human bone marrow–derived mesenchymal stem cells in the ... · stem cells have been evaluated in...

Human Bone Marrow–Derived Mesenchymal Stem Cells in the

Treatment of Gliomas

Akira Nakamizo,1,2Frank Marini,

3Toshiyuki Amano,

1,2Asadullah Khan,

1,2Matus Studeny,

3

Joy Gumin,1,2Julianne Chen,

1,2Stephen Hentschel,

1,2Giacomo Vecil,

1,2Jennifer Dembinski,

3

Michael Andreeff,3and Frederick F. Lang

1,2

1Department of Neurosurgery, 2Brain Tumor Center, and 3Department of Blood and Marrow Transplantation, University of Texas M.D.Anderson Cancer Center, Houston, Texas

Abstract

The poor survival of patients with human malignantgliomas relates partly to the inability to deliver therapeuticagents to the tumor. Because it has been suggested thatcirculating bone marrow–derived stem cells can berecruited into solid organs in response to tissue stresses,we hypothesized that human bone marrow–derived mesen-chymal stem cells (hMSC) may have a tropism for braintumors and thus could be used as delivery vehicles forglioma therapy. To test this, we isolated hMSCs from bonemarrow of normal volunteers, fluorescently labeled the cells,and injected them into the carotid artery of mice bearinghuman glioma intracranial xenografts (U87, U251, andLN229). hMSCs were seen exclusively within the braintumors regardless of whether the cells were injected intothe ipsilateral or contralateral carotid artery. In contrast,intracarotid injections of fibroblasts or U87 glioma cellsresulted in widespread distribution of delivered cellswithout tumor specificity. To assess the potential of hMSCsto track human gliomas, we injected hMSCs directly into thecerebral hemisphere opposite an established human gliomaand showed that the hMSCs were capable of migrating intothe xenograft in vivo . Likewise, in vitro Matrigel invasionassays showed that conditioned medium from gliomas, butnot from fibroblasts or astrocytes, supported the migrationof hMSCs and that platelet-derived growth factor, epidermalgrowth factor, or stromal cell–derived factor-1A, but notbasic fibroblast growth factor or vascular endothelialgrowth factor, enhanced hMSC migration. To test thepotential of hMSCs to deliver a therapeutic agent, hMSCswere engineered to release IFN-B (hMSC-IFN-B). In vitrococulture and Transwell experiments showed the efficacy ofhMSC-IFN-B against human gliomas. In vivo experimentsshowed that treatment of human U87 intracranial gliomaxenografts with hMSC-IFN-B significantly increase animalsurvival compared with controls (P < 0.05). We conclude thathMSCs can integrate into human gliomas after intravascularor local delivery, that this engraftment may be mediated bygrowth factors, and that this tropism of hMSCs for humangliomas can be exploited to therapeutic advantage. (CancerRes 2005; 65(8): 3307-18)

Introduction

There is currently no optimal treatment for glioblastomamultiforme, the most common malignant brain tumor in adults,and patients typical survive <1 year (1, 2). This poor outcomerelates at least in part to the inability to deliver therapeutic agentsto the tumor (3). Delivery problems have especially slowed thedevelopment of novel gene therapy strategies (4). Indeed, intra-tumoral injection of viral vectors has proven incapable of deliveringtherapeutic genes to many tumor cells (4), and systemic i.v. orintra-arterial administration has been limited by the neutralizingeffects of antibodies and by immune-mediated organ toxicity (5, 6).Methods for achieving widespread distribution of therapeuticagents throughout infiltrative gliomas would substantially improvebrain tumor therapy.Recent evidence suggests that stem cells are useful delivery

vehicles for brain tumor therapy. Several laboratories have shownthe potential of neural stem cells to function as delivery vehicles forbrain tumor therapy (7–10). Aboody et al. were among the first toshow that after intracranial injection neural stem cells have atropism for brain tumors that could be exploited therapeutically(7). Likewise, Ehtesham et al. have shown that locally injectedneural stem cells engineered to deliver interleukin-12 or tumornecrosis factor–related apoptosis-inducing ligand could slow thegrowth of brain tumors (9, 10). However, the clinical application ofneural stem cells will be limited undoubtedly by logistic and ethicalproblems associated with their isolation and by potentialimmunologic incompatibility due to the requirement for allogenictransplantation. Because of the significant limitations associatedwith isolating human neural stem cells, to date, only murine neuralstem cells have been evaluated in experimental settings. Theseinherent problems with neural stem cells led us to ask whetherother types of stem cells that are more readily accessible (and thusmore clinically applicable) may be used as vehicles for deliveringtherapeutic agents to brain tumors.Bone marrow is an alternative source of stem cells (11–14).

Human bone marrow–derived stem cells are well suited for clinicalapplication because they are easily obtained from patients andbecause autologous transplantation, which obviates immunologicincompatibilities, is possible (15). Of the various progenitor cellsthat exist within bone marrow, human mesenchymal stem cells(hMSC) are particularly attractive for clinical use because they areeasily isolated, can be expanded in culture, and can be geneticallymanipulated using currently available molecular techniques(14, 16–23). hMSCs are precursors that cause bone marrow stromaby differentiating into adipocytes, chondrocytes, and osteoblasts(11, 20, 24). However, MSCs have also been shown to be capable ofdifferentiating into nonmesodermal tissues, including neurons andastrocytes (25, 26).

Requests for reprints: Frederick F. Lang, Department of Neurosurgery, Universityof Texas M.D. Anderson Cancer Center, Unit 442, 1515 Holcombe Boulevard, Houston,TX 77030. Phone: 713-792-2400; Fax: 713-745-4341; E-mail: [email protected].

I2005 American Association for Cancer Research.

www.aacrjournals.org 3307 Cancer Res 2005; 65: (8). April 15, 2005

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The rationale for using bone marrow–derived stem cells fordelivering therapies to brain tumors is based on the developingcurrent concept that bone marrow is a source of circulating stemcells that are recruited from the blood into peripheral solidorgans in times of tissue stress or injury (27–31). Because themicroenvironments of solid tumors is similar to the environmentof injured/stressed tissue (32, 33), it is logical to hypothesize thatsolid tumors may provide a permissive environment for theengraftment of exogenously given hMSCs (27). In this context, wehave shown previously that systemically delivered hMSCs arecapable of integrating into human tumors grown within the lungsof nude mice (27). However, the unique features of themicroenvironment of the brain and gliomas, including theirhighly specialized vasculature and glia-derived stroma, led us toevaluate whether brain tumors would also provide a permissiveenvironment for the selective engraftment of hMSCs. Using anintracranial model of gliomas, we now show that hMSCs have atropism for human gliomas after intravascular and local deliveryand that this tropism can be exploited therapeutically byengineering hMSCs to release a soluble antiglioma factor.

