al., 2013). Further modifications to the genetic construct may offer more functionality, and toward this end, inducible ferritin constructs have been used in culture to monitor the differentiation of mesenchymal stem cells into neural cells (Song et al., 2015).

There are various sources of NPC that can be used for this purpose (Table 2): they can be isolated from the SVZ of adult rodents (e.g., Zhang et al., 2003; Jiang et al., 2005), immortalized cell lines isolated from embryonic rodent brain (e.g., Hoehn et al., 2002; Guzman et al., 2008; Obenaus et al., 2011), and finally human NPC can be isolated from human fetal tissue (e.g., Daadi et al., 2013; Ashwal et al., 2014; Song et al., 2015; Barish et al., 2017). Feridex has been the most common particle used for labeling NPCs in culture (Nucci et al., 2015). Typical iron concentrations for magnetic cell labeling range from 11.2 μg Fe/mL (Ashwal et al., 2014) to 112.3 μg Fe/mL (Song et al., 2009). The simplest method of labeling is incubating cells with particles for 3 days (Song et al., 2009) or for 1 day following trypsinization (Ashwal et al., 2014). Labeling cells with iron oxide particles using these methods had no significant effect on cell viability or on ability to differentiate and migrate (Ben-Hur et al., 2007), especially at low (but still MR-visible) doses. However, some studies with mesenchymal stem cells (Bulte et al., 2004) have found differentiation along some lineages can be impaired; other studies have seen no effect (Arbab et al., 2005b; Kassis et al., 2010). Each cell type that is labeled in this way should be evaluated for effects on cell function prior to conducting studies in vivo.

The aim of regenerative medicine is to repair damaged tissues or organs within an organism through replacement or restoration (Mason and Dunnill, 2008). Stem cells, such as NPCs in the brain, play an important role in regeneration and have been evaluated in several disease and injury models. These cell replacement techniques have been paired with MRI in the investigation of recovery from spinal cord injury (Lepore et al., 2006), reducing autoimmune damage in multiple sclerosis (Ben-Hur et al., 2007; Reekmans et al., 2011), and to restore function in stroke models (Hoehn et al., 2002; Zhang et al., 2003).

The disease that has been the most extensively researched using exogenously labeled NPCs and MRI is stroke. In the United States, stroke is the fourth leading cause of death and occurs in one out of every 19 fatalities (Kochanek et al., 2011). Over the past 10 years, stroke-attributed deaths have decreased (Go et al., 2014) thus increasing the demand for regenerative medicine treatment of survivors, who suffer from the adverse effects of stroke. Promising data has been collected in both animal models and clinical trials using exogenous NPCs to reverse or repair damage induced by stroke. Incorporation of MR imaging into the evaluation of NPCs as cell-based therapies is a way for researchers to gain an understanding of the relevant cellular processes, and to guide the development of this approach into a viable strategy for effective regenerative medicine.

In rodent models of ischemic stroke, where the middle cerebral artery (MCAO) is occluded, administration of NPCs has led to marked improvements in both behavioral and physiological markers of animals. The use of iron oxide particles to label cells has allowed researchers to use MRI to visualize the movement and incorporation of cells into the site of stroke (Hoehn et al., 2002; Zhang et al., 2003) and has demonstrated that the migration is directly triggered by the onset of disease (Figure 4; Guzman et al., 2008). Additionally, many studies have established that of exogenous NPCs labeled with magnetic particles retain normal function with the same positive effect on restoration of function as control cells without particles. In these studies, magnetically labeled NPCs have been shown to reduce the size of the lesion (Obenaus et al., 2011; Daadi et al., 2013), increase angiogenesis (Jiang et al., 2005; Li et al., 2006) and improve glucose utilization (Daadi et al., 2013) in damaged areas of the brain, and recover sensorimotor functions (Zhang et al., 2003; Daadi et al., 2009). In a large animal, porcine, model of ischemia, NPCs injected into the region of stroke decrease the immune response and reduced changes in cerebral blood perfusion and brain metabolism (Baker et al., 2017). Additionally, rodent studies have demonstrated that labeled cells that migrated into the site of the infarct have functionally incorporated into the tissue (Figure 5) and differentiated into neurons (β-tubulin and NeuN), astrocytes (GFAP), and oligodendrocytes (CNPase) (Song et al., 2009; Daadi et al., 2013). These cells did not, however, counterstain with antibodies that were specific for microglia and macrophages (OX6; Song et al., 2009). The cellular and molecular mechanisms that lead to functional improvement have not yet been established, although several hypotheses have been proposed (reviewed in Boese et al., 2018). The effects could be mediated by replacement of lost circuitry, neuroprotective function, improved vascular recovery, paracrine stimulation of surviving neurons and other cells, recruitment of endogenous progenitors, or reduced inflammation.

