저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.
Stereological Analysis on Migration
of Human Neural Stem Cells in the
Brain of Rats Bearing Glioma
by
Jae Ho Kim
Major in Neuroscience
Department of Medical Sciences
The Graduate School, Ajou University
Stereological Analysis on Migration
of Human Neural Stem Cells in the
Brain of Rats Bearing Glioma
by
Jae Ho Kim
A Dissertation Submitted to The Graduate School of Ajou University
in Partial Fulfillment of the Requirements for the Degree of
Ph. D. in Biomedical Sciences
Supervised by
Kyung Gi Cho M.D., Ph.D
Major in Neuroscience
Department of Biomedical Sciences
The Graduate School, Ajou University
This certifies that the dissertation
of Jae Ho Kim is approved.
SUPERVISORY COMMITTEE
Myung-Aae Lee
Kyung-Gi Cho
Se-Hyuk Kim
Byung-Gon Kim
Jong-Eun Lee
The Graduate School, Ajou University
December, 21st, 2009
ACKNOWLEDGMENT
끝이 보이지 않을 것만 같고 막막하기만 했던 학위과정을 보내면서 너무나 도 부족한 저를 지금 이 자리에 있게끔, 이끌어주신 저의 지도교수님인 조경기 선생님께 우선 너무나도 감사하다는 말과 존경의 마음을 드리고 싶습니다. 매 순 간 최선을 다해서 열정을 가지고 임하라고 해주신 좋은 가르치심 때문에 힘들고 포기하고 싶을 때에도 꿋꿋이 해쳐나갈 수 있었던 것 같습니다. 그리고, 많이 부 족하고 아쉬움이 남는 학위 논문이지만, 논문 심사를 맡아주신 김병곤 선생님, 이명애 선생님, 김세혁 선생님, 그리고 지금 이 연구를 잘 마무리 지을 수 있게 많은 조언과 도움을 주신 이종은 선생님께 깊이 감사 드립니다. 같이 학위과정을 하면서 힘들 때 옆에서 힘이 되어주고 버팀목이 되어준 용규, 재원, 정용, 그리고 동기이자 형으로써 많은 조언을 주신 영철이형, 정훈이형, 범수형에게 감사의 마 음을 전합니다. 그리고 힘들고 좌절할 때 많은 위로와 격려를 해준 경진이 누나, 화정이 누나, 석순이형, 혁민이형, 동훈이형, 다영이 등 여러 대학원 동료들 그리 고 일일이 감사함을 표현하지 못한 저의 주변 분들께도 고마운 마음을 전하고 싶습니다. 부족했던 제가 무사히 학위과정을 마칠 수 있었던 건, 제가 대학원을 오게 되고 학문적으로 성장하는데 있어 절대적인 도움을 주시고 전폭적인 지원 을 해주신 저의 정신적 지주 아버지, 어머니의 희생이 없었더라면 불가능 했을 것 입니다. 항상 큰 용기를 북돋아주시고 격려해주신 할머니, 비슷한 연구를 하 면서 많은 조언을 주고받은 동생 현호 등 저의 소중한 가족들에게 너무 감사하 고 깊은 존경을 마음을 전하고 싶습니다. 그리고 옆에서 따뜻한 마음의 안식처가 되어준 저의 아내 주연이와 장인, 장모님께도 힘든 시간 묵묵히 기다려주심을 깊이 감사 드립니다. 5년 간의 학위과정은 저에게 기다림과 인내, 그리고 쉽게 좌 절하지 않는 방법을 일깨워준 너무나도 소중한 시간이었습니다. 이곳에서 여러 훌륭하신 선생님들의 가르침을 잊지 않고 항상 열정을 가지고 매 순간 최선을 다해서 성실히 살아가도록 하겠습니다.
“삶을 사는 데는 단 두가지 방법이 있다.
하나는 기적이 전혀 없다고 여기는 것이고
또 다른 하나는 모든 것이 기적이라고 여기는 방식이다”
-
알베르트 아인슈타인
-
i
-ABSTRACT-
Stereological Analysis on Migration of Human Neural Stem Cells
in the Brain of Rats Bearing Glioma
Glioblastoma cells migrate away from the primary tumor site to distant sites. This migrating and infiltrating features of glioblastoma make a crucial stumbling block for successful treatment of glioblastoma. Neural stem cells (NSCs) have the potential to act as vectors for the cell-based delivery of therapeutic genes for brain tumors, particularly in malignant gliomas. According to recent studies, the combination of human NSC line expressing cytosine deaminase (F3.CD) and 5-fluorocytosine (5-FC) could be a highly effective treatment modality in the rat glioblastoma model. However, little is known about pattern of NSCs migration in the brain over time after NSC transplantation.
We investigated tropism of HB1.F3 (F3) immortalized human NSCs in rats bearing U373 human glioma in the brain. Rats received an injection of human U373MG malignant glioma cells into the striatum followed by an injection of F3 cells into the contralateral hemisphere 7 days later. We analyzed the number, distribution, and migration rate of NSC using unbiased stereology.
Approximately 10% of the injected NSCs migrated into the tumor region by 50 min after NSC injection. The number of NSCs in the tumor region increased slowly up to 5 days post-injection and increased significantly up to 15 days post-post-injection. Tumor volume was
ii
increased gradually by 15 days after inoculation. The rate of NSC migration was approximately 175 μm/min.
In particular, NSCs increased in number approximately 1.7 fold during day 1 in the absence of tumor cell inoculation in vivo. However, the proliferation of NSCs began to decline after 5 days following injection.
We identified for the first time, the rate and pattern of NSC migration to the tumor mass in vivo. These findings may provide useful information with respect to preclinical research of gene therapy for malignant glioma.