Materials and Methods

Mesenchymal stem cell isolation and culture. Human MSCs wereisolated as described previously (27) from the bone marrow of normal

individuals undergoing bone marrow harvest for allogeneic bone marrow

transplantation after informed consent according to institutional guide-

lines under an approved protocol. Briefly, mononuclear cells were sepa-rated by centrifugation over a Ficoll-Hypaque gradient (Sigma Chemical

Co., St. Louis, MO) and suspended in a-MEM containing 20% fetal bovine

serum (FBS; Life Technologies, Inc., Rockville, MD), L-glutamine, andpenicillin-streptomycin mixture (Flow Laboratories, Rockville, MD) fol-

lowed by plating at an initial seeding density of 1 � 106 cells/cm2. After

3 days, the nonadherent cells were removed by washing with PBS, and

monolayers of adherent cells were cultured until they reached confluence.Cells were then trypsinized (0.25% trypsin with 0.1% EDTA), subcultured at

densities of 5,000 to 6,000 cells/cm2, and used for experiments during

passages 3 to 4.

Mouse MSCs (mMSC) were isolated from long bones of C57BL/6-TgN(ACTbEGFP)1Osb(GFPtg) mice (The Jackson Laboratory, Bar Harbor,

ME) using the methods described by Peister et al. (34). Briefly, cells from

each long bone were plated in 40 mL complete isolation medium (seeref. 34). After 24 hours, nonadherent cells were removed and adherent cells

were washed with PBS, and fresh complete isolation medium was added

every 3 to 4 days for 4 weeks. Cells were collected by trypsinization and

replated in 30 mL complete isolation medium in 175 cm2 flasks. After 1 to 2weeks, cells were trypsinized and plated in complete expansion medium

(see ref. 34). After another 1 to 2 weeks, passage 3 cells were either frozen or

expanded further by plating at 50 cells/cm2 and incubating in complete

expansion medium.Cell lines. Glioblastoma multiforme cell lines U87 and LN229 were

obtained from the American Type Culture Collection (Manassas, VA). U251

cells were obtained from W.K. Alfred Yung (M.D. Anderson Cancer Center,

Houston, TX). U87 and U251 cells were maintained in a-MEM supple-mented with 10% FBS, and LN229 cells were maintained in a-MEM

supplemented with 20% FBS and L-glutamine. Normal human astrocytes

(NHA) were obtained from Cambrex (Walkersville, MD). The fibroblast lineC29 was obtained from Juan Fueyo (M.D. Anderson Cancer Center). Cells

were incubated at 37jC in a humidified atmosphere containing 5% CO2/

95% air.

Animal subjects.Male athymic nude mice (nu/nu) were purchased fromthe Animal Production Area of the National Cancer Institute-Frederick

Cancer Research and Development Center (Frederick, MD). All animal

manipulations were done in accordance with institutional guidelines under

approved protocols.

Intracranial xenografting of human glioma cells. Monolayers of

human glioma cell lines were detached by trypsinization, washed, and

resuspended in PBS at a concentration of 1 � 105 cells in 5 AL. Cells wereinjected into the right frontal lobe of nude mice using a guide-screw system

implanted within the skull as described previously (35). To increase

uniformity of xenograft take and growth, cells were injected into 10 animals

simultaneously using a multiport Microinfusion Syringe Pump (Harvard

Apparatus, Holliston, MA). Animals were anesthetized with xylazine/

ketamine during the procedure.

Internal carotid artery injection of human mesenchymal stem cells.hMSCs were injected into the internal carotid artery of xenograft-bearing

nude mice according to the previously described method (36). Briefly,

animals were anesthetized with ketamine/xylazine and the internal carotid

artery was surgically identified under microscopic visualization. Monolayer

cultures of hMSCs were trypsinized and suspended in 100 AL a-MEM plus

10% FBS and injected into the internal carotid artery using a prefabricated

injection cannula. Injections were done manually over 3 to 5 minutes.

Animals were monitored continuously until awaking and then daily for

neurologic deterioration related to the injection.

Human mesenchymal stem cell labeling with SP-DiI. The fluorescentdye SP-DiI (Molecular Probes, Eugene, OR) was dissolved in dimethylfor-

mamide (Sigma Chemical) to the concentration of 2.5 mg/mL as described

previously (27). SP-DiI dye was then added directly to culture medium to a

final concentration of 10 Ag/mL. hMSCs were incubated with 25 mL

medium with SP-DiI in T175 flask for 48 hours, after which time cells were

washed with PBS, incubated with dye-free medium for 4 hours, and used for

experiments.

Brain tissue/tumor preparation. At indicated time points, animals

were sacrificed by CO2 inhalation, and 6 Am serial coronal cryosections

from frozen brains were processed for light and fluorescent microscopy.

Adjacent sections were stained with H&E for visualization of the tumor

mass. In some sections, nuclei were stained with fluorescence-conjugated

antibody [4V,6-diamidino-2-phenylindole (DAPI), Molecular Probes]. Imag-

ing was done with a Nikon microscope equipped with a CCD camera.

Images were merged using Adobe Photoshop software version 7.0 (Adobe

Systems, Inc., San Jose, CA).

In vivo migration assay. The ability of hMSCs to migrate toward

gliomas was assessed in vivo by implanting U87 glioma cells [105 cells,

stably transfected with plasmid containing green fluorescent protein (GFP)

gene, gift of Charles Conrad, M.D. Anderson Cancer Center] into the frontal

lobe of nude mice as described above. Seven days later, SP-DiI-labeled

hMSCs (105 cells) were implanted in the opposite hemisphere. Migration

toward the tumor was assessed at 14 days by direct visualization using

fluorescent microscopy.

