In Vivo Cell Tracking Techniques for Applications in Central Nervous System Disorders

Exogenous labeling

Endogenous labeling

MRI

T1WI

Gd, Mn

T2WI

SPIO

MR reporter gene

Nuclear imaging

SPECT

¹¹¹In-oxine, ^99mTc-HMPAO

Reporter gene

PET

¹⁸F- FDG

Optical imaging

QD, organic dye

GFP

Luciferase reporter

BI bioluminescence imaging, ¹⁸ F-FDG ¹⁸F-fluorodeoxyglucose, FI fluorescence imaging, GFP green fluorescent protein, Gd gadolinium, In indium, Mn manganese, PET positron emission tomography, SPECT single-photon emission computed tomography, ^99m Tc–HMPAO ^99mtechnetium-hexamethylproplyleneamine

In exogenous labeling, the cellular marker (e.g., MR contrast agents, radiotracer, or fluorescence probes) is taken up into the cell or attaches to its surface. Usually, exogenous labeling is performed in vitro prior to transplantation. There are several kinds of probes for each modality and each has specific characteristics. Although cytotoxicity and labeling efficiency vary for each probe, currently, exogenous labeling is considered the first choice for clinical applications. This method has several favorable features including requiring only simple incubation with the probe according to standardized protocols, and it is capable of being applied to allogenic or autologous cells without gene transfection. However, it also has fundamental limitations when it comes to transplanted cell quantification and long-term monitoring, due to the dilution of tracer by cell proliferation and cell death, and possible transfer to other phagocytic cells such as macrophages [15]. Another problem is that the persistence of the tracer, which can be detected by imaging, does not directly indicate the viability of transplanted cells. Moreover, radiotracers for nuclear imaging have a short half-life, and fluorescent probes for optical imaging exhibit photobleaching or degradation [16–18].

On the other hand, endogenous labeling is usually performed by genetic manipulation of transplanted cells ex vivo, so that they are able to produce certain proteins that can later be used as markers. This method is free of the abovementioned problems. The inclusion of genes means that only living cells can be detected by imaging, because dead cells will no longer synthesize proteins as a marker. Moreover, there is no dilution of a tracer by cell division because a parent cell will supply each daughter cell with the same gene. Reporter genes are widely used for cell tracking in the field of nuclear imaging [19–21]. In this technique, the manipulated gene in the transplanted cell produces a particular protein that is involved in the uptake or accumulation of the tracer, and the tracer itself is then administered immediately before imaging to label target cells in vivo. However, this method is unlikely to be used for human application because of the ethical problems and possible functional changes resulting from gene transfection [22].

Alternatively, receptor-based in vivo labeling techniques are also available. However, there is the possibility that cell surface markers might change when transplanted cells undergo differentiation. Therefore, this technique has not been widely used for in vivo cell tracking to date.

8.3 Imaging Modality

The characteristics of each imaging modality are summarized in Table 8.2. Appropriate choice of imaging system is crucial for successful in vivo imaging.

Table 8.2

Comparison of each imaging modalities

Modality	MRI	Nuclear imaging	Optical imaging
Cost	High	Medium	Low
Acquisition	Minutes-hours	Minutes	Seconds-minutes
Radiation	No	Yes	No
Stability	Weeks	Minutes-days	Weeks
Labeling toxicity	Safe	Yes	Varied
Quantifiable	No	Yes	No
Sensitivity	>1,000 cells	single cell	>1.0 × 10⁵ cells
Penetration depth	No limit	No limit	<1 cm (FI), 3 cm (BI)
Resolution	10–100 μm	1–2 mm	2–3 mm
Visualization	3D, any model	3D, any model	2D, only small animal

BI bioluminescence imaging, FI fluorescence imaging, MRI magnetic resonance imaging

8.3.1 MRI

MRI has clear advantages over the other imaging modalities because it has the highest spatial resolution and is widely available in clinical situations. MRI is an excellent modality for detailed demonstration of cell location after transplantation.

Gadolinium chelates [23] and manganese [24], which are T1 contrast agents that generate positive contrast, were used in cell labeling in early studies. Superparamagnetic iron oxide (SPIO) nanoparticles have become the most widely used agents for cell labeling, because SPIO is more sensitive and biologically compatible than other contrast agents. One of the SPIO formulations has been approved by the Food and Drug Administration (FDA) for human use, and SPIO has been used in a clinical study [10, 12]. SPIO is a T2 contrast agent that generates negative contrast (Fig. 8.1).

