a biological global positioning system: considerations...

A Biological Global Positioning System: Considerationsfor Tracking Stem Cell Behaviors in the Whole Body

Shengwen Calvin Li & Lisa May Ling Tachiki &Jane Luo & Brent A. Dethlefs & Zhongping Chen &

William G. Loudon

Published online: 18 March 2010# The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Many recent research studies have proposed stemcell therapy as a treatment for cancer, spinal cord injuries,brain damage, cardiovascular disease, and other conditions.Some of these experimental therapies have been tested insmall animals and, in rare cases, in humans. Medicalresearchers anticipate extensive clinical applications ofstem cell therapy in the future. The lack of basic knowledgeconcerning basic stem cell biology-survival, migration,differentiation, integration in a real time manner whentransplanted into damaged CNS remains an absolutebottleneck for attempt to design stem cell therapies forCNS diseases. A major challenge to the development ofclinical applied stem cell therapy in medical practiceremains the lack of efficient stem cell tracking methods.

As a result, the fate of the vast majority of stem cellstransplanted in the human central nervous system (CNS),particularly in the detrimental effects, remains unknown.The paucity of knowledge concerning basic stem cellbiology—survival, migration, differentiation, integration inreal-time when transplanted into damaged CNS remains abottleneck in the attempt to design stem cell therapies forCNS diseases. Even though excellent histological techni-ques remain as the gold standard, no good in vivotechniques are currently available to assess the transplantedgraft for migration, differentiation, or survival. To addressthese issues, herein we propose strategies to investigate thelineage fate determination of derived human embryonicstem cells (hESC) transplanted in vivo into the CNS. Here,

S. C. Li : L. M. L. Tachiki : B. A. Dethlefs :W. G. LoudonCenter for Neuroscience and Stem Cell Research,Children’s Hospital of Orange County Research Institute,University of California Irvine,455 South Main Street,Orange, CA 92868, USA

S. C. LiDepartment of Neurology, University of California Irvine,Orange, CA 92862, USA

S. C. LiStem Cell Biology & Regenerative Medicine Center,Thomas Jefferson University,Philadelphia, PA 19107, USA

S. C. LiDepartment of Biological Science, California State University,Fullerton, CA 92834, USA

W. G. LoudonDepartment of Neurological Surgery,University of California Irvine,Orange, CA 92862, USA

J. LuoBeckman Coulter, Inc.,250 S. Kraemer Boulevard,P.O. Box 8000, Brea, CA 92822-8000, USA

Z. ChenBeckman Laser Institute, Department of Biomedical Engineering,The Henry Samueli School of Engineering,University of California,Irvine, CA 92697, USA

S. C. Li (*)CHOC Children’s Hospital Research Institute,University of California Irvine,455 South Main Street,Orange, CA 92868, USAe-mail: [email protected]

Present Address:L. M. L. TachikiDepartment of Radiation Oncology—Radiation Biology,Stanford University,CCSR-So. Room 1230,Stanford, CA 94305-5152, USA

Stem Cell Rev and Rep (2010) 6:317–333DOI 10.1007/s12015-010-9130-9

we describe a comprehensive biological Global PositioningSystem (bGPS) to track transplanted stem cells. But, first,we review, four currently used standard methods fortracking stem cells in vivo: magnetic resonance imaging(MRI), bioluminescence imaging (BLI), positron emissiontomography (PET) imaging and fluorescence imaging (FLI)with quantum dots. We summarize these modalities andpropose criteria that can be employed to rank the practicalusefulness for specific applications. Based on the results ofthis review, we argue that additional qualities are stillneeded to advance these modalities toward clinical appli-cations. We then discuss an ideal procedure for labeling andtracking stem cells in vivo, finally, we present a novelimaging system based on our experiments.

Keywords Stem cells . Tracking system . Biological GlobalPositioning System (bGPS)

Introduction

Stem cell therapies hold promise for the treatment ofvarious human diseases. Emerging data indicate that trans-planted stem cells have the potential to be beneficial ordetrimental to patients [1–3]. Future clinical trials willrequire determining the location and number of transplantedstem cells in vivo, over a life time [4, 5]. It is very difficultto track small numbers of cells in the body with currenttechnologies. Current imaging modalities available for invivo tracking of the biological fate of stem cells have beenproposed, including Magnetic Resonance Imaging (MRI),Positron Emission Tomography (PET), BioluminescenceImaging (BLI), and Fluorescence Imaging (FLI) withquantum dots (QD). None of these modalities, however,are optimal. Here, we discuss the advantages and disadvan-tages of current imaging technologies with respect to stemcell tracking applications, define the characteristics of anideal imaging technology, and propose a new systemspecifically for stem cell imaging during clinical trials.

Magnetic Resonance Imaging (MRI)

MRI is a widely used medical imaging technique in whichmagnetic fields are used to detect the nuclear spin ofmolecules [6]. MRI is the most readily accessible trackingmethod. Under proper conditions, it is safe and reliable aswell as available in most hospitals. MRI has been success-fully to detect and track stem cell migration, particularly incardiac, liver, [7–11] and brain disease models [12–23]. MRItechniques have been utilized to detect the presence and/ormigration of transplanted stem cells in various animalmodels [24]. Surprisingly, MRI can detect a single hepatocytegrafted into a transplanted liver upon perfusion of the

primary hepatocytes with double-labeled, green fluorescent1.63-microm iron oxide particles and red fluorescentendosomal labeling dye, injected into the spleens of recipientmice [10]. The ability to detect single transplanted cells invivo raises the interest in monitoring stem cell behavior forearly detection of transplanted stem cell initiated tumorigen-esis (teratoma formation), which may result from the use ofstem cells outside of their “therapeutic window” [25].

