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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Jan 14, 2011.
Published in final edited form as:
PMCID: PMC3021321
NIHMSID: NIHMS87626

Bimodal viral vectors and in vivo imaging reveal the fate of human neural stem cells in experimental glioma model

Summary

Transplantation of genetically engineered cells into the central nervous system (CNS) offers immense potential for the treatment of several neurological disorders. Monitoring expression levels of transgenes and following changes in cell function and distribution over time is critical in assessing therapeutic efficacy of such cells in vivo. We have engineered lentiviral vectors bearing fusions between different combinations of fluorescent and bioluminescent marker proteins and employed bioluminescence imaging and intravital-scanning microscopy in real-time to study the fate of human neural stem cells (hNSC) at a cellular resolution in glioma bearing brains in vivo. Using Renilla luciferase (Rluc)-DsRed2 or GFP-(Rluc) expressing malignant human glioma model, transduced hNSC were shown to migrate extensively towards gliomas, with hNSC populating gliomas at 10 days after transplantation. Furthermore, transduced hNSC survived longer in mice with gliomas than in normal brain, but did not modulate glioma progression in vivo. These studies demonstrate the utility of bimodal viral vectors and real-time imaging in evaluating fate of NSC in diseased models and thus provide a platform for accelerating cell-based therapies for CNS disorders.

Keywords: neural stem cell, bi-modal vector, luciferase, fluorescent proteins, glioma, in vivo imaging

Introduction

Neural stem cells (NSC) are defined by their ability to self-renew and give rise to mature progenitors of neural lineages. The ability of NSC to migrate to diseased areas of the brain (Snyder and Macklis, 1995; Aboody et al., 2000; Tang et al., 2003; Shah et al., 2005) and their capacity to differentiate into all neural and glial phenotypes (Gage, 2000) provides a powerful tool for targeting the treatment of both diffuse and localized neurologic disorders. Several studies have demonstrated the effectiveness of NSC transplantation in the treatment of neurodegenerative diseases, including spinal cord injury and brain tumors (Snyder and Macklis, 1995; Ehtesham et al., 2002; Lindvall et al., 2004; Hofstetter et al., 2005; Iwanami et al., 2005; Shah et al., 2005). Taking advantage of their homing properties, NSC have also been modified to deliver selective anti-neoplastic proteins (Ehtesham et al., 2002; Shah et al., 2005), however with mixed results. While these studies demonstrate the feasibility of NSC-based therapy, cellular delivery of therapeutic proteins via NSC grafts will likely require long-term transgene expression. In vivo assays which permit rapid assessment of the fate of transplanted NSC, transgene expression and differentiation are urgently needed to objectively compare therapeutic efficacies of different paradigms.

Several imaging modalities, including magnetic resonance imaging (MRI) and fluorescence imaging provide means of tracking transplanted cells in vivo (Lewin et al., 2000; Bulte et al., 2002; Graves et al., 2004) but these techniques are often constrained by limited sensitivity and/or retention of the label (Jendelova et al., 2004; Lee et al., 2004). Previously, we have utilized dual bioluminescence imaging to demonstrate that mouse NSC modified to express firefly luciferase (Fluc), implanted into the contralateral hemisphere of murine brain parenchyma with established gliomas migrate across the brain towards gliomas (Tang et al., 2003; Shah et al., 2005). While bioluminescence imaging is an efficient method for longitudinal comparison of cell survival and migration, it does not offer the spatial resolution to track single cell migration in vivo. Laser scanning microscopic methods e.g. confocal and multiphoton microscopy are becoming more widely used to image cellular details in vivo. Confocal and multiphoton microscopy permit in vivo detection of fluorescent reporter proteins within intact tissue and have been used to monitor numerous molecular events such as recruitment, migration, and survival of single cells (Mempel et al., 2004). In the brain, intravital confocal microscopy (IVM) has been utilized to visualize vascular microenvironment in gliomas (Vajkoczy et al., 2003; Vajkoczy and Menger, 2004; Farhadi et al., 2005), but has not been applied to monitor stem cell survival or migration in tumor models.