Fig. 8.1

T2*-weighted MR images of the rat subjected to permanent middle cerebral artery (MCA) occlusion reveal that the transplanted superparamagnetic iron oxide (SPIO)-labeled bone marrow stromal cells (BMSC) (arrow) migrate toward the cerebral infarct 2 weeks after transplantation (arrow heads)

Hoehn et al. tracked murine ES cells using MRI after direct grafting them into rat brains subjected to focal cerebral ischemia. They found that the ES cells labeled with ultrasmall SPIO started to migrate toward the lesion within a few days and that they accumulated in large numbers in the border zone of the damaged brain tissue 3 weeks after transplantation [25]. Zhang et al. intracisternally transplanted rat subventricular zone cells labeled with ferromagnetic particles into the infarcted rat brain. Serial MRI tracking revealed that the engrafted cells migrated toward the ischemic parenchyma at a mean speed of 65 ± 14.6 μm/h in the living rats [26]. As mentioned above, long-term monitoring of this technique was problematic; however, Kim et al. directly transplanted the SPIO-labeled human BMSCs (hBMSCs) into rodent brains subjected to cerebral infarction and were able to track them using MRI 10 weeks after transplantation [27]. Our group also succeeded in tracking hBMSCs up to 8 weeks after transplantation [28]. As with the nuclear imaging method detailed below, MR reporter gene assays were developed as a novel endogenous labeling technique geared toward long-term and quantifiable cell tracking study [20].

MRI is not without limitations. First, the sensitivity to detect transplanted cells is generally lower as compared to nuclear imaging and bioluminescence imaging. Although MRI could detect 100 cells when using high-field (17.6 T) MRI [29], our recent study showed that 1 × 10³ cells were needed to reach detection threshold using a clinical (3.0 T) MRI apparatus [30]. MRI is also sometimes impeded by imaging artifacts, such as an intracranial hemorrhage, thus the specificity of MR signal is not always excellent [12].

8.3.2 Nuclear Imaging

Nuclear imaging, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), is characterized by excellent in vivo sensitivities and whole-body imaging capabilities. Nuclear imaging can detect even a single cell. Therefore, this modality is useful in quantifiable analysis of transplanted cells. Brenner et al. and Aicher et al. transplanted ¹¹¹indium (In) oxine-labeled endothelial progenitor cell and hematopoietic progenitor cell into rats subjected to myocardial infarction intravenously and monitored them using SPECT [31, 32]. They reported that only 1 % of transplanted cells engrafted to the myocardial lesion. de Haro et al. transplanted intravenously ¹¹¹In oxine-labeled BMSC into rats with spinal cord injury and detected accumulation of the cells at the injured lesion using SPECT [33]. Correa et al. and Barbosa da Fonseca et al. transplanted and monitored ^99mtechnetium-hexamethylproplyleneamine oxine-labeled MNCs into a patient with cerebral infarction through the internal carotid artery [8, 9, 11].

The most commonly used radiotracer for PET is ¹⁸F-fluorodeoxyglucose, which is FDA approved. Thus, several studies have used this technique in human clinical trials related to myocardial infarction [13].

The short half-life of radiotracers is a major limitation of this method. To overcome this limitation, reporter gene assays have been used for long-term monitoring and assessment of transplanted cell viability. Cao et al. injected ES cells labeled with this method into the myocardium of adult nude rats and succeeded in monitoring viability, engraftment, and proliferation of the transplanted cells at least 4 weeks after transplantation [19, 21]. As an another approach, longer half-life tracers, for example, ⁸⁹Zr-oxinate₄, have recently detected cells up to 14 days after labeling and administration [34].

Another major disadvantage of a radiotracer is radiation toxicity to the labeled cell. Therefore, knowledge of maximum safe doses of radiotracers is crucial for clinical applications. A recent study showed that ¹⁸F-fluoro-2-deoxy-D-glucose may label cells safely at concentrations up to 25 Bq/cell without compromising cellular function [35].

8.3.3 Optical Imaging

Because of light scattering and absorption by tissue, the use of optical imaging for cell tracking is limited to only small animals. However, optical imaging has some advantages including lower cost and rapid acquisition time.

“Bioluminescence” refers to light generated by intrinsic properties of organisms in nature, such as fireflies. Bioluminescence reporter gene luciferase assays have been applied to cells through genetic manipulation before transplantation. When the luciferase substrate (luciferin) is systemically injected, light photons are produced by transplanted cells [36]. Using this technique, in one study, transplanted neural progenitor cells were monitored 21 days after stroke in both rats and mice [37].

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