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DOI: 10.1148/radiol.2413050466
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(Radiology 2006;241:822-830.)
© RSNA, 2006

Molecular Imaging

Neural Stem Cell Transplant Survival in Brains of Mice: Assessing the Effect of Immunity and Ischemia by using Real-time Bioluminescent Imaging1

Dong-Eog Kim, MD, PhD2, Kiyoshi Tsuji, MD, Young Ro Kim, PhD, Franz-Josef Mueller, MD, Hyeon-Seok Eom, MD, PhD3, Evan Y. Snyder, MD, PhD, Eng H. Lo, PhD, Ralph Weissleder, MD, PhD and Dawid Schellingerhout, MBChB

1 From the Center for Molecular Imaging Research (D.E.K., R.W., D.S.), Neuroprotection Research Laboratory (K.T., E.H.L.), and NMR Center, Department of Radiology (Y.R.K.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass; Program in Developmental & Regenerative Cell Biology, Burnham Institute, La Jolla, Calif (F.J.M., E.Y.S.); and Catholic Hematopoietic Stem Cell Transplantation Center, Catholic University of Korea, Seoul, Korea (H.S.E.). Received March 18, 2005; revision requested May 19; revision received August 4; accepted September 1; final version accepted March 6, 2006. D.S. supported in part by grants from the Radiological Society of North America Research and Education Foundation and the American Brain Tumor Association. Address correspondence to D.S., Neuroradiology Section, Departments of Radiology and Experimental Diagnostic Imaging, University of Texas M. D. Anderson Cancer Center, Box 57, 1515 Holcombe Blvd, Houston, TX 77030 (e-mail: dawid.schellingerhout{at}di.mdacc.ut.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To use bioluminescent imaging in a murine transplant model to monitor the in vivo responses of transplanted luciferase-gene-positive neural progenitor cells (NPCs) to host immunity and ischemia.

Materials and Methods: All animal studies were conducted according to institutional guidelines, with approval of the Subcommittee on Research Animal Care. Cranial windows were created in all animals, and all animals underwent NPC (C17.2-Luc-GFP-gal) transplantation into the right basal ganglia. An observational study was performed on C57 BL/6 (n = 5), nude (n = 4), and CD-1 (n = 4) mice, with bioluminescent imaging performed at days 7, 11, and 14 after transplantation. A study on the effects of ischemia was performed in a similar manner, but with the following differences: On day 9 after transplantation, the C57 BL/6 mice underwent 18 minutes of transient forebrain ischemia by means of temporary bilateral carotid occlusions (n = 6). A control group of C57 BL/6 mice underwent sham surgery (n = 6). Bioluminescent imaging was performed on the ischemic animals and control animals at days 7, 9, 11, and 14. Repeated-measures analysis of variance or Student t test was used to compare the means of the luciferase activities.

Results: In vivo cell tracking demonstrated that (a) C17.2-Luc-GFP-gal NPCs survived and proliferated better in the T-cell deficient nude mice than in the immunocompetent C57 BL/6 or CD-1 mice, in which progressive immune mediated cell loss was shown, and (b) transient forebrain ischemia appeared, unexpectedly, to act as a short-term stimulus to transplanted NPC growth and survival in immunocompetent mice.

Conclusion: Immune status and host immunity can have an influence on NPC graft survival, and these changes can be noninvasively assessed with bioluminescent imaging in this experimental model.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
During the course of cerebral ischemia, transplanted neural progenitor cells (NPCs) are stimulated to migrate and differentiate in order to effect and participate in brain repair. The mechanism of this response to pathologic conditions of the central nervous system is not completely understood at present but is likely to be a dynamic process characterized by interplay between the NPC transplant and the microenvironment of the diseased host brain (1). The details of this interplay are likely to be of importance in the future use of stem cell therapies for neurologic diseases.

The central nervous system is often thought of as an immunoprivileged environment and thus not subject to graft-host immune interactions in the same way as other organs (2,3). NPCs were reported to have low immunogenic properties without expressing major histocompatibility complex (MHC) antigens at levels above the threshold of detection and thus to be relatively inert to immune surveillance (4,5). Implantation of these cells into the central nervous system may be one of the most favorable situations for transplant survival. In fact, it was suggested that NPCs could be grafted into allogenic recipients without the need for immunosuppression (4,5).