The benefits of MRI include high resolution and 3-Danatomical imaging capability. Drawbacks include lowsensitivity and difficulty in quantifying a labeled cellpopulation [26]. MRI generally has lower sensitivity, butproduces high soft-tissue contrast and provides spectro-scopic information and functional MRI (fMRI) [27, 28].However, dilution of intracellular markers occur with each celldivision, and false signals may occur due to shedding andsubsequent sequestration of iron particles [29].

There are two general categories of cellular MRImarkers, T1 and T2/T2*, with T2 contrast being morecommonly employed in MR imaging. Gadolinium (Gd3+)contrast enhanced imaging is employed for T1 systems.Apart from Gd, Manganese is also a T1 agent [30, 31]. T2contrast agents include superparamagnetic iron oxides(SPIO) [32], ultrasmall superparamagnetic iron oxides(USPIO), and magnetodendrimers [27], micron sizedparticles of iron oxide (MPIOs) [33]. These magneticmarkers are sequestered into stem cells. They may exhibitlong-term health problems due to endocytosis of magneticparticles with CNS (Fig. 1). The clinically transferableproperties of superparamagnetic iron oxide nanoparticles(SPIO) requires modification in MR for tracking stem cellsin living organisms [4]. MRI can be used to track cellslabeled with iron particles in damaged tissues for at least16 weeks after injection and to guide tissue sectioning byaccurately identifying regions of cell engraftment. Themagnetic properties of iron-labeled donor cells can be usedfor their subsequent isolation from host tissue to enablefurther characterization [34].

Bioluminescence Imaging (BLI)

Bioluminescence imaging is a technique in which a charged-coupled device camera detects light emitted from the substrateof the enzyme luciferase. The luciferase gene is incorporatedinto for example a lentiviral vector, and this vector transfectsthe luciferase indicator transgene into the defined stem cellpopulations. Firefly luciferase, in the presence of substrate D-luciferin, oxygen and ATP, emits light at about 560 nm [35](Fig. 2). Bioluminescence imaging is safe, permitting therepeated tracking of small numbers of labeled cells.Bioluminescence imaging is typically not as sensitive to asmall number of cells as MRI or PET. This technique hasbeen little used in humans due to concern about immuno-

318 Stem Cell Rev and Rep (2010) 6:317–333

genicity of the proteins and products involved. In addition,human clinical applications are limited because of the highabsorbance and scattering of luminescence in living tissue.As such, BLI is likely to be limited to its current use inimaging transfected stem cells in small animals.

Positron Emission Tomography (PET)

PET uses positron emitting radioisotopes as probes forimaging cells in vivo [36, 37]. The decay of a radioisotopeproduces two high-energy (511 keV) gamma photons thattravel in two opposite directions, which are collected and

verified by a ring of scintillating crystals. PET scanners canbe used in conjunction with CT scanners to produce moredetailed, fused, 3-D anatomical images [38]. To date, mostclinical PET-CT studies have used radioactive metals suchas 18F-fluoro-2-deoxyglucose (18FDG) and 64Cu, as imagingmarkers [39, 40]. These markers need a chelator such asPTSM to label cells [41–43], and have been used to trackmesenchymal stem cells [38]. Alternatively, the reporter genemethod, using herpes simplex virus type 1 thymidine kinase(HSV1-tk), is a stable labeling method (Fig. 2) [44]. However,the latter method poses a dilemma because it has the potentialto alter the genetic properties of labeled stem cells. Althoughthe value of PET lies in its high-sensitivity tracking ofbiomarkers in vivo, it lacks the ability to resolve morphology.

Using PET and CT fused images, Love et al. were ableto locate and detect as few as 10,000 mesenchymal stemcells (MSC) for over a two-month period in mice [38].Potential disadvantages of PET include repeated injectionof radioactive materials into an organism with the potentialfor radioactive damage to normal tissue associated withcumulative radiation and exposure of surrounding tissues[26]. Additionally, most currently available radiotracershave short half lives which makes them less suitable forlong-term tracking. Imaging by positron emission tomog-raphy may generate stem-cell specific signals only if themarker is restricted to the stem cell and is the sole sourceproducing a signal of radioactive decay. However, PETdetects only the radioisotope. If the radiometals is releasedfrom the stem cell, the signal will reflect the location ofisotope but not the stem cell. In fact, we know thattranschelation is common.

Engineering cells with receptors for imaging is unlikelyto find its application in human patients. Furthermore, PETimaging requires quite a bit of post-processing of images toget an image and it is therefore unlikely to be of great useimmediately after implantation to track and guide theinjection of stem cells.

Fig. 2 Reporter gene methods appear to be the most effective way tostably integrate markers into cells for bioluminescence imaging andPET imaging. By inserting a gene into the cell, the marker becomesstem-cell specific and will not transfer to surrounding non-stem cells.The use of a reporter gene allows for serial transgene expression alongwith key cellular properties (differentiation, proliferation and cellviability). This tracking strategy can be integrated to track stem cellswith other modalities (BLI, PET, and MPM)

Fig. 1 Direct label of stem cellswith markers. Left: by the processof phagocytosis of SPIO (usuallycoated with dextran). Middle:USPIO are taken in by pinocy-tosis [47]. Right: for cells withoutphagocytic ability, receptor-mediated endocytosis is used tofacilitate labeling

Stem Cell Rev and Rep (2010) 6:317–333 319

Quantum Dots (QDots or QD)

Quantum dots (QDots or QD) represent one of several typesof fluorescent imaging agents. (QDots are semiconductorparticles with physical properties that enable them to emitfluorescent light from 525 to 800 nm (Fig. 3). Quantum dotsconsist of an inorganic core, a shell of metal and an outerorganic coating with a total diameter of 2–10 nm [45].Quantum dots are capable of entering stem cells throughpassive loading, receptor-mediated endocytosis or transfec-tion [46]. Passive loading has been found to be the mosteffective method because it is efficient at labeling, limitingdamage to surrounding stem cells. Quantum dots arecapable of multiplex imaging [47], single quantum dottracking [48], and 3-D imaging reconstruction [46].