In vitro migration assay. The tropism of hMSCs for tumor cells andgrowth factors was determined using an in vitro migration assay according

to previously described methods (37). hMSCs in serum-free medium

were placed in the upper well of 24 mm tissue culture Transwell plates

(12 Am, Nunc, Naperville, IL) coated with polylysine and Matrigel (1 mg/mL

in a-MEM). U87 cells, fibroblasts, or NHAs were incubated in serum-free

medium for 48 hours, and the resulting conditioned medium was aspirated

and placed in the lower well of the Transwell plates. In selected

experiments, growth factor [epidermal growth factor (EGF), vascular

endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-

derived growth factor (PDGF), or stromal cell–derived factor-1a (SDF-1a),100 mg/mL] was added to the lower compartment. In other experiments,

a cocktail of antibodies that blocked the activity of specific growth

factors [i.e., anti-PDGF-BB (Sigma Chemical), anti-EGF (Santa Cruz

Biotechnology, Santa Cruz, CA), and anti-SDF-1a (Chemicon, Temecula,

CA) each at 0.8 Ag/mL] was added to the conditioned medium from gliomas

cells. hMSCs were incubated for 48 hours at 37jC, and the migration ratio

was determined using colorimetric assay [3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide] as described previously (38) or by fixing the

membrane, staining the cells using the Hema3 staining kit (Fisher

Diagnostics, Pittsburgh, PA), directly counting the number of migrated

cells in 10 high-power fields, and calculating the mean. All experimentswere done in triplicate.

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Adenoviral vectors and human mesenchymal stem cell transfection.Adenoviruses carrying the IFN-b gene or the h-galactosidase (h-gal) wereengineered using the bacterial plasmid recombination AdEasy system

(Qbiogene, Irvine, CA) as described previously (27). To transfect hMSCs,

cells were incubated with adenoviruses at specified multiplicity of infectionfor 2 hours, cells were washed and used as described in each experiment.

IFN- expression was detected by ELISA (R&D Systems, Minneapolis, MN).

In vitro efficacy experiments of hMSC-IFN-B. For coculture experi-

ments, U87 glioblastoma cells (105 cells) were plated onto 6 cm dish alongwith increasing ratios of IFN-h-secreting hMSCs. As a control, MSCs-h-galwere used. Cells were trypsinized, stained with 0.04% trypan blue solution,

and counted using a hematocytometer. Growth curves were generated by

combining data from three independent experiments.For efficacy experiments involving Transwell plates, U87 cells were grown

as monolayers in the lower well of 24 mm tissue culture Transwell plates on

porous inserts (12 Am). hMSCs, hMSC-h-gal, or hMSC-IFN-h were plated inthe upper well of the Transwell plates. U87 cells were trypsinized and viable

cells were counted using a hemocytometer after trypan blue staining. All

experiments were done in triplicate.

In vivo efficacy experiments of hMSC-IFN-B. To evaluate the effects ofhMSCs in vivo , direct intratumoral injection of hMSC-IFN-h was

undertaken. U87 cells (5 � 105 cells in 5 AL PBS) were implanted into the

right frontal lobes of nude mice as described above. After 10 days, when

tumors where well established, hMSC-IFN-h were injected directly into thetumor using the permanently implanted guide-screw system or into the

ipsilateral carotid artery as described above. Animals were followed until

moribund at which time they were sacrificed by CO2 inhalation. In all cases,brains were removed to verify the presence of tumor as the cause of death.

Statistical methods. For migration assays, differences between groups

were determined using Fischer’s exact test. For efficacy experiments,

differences in survival among groups were determined by a log-rank test.

Results

Isolation and expansion of human mesenchymal stem cells.Bone marrow aspirates were obtained from normal human donors,isolated, and expanded according to previously described methods(27). Cells had a typical spindle shape, consistent with themorphology reported by others (11, 39–42). Although hMSCs donot have a specific antigen profile, for each culture, we verified thatisolated cells were negative for typical hematopoietic antigensCD45, CD34, and CD38 and were positive for CD44 and CD105(data not shown). The doubling time of our cultures variedbetween 30 and 40 hours and cells could be expanded to 10 to 11passages before developing a large flat morphology. Thus, based onavailable criteria, the cells used in our experiments had theproperties of hMSCs as described previously (11, 39–42).Localization of human mesenchymal stem cells in human

glioma xenografts after regional intravascular administration.To determine the extent to which hMSCs are capable of selectivelyintegrating into human gliomas after systemic delivery in vivo ,intracranial xenografts of the human glioma cell line U87 wereestablished in the frontal lobes of nude mice using a guide-screwsystem as described previously (35). Seven days after tumorinoculation when xenografts were established, hMSCs (106

suspended in 200 AL a-MEM with 10% FBS) were injected intothe carotid artery ipsilateral to the implanted tumor (n = 8). Tovisualize them on postmortem histologic sections, hMSCs werestained before injection with a fluorescent vital dye SP-DiI asdescribed in Materials and Methods. Animals were sacrificed 1 and7 days after injection, the brains were removed, and frozen sectionswere analyzed by light and fluorescent microscopy. SP-DiI-labeledhMSCs were seen exclusively within the U87 tumor mass both 1 and7 days after injection (Fig. 1). Essentially no hMSCs were seen in the

peritumoral normal brain or in the hemisphere opposite to theimplanted tumor in all animals assessed, indicating that hMSCsspecifically localize in the tumor but not in the normal brain. Thespecificity of hMSCs for U87 xenografts was seen best in wholemounts of the entire brain in which SP-DiI-labeled hMSCs wereconfined specifically within the borders of the irregularly shapedtumor but were not seen in the surrounding brain (Fig. 2).The selectivity of hMSCs for U87 gliomas was further supported

by experiments in which SP-DiI-labeled hMSCs were injected intothe internal carotid artery contralateral to the hemisphere bearingthe U87 xenograft (n = 3). Similar to the ipsilateral injection,contralateral injection resulted in the localization of SP-DiI-labeledhMSCs exclusively within the tumor, including (in one example)within small tumor nodules away from the main mass but not inthe normal brain (Fig. 3). In addition to supporting the conceptthat hMSCs are selective for gliomas compared with normal braintissue, these results also indicate that the localization within thexenograft was not merely the result of preferential blood flow to thetumor mass.To show that injected hMSCs remained as whole cells within the

xenograft, engrafted SP-DiI-labeled hMSCs were counterstainedwith DAPI, a nuclear-specific probe. Dual DAPI-positive and SP-DiI-labeled cells were seen exclusively within the xenografts, indicatingthat engrafted hMSCs were morphologically intact (Fig. 4).To determine whether hMSCs could localize to gliomas other

than U87, xenografts were established in the frontal lobe of nudemice using the U251 (n = 5) or LN229 (n = 4) glioma cell lines.After injection into the carotid artery ipsilateral to the implantedtumor, SP-DiI-labeled hMSCs were identified within the LN229and U251 xenografts (Fig. 5), suggesting that the ability tosupport the integration of hMSCs was not a unique property ofU87 cells.To show that the tropism of hMSCs for gliomas was a unique

property of these stem cells and not a property of other humancells, SP-DiI-labeled human fibroblasts were injected into the

Figure 1. Photomicrograph of brain sections showing hMSCs in U87intracranial xenograft. Section was acquired 7 days after hMSCs were injectedintra-arterially. Top, light microscopy of tumor, T (left), adjacent to tumor(center), and contralateral brain (right ). Middle, corresponding fluorescentmicroscopy view. Fluorescently labeled hMSCs (red) are seen only in the tumor.Bottom, high-power view (�100) of the tumor.