A second vital aspect in successfully imaging the fate of implanted cells is to achieve robust and prolonged reporter gene expression. Lentiviral vectors (LV) have been used in several studies to genetically modify NSC ex vivo as these viruses allow stable integration of the transgene into the host genome irrespective of their state of division (Steffen and Weinberg, 1978; Naldini et al., 1996). Recent reports have indicated that lentiviral vectors are also resistant to stem cell specific gene silencing in various types of stem cells as compared to MLV-based retrovirus vectors (Lois et al., 2002; Pfeifer et al., 2002). In the current study, we have engineered a variety of lentiviral vectors to express fusions between fluorescent and bioluminescent proteins and show that both fusion partners are fully functional. Using these vectors we show that hNSC can be efficiently transduced and tracked by dual bioluminescence imaging and IVM. Using these methods, we study hNSC kinetics and migration to malignant brain tumors. To our knowledge this is the first report describing the real time fate of human NSC in a CNS disease model at a cellular resolution.

Experimental Procedures

Generation of fluorescent/bioluminescent lentiviral vectors

We used CS-CGW transfer plasmid based lentiviral vector system (kindly provided by Dr Esteves, MGH, Harvard Medical School) (Miyoshi et al., 1998; Sena-Esteves et al., 2004). To create the LV-GFP-Fluc construct, the cDNA sequence encoding GFP and Fluc were amplified by PCR using the pEGFP-N1 vector (Clontech) and pGL3-Basic (Promega), respectively, as templates. All the restriction enzyme sequences were incorporated into the primer sequence and in addition, a four amino acid linker sequence (DEED) was incorporated into the Fluc forward primer. The resulting GFP fragment was digested with Nhe1 and BamH1 and Fluc fragment was digested with BamH1/Xho1. Both fragments were ligated in-frame into Nhe1/Xho1 digested CSCGW plasmid. Similarly, we have created LV-GFP-Rluc, LV-Fluc-DsRed2, LV-Rluc-DsRed2 lentiviral vectors. To create LV-Lacz vector the cDNA sequence encoding Lacz was amplified by PCR and the resulting Lacz fragment was digested with NheI and XhoI and ligated in-frame into NheI/XhoI digested CSCGW plasmid. Lentiviral vectors were produced by transient transfection of 293T cells (Sena-Esteves et al., 2004). Cells (15 × 106) were seeded in 150-mm2 tissue culture plates 24 h before transfection in DMEM with 10% FBS and cells were re-fed with fresh medium 4 h before transfection. Transfection was performed by calcium phosphate precipitation method using 18 μg of transfer plasmid DNA, transfer vectors constructed above and the lentiviral helper plasmids pCMVΔ8.91 (18 μg) and glycoprotein expression plasmid pVSVG (12 μg; Clontech). Cells were washed with fresh medium 16–18 h post-transfection and vector supernatants were harvested approximately 48 h after transfection. The supernatants were filtered (0.45 μm) and loaded in a Beckman Quick-Seal ultracentrifuge tube (Beckman Coulter, Fullerton, CA, USA) and centrifuged at 28,000×g for 90 min. Pellets were resuspended in PBS and stored at −80°C. Titers of the GFP and DsRed2 expressing vectors were determined by counting fluorescent transduced 293T cells. For titering Lacz vectors, cells were stained with X-gal for 6 h and the number of Lacz expressing cells was counted.

