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Jan 22, 2010 - J.D.K., J.S.R.), and Pathology (M.G.), University of Illinois College of Medicine at Peoria, Peoria, Illinois. Despite advances in clinical therapies ...
Neuro-Oncology 12(5):453 – 465, 2010. doi:10.1093/neuonc/nop049 Advance Access publication January 22, 2010

N E U RO - O N CO LO GY

Human umbilical cord blood stem cells show PDGF-D – dependent glioma cell tropism in vitro and in vivo Christopher S. Gondi, Krishna Kumar Veeravalli, Bharathi Gorantla, Dzung H. Dinh, Dan Fassett, Jeffrey D. Klopfenstein, Meena Gujrati, and Jasti S. Rao Departments of Cancer Biology and Pharmacology (C.S.G., K.K.V., B.G., J.S.R.), Neurosurgery (D.H.D., D.F., J.D.K., J.S.R.), and Pathology (M.G.), University of Illinois College of Medicine at Peoria, Peoria, Illinois

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Neural stem cells (NSCs) have a chemotactic tropism toward glioma cells. The use of NSCs as carriers of therapeutic agents for gliomas is currently being explored. Here, we demonstrate that cells isolated from the umbilical cord blood show mesenchymal characteristics and can differentiate to adipocytes, osteocytes, and neural cells and show tropism toward cancer cells. We also show that these stem cells derived from the human umbilical cord blood (hUCB) induce apoptosislike cell death in the glioma cell line SNB19 via Fasmediated caspase-8 activation. From our glioma tropism studies, we have observed that hUCB cells show tropism toward glioma cells in vitro, in vivo, and ex vivo. We determined that this migration is partially dependent on the expression levels of platelet-derived growth factor (PDGF)-D from glioma cells and have observed that local concentration gradient of PDGF-D is sufficient to cause migration of hUCB cells toward the gradient as seen from our brain slice cultures. In our animal experiment studies, we observed that intracranially implanted SNB19 green fluorescent protein cells induced tropism of the hUCB cells toward themselves. In addition, the ability of these hUCBs to inhibit established intracranial tumors was also observed. We also determined that the migration of stem cells toward glioma cells was partially dependent on PDGF secreted by glioma cells and that the presence of PDGF-receptor (PDGFR) on hUCB is required for migration. Our results demonstrate that hUCB are capable of inducing apoptosis in human glioma cells and also show that glioma tropism and hUCB tropism Received March 2, 2009; accepted June 9, 2009. Corresponding Author: Jasti S. Rao, PhD, Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Box 1649, Peoria, IL 61605 ([email protected]).

toward glioma cells are partially dependent on the PDGF/PGGFR system. Keywords: apoptosis, brain slice, cord blood stem cells, glioma Fas, glioma tropism

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he prognosis for patients with malignant gliomas remains dismal despite significant progress in clinical therapies and technologies. This is largely due to the inability of current treatment strategies to address the highly invasive nature of this type of tumor. Malignant glial cells often spread throughout the brain, making it exceedingly difficult to target and treat all intracranial neoplastic foci. As a result, that tumor recurrence is inevitable despite aggressive surgery, adjuvant radiotherapy, and/or chemotherapy.1 The use of neural stem cells (NSCs) as delivery vehicles for tumor-toxic molecules or as gene therapy tools represents the first experimental strategy aimed specifically at targeting malignant brain tumors. The origin of gliomas remains controversial.2 In the past, the neoplastic transformation of fully differentiated glia was assumed to be the mechanism of gliomagenesis. It is now known that there are both NSCs and glial progenitor cells in multiple regions of the adult brain.3 – 5 Investigators have demonstrated that NSCs possess tropism for infiltrating tumor cells and that they can be used to deliver therapeutic agents directly to tumor sites with significant therapeutic benefit. With the aim of developing these findings into a clinically viable technology that would not be hindered by ethical and tissue rejection – related concerns, the use of adult tissue-derived stem cells has recently been explored. Despite encouraging results in preclinical models, there are significant impediments that must be overcome prior to clinical implementation of this strategy. Key among these is an inadequate understanding of the specific tropic mechanisms that govern NSC migration toward invasive tumor, and the need to

# The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected].

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

refine the processes used to generate tumor-tropic stem cells from adult tissues so that this can be accomplished in a clinically practical fashion. During the embryonic development of the CNS, NSC migration is guided over long distances by gradients of chemoattractive and chemorepulsive molecules.6 It seems reasonable to speculate that soluble factors overexpressed by tumor cells are responsible for the brain tumor tropism of NSCs. Previously, researchers have demonstrated that tumor-upregulated vascular endothelial growth factor (VEGF) and angiogenic-activated microvasculature are relevant guidance signals for NSC tropism toward brain tumors.7 In the present study, we have attempted to demonstrate that adherent cells derived from human umbilical cord blood (hUCB) show tropism toward glioma cells in vitro and in vivo and induce apoptosis in human glioma cells via caspase-8 activation.