Human Mesenchymal Stem Cells in Glioma Therapy




ipsilateral carotid artery of mice bearing U87 gliomas. Fibroblastswere chosen because hMSCs were originally described as fibroblastcolony-forming cells and because fibroblasts are morphologicallysimilar to hMSCs (14). However, injections of fibroblasts into thecarotid artery consistently resulted in the death of animals (n = 8)presumably due to intravascular cellular embolization andsecondary brain ischemia, a finding suggestive of widespreaddistribution of fibroblasts in the brain without tumor specificity. Asan alternative approach, U87 tumor cells were used. In contrast tohMSCs, ipsilateral carotid injection of SP-DiI-labeled U87 tumorcells into the carotid artery of animals bearing U87 xenograftsresulted in widespread distribution of injected cells throughout thebrain with no cells colocalizing to the tumor (Fig. 6A). Thesestudies show that not all human cells are capable of localizing tohuman gliomas after intracarotid injection; thus, the capacity ofhMSCs to integrate into gliomas is a specific property of these stemcells. These studies also indicate that the observed localization ofhMSCs within human glioma xenografts was not due to the factthat human cells were injected into mice (i.e., that the results weredue to a species-specific interaction between human xenograftsand human stem cells in a mouse brain background), because notall injected human cells have the capacity to localize to humanxenografts in this model system.To further show that the observed localization of hMSCs within

gliomas was not a function of the model system (i.e., a species-specific interaction), mMSCs were harvested from the bonemarrow of C57 mice (see Materials and Methods). mMSCs werelabeled with SP-DiI and injected into the ipsilateral carotid arteryof nude mice bearing established U87 xenografts (n = 3). Similar tothe hMSCs, mMSCs were found exclusively within the xenografts7 days after injection (Fig. 6B).Human mesenchymal stem cells migrate toward gliomas

after intracranial injection. Although the above studies indicatethat hMSCs can localize to human gliomas after intravasculardelivery, it is also of interest to determine the extent to whichhMSCs have the capacity to migrate toward gliomas once withinthe brain. To better define the extent to which hMSCs are capableof migrating toward human gliomas in vivo , local delivery

experiments were carried out. Specifically, intracranial xenograftsof the human glioma cell line U87 were established in the rightfrontal lobe in mice using a guide-screw (35). Seven days aftertumor inoculation, SP-DiI-labeled hMSCs (105 cells in 10 ALmedium/FBS) were injected directly into the opposite cerebral lobe(n = 5). As a control, a group of tumor-bearing animals receivedintracranial injections of human fibroblasts (105 cells in 10 ALmedium/FBS). Animals were sacrificed 14 days after injection, theirbrains were removed, and frozen sections were analyzed by lightand fluorescent microscopy. By 14 days after administration,

Figure 3. Photomicrograph of section of entire brain showing selectivity ofhMSC engraftment for right hemisphere U87 glioma after SP-DiI–labeled hMSCs(106 cells) were injected into the contralateral (left) carotid artery. U87 cells werestably transfected with GFP and appear green under fluorescent microscopy(insets ). SP-DiI–labeled hMSCs (red ) are located within the tumor (inset ) as wellas in a smaller tumor nodule (smaller inset ) but not in brain (left lower inset).

Figure 2. Photomicrograph of a section of the entire brainshowing selectivity of hMSC engraftment for U87 gliomaafter intracarotid injection of SP-DiI-labeled hMSCs (106

cells). Light microscopy (center) and H&E staining (bottomleft ) reveals tumor in right hemisphere. Fluorescentmicroscopy (bottom right ) show fluorescently labeled cellsconforming to tumor shape.

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SP-DiI-labeled hMSCs were seen extending from the site ofinjection, across the brain between the tumor and the injectionsite, and within the tumor (Fig. 6C). In contrast, fibroblastsremained within the injection site. Thus, hMSCs have an intrinsicattraction for gliomas and are capable of migrating betweenhemispheres toward gliomas.Factors mediating tropism of human mesenchymal stem cell

for gliomas. We hypothesized that a factor released by the gliomacellsmay be a potential mediator of the tropism of hMSCs for humangliomas. To test this hypothesis, in vitro Matrigel invasion assaysusing Transwell plates were done as a surrogate assay for thetropism of hMSCs for gliomas. We first investigated if human gliomaU87 cell lines were capable of stimulating the migration of hMSCs.Thus, hMSCs were placed in the upper wells on Matrigel, andconditionedmedium fromU87 gliomas and C29 fibroblasts grown inserum-free medium were placed in the lower wells. Conditionedmedium fromNHAswas used as another control to better mimic thenormal brain milieu. A semiporous membrane (8 Am pores)separated the wells. Cell-free medium without and with 20% FCSwas also used as controls. Migration was quantified by directlyvisualizing and counting migrated cells under the microscope aftercell staining. Whereas exposure to cell-free medium or toconditioned medium from fibroblasts or NHAs resulted in lowlevels of migrating hMSCs, exposure to conditioned medium fromU87 cells produced significant hMSC migration (Fig. 7A). Theobserved differences inmigration were not due to increases in hMSCproliferation because the total number of hMSCs (invading plusnoninvading) was the same for each condition.Because exposure to 20% FCS stimulated significant hMSC

migration (Fig. 7A), we analyzed the effects on hMSC migration ofseveral growth factors (specifically PDGF-BB, EGF, basic FGF, andVEGF) that are commonly present in serum and that have beenimplicated in glioma growth. Maximal hMSC migration occurredwith exposure to PDGF-BB (100 ng/mL). Intermediate levels ofmigration were observed after exposure to EGF (100 ng/mL) andSDF-1a (100 ng/mL), whereas basic FGF (100 ng/mL) and VEGF(100 ng/mL) had no significant effect compared with serum-freemedium (Fig. 7B and C).