However, there is mounting evidence that the central nervous system may not be as immunoprivileged as originally thought. New data point to a major role for the immune system during NPC chemotaxis to sites of ischemic injury (6) and the expression of MHC antigens on transplanted and host cells in other models of traumatic cerebral injury (7). Disruption of the blood-brain barrier that occurs for up to a week following transplantation surgery could allow the unshielded NPCs to be surveyed by the host immune system (8).

Similarly, NPCs may lose their favorable immunologic profiles under certain circumstances (9). Exposure of NPCs to interferon {gamma} (IFN{gamma}) in vitro could upregulate expression of MHC antigens (4). Thus, it is not unreasonable to propose that the host immune system, along with a diseased central nervous system environment, could play an important role in NPC transplantation to the brain.

The field of stem cell biology is facing major challenges in translating advances in progenitor cell–based therapies into clinical practice. There is a need for imaging technology that can assess the status of transplanted progenitor cells, allowing the noninvasive assessment of cell survival, rejection, and anatomic location.

Thus, the purpose of our study was to use bioluminescent imaging in a murine transplant model to monitor the in vivo responses of transplanted luciferase-gene-positive NPCs to host immunity and ischemia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
C17.2-Luc-GFP-gal NPC Line
The lacZ-positive C17.2 cell line (10,11) is a clonal, multipotent murine precursor cell line (isolated from K-strain mice [12] having CD-1 x C57 BL/6J hybrid animals as founders), immortalized by means of v-myc transformation. This cell line has previously been well documented in terms of migratory ability and homing toward brain disease (13–15). These cells have also been shown to undergo differentiation into diverse neuronal and glial cell types, with replacement of degenerated or inadequately developed cells and concomitant improvement of behavioral outcomes (16–18). The C17.2-gal NPC line was further modified by stable transfection with the firefly luciferase gene (Luc) and the green fluorescent protein gene (GFP) under the control of constitutive cytomegalovirus promotors, without changing their stem cell properties (13).

In Vitro Characterization of the C17.2-Luc-GFP-gal NPC Line
The C17.2-Luc-GFP-gal (hereafter, C17.2-Luc) NPCs were plated, washed with phosphate-buffered saline, fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 minutes, permeabilized in Tris-buffered saline, or TBS, with a pH of 7.4, plus 0.05% Triton X-100 (ie, TBS-Triton) and blocked in TBS-Triton plus 5% normal goat serum for 60 minutes. Cells were then incubated with a rabbit anti-ß-galactosidase antibody (1:100, Abcam, Cambridge, England) and an anti-nestin monoclonal antibody (1:500, Chemicon International, Temecula, Canada) for 1 hour at 37°C. Mouse NeuN (1:100, Chemicon International)/MAP2 (1:50, BD PharMingen, San Diego, Calif) antibodies and a rabbit GFAP antibody (1:10, DakoCytomation, Carpinteria, Calif) were used in different sets of cells. Cells were then washed and incubated with either a goat anti-rabbit Alexa dye (488 nm) or a goat anti-mouse Alexa dye (594 nm) conjugated secondary antibody (Molecular Probes, Eugene, Ore) for 1 hour, then washed, mounted, and examined microscopically.

MHC expression was assessed (D.E.K., H.S.E., F.J.M., E.Y.S., D.S.) with a flow cytometer (FACScan; Becton Dickinson, Mountain View, Calif). A total of 1 x 106 C17.2-Luc NPCs were stained with anti-MHC class I anti-Db (clone KH-95) fluorescein-isothiocyanate (FITC) and anti-MHC class II anti-I-Ab (clone 25–9-17) FITC. As negative controls, HOPC-FITC and rat IgG2a-PE (BD PharMingen) were used. A total of 10 000 events were acquired per sample.

Stimulation and Assessment of MHC I Expression on Neural Stem Cells with Flow Cytometry
NPCs were plated in six-well plates, and all stimulation experiments (F.J.M., E.Y.S.) were performed on confluent cultures. Cells were stimulated with an eightfold serial dilution of murine IFN{gamma} (Chemicon International) with a starting concentration of 250 U/mL IFN{gamma} (final concentration for each experiment, 250/2n U/mL; n = 0, 1, 2, 3, 4, 5, 6, 7, and 8) This resulted in a concentration range between 250 and approximately 1 U/mL IFN{gamma}. An unstimulated control was analyzed with every time point in this study. Cells were stimulated for 1, 2, 3, and 4 days with the indicated concentration of IFN{gamma}. The cells were dissociated with enzyme-free dissociation buffer (Invitrogen, Carlsbad, Calif) fixed with 2% paraformaldehyde, split into four groups. The results of one sample per time point and stimulation condition was plotted (Fig 1). The results were validated with experimental replicates of days 1 and 2 (data not shown).