Materials and Methods Isolation and Characterization of Nucleated Cells from hUCB After the birthing process but prior to the release of the placenta, the umbilical cord was drained of blood by inserting an intravenous needle into the umbilical cord and the blood allowed to flow by gravity into collection bags containing anticoagulant. Soon after the collection, the cord blood was centrifuged over ficol as per the standard protocol and the buffy coat collected. Mesenchymal stem cells were separated by selectively precipitating nonmesenchymal cells with appropriate antibodies using the Rosette mesenchymal separating kit (Stem Cell Technologies, Vancouver, BC, Canada). To determine the presence of mesenchymal cells in the cellular isolate, the cells were cultured in knockout media supplemented with 10% knockout serum replacement (Stem Cell Technologies). To further determine the presence of stem cell marker proteins CD133, CD44, and STRO-1, the isolated cells were cultured on 100 mm plates to 40% confluence after which the cells were fixed in 10% buffered formaldehyde followed by incubation with 1% BSA in PBS for 1 hour at room temperature. The cells were further incubated with 1% BSA in PBS with a 1:100 dilution of the primary antibody (CD133, CD44, or STRO-1) for 1 hour at room temperature. Following incubation, the cells were washed three times with the 1% BSA in PBS solution at room temperature after which appropriate FITC-conjugated secondary antibody (1:1000 dilutions) was added and cells were incubated at room temperature for 1 hour. The cells were then washed three times with PBS at room temperature, scraped and analyzed by FACS analysis. FACS analysis was done on cells cultured for 0, 10, or 20 days to determine changes in mesanchymal cell population if any. Antibodies Antibodies were obtained from Abcam (Cambridge, Massachusetts) and Santa Cruz Biotechnology (Santa

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Cruz, California) and were used with appropriate secondary antibodies. Antibodies from Abcam are: Osteocalcin (cat. no. ab13418), Nestin (cat. no. ab5968), CD34 (cat. no. ab8158), CD133 (cat. no. ab19898), CD44 (cat. no. ab51037), Caspase-8 (cat. no. ab10450), and neurofilament 70 (NF70; cat. no. ab71979). Antibodies from Santa Cruz Biotechnology are: STRO-1 (cat. no. sc-47733), Fas (cat. no. sc-52393), and FasL (cat. no. sc-71096). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used for Western blot analysis, and appropriate FITC-conjugated secondary antibodies were used for FACS analysis.

Multipotency of Umbilical Cord Blood Cells To validate the presence of mesenchymal stem cells, cultured umbilical cord blood (hUCB) cells were differentiated to adipocytes, neural, and osteocytes in appropriate differentiating media. Adipogenic differentiation was done as follows: isolated cord blood cells were cultured in 100-mm cell-culture plates as described earlier and supplemented with adipogenic stimulatory supplements from Stem Cell Technologies. Phase contrast microscopy and selective staining with oil red dye, which selectively stains lipid tissue, were used to determine the adipogenicity of the cells followed by FACS analysis for oil red florescence (excitation 515 –560 nm; emission 620 nm), percent adipogenisity was determined for 10 different hUCB isolates and graphically represented. Osteogenic differentiation was done by culturing hUCB in osteogenic differentiating media containing 42.5 mL of Mesencult Basal Medium (Stem Cell Technologies) with the following: 7.5 mL of osteogenic supplements, 5 mL of dexamethasone (1024 M stock solution), and 250 mL of ascorbic acid (10 mg/mL stock solution). After we observed evidence of cell multilayering, b-glycerophosphate (175 mL of b-glycerophosphate [stock solution at 1.0 M]) was added. Mineralization was visualized by culturing the cells in 10 mg/mL tetracycline. We observed cells for green fluorescence using a fluorescent microscope followed by FACS analysis for the expression of osteocalcin (FITC conjugated antiosteocalcin) as per standard protocols, percent Osteocalcin-positive cells were determined and graphically represented from 10 different hUCB isolates. To determine the neural differentiation ability of the hUCB cells, the cells were cultured in neural differentiating media as per standard protocols. Briefly, undifferentiated hUCB stem cells (3  105cells) were plated in 60 mm plates containing neurobasal medium (Invitrogen, Carlsbad, California) supplemented with 25 mM glutamate. To induce differentiation, after 24 hours, the medium was replaced with neurobasal medium supplemented with 10% FBS, 2% B27-supplement (Invitrogen), 1% N2-supplement (Invitrogen), 0.5 mM L-glutamine, NGF (100 ng/mL, Invitrogen), and bFGF (10 ng/mL, Invitrogen). The cells were maintained in a humidified atmosphere with 5% CO2 at 378C and allowed to undergo differentiation for 7– 10 days. One

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

half of the medium was changed with fresh medium every 3 days. Differentiation of hUCB stem cells to neuronal cells was observed after 10 days. After complete differentiation, cells were photographed using phase contrast microscopy. To determine the presence of neural cells, early neuron marker protein Nestin (FITC-conjugated) was immunoprobed on neural differentiating cells as per standard protocols followed by FACS analysis for Nestin-positive cells. Percent Nestin-positive cells were determined, quantified, and graphically represented from 10 different hUCB isolates.

plates coated with 0.75% agar. Six to eight spheroids measuring approximately 150 mm in diameter (4  104 cells/spheroid) were selected for each group and grown in conditioned media from SNB19 cells. Forty-eight hours later, spheroids were fixed, stained with Hema-3, and photographed. The migration of cells from the center of the spheroids to monolayers was measured to calculate the migration index of the hUCB stem cells.