Quantum dots represent a fairly new advance fortracking stem cells. However, light scattering for quantumdots limits the applicability of this approach, especially tothe brain in humans. This scattering property makes it verydifficult for use in 3D localization or quantitative estima-tion of cell survival, which is clearly desired properties invivo imaging. The long-term biological health effects areunknown, and the method has not yet been used in vivo totrack stem cells in animals larger than mice.

Multimodal Imaging

Multimodal imaging is becoming more popular becauseof its improved sensitivity, high resolution, and morpho-

logical visualization. More than a decade ago, SimonCherry at the University of California, Davis, beganworking on small animal imaging system combiningmodalities with crucial qualities: MRI, with its excellentspatial resolution and high-contrast morphologic imagingof soft tissues; and PET, which can track the distributionof biologically targeted radiotracers with high sensitivity,but lacks anatomic context and spatial resolution.Physical constraints (i.e., no metal can get inside theMRI magnet) have frustrated these efforts. Cherry’sgroup has recently revolutionized the identification ofcancer biomarkers by creating an MRI-compatible PETscanner, allowing data from both modalities to beacquired simultaneously [49]. Judenhofer and colleaguesreported that they have developed a three-dimensionalanimal PET scanner that is built into a 7-T MRI for acombined molecular imaging system [50], simultaneouslyacquiring functional and morphological PET-MRI datafrom living mice. Such a PET-MRI provides a powerfultool for studying biology and pathology in preclinicalresearch and has great potential for clinical applications.Shen et al. combined MRI and PET methods to createmultimodal imaging for infarcted myocardium in rats[51]. This technique is integrated for coregistration of PET(with higher sensivitity) or SPECT images with MRI (with3-D anatomical details of MR scanning) and allowsmapping of the location and distribution of stem cells ondetailed myocardium structures. A new framework forhow whole body imaging using different techniques canbe integrated needs to be proposed and tested for stemcell tracking.

Whole body imaging using different techniques has beentested in small animals for feasibility. I (Intravenous)injections of the 111In-oxine-labeled human embryonic stem(ES) cell-derived neural progenitors and rat hippocampalprogenitors accumulated primarily into internal organs,instead of in the targeted brain [52] while intra-arterialinjection of the labeled stem cells showed a weak signal inthe ischemic hemisphere detected using SPECT/CT device.The limit of detection sensitivity of SPECT/CT device wasapproximately 1,000 111In-oxine-labeled cells.

Requirements for Clinical Applications

Definition of a Biological Global Positioning System(bGPS) for tracking stem cells. The ability to observetargeted stem cell behaviors using a system capable ofscanning for transplanted stem cells throughout the wholebody, at any given time is essential to determining whetheror not transplanted stem cells in any given clinicalintervention is beneficial or harmful in vivo. An advancedbGPS system could be employed to record viability,

Fig. 3 Quantum dots (QD) are directly loaded into stem cells andused in multplex imaging. Multiplex Imaging (Stacked Image) basedon the energy absorbed is different. Some QDs absorb less energythan others because QDs can produce different light levels at the sameexcitation wavelength QD 525, 565, 605, 655, 705, and 800 were usedto label stem cells. QDs were excited by the same wavelength, whichis why longer wavelengths are brighter [47]


differentiation state and the functionality of targeted stemcells in vivo. Specifically, bGPS can be defined as a unitdevice to locate and record at regular intervals the preciselocation of a transplanted stem cell in tissue to which it hasmigrated within the body. Additionally, a bGPS could bedesigned to record and track the differentiation state,physiological or pathological state, direction and movementof a transplanted stem cell through the body during the timein which it remains in vivo. The work flow for labeling andtracking stem cells is described in Fig. 4. Critical elementsin bGPS include:

(1) Sensitivity for single cell detection.(2) Real-time positioning: Ideally, targeted stem cells

could be monitored as they travel throughout the bodyover any given time period in order.

(3) An inducible system: Signal retrieval under the controlof an inducing mechanism to minimize the impact ofintervention on the host.

(4) Retractable: Ability to inactivate stem cells shouldeffects prove deleterious.

(5) Targeted and durable: Label detectable for the life-time of the cell and not transferable to host cells.

(6) Monitoring cell fate: Ability to track cell viability,proliferation, and differentiation state in vivo. Morespecifically, monitoring physiological changes in the stemcells, e.g., the growth rate (size of the cell), the proliferationrate (the number of the cell), and the differentiation state(the functional transformation). i.e., the label should lastfor the life time of the stem cell in vivo.

(7) Compliant with the FDA GMP guidelines for clinicalapplications.

Fig. 4 Multimodality of imaging can be applied for tracking stem cellbehavior. A work flow chart for labeling cells and introducing labeledcells into the human body include: (1) Cell is labeled using a markerfor magnetic resonance imaging (MRI), bioluminescence imaging

(BLI), positron emission tomography (PET) or fluorescence imaging(FLI). (2) Cells are cultured in vitro and ex vivo (i.e., OTS), and then(3) Injected intravenously into the human body. (4) Stem cells are thentracked in the body with a camera or scanner


An ideal procedure for labeling and tracking stem cellsin vivo is illustrated (Fig. 4). Optimal qualities for celltracking strategies were proposed by Fragioni [26]. Theseinclude biocompatibility, nontoxicity, quantification, nogenetic modification, no perturbation of stem cells, andnoninvasive imaging. Additional qualities were posited,including (1) specificity of stem cell labeling, (2) continu-ous in vivo tracking, (3) imaging in a greater depth, and (4)specificity of stem cell imaging. These four requirementsare addressed respectively with four currently used trackingmethods (Table 1).