Figure 1
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  		Figure 1: Characterization of C17.2-Luc NPC before transplantation. A, Phase-contrast light micrograph of cells, with fluorescent immunohistochemistry stains showing nestin (red) and ß-galactosidase (Beta-gal) (green). A fusion image of these fluorescent markers demostrates marker co-localization. (Original magnification, x20.) B, Expression of C57 BL/6 MHC I (tested with antibody 1) after 4 days (4d) of stimulation with 250 U murine IFN{gamma} and no stimulation control, as well as according isotype controls. Data indicate upregulation of MHC I in response to IFN{gamma} (12.8% of NPC > 2nd log vs 2.71% of NPC > 2.71%). C, Plot of percentage of MHC I–positive neural stem cells (z-axis) in relationship to duration of stimulation with IFN{gamma} (x-axis) and concentration of IFN{gamma} in the stimulation medium (y-axis). Cells were stimulated for 1, 2, 3, or 4 days with 250/2n U murine IFN{gamma} (n = 0, 1, 2, 3, 4, 5, 6, 7, and 8). This results in a dilution curve with nine dilutions between 250 and approximately 1 U IFN{gamma}. A control group with no IFN{gamma} stimulation was plotted as zero value. Values between the assessed data points are extrapolated. Percentage of MHC I–positive cells was also color-coded (color profile displayed next to chart). The chart shows a marked-up regulation of MHC I in a time- and dose-dependent fashion in response to IFN{gamma}, with a pronounced response even at low IFN{gamma} concentrations.

 
Samples were stained with a total of four different FITC-labeled antibodies: Antibody 1 was anti-major MHC class I anti-Db (clone KH-95, C57 BL/6 specific). Antibody 2 was anti-major MHC class I anti-H2-Kd (clone SF1-1.1, Balb/C specific, since the C17.2 NPC line was derived from a mixed C57 BL/6 and Balb/C background). No anti-H2-Kd–positive cells were detectable under any circumstance (data not shown), and therefore this antibody also served as a negative control. Antibody 3 was rat IgG2a-FITC (clone DD3; Chemicon International), used as an isotype control for antibody 1. Antibody 4 was mouse IgG2b-FITC (clone DD311; Chemicon International), used as an isotype control for antibody 2.

Antibody staining was assessed with a FACSort flow cytometer (Becton Dickinson). A total of 10 000 events were acquired per sample, and the data were further analyzed with the FlowJoV6 software package (Treestar, Ashland, Ore) and the Aabel 1.5.8 statistical and data visualization software package (Gigawiz, Tulsa, Okla).

Animal Numbers and Study Design
All animal studies were conducted according to institutional guidelines at Massachusetts General Hospital, with approval of the Subcommittee on Research Animal Care.

We previously reported that stem cells can be visualized in vivo and studied longitudinally in the same animal over time by means of bioluminescent imaging (14). There was excellent correlation between luciferase imaging and histologic findings. However, bioluminescent imaging is known to be affected by photon attenuation in intervening tissues such as skin, skull, or hair. This would make it difficult to compare emitted photon counts between different animal strains. Thus, we elected to use a cranial window model to control for these variables.

Immunocompromised nude mice (NU/NU mice, n = 4, female, 25 g) and two kinds of immunocompetent mice, C57 BL/6 (hereafter, C57) mice (n = 17, female, 25 g) and CD-1 mice (n = 4, female, 25 g), were used for in vivo studies. All the animals were purchased from Charles River Laboratories (Cambridge, Mass). A cranial window was surgically inserted and C17.2-Luc NPC were transplanted (see next section) through the craniotomy before inserting the glass window.

To study the proliferation and survival of C17.2-Luc NPC in response to an ischemic episode, 9 days after transplantation, C57 mice underwent 18 minutes of transient forebrain ischemia (n = 6) or sham surgery without ischemia (control group, n = 6) (D.E.K., Y.R.K., D.S.). Luciferase imaging was performed at days 7, 9, 11, and 14. For selected animals, additional imaging was performed at 1 hour, 12 hours, and 24 hours after ischemia.