Cell lines and cell culture

Mouse C17.2 neural stem cells (mNSC) were cultured as described previously (Shah et al., 2005). hNS1 is a human fetal neural stem cell line (hNSC) derived from the human diencephalic and telencephalic regions of 10-10.5 weeks gestational age from an aborted human Caucasian embryo. hNSC were immortalized using retrovirally transduced v-myc (Villa et al., 2000). Their in vitro and in vivo properties (including the absence of transformation, clonality, multipotency, stability and survival) have been described in detail elsewhere (Rubio et al., 2000; Villa et al., 2004; Navarro-Galve et al., 2005). Cells were cultured in 4:1 culturing medium (DMEM/F-12 Gibco, 0.6% D-glucose (Sigma-Aldrich), 0.5% albumax (Gibco), 0.5 % glutamine (Gibco), recombinant human FGF (40 ng/ml) (R & D Systems), recombinant human EGF (40 ng/ml) (R & D Systems), N2 supplements (Gibco) and 1% non-essential amino acids (Cellgro) and growth medium (DMEM with 5% fetal bovine serum (Sigma, St. Louis, MO)., 1 mM sodium pyruvate (Cellgro), 26 mM sodium bicarbonate). For hNSC differentiation cells were grown in differentiation medium (DMEM/F-12, 0.6% D-glucose, 0.5% albumax, 0.5 % glutamine, N2 supplements and 1% non-essential amino acids) for 2 weeks and immunohistochemistry was performed as described below. 293T/17 cells (from Dr. David Baltimore, MIT), and Gli36 human glioma cells (from Dr. Anthony Campagnoni, UCLA, CA) were grown in DMEM with 10% fetal bovine serum. mNSC were grown as described earlier (Shah et al., 2005). All cells were cultured at 37°C in a humidified atmosphere with 5% CO2 and 1% penicillin/streptomycin (Invitrogen, Grand Island, NY). hNSC, mNSC and Gli36 glioma cells were transduced or co-transduced with lentiviral vectors in a growth medium containing 12 μg/ml polybrene (Fisher Scientific) and 24 h later cells were visualized for GFP or DsRed2 expression by fluorescence microscopy and assayed for luciferase activity by bioluminescence imaging as discussed below.

In vitro bioluminescence

To determine the correlation between the number of transduced cells and the bioluminescence signal, cells were seeded in different concentrations and substrates for luciferases (1 ug/ml coelenterazine for Rluc; and 1.5 ug/ml D-luciferin for Fluc) were added to the medium. Luciferase activity was measured using a cryogenically cooled high efficiency CCD camera system (Roper Scientific, Trenton, NJ, USA). Each experiment was performed in triplicate.

Immunocytochemistry

To assess transgene expression and differentiation of hNSC into terminal cells, GFP-Fluc expressing hNSC were fixed, permeabilized and incubated with anti-nestin monoclonal antibody (clone 10C2 1:200, Chemicon International, Inc., Temecula, California) directed specifically against human nestin, for 1 h at 37°C. Cells were then washed and incubated with goat anti-mouse Alexa dye 496 nm conjugated secondary antibody (Molecular Probes, Eugene, Oregon) for 1 h, then washed, mounted and examined microscopically. Non-transduced hNSC and hNSC transduced with LV-Lacz were seeded on poly-L-lysine (50 μg/ml; Sigma) coated glass coverslips at a density of 2×104 cells/cm2. After 24 hours of proliferation, the growth factors were withdrawn and cells were allowed to differentiate for 7 days. Cultures were fixed, permeabilized and incubated overnight at 4°C with mouse monoclonal antibodies against β-III-tubulin (1:2000; Sigma) and rabbit antiserum against glial fibrillary acidic protein (GFAP) (1:200; DAKO). On the next day, the cultures were incubated with CY3-conjugated antibody (goat anti mouse; 1:500, Jackson Immunoresearch) or CY5-conjugated antibody (donkey anti rabbit; 1:300, Jackson Immunoresearch). Cell nuclei were counterstained with Hoescht 33258 (Molecular Probes) at 0.2 μg/ml in PBS and detected as described above.