Specificity of Stem Cell Labeling

The confusion between specificity and sensitivity can beclarified as follows: Specificity reflects the ability todifferentiate transplanted cells from a host cell or anartifact. In this application, sensitivity describes theminimum number of labelled cells necessary for detection;ideally this would be one cell. For example, MRI issensitive enough to detect transplanted cells (even a singleone), but not specific enough to distinguish it from ferritindeposits in host cells. For example, the blooming effect ofiron oxide particles poses a potential obstacle to locatingcells by diffusing a signal across a much larger area thanwhere a transplanted stem cell is actually located. There is athorough study on fluorine imaging by MRI to selectivelyand quantitatively detect transplanted cells and avoid manyof the artifact traps associated with ferritin labeling [53].

Specific labeling of targeted stem cells could beaccomplished through genetically labeling using a reportergene (Fig. 2). This reporter gene allows for detection ofserial transgene expression in conjunction with key cellularproperties (differentiation, proliferation and cell viability)[54–57]. This tracking strategy can be used with othermodalities including PET, BLI, and MPM. Labelingspecificity is an important requirement for tracking stemcells because it ensures that the label is present only in thestem cells of interest. Furthermore, the label should notinteract with or transfer from stem cells to surrounding non-stem cells. Stem cells would ideally retain the label for life

in order to maintain specific targeting in vivo. After alabeled cell dies, the label should spontaneously dissipateor be eliminated to prevent re-uptake by surrounding non-stem cells and to indicate that the labeled stem cell has beencleared.

Luciferase, used for bioluminescence imaging, remainsspecific to transduced stem cells because the marker gene isincorporated into the DNA of the labeled stem cell. Althoughthe marker is retained within the cell, bioluminescenceimaging can still be distorted because the surrounding livingtissue absorbs and scatters the light given off by the luciferasemarker [26].

The magnetic markers used in MRI are not neccessarilyspecific to stem cells. Iron oxide nanoparticles canpotentially transfer from stem cells to non-stem cells, suchas macrophages. Moreover, these iron particles may shed orspill from stem cells into the surrounding living tissue [6,26, 29]. To address these issues, Modo et al. (2002)established that the Gd particles remained inside labeledcells in a co-culture system [58] and the uptake of USPIOin macrophages was demonstrated in an in vitro system[59].

Depending on the mechanism used to insert the markerinto the stem cell, positron emission tomography may ormay not specifically detect only labeled stem cells. When aradiotracer is directly loaded into a cell, the possibilityexists that non-stem cells will uptake the marker [1].Alternatively, we can design an enzymatic conversion/retention of radiotracers for stable incorporation of markersinto the stem cell, eliminating the possibility of transferringmarkers from labeled stem cells to non-labeled cells.

Quantum dots might be used as markers but, incomparison, are not stem cell-specific. While passiveloading of bare quantum dots into mesenchymal stemcells provided a very effective method of delivery [46],the dots demonstrated no specificity for stem cell binding,and therefore, the possibility that quantum dots can spillout of the labeled stem cells, resulting in a loss offluorescent signal. Quantum dots, however, do not appearto be transferable from labeled stem cells to non-stemcells. When QD-labeled human mesenchymal stem cells

BLI QD MRI PET

Specificity for stem cell labeling ● × × ●Marker is nontoxic to stem cell ● ● ●a ●Marker does not alter stem cell properties ● ● ● ●Non-invasive tracking × ● ● ×

Continuous in vivo tracking for up to one month ● × ● ●Tracked at >3 in depth from skin × × ● ●Specificity for stem cell imaging ● ● × ●Quantification capability ● × × ●

Table 1 Comparison of currentlyused methods for tracking stemcells

● Indicates that tracking methodfulfills this capability

× Indicates that tracking methoddoes not fulfill this capabilitya There are reports to show thatthe marker particles are toxic forcertain functions for stem cells(see the text for details)


were perfused in animals, adjacent cardiac cells failed totake up the released quantum dots in vivo [46].

Marker is Nontoxic to Stem Cells

An essential requirement for cell labels is that they are nottoxic over time. Markers for bioluminescence labeling,small doses of radiotracers for PET, and small amounts ofquantum dots have generally not shown to be toxic tolabeled cells. Still, Lin, et al. demonstrated for fluorescentimaging that although quantum dots are generally nontoxic,escalating concentrations (>20 nmol/L) of quantum dotsprove to be cytotoxic over time [47]. A recent study foundthat commonly used iron oxide nanoparticles used for MRimaging can have toxic effects on neuronal cells [48]. Thepossibility that iron oxide nanoparticles can transfer fromlabeled cells to neuronal cells poses a hindrance to clinicalapplications of tracking stem cells through MR imaging.Modo et al. (2008) followed Gd-labeled cells over one yearand found that although there was no in vitro effect, cellslabeled with Gd did not recover as they exerted deleteriouseffects on repair [60]. Microscopy studies revealed lyso-somal compartmentalization of iron particles in humanmesenchymal stem cells up to 14 days after labeling [61].With subsequent loss of the compartmentalization of ironoxide particles, resulting in release of iron oxides into theadjacent environment clearly, clinical safety standards forthe application of labels remain to be defined.

Marker does not Alter Stem Cell Properties

While searching for ideal labeling strategies, it is essentialthat imaging must not alter the behavior or fate of markedstem cell populations. No major adverse effects of the fourimaging techniques have been shown to interfere with stemcell life span or the ability to proliferate over the timeperiod observed [62–66]. Indeed, in vitro labeling of humancentral nervous system stem cells with magnetic nano-particles does not adversely affect survival, migration, anddifferentiation or alter neuronal electrophysiological char-acteristics [67]. Amazingly, these labeled human centralnervous system stem cells transplanted either to theneonatal, the adult, or the injured rodent brain respondedto cues characteristic for the ambient microenvironment.They survive long-term and differentiate in a site-specificmanner identical to that seen for transplants of unlabeledcells. However, it was reported that a reporter gene is abetter marker for monitoring cell viability, whereas ironparticle labeling is a better marker for high-resolutiondetection of cell location by MRI [68]. No significantdifference has been reported thus far in stem cell growthand development when a marker is added to the cell [47].Time-lapse microscopy successfully captured a moment of

dramatic change in chromosome positioning during thetransition between two differentiation stages of pluripotentmouse ES cells [69]. These images following the epigeneticmarkers showed unique nuclear organization in thatmethylated centromeric heterochromatin coalesced to formlarge clusters around the nucleoli. Upon differentiation, theorganization of these heterochromatin clusters changeddramatically.