Isolation of Membrane and Cytoplasmic Protein Fraction

Umbilical cord blood cells were seeded on the upper chamber of matrigel-coated Boyden chamber inserts (Collaborative Research, Inc., Boston, Massachusetts) and placed in transwell chamber plates (Costar, Corning, New York). The cells were then allowed to invade for 24 hours with appropriate conditioned media from SNB19 cells. Cells on the lower side of the membrane were fixed, stained with Hema-3, and photographed. Invasion was quantitated and expressed as a percentage of controls. To determine the involvement of platelet-derived growth factor (PDGF)-D and PDGF-receptor (PDGFR)-a in migration, we downregulated PDGFR or PDGF in glioma cells (SNB19, U87, 4910, and 5310) and stem cells using siRNA (PDGFR: cat. no. sc-39709, PDGFR: cat. no. sc-29443, Santa Cruz Biotechnology) and quantified migration based on the above described assay. In all the cases, migration of stem cells was determined using glioma conditioned media as attractant from controls, PDGF, or PDGFR downregulated cells.

Membrane protein fractions were isolated from SNB19 cells or hUCB cells or cocultures based on standard protocols. Briefly, an extraction buffer for membrane fractions was used (0.1 M Tris [pH 7.5], 1% Triton-X 114, 10 mM ethylenediaminetetraacetic acid [EDTA], aprotinin, and phenylmethylsulfonyl fluoride). The extracts were incubated at 37 8C for 10 minutes and centrifuged to separate the lower (detergent) phase, which contains mostly hydrophobic membrane proteins; this membrane fraction was quantified, and equal quantities of protein were separated by Western blot analysis. For the isolation of the cytoplasmic protein fractions, the remainder from the membrane fraction was used as the cytoplasmic fraction. Western Blot Analysis hUCB stem cells were cocultured with human glioma cells in the following combinations: stem cells alone, SNB19 cells alone, stem cells þ SNB19 cells, and stem cells þ 4910 human xenograft cells. Cells were lysed with a Nonidet P-40 (NP-40) buffer containing 0.3% NP-40, 142 mM KCl, 5 mM MgCl2, 2 mM EDTA, 20 mM HEPES (pH 7.4), and a cocktail of protease inhibitors (aprotinin, leupeptin, and phenylmethylsulfonyl fluoride; Sigma, St Louis, Missouri). Total protein (10–50 mg) was separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, California). The membranes were blocked with 6% nonfat dry milk and probed with antihuman caspase-8 antibody. Appropriate antibody conjugated with HRP was used as the secondary antibody, and membranes were developed using enhanced chemiluminescence protocol as per the manufacturer’s instructions (Amersham, Arlington Heights, Illinois). GAPDH served as a loading control. Similarly, membrane fractions and cytoplasmic fractions were separated on a 12% SDS–PAGE gel followed by Western blot analysis for Fas and FasL as per standard protocols. Spheroid Migration Assay Umbilical cord blood cells (2  105/mL) were grown as multicellular tumor spheroids on 100 mm tissue culture

Matrigel Invasion Assay

Spheroid Invasion Assay Umbilical cord blood cells (2  105/mL) from African American (AA) or white American (WA) descent were grown as multicellular tumor spheroids on 100 mm tissue culture plates coated with 0.75% agar and labeled with DiL lipophilic dye (red). Six to eight spheroids measuring approximately 150 mm in diameter (4  104 cells/spheroid) were selected for each group and grown and confronted with spheroids made similarly from SNB19 glioma labeled with DiO (green). Spheroids were observed using a confocal laser scanning microscope. Confrontation was observed at 4 and 24 hours. Determination of the Induction of Apoptosis in Glioma Cells by Stem Cell Cocultures To determine the apoptotic cascade in SNB19 cells, SNB19 green fluorescent protein (GFP) cells were confronted with cord blood stem cells by plating in 1:1 ratio followed by incubation for 48 hours. The cocultures were monitored every 12 hours for the initiation of apoptosis as determined by the formation of apoptotic bodies. The activation of FasL was determined using immunocytochemistry with anti-FasL antibody and appropriate Texas red-conjugated secondary antibody as per standard protocols.