Implementation of MR imaging of cellular fates in stroke research has the potential for making NPC transplantation a realistic therapy for patients suffering from this disease. Through imaging we can refine our understanding of the optimal route and timing of injection, the effective dose of cells for a given lesion, rapid determination of lesion size, and the relevant cell types for reversing the damage and restoring function. In stroke, four routes of administration of cells have been used: intracerebral, intraventricular, intracisternal, and intravascular. The most common route used in animal studies is intra-cerebral injections into the striatum (Table 2) and this is also being evaluated in clinical trials. Investigation in the field of regenerative medicine originally focused on this approach because researchers wanted to limit the distance cells would have to migrate and eliminate the complications of requiring cells to penetrate the blood brain barrier (Willing and Shahaduzzaman, 2013). However, intra-cerebral injection is an invasive procedure that involves complex stereotactic surgery in patients already weakened by stroke; therefore, other routes of administration would be preferable. Intra-ventricular and intracisternal injections are less invasive and may minimize tissue damage by targeting the brain ventricles or subarachnoid spaces, respectively. Both methods have been demonstrated to allow cell migration to the site of stroke (Li et al., 2006; Obenaus et al., 2011; Ashwal et al., 2014); however, only one study compared these methods directly to intracerebral injection. In this study (Obenaus et al., 2011), more cellular migration was observed with intra-ventricular injections compared to intracerebral injections into the striatum, however, no statistics or compelling images were provided to bolster these claims.

The least invasive routes of NPCs administration are peripheral injections, either venous (IV; Song et al., 2009) or arterial (IA; Li et al., 2010). When comparing these two vascular routes of administration to intracisternal injections, Li et al. (2010) found that IA injections had the fastest and greatest dispersion of cells within the stroke area while IV injection had the slowest and least number of cells, however, animals that received IA injections also had a higher mortality. This study, while providing interesting insights into the migration of NPCs after these injection routes, did not address the full range of physiological effects, the overall localization of cells, or the cause of mortality. More in depth investigation will be required prior to drawing any conclusions and developing an effective treatment plan for patients is identified to minimize complications while providing optimal care.

The relationship between cell dose and recovery is more straight-forward. When heNSCs labeled with Feridex were injected into the striatum of rats with stroke, the number of cells administered was inversely proportional to the size of the stroke after treatment (Daadi et al., 2009, 2013). Thus, studies comparing injection routes should control for number of cells to avoid confounding results. Additionally, the relationship between the number of transplanted cells and stroke recovery is even more complicated. The more severe and larger the initial stroke volume, the less of an impact treatment with NPCs has on the outcome of adult (Daadi et al., 2013) and infant (Ashwal et al., 2014) animals with stroke. Others have determined that, paradoxically, a lower dose of NPC is more effective at achieving homing toward a tumor (Barish et al., 2017).

There are still many questions that have yet to be addressed with these models; including the best cell type and the best methodology for labeling cells, since a wide variety of each have been used, as well as, the interaction of the transplanted cells with those of the tissue. Rodent studies have used cells isolated from the SVZ of adult rats, immortalized NPC lines from rodent embryos, or those isolated from human fetuses. In humans, there are two abstracts that have used NPCs derived from human fetal tissue and demonstrate improved symptoms in patients (Kalladka et al., 2013; Qiao et al., 2014). The authors of one abstract are currently running two clinical trials (NCT01151124 and NCT02117635, ClinicalTrial.gov) using the cell line CTX0E03 DP, an immortalized human stem cell line derived from third trimester human fetal cortex (Pollock et al., 2006). These different types of cells have never been compared in terms of efficiency; therefore, generalizations between studies should be analyzed in this light. The phase I escalation trial showed no adverse effects of the cell injection and modest neurological improvements in some patients (Kalladka et al., 2016), the phase II trial has completed recruitment, but no results have been published.