iii
TABLE OF CONTENTS
ABSTRACT ··· ⅰ
TABLE OF CONTENTS ··· ⅲ
LIST OF FIGURES ··· ⅴ
ABBREVIATION ··· ⅶ
Ⅰ. INTRODUCTION ··· 1
Ⅱ. MATERIALS AND METHODS ··· 3
1. Cell culture
···
3
2. In vivo protocol of experimental tumor models ··· 3
3. In vitro and in vivo cell proliferation assays ··· 4
4. In vivo analysis of tumor-tropism of human neural stem cells ···5
5. X-gal stain ···6
6. Cell count and unbiased stereology in a tumor region of rats ···7
7 .Cell count and unbiased stereology in a entire brain of rats ···8
8. Labeling of F3 Cells with 5-bromo-2-deoxyuridine ···8
9. Hematoxylin and Eosin stain ···9
10. Beta-galactosidase immunofluoresecence ···9
11. Statistical Analysis ···10
Ⅲ. RESULTS ···11
iv
2. Migration rate of NSCs ···19
3. NSC migration studied by stereology ···23
4. Histological analysis ···28
5. Proliferation of NSCs in vitro & in vivo ···32
Ⅳ
. DISCUSSION ···37
Ⅴ. CONCLUSION ···43
RFERENCES ···45
v
LIST OF FIGURES
Fig.1. Scheme of the experimental protocol ··· 12
Fig.2. Images of NSCs in the tumor region ··· 13
Fig.3. Tumor cells inoculation ··· 14
Fig.4. Sagittal section of the transplanted site with F3 cells ··· 15
Fig.5. Sagittal section within the tumor area ··· 16
Fig.6. Coronal section 10 days after F3 cell injection ··· 17
Fig.7. Migration of NSCs into the tumor region ··· 20
Fig.8. Migration rate of NSCs ··· 21
Fig.9. Scheme of the stereological experiments ··· 25
Fig.10. NSCs migrated studied by stereology ··· 26
Fig.11. The number of NSCs in the tumor region increased with time ··· 27
Fig.12. In vitro, F3 cells labeled with a 5-bromo-2-deoxyuridine ··· 29
Fig.13. Representative sections of tumor-induced migration of neural stem cells ··· 30
vi
Fig.15. Quantification of NSC proliferation in vitro ··· 33 Fig.16. Engrafted neural stem cells in a transplantation site at time points sequential ···· 34 Fig.17. Sagittal sections were performed in the entire brain ··· 35 Fig.18. Quantification of NSC proliferation in vivo ··· 36
vii
ABBREVIATION
hNSCs; human neural stem cells
F3; HB1.F3
CNS; central nervous system
CC; corpus callosum
5-FC; 5-fluorocytosine
5-FU; 5-fluorouracil
CD; cytosine deaminase
TK; thymidine kinase
SF/HGF; scatter factor/hepatocyte growth factor
VEGF; vascular endothelial growth factor
FGF-2; fibroblast growth factor 2
SDF-1; stromal derived factor-1
CXCR4; chemokine receptor 4
PLGF; placenta growth factor
PI3K; phosphoinositide 3-kinase
PI; post injection
א
Ⅰ
. INTRODUCTION
Glioblastoma is the commonest form of primary brain tumor as well as the most aggressive, hence having the worst prognosis with less than one year of median survival (Vogel et al., 2005). Glioblastoma multiforme remain virtually untreatable and lethal (Imperato et al., 1990; Black et al., 2005; Phillips et al., 2006). Multimodal treatment including radical surgical resection followed by radiation and chemotherapy, has substantially improved the survival rate in patients from glioblastoma, however prognosis of the majority of patients remains poor (Surawicz et al., 1999).
During the past 20 years gene therapy research has advanced greatly, and over 1,340 gene therapy clinical trials have been completed or are ongoing worldwide in 28 countries, and more than 70% of these trials are in cancer gene therapy(Edelstein et al., 2007). However, clinical trials in cancer gene therapy achieved only a limited success because of the low efficiency of gene transfer by currently available vectors and the inability of these vectors to specifically target cancer cells (Crystal, 1995; Anderson, 1998; Benedetti et al., 2000; Marchisone et al., 2000).
Recently studies have shown that murine and human neural stem cells (NSCs) possess an inherent tumor-tropism that supports their use as a reliable delivery vehicle to target therapeutic gene products to primary and secondary invasive brain tumors (Aboody et al., 2000; Kim et al., 2005; Kim et al., 2006a; Lin et al., 2007; Najbauer J, 2007; Shimato et al., 2007; Aboody et al., 2008; Mercapide et al., 2009). Stem and progenitor cell-mediated gene delivery is emerging as a strategy to improve the efficacy and minimize the toxicity of current gene therapy approaches(Aboody et al., 2008).
ב
Recently we have generated a stable immortalized cell line of human NSC by introduction of v-myc oncogene, and this human NSC line, HB1.F3 (F3), was successfully utilized in NSC-based gene therapy in animal model of neurological disorders (Kim et al., 2004). There are many studies that have demonstrated the tumor-tropism of NSCs for glioma in rat brain using PET or other image programs. However, little, if any is known about the number of NSCs migrated to glioma sequentially and the rate and pattern of NSCs migration in the brain over time after NSC transplantation. Therefore, in the present study we used unbiased and objective measuring method via the stereological counting system.
The present study as well as previous studies using the same human NSCs, demonstrated that the NSCs serve as powerful and attractive delivery vehicle for tracking glioma. Ten percent of the injected NSCs could migrate into the contralateral tumor site within 1 hour. The selective and specific nature of NSC’s tumor-tropic property should serve well in suicide/immune therapy for brain tumors. In addition, transplanted NSCs migrated not only to tumor site but also other regions such as hippocampus, auditory cortex and olfactory bulb in other animal models of neurological diseases (Jeong et al., 2003; Lee et al., 2005; Yasuhara et al., 2006). The neural stem cell-based gene therapy should prove as the powerful therapeutic choice for the patients suffering from neurological diseases.
In the present study, we investigated specific tropism characteristics of human NSCs, including rate of migration, area of spread, survival pattern, and proliferation in the brain parenchyma in a rat brain tumor model. These results may provide useful information on the nature of tumor-tropism of the NSCs.
ג
Ⅱ
. MATERIALS AND METHODS
1. Cell culture
The human glioblastoma cell line U373MG was obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 4.5 g/L glucose, 110 mg/L sodium pyruvate, and 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were grown at 37 °C in an atmosphere of 95% air and 5% CO2.
HB1.F3 (F3) is an immortalized human NSC line derived from human fetal telencephalon at 15 weeks of gestation with the use of an amphotropic, replication-incompetent retroviral vector containing v-myc (Cho et al., 2002; Kim, 2004; Kim et al., 2008) F3 cells were maintained as adherent cultures in DMEM supplemented with 5% FBS, 4 mM L-glutamine, 4.5 g/L glucose, 110 mg/L sodium pyruvate, and 100 units/mL penicillin and 100 μg/mL streptomycin. F3 cells transfected with LacZ gene (HB1.F3.LacZ) were cultured under the same conditions.
2. In vivo protocol of experimental tumor models
Sprague-Dawley (SD) female rats (250g, Samtaco, Osan, Korea) were anesthetized with 10% chloral hydrate (Fluka, Germany) and placed in a stereotaxic apparatus. Three µl of PBS containing 1x106 glioma cells were intracranially transplanted at the rate of 0.2 µl /min
ד
using a 26 gauge Hamilton micro-syringe. The target was the right caudate-putaman at [antero/posterior (AP) +0.4mm, medial/lateral (ML) -3.1mm, dorsal/ventral (DV) 4 mm]. After tumor cell inoculation, the human neural stem cells transplanted opposite site of hemisphere.
3. In vitro and in vivo cell proliferation assays
In vitro cell proliferation assays were performed with a thiazolyl blue tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO). In brief, F3 cells, F3.LacZ cells, and U373MG cells grown in serum-containing medium for 3 days were isolated, and were plated in 96-well culture plates (1 × 104 cells in a 100-μL volume per well) in DMEM. After a 2-hour plating period, MTT labeling agent [0.5 mg/mL in 10 μL phosphate-buffered saline (PBS)] was added to each well at 0, 12, 24, and 36 hours and on days 2, 3, 5, and 7 and then incubated for 4 hours. A volume of 100 μL solubilization buffer [10% sodium dodecyl sulfate (Sigma-Aldrich) and 50% N,N-dimethyl formamide (Sigma-Aldrich), pH 7.4] was added to each well and allowed to incubate overnight; the absorbance of each well was then measured at 570–630 nm (Sanfeliu et al., 1999).