To document that the increase in migration of hMSCs that wasseen after exposure to conditioned medium from U87 cells was dueto the presence of PDGF-BB, EGF, or SDF-1, conditioned mediumfrom U87 cells was treated with a cocktail containing anti-PDGF-BB, anti-EGF, and anti-SDF-1 antibodies (see Materials andMethods) that are capable of blocking the activity of each growthfactor. Whereas conditioned medium from U87 cells resulted in asignificant increase in hMSC migration, treatment with theblocking antibody cocktail significantly attenuated the migrationof hMSCs through Matrigel (Fig. 7D). These results suggest thatspecific growth factors may at least in part mediate the tropism ofhMSCs for gliomas.

Figure 5. Photomicrograph of brain section showing hMSCs in U251intracranial xenograft. Section was acquired 7 days after hMSCs were injectedintra-arterially. Top, light microscopy of tumor (left ), adjacent to tumor (center),and contralateral brain (right ). Middle, corresponding fluorescent microscopyview. Fluorescently labeled hMSCs (red ) are seen only in the tumor. Bottom,high-power view (�100) of tumor.

Figure 4. Photomicrograph of U87 tumortreated with intravascularly givenSP-DiI-labeled hMSCs. Sections were alsostained with DAPI nuclear stain. Top,H&E-stained section of tumor in brain;bottom, fluorescent microscopic views.Red, SP-DiI-labeled hMSCs; blue, DAPInuclear stain. Merged images showedSP-DiI labeling in the membrane and aDAPI-positive nuclei, indicating that thecells are intact inside the tumor.





Therapeutic potential of genetically engineered humanmesenchymal stem cells on human gliomas: in vitro studies.

As a proof of principle that hMSCs are capable of delivering a

therapeutic agent to brain tumors, we transfected hMSCs with

an adenoviral vector containing the cDNA of the IFN-b gene

(Ad-IFN-h) as described previously (27). A quantified ELISAassay revealed that monolayers of these engineered hMSCs(designated hMSC-IFN-h) released IFN-h into the mediumdependent on the number of viral particles used to transfectthe cells (Fig. 8A). Based on these results, hMSCs were typically

Figure 6. A, photomicrograph of sectionof entire brain showing lack of tropism ofintravascularly given U87 cells for establishedU87 xenografts. U87 cells (containing GFP)were implanted in the frontal lobe of nude miceand SP-DiI-labeled U87 cells (106 cells) wereinjected into the ipsilateral carotid artery.SP-DiI-labeled U87 cells (red) are not foundwithin the U87 xenograft (GFP-labeled; rightinset ). However, SP-DiI-labeled U87 cells (red )are found in the brain within the oppositehemisphere (left inset ). B, photomicrograph ofU87 xenograft after treatment with MSCsobtained from mouse bone marrow (mMSCs).U87 cells were implanted into the frontal lobe,and after 7 days, SP-DiI-labeled mMSCs (106)were injected into the carotid artery. Oneweek later, brains were sectioned. Left, lightmicroscopic view of representative unstainedfrozen section showing area of tumor (�100);right, higher-power fluorescent microscopicview (�400) of area in black box. C,photomicrograph of brain showing migration ofhMSCs toward glioma. U87 cells (GFP-labeled)were implanted in the right frontal lobe (day 0),and after 7 days, SP-DiI-labeled hMSCs wereimplanted in the left lobe. Left, injection site andtumor position. Middle, high-power view ofarea within the rectangle; left, view within thetumor. After 14 days, hMSCs (red) were seenbetween injection site and tumor (middle ) aswell as in the GFP-labeled tumor (right ).

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infected with 3,000 multiplicities of infection for all subsequentexperiments.To determine whether hMSC-IFN-h are of therapeutic benefit,

U87 glioma cells were cocultured with increasing ratios (0.1-50%) ofhMSC-IFN-h. As a control, hMSCs transfected with adenoviruscontaining the h-gal cDNA (hMSC-h-gal) were cocultured with U87cells at a ratio of 2:1 (U87:hMSC). hMSC-IFN-h significantlyinhibited the growth of human gliomas even when the ratio of U87to hMSC-IFN-h was 1,000:1 (0.1%; Fig. 8B).To prove that this growth inhibition was specifically due to

the release of soluble IFN-h, hMSCs (untransfected), hMSC-h-gal,or hMSC-IFN-h were grown in the upper well of Transwellplates in increasing numbers and U87 glioma cells were grownin the lower well. A semiporous membrane (8 Am pores)separated the cells. Whereas treatment with hMSCs or hMSC-h-gal resulted in progressive increase in cell numbers, exposure tohMSC-IFN-h resulted in a dose-dependent growth inhibition ofU87 glioma cells (Fig. 8C). To verify that this effect was due torelease of soluble IFN-h, the concentration of IFN-h in themedium of the lower well was determined at each time point(Fig. 8D). There was a dose-dependent increase in the amount ofsoluble IFN-h that directly correlated with the inhibition oftumor cell growth.

Therapeutic potential of genetically engineered humanmesenchymal stem cells on human gliomas: in vivo studies.To determine if hMSC-IFN-h-induced growth inhibition alsooccurs in vivo , U87 cells (5 � 105) were implanted into the frontallobe of nude mice, and after 10 days, animals were treated with asingle intratumoral injection of PBS, hMSC-h-gal (2.5 � 105 cells),or hMSC-IFN-h (2.5 � 104 or 2.5 � 105 cells). In a group of animals,hMSC-IFN-h (2.5� 105 cells) were given s.c. Compared with animalstreated with PBS or with hMSC-h-gal, treatment with 2.5 � 105

hMSC-IFN-h resulted in a significant (P < 0.05) increase in animalsurvival (Fig. 9A). Interestingly, injection of 2.5 � 104 hMSC-IFN-hdid not prolong survival, suggesting that in this model >2.5 � 104

hMSC-IFN-h must be present within the tumor for growthinhibition to occur. Furthermore, s.c. administration of hMSC-IFN-h had no effect on survival compared with controls, suggestingthat local (intratumoral) release of IFN-h is required for anantitumoral effect.To determine whether regional delivery of hMSC-IFN-h is an

effective antiglioma approach, U87 cells were implanted into thefrontal lobe of nude mice (n = 6/group). Ten days later, animals weretreated with PBS, hMSC-h-gal (106 cells), or hMSC-IFN-h (106 cells)by injection into the internal carotid artery (Fig. 9B). A group ofanimals were also treated with human IFN-h (50,000 IU) given i.v.