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In Vivo Behavior of Cord Blood Cells in Mice Implanted with Intracranial Tumors SNB19-GFP human glioma cells were implanted intracranially in nude mice (nu/nu). After 7 days, 1  106 cord blood cells were injected via the tail vein. SNB19-GFP implanted mice were sacrificed after a total of 15 days and their brains harvested. The tissues were paraffin-sectioned as per standard protocol and then observed for GFP. The presence of the cord blood cells was verified by immunocytochemistry for the presence of CD133 as per standard protocols. To determine the fate of the cord blood cells that localized intracranially, SNB19-GFP human glioma cell implanted mice were sacrificed on day 15 or 95 after implantation. The mice brains from the survival experiments were immunoprobed for human specific NF70 antigen. Mice sacrificed on day 15 were immunoprobed for CD133 as per standard protocol with appropriate secondary HRP-conjugated antibody. Determination of Cord Blood Stem Cells Tropism toward Glioma Cells In Vivo To determine the in vivo migration of umbilical cord blood stem cells toward glioma tumors in vivo, nude mice were intracranially implanted with SNB19 glioma cells (2  106). Seven days after implantation, the mice were given subcutaneous injections of cell tracker redlabeled cord blood stem cells near the left hind flank. Mice were imaged for red fluorescence using the Xenogen IVIS imaging system every second day for 10 days. To visualize the intracranial migration of umbilical cord blood stem cells, nude mice were implanted with SNB19-GFP human glioma cells intracranially in the left hemisphere followed by the implantation of stem cells labeled with cell tracker red in the right hemisphere. Mice were sacrificed on days 3, 5, and 6, and hand sections of the brains were taken at the site of implantation and observed under a fluorescent microscope (1.25) for SNB19-GFP and stem cell tracker red fluorescence. Images were obtained with both green and red emission filters to determine the position of stem cells relative to glioma SNB19-GFP cells.

Pleasanton, California). These membranes were then placed in wells of 6-well culture plates filled with PBS. The slice culture medium consisted of 50% Eagle’s MEM with HEPES, 25% HBSS, 25% heat-inactivated horse serum (Life Technologies, Inc.), 6.5 mg/mL glucose, 100 U/mL of penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B (Fungizone). The brain slice culture was incubated at 378C under standard conditions of 100% humidity, 95% air, and 5% CO2. To determine the migration of stem cells toward glioma cells, SNB19-GFP glioma spheroids were developed and placed on one portion of the brain slice hemisphere. On the opposite slide, stem cell spheroids labeled with Q-dots red were placed with a distance of 1 cm between the glioma and stem cells spheroids. The brain slices were observed for migration by fluorescent microscopy at regular intervals. To determine the involvement of PDGF as a chemoattractant, SNB19-GFP glioma cell spheroids both with and without PDGF siRNA treatment were placed on the brain slice or gelatin gel foam infused with 1 ng of PDGF was used. Stem cell migration was observed by fluorescent microscopy. Appropriate controls were used for experimental validation. Migration of stem cells was determined by measuring the distance of stem cell migration toward glioma spheroids relative to time. Survival Study of Mice Implanted with Intracranial Tumors and hUCB Cells Stem cells were also intracranially implanted with 1  106 SNB19 glioma cells. Seven days after tumor implantation, the mice were subcutaneously injected with cord blood cells as described earlier and their survivability determined. Control mice did not receive any cord blood cells. Statistical Data Analysis All statistical analyses were performed using Graph Pad Prizm software and Image J image analysis software (NIH) for density extraction. We statistically evaluated experimental results using the Kruskal – Wallis test, the Mann– Whitney test, and the ANOVA test. Survival curves were constructed using the Kaplan – Meier method. P , .05 was considered statistically significant.

Brain-Slice Culture A slice culture of rat whole cerebrum was produced by modifying an organotypic culture method used previously.8 Brain slices were prepared from 2-day-old neonatal wistar rats. After brief anesthesia with diethyl ether, the mice were plunged into a 10% povidone-iodine solution, and the heads were cut off with scissors. The whole brains were quickly removed and placed in HBSS (Life Technologies, Inc., Rockville, Maryland). The brains were then cut vertically to the base, 1 mm inward from both rostral and caudal ends of the cerebrum, and mounted on the stage of a microslicer. Slices of 300-mm thickness were cut and transferred onto polycarbonate membrane with 8 mm pores (Nucleopore,

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Results Multipotent Character of Umbilical Cord Blood Stem Cells To validate the presence of mesenchymal stem cells in cord blood isolates, the cultured hUCB cells were differentiated to adipo, osteo, and neural cells in appropriate differentiating media as described in the Materials and Methods section. We used phase contrast microscopy and selective staining with oil red dye to observe for adipogenisity. Presence of adipogenic cells was determined by the appearance of lipid deposition as red globules

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

Fig. 1. Multipotency of human umbilical cord blood (hUCB) stem cells. To validate the multipotent characteristic of umbilical cord blood stem cells (hUCB), the isolated cells were differentiated to adipose cells in appropriate differentiating media followed by selective staining with oil red dye, which selectively stains lipid tissue, and confirmed by FACS analysis (A). To determine the osteogenic potential of the isolated hUCB cells, the cells were cultured in osteogenic differentiating media as described. Mineralization was visualized by phase fluorescent microscopy for tetracycline deposition (A) and confirmed by FACS analysis for osteocalcin. To determine the neural differentiation ability of the hUCB cells, the cells were cultured in neural differentiating media as per standard protocol. Differentiation of hUCB to neuronal cells was observed after 10 day; the cells were photographed after complete differentiation (A) and confirmed by FACS analysis for Nestin expression. To determine the culture characteristics, hUCB cells were analyzed for the expression of CD34, CD133, CD44, or STRO-1 expression over a period of 20 days (B). To determine the efficiency of differentiation of hUCB, 10 different isolates of hUCB were differentiated to adipo, osteo, or neural cells and quantified by FACS analysis (adipo, oil red; Osteo, osteocalcin; and neural, Nestin) (C).