However, Kostrua et al. (2005) showed that using ironoxide particles on mesenchymal cells impaired their abilityto differentiate into chrondrocytes [70, 71]. The inhibitoryeffect of iron oxide compounds on stem cell differentiationwas a major issue in using labeled stem cells for infarctedmyocardium repair [11]. There is, however, no demonstra-ble effect on stem cells originating from the CNS includingviability, proliferation, or multipotency [72]. Moreover, it isbecoming increasingly clear that one of the major effects ofstem cells (regardless of their niche of origin) is mediatedthrough the paracrine release of various cytokines orgrowth factors [73–77]. It should be noted that probelabeling of stem cells might alter this “paracrine effect.”There is currently little data to prove or disprove the impactof tracking agents on this important (in vivo) stem cell“pro-healing” effect.

Non-invasive Tracking

Stem cell tracking in humans ideally should be non-invasive. Both magnetic resonance scanners and fluores-cent detectors for quantum dots can image stem cellswithout procedures more invasive than stem cell injection.Once these cells are labeled in vitro and transplanted intothe body, imaging for tracking the fate of these cells isnon-invasive.

Bioluminescent imaging and PET imaging require aslightly more complicated process in which specificsubstrates must also be injected in order to insert detectablesubstrates into transplanted stem cells. These substrates,when attached to the enzymes produced by labeled stemcells, produce a signal which can be used to identify stemcells in vivo. However, there have been concerns about theeffect of iron oxide compounds on stem cell differentiation,a pivotal cell property [11].

Continuous Tracking In Vivo

In order to determine the accuracy and efficiency of stemcell therapy in vivo, stem cells must be continuouslytraceable from the time of injection to the time that theyreach a final destination. For example, stem cells should betracked for months at a time to monitor the long-termengraftment and function of stem cells migrated to adamaged region.


However, most experiments tracking stem cells havebeen conducted in small animals and last only days orweeks. For clinical applications, stem cell labeling mustremain intact for months in order to monitor theirviability, proliferation, and integration into the targetedhost microenvironment.

J.M.W. Bulte et al. tracked magnetically labeled stemcells traveling into the brain parenchyma for up to sixweeks, illustrating clinical potential of MRI [27]. However,MRI technology still calls for improvement because eachcell division of a labeled cell reduces the concentration ofsuperparamagnetic iron oxide nanoparticles within prog-eny cells. Such a progressive dilution of the cell markerthrough cell division limits the capability of this methodfor long-term tracking of stem cells. A study by Daldrup-Link et al. demonstrated that with concentrations of lessthan 20 nmol/ml, individually labeled cells cannot bedetected by MRI [78]. A threshold concentration must bemaintained in order for cells to be imaged by MRI. Thisthreshold limits the length of time that MRI can be used totrack stem cells in vivo.

Whereas the MRI technique can track magneticallylabeled cells for over a month, studies employingbioluminescence imaging have successfully tracked cellsfor up to three months [38]. Positron emission tomogra-phy tracking has also been successfully employed forover two months using the HSV1-tk reporter gene [38].Transfecting the stem cell with reporter genes appears tobe a promising method for stable integration of amarker into the target cell permitting longer periods oftracking.

The capability for continuous tracking of quantum dotsin vivo has not been tested as rigorously as that for MRI,bioluminescence imaging, or PET imaging. Althoughcurrent technology generally enables continuous in vivotracking in mice for only a few weeks [47], Rosen et al.were able to identify stem cells within a canine heartlabeled by quantum dots 8 weeks after injection ofmesenchymal stem cells by ex vivo imaging of these cells[46]. In order for quantum dots to be clinically useful,larger test subjects must be used to determine the maximumdepth that emitted fluorescence can be reliably detected invivo and repeatedly.

Tracking and Depth

MRI, BLI, PET and QD techniques can successfully imagestem cells within small rodents. However, these sametracking capabilities can not be currently translated intoimaging larger organisms, such as humans. Ideally, labeledstem cells should be traceable at depths greater than 3 in.from the skin in order to image internal organs, such as theheart or the brain.

MRI and PET medical imaging routinely exploreinternal regions of the human body, creating 3-D renditionsof the anatomy of interest. As such, MR and PET scannersare advantageous for this desired characteristic of in-depthdetection.

A major impediment to the current use of bioluminescentimaging in clinical applications is that luciferase and otherBLI markers can only be tracked to depths of 2–3 cm fromthe skin [79]. While this does not pose a problem inlaboratory experiments with small animals, such superficialimaging will not be practical with human patients. Imagingof deeper regions, such as the human brain or heart, isimpractical with current BLI markers. There is a possibilityfor deep tissue visualization with FLI and “stealth” nearinfra-red fluorescent probes (See details in the latersections).

Quantum dots may have great potential for tracking stemcells in vivo at greater depths than conventional fluorescentmarkers because of its increased intensity of light emission.Ballou et al. have reported that fluorophore signals can bedetected deep from within the liver and bone marrow of amouse using whole-body imaging [80]. Additionally,Larson et al. have been able to image quantum dot emissionsignals in capillaries hundreds of millimeters deep in theblood vessels from the skin of mice [81]. However,experiments tracking stem cells in vivo in larger animalsmust be conducted to determine the extent to whichquantum dot fluorescence can be tracked.

Specificity for Stem Cell Imaging

Specificity, the ability of an imaging method to accuratelydiscriminate between labeled stem cell and surroundingnon-specific signal noise, is a fundamental requirement forstem cell tracking.