Cell transplantation

Athymic nude mice (nu/nu; 6-7 weeks old) and SCID mice (6-8 weeks old) Charles River Laboratories, Wilmington, Massachusetts) were stereotactically implanted with LV transduced Gli36 glioblastoma cells and mNSC or hNSC. All animal protocols were approved by an institutional review board. Five sets of experiments were performed: (A) For correlation studies, GFP-Fluc or Rluc-DsRed2 hNSC (n=8 in each case) were implanted stereotaxically in the right frontal lobe (from bregma, AP: −2 mm, ML: 2 mm V (from dura): 2 mm) in varying concentrations (1×103−1×105) and 2 days later mice were imaged for Fluc or Rluc activity as described below. (B) To compare hNSC and mNSC survival in nude and SCID mice, either GFP-Fluc expressing hNSC or mNSC (5 × 105 in 4 μl PBS; n=5 in each case) were implanted stereotaxically in the right frontal lobe (from bregma, AP: −2 mm, ML: 2 mm V (from dura):2 mm) and mice were imaged on day 3, 7 and 10 for Fluc activity as described below. (C) To study migration of hNSC toward gliomas by bioluminescence imaging and hNSC fate, Gli36 or Gli36-Rluc-DsRed2 cells (5 × 104 in 4 μl PBS) were implanted stereotaxically into the right frontal lobe (from bregma, AP: −2 mm, ML: 2 mm V (from dura):1.0 mm) of SCID mice (n=10) and 2 days later, GFP-Fluc hNSC (n=8) (5 × 105 in 4 μL PBS) or saline (n=4) were injected into the right frontal lobe (from bregma, AP: −2 mm, ML: 1 mm, V (from dura):1.0 mm) of tumor-bearing mice. GFP-Fluc hNSC were also implanted into the same right frontal lobe of non-tumor bearing mice (n=4). (D) To study the influence of glioma on hNSC survival in vivo, Gli36-Rluc-DsRed2 cells (5 × 104) were mixed with GFP-Fluc hNSC (5 × 105) in 4 μL PBS and implanted stereotactically into the right frontal lobe of nude mice (n=20) and mice were imaged for hNSC survival and glioma growth as described below. (E) Finally to image cells in vivo, Gli36-GFP-Rluc glioma cells were implanted as described above and 7 days later, a small circular portion of the skull (~7 mm diameter) was removed, and the dura was gently peeled back from the cortical surface. hNSC expressing Fluc-DsRed2 (5 × 105 in 4 μl PBS) were implanted in non-tumor bearing mice and also in glioma bearing mice in a close proximity (1 mm lateral) to the gliomas.

In vivo bioluminescence imaging

Mice were imaged for Rluc activity by injecting coelenterazine (100 μg/animal in 150 μl saline) intravenously via the tail vein and 5 min later, photon counts were recorded over 5 min using a cryogenically cooled high efficiency CCD camera system (Roper Scientific, Trenton, New Jersey). To image Fluc, mice were given i.p. injection of D-luciferin (4.5 mg/animal in 150 μL saline) and photon counts were recorded 5 min after D-luciferin administration over 5 min. Images were processed and visualized as described previously (Shah et al., 2003). Mice were imaged every 2-5 days for Fluc or Rluc activity.

Intravital fluorescence microscopy

A prototype multichannel upright laser scanning fluorescent microscope (Olympus IV100, Japan) with a custom-designed stage and scanning unit for intravital observations was used (Alencar et al., 2005). The stage was equipped with a heating plate regulated by a thermostat (37 °C). Images were acquired with Fluoview imaging software (Olympus). Lasers used for excitation included a 488-nm argon laser, a 561-nm solid-state yellow laser, and a 633-nm HeNe-R laser. Emission signal was filtered using 505-525 nm, 586-615 nm, and 660-730 nm band-pass filters, respectively. Cell migration quantiation was performed using Image J analysis software.