inside the cells (Fig. 1A); control cells did not show the presence of lipid deposition. FACS analysis was used to characterize the increase in lipid deposition, which showed an increase in oil red stained cells indicative of adipogenisity (Fig. 1A). To determine the osteogenic

potential of the isolated hUCB cells, the cells were cultured in osteogenic differentiating media as described in the Materials and Methods section. From fluorescent microscopy studies (Fig. 1A), osteogenic differentiating cells grown in the presence of tetracycline as described

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in the Materials and Methods section showed fluorescence indicative of mineralization, which confirmed the presence of osteocalcin by FACS analysis and the presence of tetracycline florescence at 520 nm indicative of osteogenicity (Fig. 1A). Neural differentiation was confirmed by the presence of neuron-like structures in cultures grown in neural differentiating media with characteristic axon-like structures; control cells did not differentiate to neuron-like structures. FACS analysis for Nestin-positive cells indicated an increase in neural precursor cells indicative of neural differentiation (Fig. 1A). To determine the efficiency of differentiation in vitro, 10 different umbilical cord blood isolates were collected and allowed to differentiate to adipo, osteo, or neural cells as described in the Materials and Methods section. To determine whether the cells isolated from the umbilical cord blood show stem cell – like characteristics, the adherent cells were immunoprobed for the mesenchymal stem cell marker proteins CD133, CD44, and STRO-1, and control cells were also probed for CD34. The cells were collected after ficol centrifugation and plated on 100 mm plates followed by FACS analysis over a 20-day period. From the FACS analysis, cells positive for CD133, CD44, and STRO-1 were determined and graphically plotted in relation to the expression of CD34. From the results, it was observed that over time the expression levels of CD133, CD44, and STRO-1 increased (25%– 35%) in a 20-day culture, whereas the levels of CD34 decreased from 7 + 3% at day zero to 5 + 2% after 20 days in culture (Fig. 1B). From the results, we observed that in all cases and 45 + 3% of cells differentiated to their targets when compared with controls, indicative of a heterogeneous cell population (Fig. 1C).

Cord Blood Cells Showed Tropism toward Cancer Cells as Determined by Matrigel Invasion Assay, Spheroid Migration Assay, and Spheroid Invasion Assay Matrigel invasion assay was done as described in the Materials and Methods section. From the results, it was observed that in controls, no matrigel invasion was observed whereas invasion was observed with the SNB19 conditioned media treatment, indicating that active migration was taking place toward the conditioned media via the matrigel (Fig. 2A). Quantitative analysis revealed that with SNB19 conditioned media, up to 85% of umbilical cord blood cells migrated when compared with the control where less than 6% invasion was observed (Fig. 2B). To determine the migration ability of hUCB-derived cells, the cells were cultured as spheroids as described earlier and allowed to migrate in the presence of conditioned media from SNB19 cells. From Fig. 2A, it is clear that in the controls, migration of cells from spheroids was minimal when compared with treatment with SNB19-conditioned media. Quantitative analysis showed that hUCBderived cell spheroids migrated to about 3700 + 100 mM when compared with controls, which migrated only 750 + 28 mM, in SNB19-conditioned media

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(Fig. 2C). As described earlier, umbilical cord blood cells of AA or WA descent (2  105/mL) were grown as multicellular spheroids and confronted with SNB19 spheroids. After 4 hours of confrontation, we observed that hUCB-derived spheroids invaded the glioma spheroids and after 24 hours, complete invasion of the spheroids was observed. hUCB-derived spheroids from AA or WA descent did not show any difference in invasion characteristics (Fig. 2D). Umbilical Cord Blood Cells Induced Apoptosis in SNB19 Glioma Cells via Fas Activation To determine the interaction of umbilical cord blood stem cells with SNB19-GFP human glioma cells, the cells were cocultured in equal quantities of hUCB observed by fluorescent microscopy. Thirty-six hours after coculture, SNB19-GFP cells showed alterations in cellular morphology, including characteristic cellular blebbing and apoptotic body formation. Forty-eight hours after coculture, the formation of apoptotic bodies was more pronounced, indicating the onset of apoptosis in SNB19-GFP cells. The hUCB-derived cells did not show the presence of any apoptotic bodies (Fig. 3A). To determine whether FasL mediated the activation of apoptosis, the cocultures were immunoprobed with anti-FasL antibody and appropriate Texas red-conjugated secondary antibody. Figure 3B shows that in SNB19-GFP cocultured with hUCB-derived cells, overexpression of FasL was observed. Umbilical cord blood cells showed high expression of FasL when compared with SNB19-GFP cells alone. To determine the expression of caspase-8, SNB19 cells alone, umbilical cord blood cells alone, and SNB19 þ umbilical cord blood cocultures cell lysates (membrane fraction, cytoplasmic fraction, and whole cell lysate) were collected and fractionated on a SDS– PAGE gel followed by Western blotting as per standard protocols. Coculture cell lysates showed the presence of increased activated caspase-8, indicating the initiation of apoptosis; cytoplasmic extracts of SNB19, hUCB cells, and cocultures showed high expression levels of FasL, whereas the membrane fraction showed high expression levels of FasL in cocultures alone. Interestingly, high levels of Fas were also observed in membrane fractions of cocultures, and whole cell lysates did not show change in the expression levels of FasL or Fas when compared with controls (Fig. 3C). Quantitative analysis indicated that cocultures showed increased expression of membrane bound FasL (,2-fold) and membrane bound Fas (,1.5-fold; Fig. 3D). Quantitative analysis of caspase-8 cleavage indicated that in hUCB-SNB19 cocultures cleavage of caspase-8 was enhanced over 4-fold, indicative of apoptotic induction (Fig. 3E). hUCB Stem Cells Showed Glioma Tropism In Vivo To determine the in vivo migration of umbilical cord blood stem cells toward glioma tumors in vivo, nude mice were implanted with SNB19 glioma cells intracranially (2  106) followed by subcutaneous or

