Stable and Nondisruptive In Vitro=In Vivo Labelingof Mesenchymal Stem Cells by Internalizing Quantum Dots

Yoshimi Ohyabu,1,2* Zeenia Kaul,1,3* Tomokazu Yoshioka,1,4* Kazuki Inoue,1,5

Shinsuke Sakai,4 Hajime Mishima,4 Toshimasa Uemura,1 Sunil C. Kaul,1 and Renu Wadhwa1

Abstract

Progress in stem cell research has prioritized the refinement of cell-labeling techniques for in vitro and in vivobasic and therapeutic studies. Although quantum dots, because of their optical properties, are emerging asfavorable nanoparticles for bioimaging, substantial refinements or modifications that would improve theirbiocompatibility are still required. We report here that internalizing quantum dots (i-QDs) generated by theirconjugation with an internalizing antibody against a heat shock protein-70 family stress chaperone, mortalin,offered an efficient, genetically noninvasive, nontoxic, and functionally inert way to label mesenchymal stemcells (MSCs). The i-QD-labeled MSCs underwent normal adipocyte, osteocyte, and chondrocyte differentiationin vitro and in vivo, suggesting the potential application of i-QDs in in vivo diagnostics, regenerative andtherapeutic medicine.

Introduction

Stem cell research has progressed substantially. It hastransformed the conventional view of multipotent mes-

enchymal stem cells (MSCs) differentiating into a variety ofmesodermal lineage cell types (myocytes, adipocytes, chon-drocytes, and osteocytes) and tissues (muscle, fat, cartilage,and bone, respectively) to include their reprogramming andtransdifferentiation into other germ layers and tissues withmore specialized functions (referred to as stem cell plasticity).Most recently, induced pluripotent stem cells have been arti-ficially derived from nonpluripotent adult somatic cells bytransducing them with specific genes (Okita et al., 2007; Yuet al., 2007; Nakagawa et al., 2008). MSCs are being testedwidely in disease modeling, drug discovery, tissue engineer-ing, therapeutics, and clinical trials (Ohgushi et al., 2004;Miyahara et al., 2006; Scott and Baker, 2007; Swieszkowskiet al., 2007; Kagiwada et al., 2008).

A major challenge for both basic and clinical research is thenecessity to study these cells under conditions that model thein vivo environment. This can be facilitated by cell-labelingtechnologies that can track the fate of cells in vivo and dis-

tinguish implanted cells from the host tissues without affect-ing their normal genetic constitution and functions. Con-tinuous observation of biological processes instead of thediscrete time points holds great promise to promote under-standing of tissue functions, physiology, regeneration, andtherapeutic responses. Besides, cell labeling in vitro and in vivocan offer additional merits in gene therapeutics in which genedelivery and therapy can be monitored more closely and ac-curately. In contrast to the rapidly developing gene therapyand stem cell technologies, methods for stable cell labeling arestill limited, precluding long-term functional studies. Thesemethods include exogenously introduced radioactive probes(Stodilka et al., 2006), ultrasmall iron particles (Wilhelm andGazeau, 2008), organic fluorescent dyes (Templin et al., 2006),semiconductor nanoparticles (quantum dots) (Gao et al., 2002;Muller-Borer et al., 2007), and stably expressed fluorescentproteins (Stadtfeld et al., 2005). However, evidence regardingtheir genetic noninvasiveness and functional inertness is stilllimited. Quantum dots (QDs) have emerged as high-qualityfluorescent alternatives to conventional organic dyes. Severalmodified QDs have been generated that, in addition totheir unique spectral, physical and chemical properties, are

1National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan.2Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, Tokyo 152-8550, Japan.3Children’s Medical Research Institute, University of Sydney, Westmead, New South Wales 2145, Australia.4Department of Orthopedic Surgery, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-

8575, Japan.5Agrobiological Resource Sciences and Technology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba,

Ibaraki 305-8575, Japan.*Y.O., Z.K., and T.Y. contributed equally to this work.