Tissue processing

Immediately following the last imaging session, mice were sacrificed and brains were immersed in Tissue-Tek (Sakura Finetek, Torrence, California) on dry ice and 7 μm coronal brain sections were cut. For TO-PRO-3 (Invitrogen) nucleic acid staining, brain sections on slides were incubated with 1 μM TO-PRO-3 for 10 min and slides were washed in PBS and mounted for microscopy to be visualized for TO-PRO-3 staining, GFP and DsRed2 fluorescence on a confocal microscope (Biorad). For Nestin, GFAP, MAP-2 and Ki67 staining, sections were incubated for 1 hr in a blocking solution (0.3% BSA, 8% goat serum and 0.3% Triton-X100) at room temperature (RT), followed by incubation at 4° C overnight with following primary antibodies diluted in blocking solution: 1) anti-human nestin (clone 10C2; Chemicon), 2) anti-human GFAP (Chemicon), 3) anti-Ki67 (clone MIB-1; DAKO) and anti-MAP-2 (Chemicon). Sections were washed three times with PBS, incubated in appropriate secondary antibody and visualized using confocal microscope (LSM Pascal, Zeiss). For quantitative analysis, the number of cells immunoreactive for a specific antigen was counted in at least three non-overlapping fields in each sample, and expressed as a percentage of GFP-positive hNSC.

Stastical analysis

Data were analyzed by Student t test when comparing 2 groups and by ANOVA, followed by Dunnetts post-test when comparing greater than 2 groups. Data were expressed as mean±SEM and differences were considered significant at P<0.05.

Results

A number of different lentiviral vector constructs encoding bioluminescent and fluorescent fusion proteins were generated (Fig. 1A). To test the efficiency of these vectors, human Gli36 glioma cells and hNSC were transduced with LVs at an M.O.I of 1. All packaged vectors exhibited greater than 90% infectivity for both human Gli36 glioma cells and for hNSC (Fig. 1B). Engineered fusion proteins (GFP-Fluc; GFP-Rluc; Fluc-DsRed2; and Rluc-DsRed2) were expressed at high intensity and were retained through several passages of cells. hNSC transduced with LV-GFP-Fluc retained the characteristics of hNSC as revealed by their nestin expression and proliferation rate for over two weeks in culture (percent transduced cells: Day 3-98±5%; Day 7-93±4%; Day 15-97±5%) (Fig. 2A and 2B). Furthermore transduced hNSC retained their potential to differentiate into neuronal cells and astrocytes as revealed by βIII-tubulin (percent positive cells: transduced 12±1%; non-tranduced 10±3%) and GFAP staining (percent positive cells: transduced 37±2%; non-tranduced 32±2%) (Fig. 2C-E). These experiments demonstrate the retention of the NSC multipotency in lentiviral-transduced cells.

Fig. 1
Expression of bi-modal imaging transgenes using lentiviral vectors. A self-inactivating lentiviral system based on HIV-1 (CS-CGW;(Miyoshi et al., 1998)) was used to construct vectors: (A) fusion between: GFP and Fluc; GFP and Rluc; Fluc and DsRed2; and ...
Fig. 2
Characterization of transduced hNSC. hNSC were transduced with LV-GFP-Fluc and (A) growth of transduced and non-transduced cells in culture was compared over 15 days; (B) immuno-histochemistry was performed with anti-nestin antibody and detected with ...

In order to characterize the activity of luciferase fusion proteins, we performed correlation studies between the cell number and the bioluminescent signal both in culture and in vivo in living mice. Different concentrations of GFP-Fluc and Rluc-DsRed2 hNSC (ranging from 1×104 to 5×105) were either plated or implanted into the frontal lobe of nude mice and bioluminescence imaging was performed. The bioluminescence signal generated from both GFP-Fluc and Rluc-DsRed2 hNSC in culture (Fig. 3A and 3B) and in vivo (Fig. 3C and 3D) correlated linearly with cell number within the ranges tested. This demonstrates that we can quantify the cell number of hNSC implanted in the mouse brain based on the bioluminescence imaging signal.

Fig. 3
Bioluminescence characteristics of hNSC. Different concentrations of either NSC transduced with LV-GFP-Fluc or LV-Rluc-DsRed2 were either plated or implanted intra-parenchymally in the mouse brain. (A and B) Transduced hNSC were plated in concentrations ...