To investigate the fate of C17.2-Luc NPCs in response to the host immunity, the C57 (n = 5), nude (n = 4), and CD-1 (n = 4) mice underwent serial luciferase imaging at days 7, 11, and 14. These time points were chosen because the results of prior work had shown neuroinflammatory immune responses to hypoxic ischemic disease to be most pronounced at 2 weeks after insult (19,20).

Animals were sacrificed after the last imaging session.

Cranial Window and Cell Implantation
Surgical creation of a cranial window was performed (D.E.K., Y.R.K.) as previously described (21), with some modifications. In brief, after anesthesia was induced with intraperitoneal injection of ketamine and xylazine (90 and 10 mg per kilogram of body weight, respectively), a circular craniotomy (5 mm in diameter, center located 1 mm posterior to the bregma) was created by using a high-speed microdrill with a small steel burr (Fine Science Tools, North Vancouver, Canada), while the animal was immobilized in a stereotaxic frame (David Kopf Instruments, Tujunga, Calif). C17.2-Luc NPCs (1 x 106 in 4 µL of Hanks solution) were stereotactically injected into the right basal ganglia over 15 minutes at the following coordinates: 0 mm anterior, 2 mm lateral, and 2 mm deep. A glass coverslip (Warner Instruments, Hamden, Conn) was used to cover the craniotomy, with dental cement (Co-oral-ite, Dental Manufacturing, Santa Monica, Calif) used to achieve an airtight seal.

Transient Forebrain Ischemia
Animals were anesthetized with 1.0% halothane in a mixture of 30% oxygen and 70% nitrogen. A rectal thermometer with a heating pad (DC temperature control module; FHC, Bowdoinham, Me) was used to maintain body temperature at 36°C. By using a surgical microscope (Carl Zeiss, Oberkochen, Germany), the bilateral common carotid arteries were exposed after a ventromedial cervical skin incision. The bilateral common carotid arteries were occluded with small aneurysm clips for 18 minutes. Laser Doppler flowmetry (Periflux 5000; Perimed, Järfälla, Sweden) was used to confirm that during occlusion, cerebral perfusion was decreased to below 10% of normal preischemic baseline levels (n = 6). The clips were removed for reperfusion. After closure of the surgical wounds, animals were allowed to recover for the indicated periods before being sacrificed. Control animals undergoing sham operations were treated as described above but without bilateral common carotid artery occlusion.

In Vivo Cell Tracking with Bioluminescence Imaging
Luciferase imaging with use of C17.2-Luc NPCs (D.E.K., K.T., D.S.) was performed as previously described (14). In brief, a custom-built imaging system that used a cryogenically cooled high-efficiency charge-coupled device camera system (Roper Scientific, Trenton, NJ) was used to perform photon counting in anesthetized animals after intraperitoneal injection with D-luciferin (Biosynth, Chicago, Ill; 160 µg per gram of body weight). White-light surface images were obtained immediately before each photon counting experiment to provide an anatomic outline of the animal. Images were acquired from 5 to 15 minutes after D-luciferin administration. The distribution of luciferase activity within the brain of the animal was then measured by recording photon counts with the charge-coupled device. Image processing was performed by using a custom-written program (CMIR Image; Edward Graves, Massachusetts General Hospital, Boston, Mass). Images were displayed as a false-color photon count image superimposed on a gray-scale anatomic white-light image. Image analysis software was used to quantify stem cell–related photon emissions from the mice. These numbers were subjected to standard statistical analysis (see below). Imaging was performed at multiple time points (days 7, 9, 11, and 14 after transplantation).

Tissue Processing and Histologic Evaluation
Immediately following the final imaging session, mice were sacrificed and brains were harvested. Horizontal 30-µm brain sections (coronal in the mouse frame of reference) were acquired and stored at –80°C until processed for immunohistochemistry.

The lacZ gene product of the transplanted cells was probed with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal; Fisher Scientific, Pittsburgh, Pa) staining, and counterstaining with eosin was performed. The blue X-Gal stain indicated the presence and location of transplanted stem cells.