HUMAN GENE THERAPY 20:217–224 (March 2009)ª Mary Ann Liebert, Inc.DOI: 10.1089=hum.2008.100

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designed to have low toxicity and high hydrophilicity (Wuet al., 2003; Alivisatos et al., 2005; Arya et al., 2005; Gao et al.,2005; Michalet et al., 2005; Shah et al., 2006; Hoshino et al., 2007;Jaiswal and Simon, 2007). Despite progress, there is still muchneed to manipulate QDs to improve their inertness so thatthey can be used as reliable imaging probes in vivo. We ini-tially generated internalizing quantum dots (i-QDs) by con-jugating them to a unique internalizing antibody against aheat shock protein-70 (hsp70) family stress chaperone, mor-talin, that is upregulated and expressed on the surface of di-viding cells. We showed that i-QDs can be used for long-termimaging of cancer cells in culture (Kaul et al., 2007). Here wereport the use of i-QDs for stable, genetically noninvasive, andfunctionally inert labeling of MSCs for long-term in vitro andin vivo tracking.

Materials and Methods

Mesenchymal cells and culture

Experiments were performed in accordance with theguidelines of the Japanese government for the care and use oflaboratory animals. Rat (Fischer 344, 7 weeks old), rabbit (10-day-old Japanese White), and monkey (a newborn baby;embryonic age, 150–163 days) mesenchymal cells were de-rived from the bone marrow of their femurs (Maniatopouloset al., 1988). The epiphyses were removed and the marrowcavity was flushed with standard medium consisting of Dul-becco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St.Louis, MO) or minimum essential medium (GIBCO MEM-a;Invitrogen, Carlsbad, CA) containing 10–15% fetal bovine se-rum (FBS; Sigma-Aldrich) and antibiotic–antimycotic (GIBCO;Invitrogen), using a syringe. The cell suspension was platedinto a 75-cm2 culture flask (BD Falcon, San Jose, CA) with15 ml of standard medium and cultured at 378C in a humidi-fied atmosphere containing 95% air and 5% CO2 until nearlyconfluent. Medium was replaced 2 days after plating andnonadherent cells were removed. Medium was changed twiceper week. Human mesenchymal cells (passage 3) were pur-chased from Lonza Walkersville (Walkersville, MD) and werecultured in standard medium. All procedures were approvedby the AIST ethics review committee.

Labeling of mesenchymal cells

i-QDs were prepared by conjugation of Qdot 655 with aninternalizing anti-mortalin monoclonal antibody, using anantibody conjugation kit (Q22021MP; Invitrogen) as de-scribed (Kaul et al., 2007). To ensure the conjugation, humanosteosarcoma cells were stained with QD–antibody conjugateas described earlier (Kaul et al., 2007). For generating i-QD-labeled mesenchymal cells, QD–antibody conjugates (*0.2–0.4 mg=ml) were added to cell culture medium for 24 hr.QD-labeled cells were subcultured for differentiation and cells(2.0�104) were seeded on 15-mm coverslips for analysis bymicroscopy. After 24 hr, when cells had attached to the surfaceand spread well, they were washed with cold phosphate-buffered saline (PBS) and fixed with a prechilled methanol–acetone (1:1, v=v) mixture for 5 min on ice. Fixed cells werewashed with PBS followed by mounting on glass slides withFluoromount (Difco=BD Diagnostic Systems, Sparks, MD) andwere examined with a microscope (Axiovert 200 M; Carl Zeiss,

Thornwood, NY) attached to an AxioCam MRm monochromecharge-coupled device camera (Carl Zeiss) and processed withAxioVision version 4.4 (Carl Zeiss) equipped with Qdot filterset XF 305-1 (excitation filter [425DF45], dichroic [475DCLP],and emission filter [655DF20]; Omega Optical, Brattleboro,VT). Optical sectioning was performed under�40 and�63 oilimmersion objectives. Fourteen and 26 sequential optical sec-tions were cut. Z-Stacks were acquired under the�63 objectiveand analyzed to perform a cut-view analysis.