To compare human and mouse NSC, transduced cells were implanted into the frontal lobe of mice and followed in real time. In vitro bioluminescence imaging revealed similar Fluc expression levels of both mNSC and hNSC (Fig. 4A). At all time points studied (3, 7 and 10 days) in grafted nude mice, the bioluminescence signal was higher in mNSC (Day 3-100%, Day 7-92±6%, Day 10-75±5%) than in hNSC (Day 3-83±4%, Day 7-35±2%, Day 10-17±1%) (Fig. 4B). When hNSC were implanted in SCID mice, the detected Fluc signal substantially increased (Day 3-88±6%, Day 7-55±3%, Day 10-31±2%) as compared to nude mice but it did not reach the levels obtained for mNSC in nude mice. As SCID mice are further immune compromised than nude mice, these results may implicate immune rejection as a factor in hNSC survival in the brain.

Fig. 4
Bioluminescence imaging of NSC in mice brains. (A) hNSC or mNSC transduced with LV-GFP-Fluc were plated and 24 hrs later imaged for bioluminescence. The photon intensities from mNSC and hNSC expressing GFP-Fluc are plotted as percent relative to hNSC. ...

Next, to determine the effect of gliomas on hNSC survival, we implanted either GFP-Fluc hNSC or a mix of GFP-Fluc hNSC and Rluc-DsRed2 glioma cells in the brain parenchyma and followed both the hNSC and glioma burden in real time by dual bioluminescence imaging (Fig. 5A). As shown in the summary graph and representative images (Fig. 5B), there was a considerable increase in hNSC cell numbers in the presence of glioma as compared to the hNSC implanted without glioma cells (Day 10 hNSC: without 15±10% and with gliomas 120±20%) (*p<0.05 vs. hNSC). While the presence of gliomas influenced hNSC net yield, hNSC did not influence the proliferation of glioma or changes in the glioma burden over time as revealed by Rluc bioluminescence imaging (Fig. 5C). The presence of a reduced number of GFP expressing hNSC in day 7 and 10 as compared to day 3 brain sections from mice without gliomas confirmed their reduced survival (Fig. 5D (a,c,e)). Furthermore, the presence of a very small number of GFP expressing hNSC in day 3 and 7 brain sections and their absence in day 10 brain sections from deeper tissues confirmed that hNSC did not migrate deeper into the parenchyma (Fig. 5D (b,d,f). These results indicate that glioma cells or host response modulates hNSC survival either through secretion of growth factors or by inhibition of molecules involved in foreign cell rejection.

Fig. 5
hNSC survival in mice bearing gliomas. hNSC expressing GFP-Fluc alone or mixed with Gli36 glioma cells expressing Rluc-DsRed2 were implanted intraparenchymally in SCID mice and the presence of hNSC and glioma cells was imaged by dual bioluminescence imaging: ...