In vivo cell proliferation assays were performed with HB1.F3 cell labeled with 10μM BrdU (1 x 105 cells) injected into the left forebrain (~3.1 mm lateral and 0.4 mm anterior to bregma, at a 3-mm depth from the skull surface) of 8-week-old female Sprague-Dawley rats. The rats (N=20) were received intraperitoneally of 50mg of BrdU/kg of body weight at a concentration of 10mg/ml BrdU (Roche) in sterile 0.9% NaCl solution every 2 days until 24
ה
hrs before perfusion. After 50 min as well as on days 1, 5, and 15, brains were removed and fixed in 4% paraformaldehyde overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Coronal cryosections (50-μm-thick) of the brain were prepared on a cryostat.
4. In vivo analysis of tumor-tropism of human neural stem cells
U373MG human glioblastoma cells were incubated in 10 μM PKH26 (Sigma-Aldrich) in diluent C solution for 4 min, cells were washed by repeated centrifugation at 400 × g for 10 minutes then harvested for transplantation (Hemmrich et al., 2006). U373MG cells labeled with PKH26 were injected into the striatum of 8-week-old female Sprague-Dawley rats. Rats were anesthetized with 10% chloral hydrate and received 1 × 106 tumor cells in 3 μL Hanks’ balanced salt solution (HBSS; Hyclone, Logan, UT) stereotactically via a 26-gauge Hamilton syringe into the right forebrain (~3.1 mm lateral and 0.4 mm anterior to bregma, at a 4-mm depth from the skull surface). Seven days after tumor cell injection, F3 cells (1 × 106 cells in 3 μL HBSS) were labeled with Hoechst 33258 (10 mg/mL, Molecular Probes, Eugene, OR) for 5 min and injected stereotactically into the opposite hemisphere at the same coordinates.
The migration of F3 cells into the brain parenchyma was observed between 6 hours and 10 days. At each time point (40 and 50 min; 24, 30, 36, and 42 hours; and days 2, 3, 5, 7, and 10; Fig. 1), brains were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Coronal
ו
cryosections (30-μm-thick) were generated. Presence of F3 cells in the transplantation area, the tumor region, as well as the corpus callosum, hippocampus, and auditory cortex was determined but not in the cerebellum. Number of migrated NSCs and the pattern of migration were analyzed with the Computer-Assisted Stereological Toolbox system (CAST-Grid, version 2.1.4; Olympus Denmark, Ballerup, Denmark).
5. X-gal stain
For immunohistochemical studies, the brains of wild-type rats and experimental group with successive tumor cell-NSC injection were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48 hrs. Tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and frozen at -20 °C until sectioning. Sagittal cryosections (50 μm-thick) were then prepared on a cryostat. Sections were rinsed three times in PBS for 5 min each, and incubated overnight in X-gal staining solution consisted of PBS containing 2 M MgCl2, 0.2 M potassium ferricyanide, 0.2 M potassium ferrocyanide, and 40 mg/mL X-gal (Amresco, Solon, OH) at 37 °C(Buckner et al., 1999) Sections were counterstained with eosin Y solution, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount (Fisher Scientific, Seoul, Korea). Sections were also stained with Harris hematoxylin solution for 2 min and followed in eosin Y solution for 30 sec, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount solution.
ז
6. Cell count and unbiased stereology in a tumor region of rats
The rats (N=25) were transplanted with human NSC line expressing LacZ gene (F3.LacZ) contra-lateral side of tumor inoculation after human glioblastoma cell line (U373MG) injection. To perform unbiased stereological estimation, LacZ-positive cells in the tumor region were counted with an optical fractionators (West, 1993). The CAST-grid system was equipped with an Olympus BX51 microscope, a motorized microscope stage run by an IBM-compatible computer, and a microcator (Prior Scientific Instruments, Cambs, UK) connected to the stage to provide distance information for the z-axis(Kim et al., 2006b; Garcia et al., 2007). The x-and y-frame sliders are used to specify the number of frames per unit in the horizontal and vertical directions. The percentage refers to the area of the counting frame compared to the screen area. If more than on frame is present the percentage refers to the total area of the frames. The tumor region was delineated with a 1.25× objective and a counting grid of 300 × 300 μm. An unbiased counting frame of known area (48.6 × 36.1 μm = 1757 μm2) superimposed on the image was placed randomly over the first counting area and moved systemically over all counting areas until the entire delineated area was sampled. Stereological cell counting method was 3-D image system and cells on green line were counting but cells on red line were non-counting. The areal sampling fraction was 1.95%. The section thickness was 50 μm.
ח
7. Cell count and unbiased stereology in a entire brain of rats
The entire brain, with the exception of the cerebellum, was assayed with a 1.25× objective and a counting grid of 1000 × 1000 μm. An unbiased counting frame of known area (60.0 × 44.6 μm = 2673 μm2) superimposed on the image was placed randomly over the first counting area and sampled as described above. The areal sampling fraction was 0.27%. Actual counting was performed with a 100× oil-immersion objective. Estimation of the total number of BrdU-positive cells was calculated according to the optical fractionator formula (West, 1993). More than 200–300 points over all sections of each specimen were analyzed. Tumor volume was estimated by Cavalieri’s principle (Larsen et al., 1998). Tumor cells were implanted 3.1 mm lateral and 0.4 mm anterior to bregma, at a 4-mm depth from the skull surface. The entire area of the tumor was assayed. The section thickness was 30 μm.
8. Labeling of F3 Cells with 5-bromo-2-deoxyuridine
For in vitro 5-bromo-2-deoxyuridine (BrdU) labeling experiments, F3 cells were incubated in feeding medium containing 10 μM BrdU (Sigma-Aldrich) for 3 hrs, fixed, permeabilized, and processed for immunostaining with anti-BrdU antibody.
Tissue sections were incubated in 2 N HCl at 37 °C for 40 min, placed in a borate buffer for 20 min, followed with 0.3% H2O2 in PBS for 30 min, an overnight incubation at 4°C in a 1:200 dilution of mouse monoclonal anti-BrdU (Roche) and 1 hr incubation in HRP-conjugated goat-anti-mouse IgG (Dako, Glostrup, Denmark)(Short et al., 1997). Sections
ט
were incubated with chromagen 3, 3’-diaminobenzidine for 3 min, counterstained with hematoxylin for 1 min, dehydrated in an ethanol series, cleared in xylene, and mounted on slides with Permount(Kuhn et al., 1996).
9. Hematoxylin and Eosin stain
The sections were dried in room temperature, and sections were rinsed with running tap water for a 5 min. Sections were stained with Harris hematoxylin (sigma, St. Louis, MO) for a 2 min and rinsed with a running tap water. The sections were dipped with 0.5 % HCl in 70 % ethanol and rinsed with running tap water. Thereafter, the sections were dipped with ammonia in distilled water and rinsed with a running tap water, hence, the sections stained with Eosin Y solution (sigma-aldrich) for 30 sec. Finally, the sections were dehydrated with 70 % ethanol, 95 % ethanol, 100 % ethanol, and xylene (DAE JUNG). The sections were covered with cover-glasses using mounting solution (Fisher Scientific)(Titford and Horenstein, 2005) .