Differentiation of i-QD-labeled MSCs to osteoblasts,chondrocytes, and adipocytes

Osteogenic culture. i-MSCs were subcultured by trypsi-nization, and 3.1�103 cells=cm2 of tissue culture surface areawere plated in standard medium. The cells were allowed toadhere to the surface for 24 hr at 378C, in a humidified at-mosphere containing 5% CO2. Standard medium was re-placed with osteogenesis induction medium consisting ofDMEM containing 10% FBS, antibiotics, and osteogenic sup-plement (10 mM b-glycerophosphate [Sigma-Aldrich], l-ascorbic acid phosphate [50 mg=ml; Wako, Osaka, Japan], and10 nM dexamethasone [Sigma-Aldrich]). Differentiating cellswere refed with fresh osteogenesis induction medium every3–4 days for the next 2–3 weeks. Control cells were refed withstandard medium at the same time. Osteogenesis was exam-ined by alizarin red staining on day 21. Cells were fixed with10% formalin–PBS for 15 min, stained with 1% alizarin red(pH 4.0) for 2 min, washed with deionized water, and thenexamined under the microscope.

Chondrogenic culture. The chondrogenesis of i-MSCswas achieved by two representative culture systems.

Pellet culture: Cells were subcultured by trypsinization,and resuspended in chondrogenesis induction mediumconsisting of DMEM containing 10% FBS, antibiotics, andchondrogenic supplement (ascorbic acid [50 mg=ml; Wako],l-proline [40 mg=ml], ITS culture supplement [BD Biosci-ences], 100 nM dexamethasone, and transforming growthfactor-b [TGF-b, 10 ng=ml; Sigma-Aldrich]). One milliliter ofthe cell suspension (2.5�105 cells) was seeded into a 15-mlconical tube (BD Biosciences), centrifuged at 150�g for 5 min,and subjected to static culture in the same medium at 378C.The medium was replaced with fresh chondrogenesis induc-tion medium every 3–4 days.

RWV reactor culture: Cells were cultured in bioreactormedium consisting of DMEM containing 10% FBS, ascorbicacid (50 mg=ml; Wako), l-proline (40 mg=ml), ITS culturesupplement (BD Biosciences), 100 nM dexamethasone (Sigma-Aldrich), TGF-b (10 ng=ml; Sigma-Aldrich), and antibiotic–antimycotic (GIBCO; Invitrogen) followed by seeding(1�107 cells) in the 12 discoidal vessels of a rotating wallvessel (RWV) reactor (RCCS-4D system with 10-ml disposablevessels; Synthecon, Houston, TX) equipped with a CO2 in-cubator, and rotatory culture was performed for 4 weeks withregular changing of medium every 3 days as described earlier(Ohyabu et al., 2006).

Histochemical analysis. On day 28 of differentiation, cellaggregates were fixed overnight with 4% paraformaldehydein PBS, and stained with toluidine blue and safranin O

218 OHYABU ET AL.

(Waldeck, Division Chroma, Muenster, Germany). Histo-chemical staining was observed with an optical microscope(IX70; Olympus America, Melville, NY) equipped with acharge-coupled device (CCD) camera (MicroPublisher 5.0;Nippon Roper, Tokyo, Japan).

Glycosaminoglycan content. On day 28 of differentiation,cell aggregates were digested with proteinase K overnight at608C. Glycosaminoglycan (GAG) content was measured bythe dimethylmethylene blue (DMMB) method, using a Bly-scan glycosaminoglycan assay kit (Biocolor, Carrickfergus,

FIG. 1. (A) Labeling of rat (i-RaMC), rabbit (i-RbMC), monkey (i-MoMC), and human (i-HuMC) MSCs by internalizingmortalin–antibody-conjugated quantum dots (i-QDs). Highly efficient internalization was observed in each case. Almost allthe cells in culture showed uptake of i-QDs. (B) Phase-contrast, fluorescence, and overlay images are shown. (C and D)Uptake of i-QDs within cells was confirmed by serial optical sectioning: (C) Z-stacking and (D) cut view mode.

i-QD LABELING OF MSCs 219

UK). DNA quantification was carried out with a fluorometricassay (PicoGreen dsDNA quantitation kit; Invitrogen) in ac-cordance with the manufacturer’s protocol. Activity was ex-pressed as micrograms of GAG per microgram of DNA.