MRI has poor specificity because signal loss or gaindoes not solely correlate with superparamagnetic iron oxide(SPIO) MRI imaging contrast agent from a labeled cell.With a negative contrast, it may become difficult todistinguish between a signal loss from a labeled cell orthe innately low signal in tissue [51]. Additionally, Mani etal. describes how cardiac imaging is difficult with MRIbecause signal loss may not only be attributed to magnet-ically labeled stem cells, but to motion, partial volumeaveraging effects, and necrosis [29]. MRI is also notsensitive enough to discriminate between a labeled stemcell signal and products of ferritin deposition [29]. Positivecontrast, on the other hand, is not necessarily better thannegative contrast [82]. In neural imaging, hemorrhages,extracellular SPIO or other iron-enriched brain regions canbe mistaken for a positive signal.

Bioluminescent markers are advantageous for produc-ing stem cell-specific images. There is an inherently low


background luminescence from tissue [35], makingbioluminescent markers easy to identify in vivo. Incomparison, negative or positive contrast signals fromMRI may be attributed to activity in the surroundingtissue, which makes it difficult to distinguish stem cellpresence.

Quantum dots, like bioluminescent markers, have usefulqualities for producing stem cell-specific images. Theirfluorescent signals have very narrow emission spectra, sothe wavelength is specific to a particular-sized quantum dot.In some studies, there was no background autofluorescenceto interfere with the quantum dots signal, and when therewas autofluorescence, programs can be used to eliminate itfrom the image. Furthermore, their fluorescence is muchstronger than organic fluorophores [6].

Quantification Capability

Ideally, a linear relationship should exist between thenumber of stem cells bearing a label and the generatedintensity or concentration of the emitted signal. Thisrelationship should follow a predictable course over thelifespan of the label in order to derive absolute values.Quantification is important to differentiate where themajority of stem cells are located in comparison to whereminor populations of stem cells are found within anorganism. Daldrup-Link et al. found no significant correla-tion between the number of labeled cells and MR imagingdata between 1×107 cells and 3×108 cells [78]. Theyproved that quantification with MR imaging is limited forstem cell tracking and a refinement is needed.

Quantification with bioluminescence imaging has beenperformed. Togel et al. has found a linear relationshipbetween photon emission by the cell markers and thenumber of stem cells [83]. PET is the only other trackingmethod of the four in which there is a correlation betweensignal intensity and population of labeled stem cells.

A Novel Practical Device Complementary to Establisha GPS-like Tracking System for TrackingStem Cells In Vivo

An estimated 18,820 new cases of brain and other CNScancers will be diagnosed in the United States each year,and more than 12,000 will die from the disease (data fromthe US National Cancer Institute). Brain tumors are now theleading cause of cancer-related deaths in children under age15 [84]. Malignant brain tumors represent a major pediatricand adult health care threat with long-term prognosisremaining dismal [85]. New approaches to the treatmentof brain tumors are desperately needed. Public enthusiasmhas fueled interest in stem cell research as a promisingapproach such that stem cell therapy has been regarded as

an eternal wonder medicine of “cure-it-all,” and novelapplications for stem cell therapy have been proposed forthe treatment of malignant gliomas as they “surround” theinvading tumor border while “chasing down” infiltratingtumor cells [86, 87].

One major obstacle is to better understand how stemcells access the brain tumor and move within it duringtumor initiation and progression. Aboody and colleaguesvisualized and quantitatively analyzed the spatial distribu-tion of tumor-tropic NSCs in a mouse model of orthotopicglioma in order to predict the therapeutic efficacy of arepresentative NSC-based glioma therapy [88]. However,this single-color-dye-loaded and confocal microscopy-based method is limited. Multiphoton excitation fluorescentmicroscopy (MPM) is a laser-based technology that allowssubcellular resolution of native tissues in situ [89, 90]. Ascompared with other in vivo imaging techniques, multiphotonmicroscopy is uniquely capable of providing a window intocellular and subcellular processes in the context of the intact,functioning animal. In addition, the ability to collect multiplecolors of fluorescence from the same sample makes in vivomicroscopy uniquely capable of characterizing up to threeparameters from the same volume, supporting powerfulcorrelative analyses [91, 92].

We have developed and applied this technology previ-ously to the structural and photochemical imaging ofhuman pathological conditions [93–96]. This system hasmost recently been applied to cultured glioma cells andexperimental gliomas ex vivo [97–100]. These studiesdemonstrated that high microanatomical definition of thetumor, invasion zone, and normal adjacent brain can beobtained down to single-cell resolution in unprocessedtissue blocks. More intriguingly, they can use multiphotonexcitation and four-dimensional microscopy to generatefluorescence lifetime maps of murine brain anatomy,experimental glioma tissue, and biopsy specimens ofhuman glial tumors. In murine brain, cellular and noncel-lular elements of the normal anatomy were identified.Distinct excitation profiles and lifetimes of endogenousfluorophores were identified for specific brain regions.Intracranial grafts of human glioma cell lines in mousebrain were used to study the excitation profiles andfluorescence lifetimes of tumor cells and adjacent hostbrain [99]. These studies demonstrated that normal brainand tumor could be distinguished on the basis of fluores-cence intensity and fluorescence lifetime profiles. Humanbrain specimens and brain tumor biopsies were alsoanalyzed by multiphoton microscopy, which demonstrateddistinct excitation and lifetime profiles in glioma specimensand tumor-adjacent brain. This study demonstrates thatmultiphoton excitation of autofluorescence can distinguishtumor tissue and normal brain based on the intensity andlifetime of fluorescence [99]. Wilson et al. (2009) used


MPM to visualize the behavior of infiltrating CD8+ T cellsin cerebral cortex by using reticular fiber conduits to movewithin the brain [101]. We and others have worked ontechnical developments in this technology that may providea means for in situ tissue analysis, which might be used todetect a residual tumor at the resection edge.