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

Fig. 2. In vitro glioma tropism of hUCB. To determine the invasive ability of hUCB in the presence of glioma conditioned media, matrigel invasion assay was performed as described previously (A). Invasion was quantified and expressed as a percentage of the sum of cells in the upper and lower wells (B). To determine the migration ability of umbilical cord blood cells, spheroid migration assay was performed. The migration of cells from the center of the spheroids to monolayer was measured using a microscope calibrated with a stage and ocular micrometer and used as an index of cell migration (A). Quantitative analysis was done to determine the migration potential of hUCB spheroids in the presence of glioma conditioned media (C). To determine whether umbilical cord blood cells of AA descent or WA descent had any influence in glioma tropism, spheroids were grown from hUCB cells from AA or WA and confronted with SNB19-GFP spheroids as described. Spheroid invasion was observed with confocal laser scanning microscopy and images obtained at 0, 4, and 24 hours (D).

contralateral implantation of stem cells as described in the Materials and Methods section. Mice were imaged for red fluorescence using the Xenogen IVIS imaging system every second day for 10 days (Fig. 4A). By day 10, we observed that most of the fluorescence accumulated in the brains of the mice. Quantitative analysis revealed that stem cells fluorescence was at a maximum at day 10 in the cranial region (Fig. 4B). To visualize the intracranial migration of umbilical cord blood stem cells, images were obtained with both green and red emission filters of mice brain sections implanted with SNB19-GFP and cell tracker red-labeled stem cells and merged. From the results, we observed that the migration of the stem cells intracranially was initiated before the third day and completed within 6 days (Fig. 4C). To determine the presence of intracerebrally implanted stem cells with SNB19-GFP, histological brain sections were immunoprobed for CD133 as described in the Materials and Methods section with appropriate Texas red secondary antibody. Figure 4D shows that the human-specific CD133-positive cells colocalized with SNB19-GFP human glioma cells after 30 days. Control

mice did not show CD133 expression. Figure 4E shows the expression of human-specific CD133 in mice sacrificed on day 15. CD133 expression was observed in treated mice, whereas control mice did not show any expression of CD133. Mice sacrificed on day 95 were immunoprobed for human-specific NF70. Control mice did not show expression of NF70, whereas mice treated with umbilical cord blood cells did express NF70, indicating the presence of cells of human origin (Fig. 4E). Figure 4F shows the survival of nude mice after intracranial implantation of human glioma xenograft cells followed by tail vein injection of hUCB-derived cells. Control mice survived for only a maximum of 30 days, whereas treated mice survived until the termination of the study at day 95, 8 mice were used per group.

hUCB Stem Cells Require PDGF/PDGFR for Efficient Glioma Tropism To determine the involvement of PDGF-D and PDGFR-a, we used the brain slice culture method to

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Fig. 3. Induction of apoptosis in glioma cells by human umbilical cord blood cells. To determine hUCB cell and glioma cell interaction mediated apoptosis, SNB19-GFP cells were plated with hUCB stem cells (1:1) and incubated for 48 hours. The cocultures were monitored at 12, 24, 36, and 48 hours and observed for the formation of apoptotic bodies (A). To determine whether FasL mediated the activation of apoptosis, the cocultures were immunoprobed with anti-FasL antibody and appropriate Texas red-conjugated secondary antibody (B). Expression levels of Fas and FasL were determined in membrane fractions (MF) and cytoplasmic fractions (Cyto) of controls and cocultures (stem cells alone, SNB19 cells alone, stem cells þ SNB19 cells) as described in the Materials and Methods section, by a standard Western blot analysis. Expression levels of whole cell fraction of Fas (w) and FasL (w) were also determined. Activation of caspase-8 was determined by Western blot analysis of whole cell lysates (C). Quantitative analysis of cytoplasmic and membrane fraction of Fas and FasL (D) and cleaved caspase-8 (E) was done. GAPDH served as a loading control. Data represents are mean + SD values of 4 independent experiments (SC ¼ hUCB).