Adipogenic culture. i-MSCs were subcultured by trypsi-nization, and 2.1�104 cells=cm2 of surface area were plated instandard medium and incubated at 378C in a humidified at-mosphere of 5% CO2 with regular changing of medium every2–3 days. Culture at 100% confluence was incubated withadipogenesis induction medium, consisting of DMEM con-taining 10% FBS, antibiotics, and adipogenic supplement(0.5 mM 3-isobutyl-1-methylxanthine [IBMX; Wako], 10 mMinsulin [Sigma-Aldrich], 1 mM dexamethasone, and 200mM

indomethacin [Sigma-Aldrich]). Adipogenesis medium waschanged every 3–4 days for 2 weeks. Adipogenic differentia-tion was tested as described below. Mesenchymal stem cellswithout i-QDs were used as a control and were treated inparallel.

Oil red O staining. On day 7 of differentiation, cells werefixed by adding 10% formalin–PBS directly to the medium(1:5) for 20 min followed by changing the buffer–medium tobuffer only. Cells were incubated with fixation buffer for 1 hr.Fixed cells were stained with oil red O for 15 min, and thengently washed with deionized water. After observing thestained cells, extraction solution from a lipid assay kit(Primary Cell, Japan) was added to the wells. The extent of

FIG. 2. (A) Differentiation of control MSCs (without QD; c-MSCs) and i-QD-labeled rat MSCs (i-MSCs) to adipocytes andosteoblasts. Differentiated i-MSCs were confirmed for the presence of i-QD (panel d, adipocytes; panel f, osteoblasts). (B)Differentiated adipocytes and osteoblasts were stained with oil red O (panel a) and alizarin red (panel b), respectively. (C andD) Differentiation of control and i-QD-treated cells to adipocytes was quantitated by oil red O staining (C). c-MSCs andi-MSCs showed no significant differences in the amount of oil red O staining (D).

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adipogenic differentiation was calculated by measuring ab-sorbance of the extract at 540 nm, using a microplate reader(VersaMax; Molecular Devices=MDS Analytical Technolo-gies, Sunnyvale, CA).

In vivo differentiation

Cultured bone transplantation rat model. b-Tricalciumphosphate (b-TCP) cubic blocks (2�2�2 mm; average porediameter, 200 mm) were kindly provided by Olympus Optical(Tokyo, Japan). After 3 weeks of primary culture, controlMSCs (c-MSCs) and i-QD-labeled MSCs (i-MSCs) were tryp-sinized and harvested by low-speed centrifugation, andresuspended in fresh medium at a concentration of 2�106

cells=ml. b-TCP blocks were soaked in the cell suspension andincubated for 2 hr in a CO2 incubator. The blocks weretransferred to 24-well plates (a single block per well) andcultured in osteogenic medium for 24 hr. As in the in vivo bonedefect model, the lateral side of the distal end of rat femoralshafts was exposed and incised surgically. Bilateral bone de-fects were created by making a hole (2 mm in diameter and3 mm deep) on the coronal face at the distal end of each femur.b-TCP–MSC composites (2 mm3) were implanted into thehole, followed by suturing of the wounds. To make stringentcontrols, we implanted control and i-QD-labeled MSC com-posites in the same rat, using the right side for i-MSCs and theleft side for c-MSCs. Differentiation of the implanted MSCswas examined on day 26 and at 8 weeks by histologic analysisas described below.

Histologic analysis. Cells=b-TCP composites and bonetissues were collected on day 26 and 8 weeks postimplanta-tion. Unfixed tissues were immediately embedded in 4%carboxymethyl cellulose, rapidly frozen in liquid nitrogen,and stored at �708C until cryostat sectioning (Leica CM3050S; Leica Microsystems, Wetzlar, Germany). Serial sectionswere stained with hematoxylin and eosin (H&E), and thecartilage marker safranin O, and were observed with anOlympus IX70 microscope equipped with a CCD camera.Digital images were analyzed with MetaMorph software(Molecular Devices=MDS Analytical Technologies). Thepresence of QDs in differentiated tissues was examined with amicroscope (Carl Zeiss) equipped with a specific Qdot filter.Whole section imaging to localize quantum dot-labeled cellswas done with a BIOREVO imaging system (BZ-9000; Key-ence, Tokyo, Japan).