10. Beta-galactosidase immunofluorescence
For immunohistochemical studies, the brains of wild-type rats and experimental group with successive tumor cell-NSC injection were removed and fixed in 4% paraformaldehyde in 0.1M phosphate buffer overnight at 4 °C and soaked in 30% sucrose for an additional 48
י
hrs. Tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek,Torrance,CA) and frozen at -20 °C until sectioning. Sagittal cryosectons (50 μm-thick) were then prepared on a cryostat. Prior to the stain, sections were dried in 20 °C for 30 min. sections were rinsed three times in PBS for 15 min each, and blocked with 10% horse serum in PBS for 1 hrs. Sections were incubated overnight at 4 °C in a 1:200 dilution of rabbit-anti-beta-galactosidase (sigma) thereafter sections were rinsed with three times in PBS for 15 min each. Sections were incubated at 20 °C in a 1:200 dilution of rabbit-rhodamine-conjugated 2nd antibody (sigma) for a 2 hrs. Stained cells were viewed with a confocal laser scanning microscope (Olympus, Tokyo, Japan).
11. Statistical Analysis
Statistical analysis was performed in SPSS (version 10.0, SPSS, Chicago, IL). Spearman’s correlation calculation was used to determine the relationship between the time elapsed and the number of migrated NSCs in the tumor region. To calculate the number of migrated cells, we counted each section stained with X-gal. The numbers of migrated cells into the tumor region were compared with each time point. The significance level for this test was set at 0.01. The comparison of the number of transplanted NSCs in rat brain parenchyma without tumor induction over time was performed using SPSS (version 11.5, SPSS, Chicago,IL) followed by paired t-test. A p value less than 0.05 were considered significant.
אי
Ⅲ. RESULT
1. Tumor-tropism pattern of NSCs
Tumor-tropism of F3 human NSCs toward U373MG human glioblastoma cells in rat brain was determined at various time points after transplantation of NSCs into brains that were previously implanted with glioblastoma cells. Migration of Hoechst 33258-labeled F3 cells into the tumor region was not observed at 30 min after injection (data not shown). The presence of F3 cells was detected in the tumor region at 40 min post injection (PI), and at 50 min PI, F3 cells were found throughout the tumor region. The accumulation of F3 cells in the tumor region increased up to 2 days PI and then appeared to decrease in number by 10 days PI (Fig. 2A). F3 cells also migrated extensively into the brain parenchyma, tracking to the tumor as well as tumor satellites (Fig. 2A). F3 cells well visualized in high magnification (Fig. 2B), and F3 and U373 cells well co-localized at 30 hours after NSCs injection in a tumor region (Fig.2C). Tumor volume increased significantly at 22 days PI as compared with 8 days after tumor cell inoculation (Fig. 3).
In sagittal brain sections, F3 cells were found migrating far from the injection site, but not to the cerebellum, at 1 day PI (Fig. 4E). F3 cells (blue) were observed in the area of the tumor (Fig.5a), as well as in the corpus callosum, hippocampus (Figs. 5B, C) but not found in a cerebellum (Figs.5D), at 1 day PI. Ten days PI, F3 cells were still observed in the hippocampus and auditory cortex (Fig. 6).
בי
Fig.1. Scheme of the experimental protocol. 1 × 106 U373MG cells labeled with PKH26 were injected into the right striatum of rats. Seven days later, when the tumors were well established,1 × 106 HB1.F3 neural stem cells (NSCs) labeled with Hoechst 33258 were
injected into the opposite striatum, and the rats were killed at various time points after HB1.F3 (F3) cell implantation.
גי
Fig.2. Images of NSCs in the tumor region.
(A):F3 NSCs (blue) in the tumor mass (red) at each time point (40 and 50 minutes; days 2, 3,
5, and 10) after NSC injection. (Bar indicates 100 μm). (B): F3 NSCs (blue) (Bar indicates 50 μm). (C): Merged F3 NSCs (blue) and U373 cells (red) at 30 hours after injected NSCs (bar indicates 100 μm).
די
Fig.3. Tumor cells inoculation
(A): Tumor size at 8 days after tumor cell inoculation (hematoxylin and eosin staining,
וט
Fig.4. Sagittal section of the transplanted site with F3 cells.
U373MG cells (red) were injected into the right hemisphere and F3 cells (blue; white arrows) were injected into the left hemisphere 7 days later. Sagittal sections are shown 24 hours after F3 cell injection (hematoxylin and eosin staining). Sagittal section of the F3 transplantation site at 24 hours (magnification, ×200; bar indicates 100 μm); (A): The anterior end of the F3 injection site. (B): F3 injection site. (C, D): The posterior end of the HB1.F3 injection site. (E): Cerebellum.
זט
Fig.5. Sagittal section within the tumor area (hematoxylin and eosin staining).
U373MG cells are labeled with PKH26 (red) and F3 cells are labeled with Hoechst 33258 (blue); (A): merged pictures of j and k. (B): corpus callosum. (C): hippocampus. (D): cerebellum. (E): U373 cell (red) in a tumor region. (F): F3 cell (blue) in a tumor region (magnification, ×200; bar indicates 100 μm).
חי
Fig.6. Coronal section through at 10 days after F3 cell [neural stem cell (NSC)] injection. (A): Hematoxylin and eosin staining. NSCs are visible in the hippocampus and
auditory cortex in the hemisphere of the F3 injection. (B): F3 cells are labeled with Hoechst 33258 (blue) a: Hippocampus on the F3 injection side. b: Hippocampus on the tumor side.
c: Auditory cortex on the F3 injection side. d: Auditory cortex on the tumor side. (Scale
טי
2. Migration rate of NSCs
To investigate the pattern of migration and the number of NSCs that migrated into the tumor mass, F3 cells encoded with LacZ gene were utilized. F3.LacZ cells migrated along the corpus callosum to the contralateral hemisphere 1 day PI and F3 LacZ cells found in corpus callosum as well as tumor region (Figs.7, 8A). The distance between the injection site and the tumor site was approximately 7 mm (Fig. 8B). Hoechst 33258-labeled NSCs were first detected in the tumor site 40 min PI (Fig.2A) but not found at 10 min and 30 min PI. The migration rate of the NSCs was calculated to be approximately 175μm/min (Fig.8B).
כ
X100a
c.c
Tumor
A
B
Fig.7. Migration of NSCs into the tumor region.
(A):F3 cells (LacZ-labeled) migrate to the tumor region along the corpus callosum. Rats were killed at 1 day after F3.LacZ cell (1 × 106 cells) injection, and the corpus callosum was stained with X-gal. (B): Migrating F3.LacZ cells in the tumor region. (Scale bar, 100 μm; C.C indicates a corpus callosum).
בכ
Fig.8. Migration rate of NSCs.
(A): F3.LacZ cells (5 × 106 cells, white arrows) were injected on the contra-lateral side and animals were stained at 1 day later; beta-galactosidase staining of the corpus callosum was performed as described in the Methods. (Bar indicates 100 μm). (B): Atlas of rat brain with tumor taken from (Paxinos G, 1998). Hoechst 33258 labeled with F3 cells migrated in the tumor region from 10 min to 40 min PI. The calculated migration rate of F3 cells is shown.
גכ
3. NSC migration studied by stereology
We designed experiment of the stereological analysis (Fig. 9). To evaluate the migration patterns of the NSCs, we counted the numbers of F3.LacZ cells in the tumor region 50 min and on 1, 3, 5, 10, and 15 days PI (Fig.10). F3.LacZ cells were injected at a depth 1.0 mm above that of the tumor cells because NSCs migrate readily into the tumor site along the corpus callosum. Most of rats died 3 -4 weeks after tumor cell inoculation; therefore we observed migration patterns of F3.LacZ cells up to 15 days after tumor cell inoculation. Approximately 10% of injected F3.LacZ cells migrated into the tumor region by 50 min after injection (Fig.11).