Figure 7. A, hMSC invasion in response to conditioned medium of the glioma cell line U87 compared with fibroblasts (C29 cells; CCD) and NHAs. Whereas mediumalone and conditioned medium from fibroblasts or NHAs did not promote hMSC invasion into Matrigel, conditioned medium from U87 cells (P < 0.05) and FCS(P < 0.001) enhanced hMSC invasion. B, effects of growth factors on hMSC invasion. Increased invasion is seen with PDGF-BB and EGF but not FGF and VEGF.a-MEM is negative control and a-MEM + FCS is positive control. C, effect of SDF-1a on hMSC invasion. SDF-1a and EGF had similar effects on promotinghMSC invasion of Matrigel. PDGF is positive control and a-MEM is negative control. D, effects on hMSC invasion after of inhibiting PDGF-BB, EGF, and SDF-1a.Conditioned medium form U87 cells was mixed with a cocktail containing anti-PDGF, anti-EGF, and anti-SDF-1a antibodies. Treatment with medium alone (a-MEM)was a negative control and treatment with 20% FCS was the positive control.





and another group received a s.c. injection of hMSC-IFN-h(106 cells). Only intra-arterial treatment with hMSC-IFN-h signifi-

cantly extended the survival of the animals (P < 0.05). Neither

systemic treatment with IFN-h (not carried by hMSCs) nor s.c.

injection (i.e., distant from the tumor) of hMSC-IFN-h altered animal

survival compared with controls. Thus, hMSC-IFN-h are effective

against intracranial gliomas when delivered regionally.

Discussion

In this study, we provide evidence that human bone marrow–derived MSCs can localize to human gliomas after regional intra-arterial delivery and can migrate toward human gliomas after localintracranial delivery. We also show that the tropism of hMSCs forgliomas may be mediated at least in part by specific growthfactors/chemokines. Most importantly, in vitro and in vivo efficacy

Figure 8. A, expression of IFN-hin the medium of Ad-IFN-h-transfected hMSCs. Monolayers ofhMSCs were plated and infectedat the doses (multiplicity ofinfection) shown. After 24 hours,cells were assayed using aquantitative ELISA assay forIFN-h. A dose-dependent releaseof IFN-h into the medium is seen.B, effects of hMSC-IFN-h onsurvival of U87 gliomas based onin vitro coculture experiments.U87 cells (105) were coculturedwith the indicated percentage ofhMSC-IFN-h. As a control, U87was cocultured with hMSC-h-gal(50%). Cells were counted 3, 5,and 7 days after plating. Adose-dependent decrease in U87survival is evident after treatmentwith hMSCs capable of producingIFN-h. Dark circle, 50% hMSC-h-gal; dark square, 0.1% hMSC-IFN-h; small circle, 1% hMSC-IFN-h; triangle, 10% hMSC-IFN-h;open square, 50% hMSC-IFN-h.C, effects of hMSC-IFN-h onsurvival of U87 gliomas based onin vitro Transwell experiments.U87 cells (3 � 105) were plated onthe lower well (day �2), and after48 hours, hMSC-IFN-h wereplated in the upper well atindicated cell numbers (day 0).U87 cells were assayed forviability 5 and 7 days after hMSCswere plated. hMSC-IFN-h resultedin a significant decrease in U87cell survival. D, amount of IFN-hdetected in the medium ofcells treated under the protocoland conditions described in (C ).Medium was collected on day 7before cell counting. Theconcentration of IFN-h wasdetermined for each conditionusing an ELISA assay.

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studies show that hMSCs can be engineered to release a solublefactor (e.g., IFN-h) and that these engineered hMSCs can beexploited to therapeutic advantage against gliomas.The finding that hMSCs localize to human gliomas is of

interest because it suggests that the capacity for integration intotumors is an intrinsic property of these stem cells. Thisobservation is consistent with the hypothesis that the intra-tumoral integration of exogenously delivered hMSCs is arecapitulation of the natural recruitment of endogenous, circu-lating hMSCs to aid in the process of stroma formation and tissueremodeling and suggests that hMSCs may contribute to thestroma of tumors (27, 30). Initial work from our group showedthat bone marrow–derived hMSCs are capable of integrating intothe stroma of metastatic melanoma grown either s.c. or in thelungs of nude mice (27). We now show that human gliomasgrown in the brain of nude mice also support the engraftment ofhMSCs delivered by an intravascular route. This finding in braintumors is surprising because the stroma of primary brain tumorsis composed of glial/astrocytic cells (ectodermal origin) and isthus distinct from the fibroblast-based (mesenchymal) stroma of

most systemic (extracerebral) cancers. However, it has beenshown that MSCs are capable of differentiating into glial cells(25, 26), including astrocytes, and it is thus possible that thisproperty may explain the intrinsic capacity of hMSCs to integrateinto the stroma of gliomas. Whether hMSCs that have localized togliomas differentiate into astrocytes in our system is currentlyunder investigation. Alternatively, human gliomas, similar to othercancers, require the elaboration of mesodermal elements,specifically endothelial cells and pericytes. It has been suggestedthat MSCs are a main source of pericytes within the bone marrowstroma (11, 16, 17, 40, 43); thus, hMSCs may integrate intogliomas to contribute to the mesenchymal elements of the tumor.In support of this concept is the observation that animals bearingU87 xenografts that received hMSC-h-gal (i.e., nonsecretinghMSCs) survived for shorter times than did animals who receivedsaline treatments in our experiments (see Fig. 9). Thus, hMSCsmay localize to tumor under physiologic conditions to assist withtissue repair and in so doing provide a microenvironmentconducive to improved tumor growth. Regardless of theirphysiologic role within tumors, this present study plus our

Figure 9. A, survival of mice after intracranial intratumoralinjection of hMSC-IFN-h into established U87 gliomas. U87 cells(105) were implanted into the frontal lobe of nude mice. After 10days, tumors were injected with a single dose of hMSC-IFN-h,hMSC-h-gal, or PBS (n = 5/group). A significant increase insurvival is evident with treatment with 2 � 105 hMSC-IFN-h (star).Diamond, PBS; triangle, 2 � 105 hMSC-h-gal; cross, 2 � 105

hMSC-IFN-h s.c.; square, 2 � 104 hMSC-IFN-h; star, 2 � 105

hMSC-IFN-h. B, survival of mice after intra-arterial treatmentwith hMSC-IFN-h. U87 cells (105) were implanted into the frontallobe of nude mice (n = 6/group). After 10 days, animals weretreated with hMSC-IFN-h or controls by injection of 106 cells intothe carotid artery. Treatment with hMSC-IFN-h significantlyincreased survival. Circle, PBS; triangle, IFN-h i.v.; diamond,hMSC-IFN-h i.v.; square, hMSC-IFN-h flank; cross, hMSC-h-galintra-arterial; dark circle, hMSC-IFN-h intra-arterial.