We have devised a comprehensive GPS-like trackingsystem for stem cell delivery and tracking in vivo withcombination of genetically labeled stem cells and in vivoimaging technology (Figs. 5, 6, 7, 8, 9). These geneticallylabeled stem cells are tagged with constitutive or develop-mentally regulated viable fluorescent markers for tracking atransplanted stem cell, in vitro or in vivo by the innovativeGPS-like tracking system [93, 94, 102–104]. In vivotracking relies upon nanotechnology coupled with anoptical fiber detector camera probe surgically implanted,providing continuous circumferential visualization within aradius of 500 nm.

The miniaturized probes are the most critical componentsfor the in vivo multiphoton microscope (MPM) imagingsystem (Figs. 7, 8, and 9). In the past few years, we havedeveloped a number of miniaturized probes for OCT andMPM imaging [93, 94, 104–106]. Figures 7, 8 and 9 showMEMS-based and needle-based miniature probes. Thefiber-based MPM system offers three advantages over thecurrent prototype system. The fiber-based femotosecondpulse (FBFP) source is much more compact and robust thanthe Ti:sapphire laser, which is ideal for in vivo imaging[107, 108]. In addition to the MPM system and probedevelopment, the fiber based MPM imaging system can becombined with the optical coherence tomography (OCT)system to enable simultaneous monitoring and evaluatingof the therapeutic effect of stem cell treatment [107, 109].

Imaging and tracking stem cell distribution in in vitro, exvivo and in vivo systems is essential for defining basic stem

cell behavior and for optimizing stem cell therapy. MPM,which includes multiphoton excited fluorescence andharmonic generation, has been widely used for biologicalimaging with molecular contrast and sensitivity [110–112].Several groups have shown in ex vivo studies that MPMcan be used to identify the tumor, invasive zone, andnormal adjacent brain with a single-cell resolution [99]. Ithas been found that normal and brain tumors can bedistinguished by either fluorescent intensity or lifetimesprofile from endogenous fluorophores. The intrinsic contrastof MPM imaging technology, combined with fluorescent-tagged stem cells, would allow imaging, identifying,tracking and evaluating interactions of tumor cells, andtransplanted stem cells.

To study the in vivo interaction of stem cells with braintumors, two fluorescent dyes can be simultaneously infusedintravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) for labelingthe stem cells and one of low molecular weight (sulforhod-

Fig. 5 Schematic diagram of the fiber-based MPM system. The double-clad PCF has a core with a diameter of 16 μm and an inner cladding witha diameter of 163 μm. Because of the large inner cladding andmultimodepropagation with a large numeric aperture, a much higher collection

efficiency can be achieved in comparison to the conventional single modefiber. In addition, the use of a large core double clad PCF minimizes thepulse broadening due to the nonlinear effect. The scanner and A/D boardare controlled and synchronized by a PC

Pump (976 nm)

Double cladding Yb-doped fiber

Birefringentquartz plat

wave plates

Output

CollimatorCollimator Isolator

Pump/signal combiner

Fig. 6 Schematic of the femtosecond fiber laser oscillator. The pumplaser has a central wavelength of 976 nm and maximum power of20 W. The pump power is delivered to the cladding of the DC gainfiber through a home-built pump-signal combiner. The Yb-dopeddouble cladding gain fiber has a core diameter of 10 mm and innercladding of 105 mm [116]


amine B, 559 Da, red fluorescence) for labeling the tumorcells. A two-photon microscope can be directed through acranial window, and obtain separate images of the two dyesin the cortex. The gains of the two channels may beadjusted so that the signals coming from within the vesselsare equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gives images inwhich the vasculature is invisible and the sulforhodamine Bin the parenchyma will be imaged with high resolution asdescribed previously [113].

However, current commercial MPM has limited capabil-ity for in vivo imaging because of the lack of a compact andflexible miniature probe. We have developed a fiber basedMPM system that is compact and robust for in situ imagingand tracking stem cell migration, differentiation, andsurvival in animal and human subject. There are a number

of obstacles in translating the MPM technology for clinicalapplications, including the compact and robustness of thefemtosecond source, efficient excitation light delivery andsignal collection, and probe miniaturization. Our fiberbased MPM addresses these limitations: (1) the develop-ment of a fiber-based femtosecond-pulse (FBFP) sources asa stable and compact light source, (2) design anddevelopment of a MPM system that integrates a FBFPsource, a double clad fiber, and a MEMS probe, and (3)design and development of a miniature scanning probes.

Fiber-Based MPM System Design

The schematic diagram for the endoscopic MPM is shownin Fig. 5. A short pulse beam generated from the FBFPsource first goes through a grating-based pulse stretcher

Electrical WireMEMS Scanner

PCF

Lens

Outer Sheatha

b c

Fig. 7 a Schematic of theMEMS-based miniature probe;b picture of the MEMS scanner;c picture of packaged probe

Fig. 8 a Schematic of theneedle-based miniature probe;b picture of the probe; c pictureof the tip of the probe


which can negatively pre-chirp the femtosecond pulses inthe pre-chirp unit to compensate for pulse broadeningcaused by positive dispersion in the double clad PCF core.The pre-chirped pulse passes through a dichroic mirror andis coupled into the double clad PCF. The short pulse lightcoupled out from the double clad PCF may be focused onthe tissue through a miniature probe. The fluorescent orharmonic generation signal can be collected through thelens in the miniature probe and coupled into both the coreand inner clad modes of the fiber. The collected MPMsignal propagates through the double clad fiber and passesthrough the dichroic mirror that removes the excitationbeam. The MPM signal detected by the photomultiply(PMT) is amplified and digitized.

Design and Development of a Fiber-based Femtosecond-pulse (FBFP) Source for MPM

One of the key components for MPM is the short pulselaser. Efficient generation of a nonlinear signal requiresfocus of the excitation laser in both space and time.Therefore, a short pulse source is essential for efficientMPM signals. Currently, most MPM systems use a Ti:sapphire laser. The Ti:sapphire laser offers short pulsewidth, high power, high repetition rate and wavelengthtunability from 710 nm to 1,000 nm. However, it also hasthe disadvantages of being bulky, expensive and notmaintenance-free. Recent advances in the development offiber lasers have made an inexpensive, portable andmaintenance-free laser source available.