Another application of magnetically labeled exogenous NPCs is their use in the detection or targeted treatment of glioma. Gliomas arise within the nervous system and are classified by histopathology and immunohistochemistry (Tsankova and Canoll, 2014). GBM is a particularly aggressive primary brain tumor with a grim prognosis for patients; the average lifespan is less than 12 months (Towner et al., 2011). Because the brain is an immune-privileged area within the body and is protected by the blood–brain barrier, detection and treatment of these tumors presents a particular challenge. Research into the use of NPCs in the treatment of glioma has made progress in harnessing the unique characteristics of NPCs in migrating toward glioma cells and the power of cell tracking via MRI.

Early studies used histological methods to demonstrate that (1) NPCs actively migrate into the tumor itself (Aboody et al., 2000) and (2) the mere presence of NPCs could act to impede the growth and proliferation of tumors (Glass et al., 2005). Both results were later replicated using MRI-based cell tracking. Brekke et al. (2007) used gadolinium-based particles to determine that NPCs injected contralateral to the tumor site migrated across the corpus callosum to the site of the tumor (Figure 6) and that tumor doubling time was significantly decreased, probably due to reduced edema formation (Brekke et al., 2007). Others have explored different routes of administration and found migration to the tumor following IV (Gutova et al., 2013), intracisternal (Zhang et al., 2004) and intracerebral (Thu et al., 2009; Chaumeil et al., 2012) injections of NPCs, demonstrating that cells also penetrate the tumor with these routes. The effects of NPC presence on tumor growth, however, were trivial and did not greatly alter the course of the disease.

The next step in the progression of the NPCs/glioma research focused on modifying NPCs to deliver agents to the site of the tumor and have a greater impact on tumor growth. Cells can be altered to (1) deliver specific cytokines, (2) impact angiogenesis, or (3) to function as “suicide cells” that deliver prodrugs to the effective area (Kwiatkowska et al., 2013). MRI labeling of NPCs with MPIOs has been used in prodrug delivery studies to optimize the timing of the activation of the suicide cell. Researchers have established cell lines that can produce an enzyme that can convert a prodrug into a chemotherapeutic agent for the purpose of directed delivery (Aboody et al., 2013). The effectiveness of this method has been verified in combination with iron labeling to demonstrate the advantages of using the combination, and patients were recruited into a phase I clinical trial (NCT01172964, ClinicalTrial.gov). This trial has completed their recruitment, and while no results have been published, two additional trials have been launched (NCT02015819 and NCT02192359) to test the addition of the combination of Leucovorin or to modify cells to sensitize them to the drug Irinotecan (Mutukula and Elkabetz, 2017).

Another novel approach in the use of MRI and magnetically labeled NPCs in the treatment of GBM is their possible use in the detection of tumor recurrence. Recurrence of tumors in GBM patients is a universal trait of the disease and is believed to be a major contributor to the poor survival of patients (Park et al., 2010). New treatments designed to reduce tumor size have been shown to increase the migration of tumor cells and the formation of recurrent masses further from the original site (Ogura et al., 2013). One suggestion is to use MPIO-labeled stem cells as a diagnostic tool for identifying small masses of tumor (<1 mm²) that are not detectible with current MRI methods (Bennewitz et al., 2012). While the authors suggested the use of mesenchymal stem cells, NPCs might provide a more attractive alternative. Indeed, there is already some anecdotal data showing the ability of NPCs to detect individual glioma cells (Zhang et al., 2004), however, this is based on immunohistochemistry demonstrating a single NPC near a glioma cell within the corpus callosum. This hypothesis merits further investigation but it is imperative to examine the signals that recruit cells to the tumor and the ability of NPCs to home to early tumors and micro-metastases in a more prospectively designed experiment.

Additionally, others suggested the use of NPCs as a method to infect remaining tumor cells with a conditionally replicative adenovirus after surgical resection of the mass. In a preliminary study, Morshed et al. (2015) imaged NPCs carrying a cytotoxic adenovirus infiltrating the margins of the tumor. This study is an initial step but shows a promising new avenue for the combination of MRI and NPCs in the treatment of GBM.