The number of F3.LacZ cells in the tumor region increased slowly up to 5 days PI and increased dramatically between 5 to 15 days PI. Changes in tumor volume showed a similar pattern (Fig. 11). Average tumor volume was 7.0 ± 1.5 mm3 (n = 3) at 50 min PI, 7.7 ± 0.8 mm3 (n = 3) on day 1, 17.0 ± 1.5 mm3 (n = 3) on day 3, 26 ± 4 mm3 (n = 5) on day 5, 66 ± 16 mm3 (n = 5) on day 10, and 230 + 56 mm3 (n = 2) on day 15 PI. The density of
LacZ-positive
cells in the tumor region increased up to 1 day PI and decreased thereafter up to 15 days PI (Fig. 11), indicating that the tumor growth rate was greater than the NSC migration rate in vivo. Average tumor volume was 7.7 + 0.8 mm3 8 days PI of U373MG cells but was 230 + 56 mm3 22 days PI (Fig. 3). Thus, the tumor volume increased approximately 30 fold over 14 days. By 15 days after F3.lacZ cell injection, F3.lacZ cells were found in the tumorדכ
region as well as along the corpus callosum and in the hippocampus and auditory cortex. Thus, NSCs survived until 15 days after transplantation and migrated to various brain areas as well as to the tumor mass.
הכ
Fig.9. Scheme of the stereological experiments. 1×106 U373MG cells were transplanted into the right striatum of rats. Seven days later, when the tumors were well established, 1 × 106 F3 cells transfected with LacZ gene (HB1.F3.LacZ) were injected into the opposite striatum and the rats were killed at various time points after HB1.F3.LacZ cell implantation.
וכ
Fig.10. NSCs migration studied by stereology.
(A): Hematoxylin and eosin staining of a coronal section containing the tumor (magnification: x12.5): (B) F3.LacZ cells were counted only the cells marked with red triangles in the tumor region with the use of a computer-assisted stereological toolbox system as described in the Methods (magnification: x1000, Scale bar, 100 μm).
זכ
Fig.11. The number of NSCs in the tumor region increased with time.
(A): The numbers of F3.LacZ cells were increased in the tumor region with time. (B): Tumor volume also increased with time (C): The density of NSCs decreased inversely with time. Black bars: NSCs implanted after tumor cell inoculation; white bars: inoculation with tumor cells only. (D): Correlation between elapsed time and number of migrating NSCs in the tumor region (Spearman’s rho 0.909; p < 0.001).
חכ
4. Histological analysis
Prior to in vivo histological analysis, when F3.NSCs labeled with 5-bromo-2-deoxyuridine in vitro, BrdU was successfully observed in vitro by DAB stain (Fig.12). To determine if tumor cells release tropic chemotactic signals that cause NSC migration, NSCs were injected in the hemisphere opposite of the tumor site. In control animals, F3 NSCs were implanted in the absence of tumor cell inoculation. F3 NSCs were found in both ipsi- and contra-lateral hemispheres in tumor cell-injected brains but only in the ipsi-lateral hemisphere where F3 NSCs were injected in controls in the absence of tumor cells (Figs. 13, 14).
טכ
Fig.12. In vitro, F3 cells labeled with a 5-bromo-2-deoxyuridine (BrdU).
F3 cells were incubated in feeding medium containing 10 μM BrdU for 3 hrs. (A): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x100), (B): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x200), (C, D): F3 cells labeled with a 5-bromo-2-deoxyuridine in vitro (magnification x400).ל
Fig.13. Representative sections of tumor-induced migration of neural stem cells.
(NSCs; F3 cells); Long-range attraction of NSCs labeled with 5-bromo-2-deoxyuridine (BrdU; brown; black arrows) from the left hemisphere (L) across the corpus callosum (cc) in response to a local microinfusion of U373MG cells (0.2 μL/ minute) in the right hemisphere (R) (1 day).
אל
Fig.14. Representative sections of tumor-induced migration of neural stem cells.
(NSCs; F3 cells); Representative coronal brain sections from different rats with NSCs labeled with BrdU (brown; black arrows) injected in the left hemisphere in the absence of tumor cell inoculation (Scale bar, 2 mm).
בל
5. Proliferation of NSCs in vitro & in vivo
We also investigated the proliferation rate of F3 cells both in vitro and in vivo. In vitro, the doubling time of NSCs was approximately 24 hr, and the proliferative activity of F3, F3.LacZ, and U373MG cells was active for 2 days and decreased thereafter (Fig. 15).
In the case of in vivo, injected F3 cells were continuously found from 50 min to 15 days PI in a same transplantation site (Fig.16). The presence of F3 NSCs was observed in various regions of the whole brain at 15 days after transplantation (Fig. 17). We counted number of BrdU-positive F3 cells in the entire brain, including the cerebellum, in vivo. F3 cells increased in number over time up to 5 days PI and decreased thereafter (Fig. 18). The number of transplanted F3 cells was increased approximately 1.7 fold during the first 24 hrs.
גל
Fig.15. Quantification of NSC proliferation in vitro.
The graph shows that the proliferative activity of all three cell lines (F3, F3.LacZ and U373MG) increased up to 2 days and decreased thereafter in vitro.
דל
Fig.16. Engrafted neural stem cells in a transplantation site at time points sequential.
(A): Engrafted F3 cells labeled with 5-bromo-2-deoxyuridine in
transplantation site at 50 min after injection. (B): Engrafted F3 cells labeled with
5-bromo-2-deoxyuridine in transplantation site at 1 day after injection. (C):
Engrafted F3 cells labeled with 5-bromo-2-deoxyuridine in transplantation site at
5 day after injection. (D): Engrafted F3 cells labeled with
הל
Fig.17. Sagittal sections were performed in the entire brain.
Transplanted HB1.F3 cells labeled with 5-bromo-2-deoxyuridine (black arrows) were dispersed in a whole brain parenchyma after 15 days injection Scale bar, 100 μm.
ול
Fig.18. Quantification of NSC proliferation in vivo.
(A): The histogram shows that the proliferative activity of the F3 cells increased by 5 days, thereafter NSCs decreased until 15 days after NSC injection in vivo. (B): Numerical value of statistics (n=5 per condition, mean±SD.).*p <0.05; **p<0.01 verse other groups by a paired t-test.
זל
Ⅳ
. DISCUSSION
Because of their high rate of cell proliferation and diffuse infiltrating properties into surrounding brain parenchyma, glioblastomas are known to be fatal. Indeed, radical surgical resection is practically impossible because of the disseminated infiltration and growth beyond the tumor boundaries, visible even on modern neuro-radiological imaging(Giese and Westphal et al., 1996). Therefore, selective targeting to treat the infiltrating tumor cells may be the goal for a new therapeutic approach. NSCs distribute throughout the primary tumor bed and migrate together with widely outgrowing tumor microsatellites after intratumoral implantation. Moreover, when NSC is implanted intracranially at sites distant from the tumor, they migrate through the normal parenchyma and localize in the tumor sites, known as the ‘chasing down’ phenomenon(Aboody et al., 2006).