In order to investigate the migratory capabilities of hNSC towards gliomas when implanted at distinct sites, we utilized dual bioluminescence imaging and intravital microscopy. GFP-Fluc expressing hNSC were implanted at a 1 mm distance from established Gli36-Rluc-DsRed2 gliomas in SCID mice. Bioluminescence imaging (Fig. 6A-C) was indicative of the migration of hNSC towards gliomas (Fig. 6D). Intravital microscopy on Fluc-DsRed2 hNSC implanted at 1 mm distance from established GFP-Rluc gliomas (Fig. 6E) confirmed the robust migration of Fluc-DsRed2 hNSC towards gliomas at day 7 after hNSC implantation (Fig. 6F and supplementary video). At day 10 hNSC had completely homed to the glioma site and were seen to penetrate gliomas (Fig. 6G). Quantification of hNSC-DsRed2 fluorescence intensity at the hNSC implantation site and glioma site, further confirmed the robust migration of hNSC within 10 days of implantation (Fig. 6H). Intravital microscopy on non-tumor bearing mice implanted with Fluc-DsRed2 hNSC confirmed the reduction of hNSC in SCID mice (Fig. 6I-K). Fluorescence confocal microscopy on brain sections from mice 10 days post-implantation revealed the presence of DsRed2 hNSC specifically in the GFP positive gliomas and not in the surrounding normal brain tissue (Fig. 7A-G). For immunohistochemical analysis, brain sections from mice bearing gliomas and implanted with hNSC expressing GFP-Fluc 10 days post-implantation revealed a robust expression of neural stem cell marker, nestin in hNSC (98±0.4%) (Fig. 7H,L,P) and not in the normal mouse brain. Furthermore, only a very small percentage of GFP expressing hNSC stained for the proliferation marker Ki-67 (2±0.2%) (Fig. 7I,M,Q). hNSC did not stain for astrocytic marker, GFAP (Fig. 7J,N,R) or neuronal marker, MAP-2 (Fig. 7K,O,S). In contrast, robust Ki67 staining was seen in glioma cells (Fig. 7I,M,Q) and GFAP (Fig. 7J,N,R) and MAP-2 (Fig. 7K,O,S) expression was seen in normal brain. These results show that hNSC implanted in the mice with established glioma migrate specifically into the gliomas, a majority of them do not proliferate and remain in an un-differentitated state.

Fig. 6
hNSC migrate into gliomas in vivo. (A-D) Bioluminescence imaging of mice implanted with GFP-Fluc expressing hNSC in mice with established Rluc-DsRed2 gliomas. Fluc images of mice on day 3 (A), day 7 (B) and day 10 (C) and Rluc image on day 10 (D) are ...
Fig. 7
hNSC are present specifically in the gliomas in an undifferentiated state. Mice implanted with Gli36-GFP-Rluc glioma cells stereotactically into the right frontal lobe were implanted with Fluc-DsRed2 hNSC 2 days later. Mice were imaged by intravital microscopy ...

Discussion

We have created a number of lentiviral vectors bearing novel fusions for in vivo multimodality imaging of NSC. We show that transduced human NSC have enhanced surviving ability in immune-compromised mice bearing gliomas, migrate efficiently towards established gliomas and do not effect glioma growth in vivo.

One of the major obstacles in the in vivo evaluation of stem cell therapies has been the challenge to visualize cell populations in the same animal over time. In order to engineer efficient stem cell therapies, the specifications of the maximal local cell delivery, the integration of delivered cells into the host organ structure, and the long-term survival and potential growth of the delivered cells is of prime importance. An efficient and robust way to follow cells both in culture and in vivo is to transduce them with lentiviral vectors expressing fusions of bioluminescent and fluorescent marker genes. These vectors have the ability to integrate transgenes into the genome of dividing and non-dividing cells (Naldini et al., 1996) and provide means of efficient long-term expression in cells and their progeny without using any antibiotic selection marker. The fluorescent marker then serves to determine the efficiency of transduction, and in conjunction with the bioluminescent marker, serve as an in vivo cell tracking protein. Other studies have used lentiviral vectors expressing either the fluorescent proteins to transduce neural stem cells in culture and in vivo (Consiglio et al., 2004; Vroemen et al., 2005) and shown that lentivirus transduction of human neural stem cells has no detrimental effect on stem cell differentiation (Okada et al., 2005). In the current studies, we have shown that NSC and glioma cells transduced with dual imaging lentiviral vectors showed sustained and robust expression of these markers while maintaining the characteristic features of NSC. A very high expression of both the bioluminescent and fluorescent proteins is due to the fact that both the proteins are expressed as a single transcript under the robust CMV promoter. This is in contrast to the majority of other viral vectors in which bioluminescence imaging reporters are expressed under the CMV promoter and fluorescent reporters are translated via an IRES which typically leads to bioluminescent proteins being expressed at higher levels than the fluorescent proteins. In the present study, bioluminescence imaging allowed real-time imaging of fate of NSC in individual mice over successive time intervals. In contrast to the other studies that have either imaged the fate of gliomas or NSC simultaneously, our current studies give an insight into the survival and influence of hNSC and glioma cells when implanted in the vicinity of each other. This ability to image both the fate of NSC and gliomas in vivo is critical in assessing the efficacy of gene delivery and in quantitating therapeutic effects. However, bioluminescent imaging is limited by a lower spatial resolution compared to other modalities such as intravital microscopy. We explored the possibility of employing intravital microscopy that offers higher spatial resolution and have shown that the migration of neural stem cells to glioblastomas can be imaged at a single cell resolution thus proving highly useful to study migratory speed and to gain a more complete insight into tumoral homing of NSC. Furthermore, we show that fusions between fluorescent and bioluminescent proteins offer possible means to circumvent limitations of bioluminescence imaging, as combined use of bioluminescence imaging and intravital microscopy can be complementary and can be used to address different questions simultaneously.