Beyond the lack of consistency with regards to techniques for cell administration in the preclinical and clinical studies mentioned above, the tracking of exogenously labeled NPCs is limited by factors related to health of the injected cells and the persistence of the loaded particles in cells after transfer. Selection of cells and particles within an experiment is, therefore, an important component of the quality control in tracking studies.

The health of cells being injected into patients is important for the transfer of therapeutic effect as well as for both the short and long-term ability to monitoring cells in vivo over time. Immediately following injection of cells, there is a possibility of the grafted cells being rejected, and this has been examined in a handful of models. Mesenchymal stem cells (MSC) can be obtained from patient and the use of such autologous grafts should reduce the likelihood of rejection (Akgun et al., 2015). MSC can be harvested in the operating room, cultured and labeled, then reintroduced into the targeted area of the patient (Callera and de Melo, 2007; Kassis et al., 2010). The importance of using autologous MSCs has not been definitively established (Kode et al., 2009). For ischemic cardiomyopathy, allogeneic, and autologous MSC produced similar positive therapeutic results, and neither triggered an immune response (Hare et al., 2012). In contrast, NPCs cannot easily be harvested from patients; therefore, these cells are collected from donors, i.e., fetal cadavers. The sex of the NPC donor was found not to be relevant to the success of the graft in a rat study (Ashwal et al., 2014), however, other parameters have not been fully examined. Taken together these studies suggest that the risk of rejection is low. However, a recent article, Bago et al. (2017) demonstrated the possibility of using donor fibroblasts to trans-differentiate into NSCs and allowing for autologous cells to be cultured into cytotoxic agents against glioblastoma.

A major criticism of using iron oxide nanoparticles for cell tracking is that the signal is retained even after the labeled cells are no longer viable. MRI does, however, have the potential to differentiate between live and dead cells. In immunosuppressed rats, there was a clear difference in the MRI appearance and image intensity and graft size over time between live and dead NPCs (Guzman et al., 2007). When live cells were injected, the size of the hypointensity was constant with time, while transfer of dead cells led to a decrease (Guzman et al., 2007). In a different study of immunocompetent rats, live cells were observed to migrate leading to an increase in the size of the hypointense region, and since dead cells don’t migrate the size of the hypointense region remained constant (Flexman et al., 2011). In a model where animals rejected grafted cells, hypointensity persisted in rejected grafts since the label from dead cells is likely taken up by microglia (Berman et al., 2011). In grafts that survived, the signal is lost over time presumably due to the label being diluted during cell division, (Berman et al., 2011). In another study, when SPIO-labeled NPC were injected into the rat brain and subsequently rejected, the hypointensity persisted at the site of injection in both cases, but again no migration was seen (Bernau et al., 2015). This demonstrates that with proper image processing, the appearance of the MR scan could be used to determine the success of a graft. This would comprise an important diagnostic and prognostic tool that could potentially indicate if a NPC transplant had been successful, or if another graft is required. However, to generate that data to inform these algorithms will require more research using disease models in which cell migration away from the site of injection is expected.

The introduction of stem cells into patients raises the possibility of long term complications due to development of tumors from the transplanted cells. A major safety concern with the use of embryonic stem cells is the formation of benign teratomas (Blum et al., 2009). The formation can be reduced by differentiating the cells beyond the pluripotent state in culture prior to transfer; however, differentiation is usually not 100% leaving stem cells and the potential risk. In addition, other factors can influence the formation of tumors from stem cells (Erdo et al., 2003). When NPCs selected from human embryonic stem cells were injected into mouse eyes, teratomas formed in 50% of animals at 8 weeks post-transplant (Arnhold et al., 2004). Tissue devoid of pluripotent stem cells, derived from either fetal or adult cells, may provide a safer mode of cell generation while still maintaining the cells’ potency to differentiate (Pollard et al., 2006; Kosztowski et al., 2009). A majority of the studies in stroke models use fetal or adult cell lines and none report any incidence of tumor formation. In one study, cells implanted into animals were successfully monitored for more than a year (58 weeks) with no report of tumors (Obenaus et al., 2011).

Another obstacle in monitoring exogenously labeled cells is the loss of signal over time due to changes in the cells, particles or both. Reduction in signal has been attributed to several different mechanisms: (1) degradation of the particles over time, (2) dilution of the contrast agent, per cell, during cell proliferation, or (3) release of the particles to endogenous cells upon death of the transplanted NPC. Any of these alone, or in combination, can significantly disrupt plans for prolonged in vivo imaging of NPC migration and distribution. By understanding these mechanisms and the vulnerable points in a cell tracking study, they can in part be circumvented.