Previous studies have demonstrated that a significant reduction in tumor size by CD-encoded F3 NSCs with 5-FC pro-drug application in medulloblastoma model (Marchisone et al., 2000; Kim et al., 2006a), and F3 NSCs transfected with PEX, fragment of human metalloproteinase-2, and an inhibitor of tumor proliferation, have reduced glioblastoma tumor size (Anderson et al., 1998; Kim et al., 2005). NSCs encoded with Herpes simplex virus thymidine kinase (HSV-tk) gene also resulted in decreases in tumor volume (Costantini et al., 2000; Pulkkanen and Yla-Herttuala et al., 2005), indicating that NSCs can be used as highly effective therapeutic tools for brain tumors (Forsyth and Cairncross et al., 1995; Flax
חל
et al., 1998; Noble et al., 2000; Bourbeau et al., 2004; Glass et al., 2005; Hadaczek et al., 2005).
Previously we have demonstrated the potential of human NSCs as an effective delivery system to target and disseminate therapeutic agents to glioma and we used gene-modified human NSCs as a new tool for gene therapy of glioblastoma. There are many studies which reported the tumor-tropism of NSCs for glioma in rat brain using PET or other image programs. However, little, if any, is known about the number of NSCs migrating to glioma at various time points. In the present study, therefore we used unbiased and objective measuring method via the stereological counting system. These results may provide useful information on the nature of tumor-tropism of the NSCs.
In the present study, we injected human NSCs into the rat’s opposite hemisphere to the tumor site and observed the migration of NSCs to the tumor area. To identify the migration of NSCs from the transplantation site to the tumor region, we observed extensive migration of BrdU-labeled NSCs along the corpus callosum and in the tumor mass. The corpus callosum appears to be the main pathway for NSC migration into the tumor mass, consistent with a previous study (Shah et al., 2005). We calculated the migration rate for NSCs as approximately 175 μm/min. NSCs were clearly tumor-tropic, but smaller number of NSCs migrated to areas other than the tumor site such as hippocampus and auditory cortex.
Migration of NSCs and tumor regression in response to gene targeting has been studied by real-time imaging with dual luciferase/substrate imaging (Shah et al., 2005). However, little is known about the number of NSCs that migrate to the tumor site over time, the rate of
טל
migration from the injection site to the tumor region, where NSCs migrate within the brain parenchyma, and how long NSCs remain in these regions. In the present study, the number of NSCs observed in the tumor region 15 days PI was approximately 10% greater than the number of injected NSCs. The number of NSCs in the tumor region increased slowly up to 5 days after injection and increased dramatically thereafter up to 15 days. Tumor volume was gradually increased by 15 days after inoculation. The density of LacZ-positive human NSCs in the tumor region increased during the first 24 hrs after injection and decreased thereafter up to 15 days after injection, indicating that the tumor growth rate as faster than the NSC migration rate in vivo.
Although we did not measure the number of proliferated NSCs which were migrated into the tumor region, we supposed that most of NSCs in the tumor are the migrated NSCs from injection site rather than proliferated NSCs. Because of the total number of NSCs in the tumor at 15 days after injection was not much more than the number of total injected NSCs. It means that NSCs were not proliferating continuously after injection of NSCs. NSCs and neural progenitor cells have the biologic potential to differentiate into CNS cell types, including neurons, astrocytes, and oligodendrocytes. Multipotent NSCs are found in the developing and adult mammalian CNS, including that of humans (Culver et al., 1992; Hurelbrink et al., 2002; Hemmrich et al., 2006). The human NSC line F3 carries the normal human karyotype (46XX) and has the ability to self-renew, differentiate into cells of neuronal or glial lineage, and integrate into the damaged CNS upon transplantation in animal models of various neuronal diseases (e.g., Parkinson’s disease, Huntington disease,
מ
stroke, and brain tumors). This cell line was generated from the human fetal telencephalon with the use of a retroviral vector encoding v-myc gene (Kim et al., 2004; Lee et al., 2007; Kim et al., 2008). A number of gene therapy trials have been performed in an attempt to improve survival rates for patients with glioblastoma with only limited gains. Nonetheless, gene therapy will continue to be used in combination with classical treatment strategies. Gene therapy can modify the genetic make-up of target cells, which is not possible with other therapeutic modalities. Gene therapy that involves suicide genes such as cytosine deaminase (CD) or thymidine kinase (TK) is an attractive approach for the treatment of cancer. The CD gene locally converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which interferes with DNA synthesis and results in the death of dividing cells (Culver et al., 1992; Barba et al., 1993; Barresi et al., 2003). Similarly, the TK gene converts the non-toxic drug ganciclovir into a phosphorylated metabolite that acts as a potent killer of dividing cancer cells(Moolten et al., 1986). To deliver these therapeutic genes to tumors, viral and non-viral delivery vectors are necessary. An optimal gene delivery system would comprise a vector capable of encoding large DNA inserts and efficiently transducing the target tissue, resulting in the selective targeting of the expression of the therapeutic gene for a sufficient duration. In addition, the vector should be easily manufactured, able to be delivered systemically, and be non-cytotoxic, preferably allowing for repeated administration.
NSCs have recently been recognized for their ability to migrate throughout the adult CNS and become normal constituents of the host cytoarchitecture (Snyder et al., 1995; Hurelbrink et al., 2002). Since the reports of the ability of NSCs to migrate into glial tumor
אמ
masses (Snyder et al., 1995; Ourednik et al., 1999; Aboody et al., 2000), several studies have investigated the mechanism involved. There are many molecules and factors associated with the tropism of NSCs for tumor cells. Tumor cells secrete various chemoattractant molecules, such as vascular endothelial growth factor (VEGF), scatter factor/hepatocyte growth factor (SF/HGF), fibroblast growth factor 2 (FGF-2), stromal derived factor-1 (SDF-1), and chemokine receptor 4 (CXCR4) (Machein and Plate et al., 2000; Heese et al., 2005; Schmidt et al., 2005; Ghosh and Maity et al., 2006; Ratajczak et al., 2006; Menon et al., 2007; Bao et al., 2009).
It is well known that certain angiogenic factors, such as VEGF, SF/HGF, and placenta growth factor (PLGF), are associated with the attraction for NSCs (Schmidt et al., 2005). In addition, NSCs express CXCR4, and the inhibition of CXCR4 dramatically decreases the capacity of NSCs to migrate toward the tumor(Ehtesham et al., 2004). Human epidermal growth factor receptor (HER2) protein enhances tumor metastasis, and HER2 is regulated by CXCR4. Thus, CXCR4 is related to tumor growth(Li et al., 2004). Hypoxia and hypoxia-inducible factor 1 (HIF-1) play critical roles in glioblastoma and are also associated with both VEGF and CXCR4(Zagzag et al., 2006). A recent study using F3 human NSCs has demonstrated that the human glioma cell lines U87 and U251 produce HGF and VEGF, which act as potent chemoattractants for F3 human NSCs. These growth factors, HGF and VEGF, stimulate receptor tyrosine kinase signaling that leads to the activation of phosphoinositide 3-kinase (PI3K), Inhibition of the PI3K pathway significantly inhibited the chemotactic cell migration towards all growth factors tested (HGF, VEGF and EGF),
במ
suggesting that the growth factors produced by brain tumors converge on the PI3K signaling pathway(Kendall et al., 2008).