previous work (27) suggest that hMSCs seem to have the capacityto engraft themselves into a variety of histologically disparatetumors, including gliomas, and thus may be a cellular vehicle thatis universally applicable for delivery of therapeutic agents to mosttumor types.Several lines of evidence suggest that the localization of hMSCs

to gliomas was not merely a consequence of the in vivo modelsystem used in our studies. First, the integration of hMSCs is seenwith several xenografts, including the U87, U251, and LN229 celllines. These cell lines are genetically and phenotypically distinct. Inaddition, the LN229 cell line is fairly invasive and growsphenotypically similar to invasive gliomas in situ . Second, theengraftment of gliomas by hMSCs was seen after injecting the stemcells into the carotid artery either ipsilateral or contralateral to thehemisphere harboring the tumor. Thus, the results are not merely afortuitous outcome of cerebral blood flow patterns but rather aredue to a selectivity of hMSCs for the tumor compared with thenormal brain. Third, intra-arterial injection of human fibroblastsresulted in death of most animals presumably due to widespreadarterial occlusion. Furthermore, intracarotid injection of U87tumor cells did not result in localization of delivered U87 cellswithin established U87 xenografts. Thus, the capacity to localize toa glioma after intravascular delivery seems to be a specific propertyof hMSCs and does not seem to be a function of all human cells.Fourth, similar to hMSCs, mMSCs that were delivered intravascu-larly also were capable of localizing to human gliomas. Takingthese findings together, it is unlikely that the localization of hMSCswithin human gliomas grown in nude mice was merely the result ofa species-specific (human cell-human cell) tropism. Instead, hMSCsseem to have an intrinsic, cell-specific capacity to localize tohuman gliomas.The isolation of MSCs from human subjects is an important

aspect of our study. In fact, to our knowledge, this is the firstdemonstration thatMSCs obtained from the bonemarrow of humansubjects can engraft themselves into human gliomas. AlthoughNakanura et al. reported recently the use of MSC in glioma therapy,these investigators isolated the stem cells from rat species (44). Thisis important because differences between nonhuman (i.e., murine orrat MSCs) and human MSCs have been noted (42). In addition, allstudies using neural stem cells have by necessity relied on murine-derived neural stem cells (7–10). Because the MSCs used in ourstudies were derived from human subjects, our results support theapplication of these cells in an autologous transplantation setting inpatients.Although we show that hMSCs can migrate toward gliomas after

intracranial delivery, an important aspect of our study from theperspective of clinical application is the use of intravasculardelivery. Although direct intratumoral (intracranial) delivery hasbeen reported for rat MSCs (44) and mouse neural stem cells(4, 7–10), intravascular delivery has the advantage that it obviatesinvasive surgical interventions and that repeated injections over anextended period are clinically feasible. To our knowledge, no otherreport has shown the propensity of hMSCs to localize to braintumors after intravascular delivery. It should be noted, however,that we originally sought to deliver hMSCs systemically by tail veininjection but found that the majority of cells were filtered by thelung and only rare hMSCs were integrated into the tumor (at leastwhen examined 7 days after injection; data not shown). Aboody etal. reported that after tail vein injection murine neural stem cellslocalized to tumors, albeit with low efficiency (45). In our studies,filtering of hMSCs within the lungs of nude mice may have been

due at least in part to species incompatibilities. Whether trappedpulmonary hMSCs eventually recirculate and localize to intrace-rebral tumors is currently under investigation. Whether similarintrapulmonary entrapment will also occur in patients treated withhMSCs will ultimately require studies in human subjects.Our results indicate that the tropism of hMSCs for gliomas may

be mediated at least in part by growth factors/chemokines. Thisobservation is consistent with the concept that hMSCs areattracted to the tumor milieu because tumors mimic tissue injury(31–33). Similar to damaged tissue, human gliomas express EGF,PDGF, VEGF, and FGF as well as the chemokine SDF-1a (see refs.46, 47 for review). Despite this wide array of growth factors withintumors, however, we show that there is selectivity of hMSCs forspecific factors. Whereas FGF and VEGF had little effect on hMSCmigration, PDGF, EGF, and SDF-1a enhanced hMSC tropism.Moreover, a cocktail of antibodies that block PDGF-BB, EGF, andSDF-1a was able to attenuate the migration of hMSCs towardconditioned medium derived from U87 cells. Indeed, hMSCs areknown to express EGF and PDGF receptors on their surface (43). Itshould be noted, however, that the in vitro invasion assay employedin this study may not directly mimic the in vivo conditionsnecessary for migration of hMSCs from the vasculature to thetumor. In vivo models that better recapitulate this process arecurrently under development. Further elucidation of the mecha-nism underlying the tropism of hMSCs for gliomas may provideinsights into methods for increasing the efficiency of theengraftment process.Our studies of IFN-h indicate that hMSCs can be used to deliver

a diffusible molecule that can be released form hMSCs to achievetumoricidal effects. IFN-h is a particularly good choice to show theproof of principle of this approach because phase I clinical trials ofrecombinant human IFN-h have shown that although responses dooccur, systemic delivery of high doses of IFN-h is associated withtoxicity and a narrow therapeutic index that limits the overallefficacy (48–52). Because IFN-h functions physiologically as aparacrine factor, its antitumoral effects can be enhanced and itstoxicity is reduced if it is given locally (53, 54). We reasoned thathMSCs engineered to produce IFN-h would provide a high degreeof local intratumoral delivery, with reduced systemic toxicity. Inthis context, we used an adenoviral vector to transfer the IFN-bgene into hMSCs and found that these engineered hMSCs (hMSC-IFN-h) released high levels of IFN-h and were capable of directlykilling human glioma cell lines grown in vitro . Indeed, in Transwellexperiments in which hMSC-IFN-h were physically separated fromthe glioma cells, there was a dose-dependent tumoricidal effect, theamount directly correlated with the concentration of soluble IFN-hreleased by the engineered hMSCs. Most importantly, regionaldelivery of hMSC-IFN-h by injection into the internal carotid arteryin vivo significantly extended the survival of animals harboringestablished intracranial gliomas. These results were due to the localdelivery of IFN-h by the engrafted hMSCs because IFN-h given i.v.did not extend animal survival compared with saline-treatedcontrols, and hMSC-IFN-h implanted s.c. (i.e., at a site distant fromthe tumor) also had no effect. Thus, these studies provide the proofof principle that hMSCs can be engineered to release a solublefactor into brain tumors. Although our studies suggest that IFN-h isitself a good therapeutic agent worthy of assessment in patientswith glioma, the same approach can be exploited in the delivery ofother agents with antitumor activity.Comparing the experiments in which hMSC-IFN-h were directly