track the migration of the stem cells toward SNB19-GFP glioma spheroids. Stem cells were labeled with Q-dot red, and spheroids were developed as described in the Materials and Methods section. The brain slice culture

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method was used to track migration of cells in real-time. From the results, it is observed that untreated glioma spheroids caused attraction of stem cells, whereas glioma cells downregulated for PDGF retarded

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

Fig. 4. Determination of tropism of umbilical cord blood stem cells toward glioma cells in vivo. To determine the in vivo migration of umbilical cord blood stem cells toward glioma tumors in vivo, nude mice were intracranially implanted with SNB19 glioma cells (2  106) followed by the subcutaneous implantation of cell tracker red-labeled cord blood stem cells as described. Mice were imaged for red fluorescence using the Xenogen IVIS imaging system for 10 days (A). Quantitative analysis was done to determine the migration of stem cells toward the cranium (B). To determine the intracranial migration of hUCB cells, nude mice were intracranially implanted with SNB19-GFP human glioma cells in the left hemisphere followed by implantation of stem cells labeled with cell tracker red in the right hemisphere. Mice were sacrificed on days 3, 5, and 6, brains harvested, free-hand sectioned at the region of implantation, and observed under a fluorescent microscope (1.25) for visualization of SNB19-GFP green fluorescence and stem cell tracker red fluorescence. Images were obtained with both green and red emission filters and merged (C). Nude mice (nu/nu) were intracranially implanted with SNB19-GFP human glioma cells followed by the subcutaneous implantation of cord blood stem cells as described in the Materials and Methods section. SNB19-GFP implanted mice were sacrificed 15 days after implantation, the brains harvested, fixed, and paraffin sectioned as per standard protocols. The intracranial tumors were seen as green GFP fluorescence stem cells and were immunoprobed for CD133 and visualized with appropriate Texas red secondary antibody (D). The presence of the cord blood cells was also determined by immunocytochemistry for the presence of CD133 using HRP-conjugated secondary antibody (E). To determine the fate of the cord blood cells that localized intracranially, mice were sacrificed on day 15 or 95 after implantation. The mice brains were immunoprobed for human-specific NF70 antigen (E). Survival graphs were plotted for control and treated mice (F).

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Fig. 5. PDGF and PDGFR are required for efficient stem cell glioma tropism. Brain slices were prepared from 2-day-old neonatal wistar rats and cultured as described in the Materials and Methods section. SNB19-GFP spheroids were cultured and placed on brain slice cultures on one hemisphere with Q-dot red-stained stem cell spheroids placed on the adjoining hemisphere 1 cm apart (A and B). SNB19-GPF spheroids were also developed from cells downregulated for PDGF using siRNA targeting PDGF and placed on brain slices as before (A). To determine the involvement of PDGF, gelatin gel foam saturated with 1 ng of PDGF was placed on brain slices instead of SNB19 glioma spheroids and served as attractant for stem cells (B). Quantitative analysis of invasion by stem cells under different condition using conditioned media from glioma cells (SNB19, U87, 4910, or 5310) downregulated for PDGF or PDGFR, invasion was quantified as percent of controls (C). Stem cells were downregulated for PDGF or PDGFR and their invasion toward normal glioma conditioned media determined and expressed as percent of controls (C). Migration of Q-dot red-stained stem cell spheroids was determined and graphically represented relative to time in controls and various treatment conditions (D). (SC, stem cell spheroids; G, glioma spheroids).

migration of stem cells (Fig. 5A). From the quantitative analysis, it is observed that after 96 hours, stem cell migration was more than 8 mm when compared with attraction of PDGF infused gel where stem cells migrate less than 6 mm. Glioma cells downregulated for PDGF showed migration of about 4 mm, whereas stem cells alone showed migration of about 1.5 mm after 96 hours (Fig. 5D). From the matrigel invasion assays, it was observed that when condition media from glioma cells downregulated for PDGF was used, between 40% and 45% decrease in stem cell migration was observed, whereas when the PDGFR was downregulated in glioma cells, between 25% and 30% decrease in stem cell migration was observed. Similarly, when

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stem cells were downregulated for PDGF, up to a 40% decrease in migration was observed, whereas when the PDGFR was downregulated, up to a 70% decrease in migration was observed (Fig. 5C).