In the present study, we measured numbers of proliferating NSCs both in vitro and in vivo. NSCs showed an increase in proliferating activity until 2 days and a decrease thereafter in vitro. In particular, NSCs increased in number approximately 1.7 fold during day 1 in the absence of tumor cell inoculation, indicating that, in addition to the continuous migration of NSCs from the transplantation site to tumor region, migrating NSCs underwent division in the tumor region soon after injection. However, the proliferation of NSCs began to decline after 5 days following injection. Therefore, NSCs may play important roles as vehicles for the delivery of suicidal genes into the tumor region. Results of the present study should contribute to the development of preclinical strategies for gene therapy in malignant glioma.
גמ
Ⅴ
. CONCLUSION
1. F3 cells were first detected in the contra-lateral side at tumor region 40 min PI .
2. HB1.F3 migrated into tumor region approximately 10 % of total implanted cells in 50 minutes after NSCs injection.
3. In vitro, the doubling time of NSCs was approximately 24 hr, and the proliferative activity of F3, F3.lacZ, and U373MG cells was active for 2 days and decreased thereafter.
4. In vivo, injected F3 cells were continuously found from 50 min to 15 days PI in a same transplantation site. The presence of F3 NSCs was observed in various regions of the whole brain at 15 days after transplantation.
5. F3 cells increased in number over time up to 5 days PI and decreased thereafter.
6.
The number of transplanted F3 cells was increased approximately 1.7 fold during the first 24 hrs in the absence of tumor cell inoculation in vivo. However, the proliferation of NSCs began to decline after 5 days following injection.דמ
8. Several NSCs were observed in corpus callosum & hippocampus but not
found in cerebellum and they were still observed in hippocampus and auditory cortex until 10 days after NSCs implantation.
9. We found that the number of the NSCs increased slowly in a tumor region during 5 days after F3 injection, thereafter the F3 number started to increase dramatically by 15 days. The density of lacZ positive cells in a tumor region was increased until 1 day after injection and thereafter reduced by 15 days after F3 injection.
10. The velocity of NSCs was approximately 0.0175 cm/min.
11. F3 were found in both ipsi- and contra-lateral hemispheres in tumor cell-injected brains but only in the ipsi-lateral hemisphere where F3 NSCs were injected in controls in the absence of tumor cells.
המ
REFERENCES
1. Aboody KS, Najbauer J, Da
n
ks MK : Stem and progenitor cell-mediated tumor selective gene therapy. Gene Ther 15:739-752,20082. Aboody KS, Najbauer J, Schmidt NO, Yang W, Wu JK, Zhuge Y, Przylecki W, Carroll R, Black PM, Perides G : Targeting of melanoma brain metastases using engineered neural stem/progenitor cells. Neuro Oncol 8:119-126,2006
3. Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PM, Breakefield XO, Snyder EY : Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci U S A 97:12846-12851,2000
4. Anderson WF : Human gene therapy. Nature 392:25-30,1998
5. Bao PH, Wang M, Su J, Liu MY, Zhang HX : [Tropism of bone marrow mesenchymal stem cells in neurogenic differentiation for C6 conditioned medium and SDF-1alpha.]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 25:794-797,2009
ומ
tumors. J Neurosurg 79:729-735,19937. Barresi V, Belluardo N, Sipione S, Mudo G, Cattaneo E, Condorelli DF : Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy. Cancer Gene Ther 10:396-402,2003
8. Benedetti S, Pirola B, Pollo B, Magrassi L, Bruzzone MG, Rigamonti D, Galli R, Selleri S, Di Meco F, De Fraja C, Vescovi A, Cattaneo E, Finocchiaro G : Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 6:447-450,2000
9. Black PM LJ : Cancer of the Nervous System. Blackwell, Oxford,2005
10. Bourbeau D, Lavoie G, Nalbantoglu J, Massie B : Suicide gene therapy with an adenovirus expressing the fusion gene CD::UPRT in human glioblastomas: different sensitivities correlate with p53 status. J Gene Med 6:1320-1332,2004
11. Buckner FS, Wilson AJ, Van Voorhis WC : Detection of live Trypanosoma cruzi in tissues of infected mice by using histochemical stain for beta-galactosidase. Infect Immun 67:403-409,1999
זמ
12. Cho T, Bae JH, Choi HB, Kim SS, McLarnon JG, Suh-Kim H, Kim SU, Min CK : Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport 13:1447-1452,2002
13. Costantini LC, Bakowska JC, Breakefield XO, Isacson O : Gene therapy in the CNS. Gene Ther 7:93-109,2000
14. Crystal RG : Transfer of genes to humans: early lessons and obstacles to success. Science 270:404-410,1995
15. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM : In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256:1550-1552,1992
16. Edelstein ML, Abedi MR, Wixon J : Gene therapy clinical trials worldwide to 2007--an update. J Gene Med 9:833-842,2007
17. Ehtesham M, Yuan X, Kabos P, Chung NH, Liu G, Akasaki Y, Black KL, Yu JS : Glioma tropic neural stem cells consist of astrocytic precursors and their migratory capacity is mediated by CXCR4. Neoplasia 6:287-293,2004
חמ
18. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY : Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol 16:1033-1039,1998
19. Forsyth PA, Cairncross JG :) Treatment of malignant glioma in adults. Curr Opin Neurol 8:414-418,1995
20. Garcia Y, Breen A, Burugapalli K, Dockery P, Pandit A : Stereological methods to assess tissue response for tissue-engineered scaffolds. Biomaterials 28:175-186,2007
21. Ghosh S, Maity P : Vascular endothelial growth factor immunoneutralization in combination with cisplatin reduces EAC tumor growth. Int Immunopharmacol 6:1550-1559,2006
22. Giese A, Westphal M : Glioma invasion in the central nervous system. Neurosurgery 39:235-250; discussion 250-232,1996
23. Glass R, Synowitz M, Kronenberg G, Walzlein JH, Markovic DS, Wang LP, Gast D, Kiwit J, Kempermann G, Kettenmann H : Glioblastoma-induced attraction of
טמ
endogenous neural precursor cells is associated with improved survival. J Neurosci 25:2637-2646,2005
24. Hadaczek P, Mirek H, Berger MS, Bankiewicz K : Limited efficacy of gene transfer in herpes simplex virus-thymidine kinase/ganciclovir gene therapy for brain tumors. J Neurosurg 102:328-335,2005
25. Heese O, Disko A, Zirkel D, Westphal M, Lamszus K : Neural stem cell migration toward gliomas in vitro. Neuro-oncol 7:476-484,2005
26.Hemmrich K, Meersch M, von Heimburg D, Pallua N : Applicability of the Dyes CFSE, CM-DiI and PKH26 for Tracking of Human Preadipocytes to Evaluate Adipose Tissue Engineering. Cells Tissues Organs 184:117-127,2006
27. Hurelbrink CB, Armstrong RJ, Dunnett SB, Rosser AE, Barker RA : Neural cells from primary human striatal xenografts migrate extensively in the adult rat CNS. Eur J Neurosci 15:1255-1266,2002
.