injected in the tumor with those in which hMSC-IFN-h were

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delivered intravascularly provides insight into the number ofhMSCs that may integrate into a tumor after intravascular delivery.Specifically, we found that a significant increase in animal survivalrequired a direct intratumoral injection of at least 2.5 � 105 hMSC-IFN-h; intratumoral injection of 2.5 � 104 did not extend animalsurvival (Fig. 9A). In this context, it is reasonable to assume the atleast 2.5 � 105 cells integrated into the tumor after intravasculardelivery; otherwise, we would not have observed the reportedincrease in survival that occurred after administering hMSC-IFN-hby this route. Because we saw a significant increase in survival afterintra-arterial injection of 106 hMSCs, we estimate that at least 25%of the cells (2.5 � 105) must have integrated into the tumor.As mentioned above, these in vivo studies revealed that animals

bearing U87 xenografts that received hMSC-h-gal (i.e., nonsecretinghMSCs) had shorter survival than did animals who received salineinjections. Although this observation implies that hMSCs may havea role in tumor development under physiologic conditions (seeabove), the fact that animal survival was increased when hMSCswere engineered to secrete IFN-h indicates that this physiologic rolecan be exploited and tumor growth is reversed when a protein withantiglioma effects is released by the hMSCs. From a therapeuticperspective, these observations imply that relatively pure popula-tions of stem cells engineered to carry a therapeutic gene (i.e.,populations free of untransfected cells) will be needed to achievemaximal antitumoral effects. Methods for maximizing transfectionof therapeutic genes to hMSCs and for separating transfected from

nontransfected cells are challenges for the ultimate application ofthis and other stem cell approaches to tumors.Although our studies have focused on bone marrow–derived

hMSCs, recent work has suggested that other cells in the bonemarrow may also be useful as delivery vehicles for brain tumors(55). Specifically, Fine et al. reported recently that a neural stem-like cell could be isolated from the bone marrow and that thesebone marrow–derived stem cells can be used to deliver therapeuticbiological agents to brain tumors (55). Interestingly, using cDNAmicroarray technology, these investigators showed that the profileof expressed genes of bone marrow–derived hMSCs (i.e., the samecells used in our studies) is distinct from the expression profile ofbone marrow–derived neural stem-like cells used in their studies,indicating that the two cell populations are unique. Thus, the bonemarrow may be a rich source of several stem cell populations thatmay be useful in the treatment of brain tumors. Future studiescomparing the functional properties of these distinct bonemarrow–derived stem cell population will be of great interest forthe clinical application of this type of stem cell therapy.

Acknowledgments

Received 6/8/2004; revised 12/13/2004; accepted 1/25/2005.Grant support: Anthony Bullock III Foundation and Elias Family Fund for Brain

Tumor Research (F.F. Lang).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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Photomicrograph Showing Migration ofhMSCs toward Glioma

In the article on human bone marrow–derived mesenchymalstem cells and gliomas in the April 15, 2005 issue of CancerResearch (1), Fig. 6C did not indicate that the middle figure was amontage created from five individual photomicrographs takenfrom regions between the stem cell injection site and the implantedtumor xenograft. For illustration purposes, the individual figureswere postprocessed to highlight the red stem cells and theindividual photomicrographs were placed on a black background,giving the incorrect impression that this was a single image. Thesedata acquisition methods were not described in the figure legendor in Materials and Methods. Any manipulation of scientificillustrations is inconsistent with both previous journal standards

and recently defined standards for electronic image collection,compilation, and alteration (2), and is not acceptable.The authors have now corrected the figure to show the fiveindividual photomicrographs, thereby eliminating the montage; theauthors also have revised the figure legend. The new figure is free ofany postprocessing, except where red and green images weremerged with each other. This corrected version of the figureappears below. The authors note that the correction does not altertheir conclusion that hMSCs are capable of migrating toward thetumor after intracranial injection.

1. Nakamizo A, Marini F, Amano T, Khan A, Studeny M, Gumin J, Chen J, Hentschel S,Vecil G, Dembinski J, Andreef M, Lang FF. Human bone marrow-derivedmesenchymal stem cells in the treatment of gliomas. Cancer Res 2005;65:3307–18.

2. Editorial. Nature 439, 891–892 (23 February 2006).

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-66-11-COR1

Figure 6. Photomicrographs showingmigration of hMSCs toward glioma. U87cells (gfp labeled) were implanted inthe right frontal lobe (day 0) andSP-DiI-labeled hMSCs were implanted inthe left lobe (day 7). Top, photomicrographof brain section taken 14 days after cellinjection. The boxes show the locationof the images represented at the bottom.The numbers in the box correspond to thephotomicrographs at the bottom. Thecorner where the number is located inthe box at the top is the same cornerof the corresponding photomicrographat the bottom. Note the overlap ofphotomicrographs 1, 2, 4, and 5. Bottom,individual photomicrographs taken underthe fluorescent microscope from regionsshown on the top. Images 1 to 3, 4R, and5R were taken with the red fluorescentfilter. Images 4G and 5G were taken withthe green filter. No green tumor cellswere seen in the left brain (not imaged).4R, 4G, 5R, and 5G were merged usingPhotoShop to produce images 4Mand 5M, respectively. Specifically, thecorresponding red and green images weremerged by copying the red image onto thegreen image and then using the LayerStyle function and adjusting the ChangeBlend option, thus producing a mergedpicture. Red-fluorescent hMSCs are seenaround the injection site (1 ), in between theinjection site (2 and 3 ), and within thegreen-fluorescent tumor (4R, 4G, 4M, 5R,5G, and 5M ).

www.aacrjournals.org 5975 Cancer Res 2006; 66: (11). June 1, 2006

Correction

2005;65:3307-3318. Cancer Res Akira Nakamizo, Frank Marini, Toshiyuki Amano, et al. the Treatment of Gliomas

Derived Mesenchymal Stem Cells in−Human Bone Marrow

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