We have collaborated with Dr. Wise’s group at CornellUniversity in developing a fiber laser for in vivo opticalimaging [114, 115]. We have recently developed a compactfiber laser that meets the requirement of MPM imaging.The schematic of the fiber laser is shown in Fig. 6 [116].The pump laser has a central wavelength of 976 nm and a

maximum power of 20 W. The pump power is delivered tothe cladding of the double cladding gain fiber through ahome-built pump-signal combiner. The Yb-doped doublecladding gain fiber (Liekki DC1200-10/125) has a corediameter of 10 mm and inner cladding of 105 mm. Thefiber laser can generate a 120 fs pulse with an averagepower larger than 2 W. The high output power of the fspulse of the fiber laser is achieved by the following method.

Fig. 9 a Schematic of theendoscopic MEMS probe;b picture of the probe

Fig. 10 MPM images of rat tail tendon (a) and fish scale (b) usingrotational probes. The image is unfolded with the horizontal directionscanned by a translation stage, and the vertical direction scanned bythe rotational probe. The images are expressed in 8-bit pseudocolor,and the scale bar is 25 µm [105]


First, we used a larger core (10 µm) single mode fiberinstead of a 6 µm fiber. The 10 µm diameter core fibermatches the core diameter of the doped double clad gainfiber. Hence, the splice loss was reduced. Second, a morepowerful pump laser was used. The pump laser used couldprovide a maximum power of 20 W. The desired outputpulse had a pulse width of 120 fs and a wavelengthbandwidth of 40 nm.

Design and Development of MPM Scanning Probes

The miniaturized probes are the most critical componentsfor an in vivo MPM imaging system. A number ofminiature scanner probes can be developed to meet theneeds of in vivo MPM imaging for tracing stem celltherapy. In the past few years, Dr. Chen’s group hasdeveloped a number of miniaturized probes for OCT andMPM imaging [93, 94, 104–106, 117]. MEMS-based andneedle-based miniature probes developed in Dr. Chen’slaboratory are shown in Figs. 7 and 8.

In addition to the 2-D linear scanner, deep tissue may bebetter imaged through a cranial window with a cylinderedgeometry. A rotational probe may be optimal to image arelative large area through the cranial window. We havedeveloped a rotational probe based on a miniaturizedrotational MEMS motor (Fig. 9) [105, 118, 119]. TheMEMS micromotor is mounted in a backward configura-tion toward the proximal direction, and a 45° prism is usedto deflect optical light toward the sample. We have builtand tested a prototype device with a motor with a diameterof 2.2 mm. A GRIN lens with a diameter of 1 mm was usedsince it was readily available with 3° cleaved angles.Imaging was performed by pulling the whole probe with alinear translational stage while the MEMS motor rotated tocreate a 3-D helix scan. Figure 10 shows the preliminaryresults of the MPM signal acquired through such aminiature rotational probe [105].

Signal and Imaging Processing

In addition to the intensity measurement, appropriatespectral filter can be included in the MPM detectionsystem so that multiple spectral channel images can beacquired simultaneously. Furthermore, time-correlatedsing-photon counting can be developed to enable flourescencelifetime imaging.

To study the in vivo interaction of stem cells with braintumors, two fluorescent dyes are simultaneously infusedintravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) for labeling thestem cells and one of low molecular weight (sulforhodamineB, 559 Da, red fluorescence) for labeling the tumor cells. Atwo-photon microscope can be directed through a cranial

window, and obtain separate images of the two dyes in thecortex. The gains of the two channels are adjusted so thatthe signals coming from within the vessels are equal.Subtraction of the image of the fluorescein-dextran fromthat of the sulforhodamine B gives images in which thevasculature is invisible and the sulforhodamine B in theparenchyma can be imaged with high resolution asdescribed previously [113].

Conclusion

(1) We have defined the problems of tracking the migrationand fate of transplanted stem cells. (2) We have alsodefined the ideal qualities of a stem cell tracking system forclinical use. (3) None of the four stem cell trackingmethods fulfill all of the requirements needed for clinicalapplication at this time. Bioluminescence, despite its highsensitivity and specificity, proves problematic in animalsbigger than mice. Studies with quantum dots providebenefits. This labeling system is in its early develop-mental stage, and further development is necessary ifquantum dots were to be used in clinical function. Tocreate the optimal in vivo imaging modality, multimodalmarkers will provide the benefits of each differentlabeling technique. MRI dual-imaging might prove tobe the most favorable combination because MRI iswidely used in hospitals. State of the art confocal andmultiphoton microscopy (MPM) and optical coherencetomography (OCT) may be integrated to complementMRI in order to create a biological global positioningsystem to track stem cell migration, differentiation, andsurvival in vivo.

Acknowledgments Support for this work came from the CHOCFoundation for Children, CHOC Neuroscience Institute, CHOC HeartInstitute, the Austin Ford Tribute Fund and the WM Keck Foundation(to S. C. L.), and NIH EB-00293 and CA 91717 (to Z. C.). We thankLong Vu, Vic Keschrumrus, Julyana Acevedo (University of SouthernCalifornia), Shi (Sherrie) Yu (Rice University), and Tiffany Dao(Stanford University) for their helpful discussions. We thank MariaMinon, MD, Anthony C. Chang, MD-MPH-MBA (CHOC); SaulPuszkin, PhD (Mount Sinai School of Medicine); Michael P. Lisanti,MD-PhD (Thomas Jefferson University); Richard G. Pestell, MD-PhD(Thomas Jefferson University); Joan S. Brugge, PhD (HarvardUniversity); Robert A. Koch, PhD (California State UniversityFullerton) for their support and enthusiasm.

Conflict of Interest All the authors declared no financial conflict ofinterest.

Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which per-mits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.


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