The degradation of particles is dependent on the chemical and physical properties of the agent used. Particles enter the cells through endocytosis and are then exposed to the acidic environment of the lysosome, leading to degradation of the agent over time. By mimicking the conditions inside of lysosomes (lysosomal conditions: pH 4.5, sodium citrate buffer) it was shown that ferumoxides (Feridex) release free iron after 7 days, and when evaluated in cultured MSCs, the particles aggregated within the lysosomes and began degradation after 5 days (Arbab et al., 2005a). Poly (lactic co-glycolic acid) (PLGA) encapsulated iron oxide nanoparticles persist for a longer period of time under these conditions but undergo a distinct burst and plateau phase; particles degrade by approximately 80% over a 12-week period (Nkansah et al., 2011). There have been no comprehensive studies published that directly examine and compare the dissolution of different particle types in NPC, either in vitro or in vivo.

Cells can also be transfected with DNA encoding modified forms of the iron storage protein, ferritin, and then transplanted into the brain. The goal would be to increase specificity of the markers and ensure iron persistence over time, while still having the signal inextricably linked to cell viability. When ferritin- or luciferase-transfected (negative control) cells were transplanted into the striatum of healthy rats, similar hypointensities were observed in the brains of these animals (Bernau et al., 2015). This unexpected result is likely due to the fact that injection of either transfected cell type caused hemorrhage at the site of injection. Moreover, the signal loss persisted even when the cells were rejected (observed after withdrawal of an anti-rejection agent), and no migration of the cells was observed this study (Bernau et al., 2015). At the present time, there are no genetically encoded MR reporters that can be transfected into exogenous cells, or endogenous cells, and used for spatiotemporal MR imaging of cell migration in vivo.

When NPCs proliferate and differentiate in vitro or in vivo, particle concentration per cell is diluted with each division. Differentiation can involve asymmetric cell division with different amounts of contrast agent being retained within each daughter cell (Walczak et al., 2007). Labeling cells with Feridex does not seem to influence the ability of NPCs to differentiate (Cohen et al., 2010). However, the rate of differentiation in vivo will vary with the tissue environment, i.e., disease model, and the cell type used. In shiverer mouse models of hereditary demyelination, iron oxide loaded cells were not detectable by MR after 6 days presumably due to dilution of the agent with cell division (Walczak et al., 2007). In contrast, similarly labeled cells transferred into models of glioma and stroke could be monitored for weeks without the loss of signal. It is important to note that these anecdotal studies have not been supported, to date, with studies directly examining either cell division rates or the differentiation of transplanted cells in glioma or stroke models. Cell division is typically assessed ex vivo using a dye, CFSE, which is diluted with cell division and flow cytometry to assess dye concentrations—such experiments are labor intensive and expensive.

Death of transplanted cells can also confound imaging studies. When implanted NPCs die, particles are released and made available to endogenous cells that can pick up the contrast and shuttled it away from the site of injection, reducing the MRI signal. The mononuclear phagocytic system is the major route of clearance for circulating particles and those found within many organs including the brain, it internalizes and metabolize the free-floating particles (Arami et al., 2015). Excess particles from intraventricular injections are taken up by macrophages and leads to the buildup of iron in macrophage-rich organs including the choroid plexus, liver and spleen (Gorman et al., 2018). Alternatively, the contrast can be taken up by resident microglia to create a false positive signal that persists within the brain. In a variety of different disease models, exogenous NPCs have been shown to primarily differentiate into neurons, astrocytes, and oligodendrocytes and not microglia or macrophages. In a study looking at the injection of dead NPCs, it was demonstrated that particles are found in astrocytes that accumulate at the site of injection and not within cells that migrate (Flexman et al., 2011). This suggests that moving particles may be indicative of live NPC, and stationary particles may have been released by the death of their original host. Nonetheless, there are a number of patterns of iron oxide distribution in the brain following transfer of labeled cells, and these patterns are indicative of the fates and function of both transplanted and endogenous cells. Working out the significance of the hypointensity patterns in MR images may be possible and become useful in guiding the development of cell-based therapies for brain injury and disease.