Cultured cartilage aggregate transplantation rabbitmodel. Ten-week-old Japanese White rabbits (Tokyo Ex-perimental Animals, Tokyo, Japan) were used in this study.All operations were performed under sterile conditions andwere approved by the university committee. The left knee wasopened via a medial parapatellar approach and the patellawas dislocated laterally to expose the patellar groove of thefemur. A hand drill, with depth-controlled bits, was used tocreate cylindrical defects (5�5 mm in area and 4 mm in depth),along the midline of the patellar groove, that did not regen-erate spontaneously. Three-dimensional cartilage aggregates

FIG. 3. Differentiation of i-QD-labeled rabbit MSCs (i-RbMSC) to chondrocytes and cartilage tissues. (A) i-QDs weredetected both in chondrocytes and cartilage. The efficiency of chondrogenesis among c-MSCs and i-MSCs was examined by(B) safranin O and toluidine blue staining and on the basis of (C) glycosaminoglycan (GAG) content.

i-QD LABELING OF MSCs 221

were implanted in flap-free transfer mode. The animals werethen caged and allowed to move freely without any splintingafter recovery, before they were killed on day 26 and 8 weekspostoperation. On sacrifice, the upper half of the reparativetissue was cut along the coronal plane from the distal femur.Differentiation of tissue was examined by histologic analysisas described previously.

Results and Discussion

As mortalin is a highly conserved protein, we tested whe-ther mortalin antibody-based i-QDs might be used in multiple

species. i-QDs were prepared by conjugation of Qdot 655 withan internalizing anti-mortalin monoclonal antibody, using anantibody conjugation kit (Q22021MP; Invitrogen) as de-scribed (Kaul et al., 2007; Shiota et al., 2007). For generating i-QD-labeled mesenchymal cells, Qdot–antibody conjugates(0.2–0.4 mg=ml) were added to cell culture medium for 24 hr.QD-labeled cells were subcultured for three kinds of differ-entiation, that is, (1) adipogenic differentiation, (2) osteogenicdifferentiation, and (3) chondrogenic differentiation, by tworepresentative culture systems: pellet culture and RWV reac-tor culture as described in Materials and Methods. As shownin Fig. 1A, MSCs from rat, rabbit, monkey, and human were

FIG. 4. In vivo differentiation of i-QD-labeled (A) rat (cultured bone transplantation model) and (B) rabbit (culturedcartilage aggregate transplantation model). In both cases, normal bone structures were formed as seen by histochemistry (A,panel a; and B, panels g and j) and substantial QD fluorescence was retained even after differentiation (A, panels b and c; andB, panels d, h, and k). Low- and high-magnification images of rat osteoblasts in b-tricalcium phosphate (b-TCP) blocks (A,panel b) and bone (A, panel c) formed from transplanted b-TCP block-supported osteoblasts, and rabbit endochondral tissueformed from implanted cartilage at 26 days (B, panels g, h, and i) and 8 weeks (B, panels j, k, and l), are shown. A strong QDsignal (B, panels d, h, i, k, and l) was observed in regenerated bone sections as confirmed by safranin O staining (B, panels gand j) 26 days and 8 weeks after transplantation.

222 OHYABU ET AL.

all labeled efficiently with i-QDs. In each case, we observedthat nearly 100% of cells took up i-QDs that were thendetected on serial sectioning of cells (Fig. 1B–D). We nextexamined whether i-QD-labeled MSCs (i-MSCs) undergo theexpected level of in vitro and in vivo differentiation. MSCsuntreated with i-QD were used as a control (c-MSCs) in par-allel to i-MSCs that were confirmed to possess i-QDs (Fig. 2A).The cells were subjected to osteocyte and adipocyte differ-entiation regimens and were stained with respective markers;oil red O for adipocytes and alizarin red for osteoblasts (Fig.2B). We found that rat i-MSCs underwent adipogenesis andosteogenesis with the same efficiency and time course asc-MSCs (Fig. 2B). There were no differences in alizarin redstaining intensities at 1 or 2 weeks of differentiation (Fig. 2Band data not shown). Similarly, oil red O staining, an estab-lished marker of adipocytes, revealed that the i-MSCs werenot affected in their adipogenic potential (Fig. 2C and D).Similar results were obtained for monkey and human MSCs(see Supplementary Fig. 1 at www.liebertonline.com=hum;and data not shown). We next induced differentiation ofrabbit i-MSCs and found no differences in safranin O andtoluidine blue staining, or in GAG content, between i-MSCsand c-MSCs (Fig. 3). Of note, in all cases, differentiated cellsand tissues retained strong QD labeling.