We have previously shown that mouse and human NSCs are attractive candidates for delivering therapeutic proteins and can be used to target both the primary tumor mass and invasive tumor foci (Martinez-Serrano and Bjorklund, 1997; Aboody et al., 2000; Martinez-Serrano et al., 2001; Shah et al., 2005). In this study, we demonstrate that hNSC implanted into mice with an established glioma migrate preferentially towards the glioma (1 mm away), and co-localize with the tumor by 10 days post-implantation. Several studies have demonstrated the migratory properties of hNSC in mice. Aboody et al. (Aboody et al., 2000) showed that hNSC implanted in the contralateral hemisphere of mice with a glioma had sufficient migratory properties to cross the corpus callosum and infiltrate the glioma by one week post-implantation. Rubio et al (Rubio et al., 2000), using rats, also demonstrated that hNSCs can colonize the neostriatum from a single, centrally placed deposit in as short time as one month. While the specific factors responsible for mediating hNSC homing to gliomas is still unclear, studies have suggested that factors such as VEGF and SDF-1 are upregulated by gliomas and function as chemoattractants (Hong et al., 2006). Additionally, evidence from Ziu et al. (Ziu et al., 2006) suggests that the extracellular matrix of gliomas may play a role in NSC migration and survival by creating a permissive environment for NSC. While further research will be required to fully understand the factors which regulate NSC migration, non-invasive serial imaging of hNSC using bioluminescent imaging or IVM may serve as a powerful tool for investigating the migratory signals responsible for the homing property of hNSC to gliomas.

Our studies reveal the persistence of hNSC in the brains of tumor bearing mice as compared to normal mice. A number of studies have shown that the tumor microenvironment, exerts profound immunosuppressive activity on antigen-presenting cells (APC) and T-effector cells by secretion of bioactive growth factors and cytokines, such as VEGF, transforming growth factor-h, or interleukin (IL)-10 (Maeurer et al., 1995); (Vieweg et al., 2007). Moreover, tumors are infiltrated by regulatory T cells (Tregs) and myeloid suppressor cells (MSC) that actively inhibit T-cell responses at the tumor site through direct cell-cell contact (Zou, 2006), secretion of nitric oxide (NO), or reactive oxygen species (Kusmartsev et al., 2004); (Vieweg et al., 2007). All these factors favor conditions that allow tumors and might also allow tumor associated hNSC in our model to escape immune recognition and foster their proliferation and survival.

In conclusion, we have employed engineered LVs and novel imaging methods to dissect the homing properties of neural stem cells and the influence of gliomas on their survival in mouse brains. Using this study as a template, advances can be made in the way neural stem cells can be engineered with therapeutic agents and both the target (e.g. glioma) and the delivery vehicles (neural stem cells) can be followed at cellular resolution in mouse models of CNS disease.

Acknowledgements

This work was supported by P50 CA86355 (RW, KS, XOB), American Brain Tumor Association grant (KS), Goldhirsh foundation (KS), P01 CA69246 (RW). Work at AMS group was funded in part by Foundation La Caixa, Spain. We would also like to Dr. Pradeep Bhide and Dr. Stephen Yip for their helpful comments.

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