28. mperato JP, Paleologos NA, Vick NA : Effects of treatment on long-term survivors with malignant astrocytomas. Ann Neurol 28:818-822,1990
נ
29. Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK : Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke 34:2258-2263,2003
30. Kendall SE, Najbauer J, Johnston HF, Metz MZ, Li S, Bowers M, Garcia E, Kim SU, Barish ME, Aboody KS, Glackin CA : Neural stem cell targeting of glioma is dependent on phosphoinositide 3-kinase signaling. Stem Cells 26:1575-1586 ,2008
31. Kim SK, Cargioli TG, Machluf M, Yang W, Sun Y, Al-Hashem R, Kim SU, Black PM, Carroll RS : PEX-producing human neural stem cells inhibit tumor growth in a mouse glioma model. Clin Cancer Res 11:5965-5970,2005
32. Kim SK, Kim SU, Park IH, Bang JH, Aboody KS, Wang KC, Cho BK, Kim M, Menon LG, Black PM, Carroll RS : Human neural stem cells target experimental intracranial medulloblastoma and deliver a therapeutic gene leading to tumor regression. Clin Cancer Res 12:5550-5556,2006a
33. Kim SU : Human neural stem cells genetically modified for brain repair in neurological disorders. Neuropathology 24:159-171,2004
אנ
Park IH : Production and characterization of immortal human neural stem cell line with multipotent differentiation property. Methods Mol Biol 438:103-121,2008
35. Kim SY, Choi KC, Chang MS, Kim MH, Kim SY, Na YS, Lee JE, Jin BK, Lee BH, Baik JH : The dopamine D2 receptor regulates the development of dopaminergic neurons via extracellular signal-regulated kinase and Nurr1 activation. J Neurosci 26:4567-4576,2006b
36. Kuhn HG, Dickinson-Anson H, Gage FH : Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16:2027-2033,1996
37. Larsen JO, Gundersen HJ, Nielsen J : Global spatial sampling with isotropic virtual planes: estimators of length density and total length in thick, arbitrarily orientated sections. J Microsc 191:238-248,1998
38. Lee HJ, Kim KS, Kim EJ, Choi HB, Lee KH, Park IH, Ko Y, Jeong SW, Kim SU : Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model. Stem Cells 25:1204-1212,2007
בנ
39. Lee ST, Chu K, Park JE, Lee K, Kang L, Kim SU, Kim M : Intravenous administration of human neural stem cells induces functional recovery in Huntington's disease rat model. Neurosci Res 52:243-249,2005
40. Li YM, Pan Y, Wei Y, Cheng X, Zhou BP, Tan M, Zhou X, Xia W, Hortobagyi GN, Yu D, Hung MC : Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 6:459-469,2004
41. Lin D, Najbauer J, Salvaterra PM, Mamelak AN, Barish ME, Garcia E, Metz MZ, Kendall SE, Bowers M, Kateb B, Kim SU, Johnson M, Aboody KS : Novel method for visualizing and modeling the spatial distribution of neural stem cells within intracranial glioma. Neuroimage 37 Suppl 1:S18-26,2007
42. Machein MR, Plate KH : VEGF in brain tumors. J Neurooncol 50:109-120,2000
43. Marchisone C, Pfeffer U, Del Grosso F, Noonan DM, Santi L, Albini A : Progress towards gene therapy for cancer. J Exp Clin Cancer Res 19:261-270,2000
44. Menon LG, Picinich S, Koneru R, Gao H, Lin SY, Koneru M, Mayer-Kuckuk P, Glod J, Banerjee D : Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow
גנ
cells. Stem Cells 25:520-528,200745. Mercapide J, Rappa G, Anzanello F, King J, Fodstad O, Lorico A : Primary gene-engineered neural stem/progenitor cells demonstrate tumor-selective migration and antitumor effects in glioma. Int J Cancer,2009
46. Moolten FL : Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46:5276-5281,1986
47. Najbauer J DM, Schmidt NO, Kim SU, Aboody KS : Neural stem cell-mediated therapy of primary and metastatic solid tumors. Progress in Gene Therapy, Autologous and Cancer Stem Cell Gene Therapy;World Scientific Vol 3,2007
48. Noble M : Can neural stem cells be used to track down and destroy migratory brain tumor cells while also providing a means of repairing tumor-associated damage? Proc Natl Acad Sci U S A 97:12393-12395,2000
49. Ourednik V, Ourednik J, Park KI, Snyder EY : Neural stem cells -- a versatile tool for cell replacement and gene therapy in the central nervous system. Clin Genet 56:267-278,1999
דנ
50. Paxinos G WC : The rat brain. Academic Press,1998
51. Phillips HS, Kharbanda S, Chen R, Forrest WF, Soriano RH, Wu TD, Misra A, Nigro JM, Colman H, Soroceanu L, Williams PM, Modrusan Z, Feuerstein BG, Aldape K : Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell 9:157-173,2006
52. Pulkkanen KJ, Yla-Herttuala S : Gene therapy for malignant glioma: current clinical status. Mol Ther 12:585-598,2005
53. Ratajczak MZ, Zuba-Surma E, Kucia M, Reca R, Wojakowski W, Ratajczak J : The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis. Leukemia 20:1915-1924,2006
54. Sanfeliu C, Wright JM, Kim SU : Neurotoxicity of isoniazid and its metabolites in cultures of mouse dorsal root ganglion neurons and hybrid neuronal cell line. Neurotoxicology 20:935-944,1999
הנ
KS, Carroll RS : Brain tumor tropism of transplanted human neural stem cells is induced by vascular endothelial growth factor. Neoplasia 7:623-629,2005
56. Shah K, Bureau E, Kim DE, Yang K, Tang Y, Weissleder R, Breakefield XO : Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Ann Neurol 57:34-41,2005
57. Shimato S, Natsume A, Takeuchi H, Wakabayashi T, Fujii M, Ito M, Ito S, Park IH, Bang JH, Kim SU, Yoshida J : Human neural stem cells target and deliver therapeutic gene to experimental leptomeningeal medulloblastoma. Gene Ther 14:1132-1142,2007
58. Short BG, Zimmerman DM, Schwartz LW : Automated double labeling of proliferation and apoptosis in glutathione S-transferase-positive hepatocytes in rats. J Histochem Cytochem 45:1299-1305,1997
59. Snyder EY : Immortalized neural stem cells: insights into development; prospects for gene therapy and repair. Proc Assoc Am Physicians 107:195-204,1995
60. Surawicz TS, McCarthy BJ, Kupelian V, Jukich PJ, Bruner JM, Davis FG : Descriptive epidemiology of primary brain and CNS tumors: results from the
ונ
Central Brain Tumor Registry of the United States, 1990-1994. Neuro Oncol 1:14-25,1999
61. Titford ME, Horenstein MG : Histomorphologic assessment of formalin substitute fixatives for diagnostic surgical pathology. Arch Pathol Lab Med 129:502-506,2005
62. ogel TW, Zhuang Z, Li J, Okamoto H, Furuta M, Lee YS, Zeng W, Oldfield EH, Vortmeyer AO, Weil RJ : Proteins and protein pattern differences between glioma cell lines and glioblastoma multiforme. Clin Cancer Res 11:3624-3632,2005
63. West MJ : New stereological methods for counting neurons. Neurobiol Aging 14:275-285,1993
64, Yasuhara T, Matsukawa N, Hara K, Yu G, Xu L, Maki M, Kim SU, Borlongan CV : Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson's disease. J Neurosci 26:12497-12511,2006
65. Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Mendez O, Yee H, Voura EB, Newcomb EW : Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion. Lab Invest