Discussion The prognosis for patients with malignant glioma, the most common primary intracranial neoplasm, remains dismal despite significant progress in therapies and technologies. This is largely due to the inability of current treatment strategies to address the highly invasive nature of this type of tumor. Malignant glial cells often

Gondi et al.: PDGF-D dependent glioma tropism of cord blood stem cell glioma

disseminate throughout the brain, making it difficult to target and treat all intracranial neoplastic foci.1 An increasing number of studies suggest that stem cells derived from distinct organs and compartments can adopt differentiation traits of heterotopic tissues. These include proposed fate shifts from hematopoietic to neural transition,9 – 19 bone marrow-derived cells to myocardial,20 to skeletal muscle,21 and hepatocyte22 as well as multigerm layer differentiation of neural, mesenchymal,23 and bone marrow-derived stem cells.24 These and similar findings have raised expectations that stem cells derived from adult regenerative tissues may be exploited for cell replacement in nonregenerative organs.25 hUCB have long been known to contain a fraction of hematopoietic stem and precursor cells that are successfully used for the treatment of leukemia and other diseases associated with the hematopoietic system.26,27 Similar to bone marrow-derived stem cells, hUCBderived stem cells have been proposed as a highly plastic donor source capable of differentiation to neural and other cell types.28 – 32 Researchers have reported that after transplantation into the developing rat brains, hUCB-derived cells express neural antigens.33 Furthermore, several studies point to a potential therapeutic effect of grafted hUCB cells in animal models of stroke,34,35 amyotrophic lateral sclerosis,36 and traumatic brain injury.37 It has been reported that NSCs migrate toward or track glioma cells in the brain, and this tropism is to some extent due to angiogenic factors.7 In the present study, we demonstrate that cells derived from hUCB possess adipogenic, osteogenic, and most importantly, neuronal differentiation ability. Previous studies have demonstrated that neuronal stem cells migrate toward glioma cells mediated by SDf1a and VEGF.38 Similarly, in our study, hUCB-derived cells show increased migration and invasion toward cancer cells. Our matrigel experiments show that the invasive character of the umbilical cord blood cells explains the ability of these cells to cross the blood-brain barrier when injected subcutaneously as demonstrated by our colocalization studies. From our spheroid invasion experiments, it is clear that the hUCB-derived spheroids invade SNB19-GFP spheroids, indicating their tropism for glioma cells. From the brain slice culture experiments, it is evident that stem cell glioma tropism is dependent on the levels of PDGF secreted by the glioma cells. The downregulation of PDGF in glioma cells did not completely inhibit the migration of the stem cells toward the glioma but significantly retarded it, indicating that PDGF alone may not be the only chemoattractant for stem cells, other chemoattractants may also be involved. From the matrigel studies, similar conclusions can be drawn where migration of the stem cells toward glioma conditioned media was retarded when glioma cells were downregulated for PDGF. Migration of stem cells was retarded when PDGFR-downregulated glioma cell condition media was used but was significantly less than when PDGF was downregulated, indicating that secretion of PDGF by the glioma cells had

significant influence on stem cells as a chemoattractant. Interestingly, when we downregulated PDGFR in stem cells, migration of the stem cells was significantly reduced by up to 70% and by up to 40% when PDGF was downregulated, indicating that PDGFR on the stem cells is important at contributing to the migration ability of stem cells toward glioma cells. Further evidence for the involvement of PDGF can be drawn from the fact that when PDGF saturated gelatin gel was used as a chemoattractant in the brain slice experiments, migration of stem cells toward the PDGF gradient on brain slice cultures was observed, confirming the above observation that PDGF is a chemoattractant for hUCB. From our animal survival studies, we observed that mice implanted with SNB19 human glioma tumors cells survived for a maximum of only 30 days whereas mice treated with hUCB survived until the termination of the study (95 days), indicating that these cells were capable of repairing or replacing the glioma tissue as confirmed by the presence of human specific NF70 antigen in mice brains. Western blot analysis and immunohistochemical localization of FasL indicate that the hUCB overexpress FasL; and as seen from the coculture studies, induction of apoptosis in SNB19-GFP cells is initiated by hUCB and is probably mediated by the activation of Fas. Western blot analysis of the coculture experiments demonstrated that caspase-8 is activated in cocultures, indicating the activation of Fas via FasL. These studies indicate that hUCB probably induce apoptosis in the glioma cells and replace them as demonstrated by the animal survival experiments where after 95 days, mice were sacrificed and brain sections immunoprobed for NF70 antigen. These results indicate the presence of human cells within the normal brain parenchyma, suggesting that cells derived from the human umbilical cord are capable of integrating into normal mouse brain. Previous studies on live injury sites suggest that hUCB cells differentiated into hepatocyte-like cells in the mouse liver, but the liver injury was essential for this process39 drawing a parallel with our glioma studies where gliomas can be considered similar to a neverhealing wound.40 Various in vivo studies demonstrated a migration tendency of NSCs toward gliomas, making these cells a potential carrier for delivery of therapeutic genes to disseminated glioma cells. Our study demonstrates that stem cells alone are capable of targeting glioma cells and need not be limited to only being carriers of therapeutic agents. This raises the therapeutic potential of hUCB in the treatment of glioma. In conclusion, it is inferred that PDGF –PDGFR system is involved in hUCB stem cell glioma tropism and that hUCB stem cells are capable of retarding glioma progression in nude mouse brains.

Acknowledgments The authors thank Shellee Abraham for preparing the manuscript and Diana Meister and Sushma Jasti for

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the manuscript review. We thank Noorjehan Ali for the technical assistance.

Funding This research was supported by the National Institute of Neurological Disorders and Stroke NS057529. Contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

Conflict of interest statement. None declared.

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