We next used cultured bone transplantation rat and rabbitmodels to examine the effect of i-QDs on the differentiationpotential of MSCs in vivo. In the rat model, b-tricalciumphosphate (b-TCP) cubic blocks (2�2�2 mm; average porediameter, 200 mm) were used to culture c-MSCs and i-MSCsthat were transferred to osteogenic medium. Bilateral bonedefects were created by making a hole (2 mm in diameter and3 mm deep) on the coronal face at the distal end of each femur.b-TCP–MSC composites (2 mm3) were implanted into the holefollowed by suturing of the wounds. Differentiation of theimplanted MSCs was examined on day 26 and 8 weekspostoperation by histologic analysis. In the rabbit model, theleft knee patella was dislocated laterally to expose the patellargroove of the femur. Three-dimensional cartilage aggregatesthat were formed in vitro were implanted in flap-free transfermode. The animals were then caged and allowed to movefreely without any splinting after recovery, before they werekilled on day 26 and 8 weeks postoperation and examined byhistologic analysis. In both rat and rabbit models (Fig. 4), wefound that c-MSCs and i-MSCs differentiated to form bonematrix with the same efficiency and time course. Hematoxylinand eosin staining of rat bone sections 3 weeks after implan-tation revealed extensive bone formation (Fig. 4A, panels a–c)and the presence of i-MSCs in the newly formed bone struc-ture. Again, the QD signals remained strong. The results alsosuggested that regenerated bone in the porous area of thescaffold was derived mostly from implanted donor cells andtheir derivative populations. Histochemical analysis of therabbit model (Fig. 4B) also showed that i-MSCs underwentnormal differentiation into bone structures; at 4 weeks, thedefects were filled with reparative tissue that resembled hy-aline cartilage (Fig. 4B, panels d–l). Furthermore, thick endo-chondral and osseous tissues were observed in the deeperportion of the reparative region, demonstrating clear boneformation in vivo. Importantly, the chondrogenic differentia-tion of c-MSCs and i-MSCs was equally efficient and followedthe same time course. From these data, it was concluded thati-QDs can serve as a nontoxic, genetically noninvasive, func-

tionally inert, sensitive, long-term imaging tool for in vitro andin vivo studies in multiple mammalian species.

Fluorescence-based labeling and imaging of cells has led tohighly significant findings, increasing our basic understand-ing of tissue function, development, and regeneration, and ofdisease and therapeutics. For example, neurogenesis in theadult brain was demonstrated with lentivirus-mediated greenfluorescent protein (GFP) labeling of neuronal cells (Consiglioet al., 2004) and initiated the development of gene therapystrategies that would allow neuron replacement and regen-eration in neurologic disease or brain damage. Cell labelingconstitutes an extremely important aspect of cybrid studiesand disease models that form an essential branch of genetherapy (Trimmer et al., 2003). Successful gene therapy is ex-pected to employ parallel development of in vitro and in vivocell labeling techniques that would coordinate gene therapy toits vital outcomes including cell survival, migration, differ-entiation, and cell fate decisions. From the technical point ofview, the i-QDs described herein and their future modifiedforms can potentially complement the lentiviral vectors orthymidine analogs that are used to label and study cellprogeny in development, regeneration, and gene therapysettings.

Acknowledgments

The authors thank Roger R. Reddel (CMRI, Australia) forreviewing the manuscript. This study was partly supportedby grants-in-aid for scientific research from the Japan Societyfor the Promotion of Science, Japan and from the Japan Sci-ence and Technology Agency ( JST).

Author Disclosure Statement

The authors declare that no competing financial interestsexist.

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Address reprint requests to:Dr. Renu Wadhwa or Dr. Toshimasa Uemura

National Institute of Advanced Industrial Scienceand Technology (AIST)

1-1-1 Higashi, Central 4Tsukuba, Ibaraki 305-8562, Japan

E-mail: renu-wadhwa@aist.go.jp or t.uemura@aist.go.jp

Received for publication July 13, 2008;accepted after revision November 29, 2008.

Published online: February 27, 2009.

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