For immunostaining to demonstrate inflammatory cells around the implantation site (D.E.K., K.T., H.S.E., E.H.L., D.S.), the sections were incubated with a mouse anti-mouse CD45 (leukocyte common antigen) antibody conjugated with FITC (1:64, BD PharMingen, San Diego, Calif), or anti-Mac-1 antibody conjugated with phycoerythrin (1:500, BD PharMingen) at 4°C overnight in a humid chamber. After the washing with phosphate-buffered saline, nuclear counterstaining was performed by using 4',6-diamidine-2-phenylindole (DAPI; DakoCytomation, Carpinteria, Calif). Fluorescent stain indicated the presence and location of the various types of immunocytes.

Statistical Analysis
Data are presented as mean ± standard error. Repeated-measures analysis of variance and Dunnet post hoc test were used to compare means of the photon counts acquired at multiple time points in three different groups of the C57, CD-1, and nude mice. The Student t test was used to compare the mean of the luciferase photon count percentages (normalized percentages relative to the baseline photon counts at day 7) of the C57 mice at day 14 between the ischemia and sham surgery groups. P < .05 was considered to indicate a statistically significant difference. A statistical software package (SPSS, version 8.0; SPSS, Chicago, Ill) was used for data analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
C17.2-Luc NPCs Express Nestin but Not MHC I Antigens during Resting (Unstimulated) Conditions
C17.2-Luc NPCs were nestin-positive and expressed ß-galactosidase as a marker gene. Cells did not express differentiated neuronal or glial markers such as NeuN, MAP2, and GFAP. Flow cytometry analysis showed that they did not express anti-C57 BL/6 class I or II MHC markers (Fig 1).

C17.2-Luc NPCs Express C57 BL/6 MHC I Antigens at Stimulation with IFN{gamma}
C17.2-Luc NPCs were stimulated with escalating doses of IFN{gamma} and showed a marked time- and dose-dependent upregulation of C57 BL/6 MHC I antigen (antibody 1) (Fig 1). Similar experiments were performed with SDF1-{alpha}, but no upregulation of MHC I was observed (data not shown). The MHC I expression of unmodified C17.2 cells and C17.2-Luc transfected cells were compared, and no significant difference in baseline and stimulated MHC I expression of C57 BL/6 MHC I was demonstrated (data not shown). Since the C17.2 was isolated from a mixed background (C57 BL/6 and Balb/C), and thus could have been also positive for the Balb/C MHC I antigen, we tested for this epitope as well and found the cells to be negative for this strain-specific epitope in any circumstance (tested with antibody 2). We validated both antibodies (antibodies 1 and 2) with the appropriate isotype controls in every experimental condition and did not observe any binding of the control antibodies 3 and 4.

Survival and Proliferation of C17.2-Luc NPCs are Higher in Immunocompromised Mice than in Immunocompetent Mice
To determine the influence of immune systems on cell survival, we compared NPC profiles in C57, CD-1, and nude mice. In all of the C57 and CD-1 mice, the initial luciferase activities (90 ± 77 and 112 ± 59, respectively) at day 7 decreased to below the threshold of detectability over the next 7 days. However, the mean luciferase activity in the nude mice increased from 369 ± 205 to 1644 ± 805 (P < .05 vs C57 and CD-1 mice at day 14; repeated-measures analysis of variance and Dunnet post hoc test) (Fig 2).


Figure 2
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  		Figure 2: Survival and proliferation of C17.2-Luc NPCs in immunocompromised and immunocompetent mice. C57, CD-1, and nude mice are shown, with time after NPC implantation indicated in days (eg, D14). Top left, Representative animals show typical luciferase imaging results, with NPC-related photon counts showing an increasing trend in nude mice but a decreasing trend in CD-1 and C57 mice. Bottom left, Quantitative analysis shows that in most C57 and CD-1 mice, initial luciferase activities (90 ± 77 and 112 ± 59, respectively) at day 7 decreased to below the imaging threshold over 7 days. However, the mean luciferase activity in the nude mice increased from 369 to 1644 (P < .05 vs C57 and CD-1 mice at day 14; repeated-measures analysis of variance and Dunnet post hoc test). Right, Consistent with corresponding luciferase imaging, in the C57 and CD-1 mice, X-Gal–positive C17.2-Luc NPCs are observed to be fewer in number than in the nude mouse. In addition, cells that are not X-Gal–positive cluster densely around the implantation sites (arrow).

 
Inflammatory Cells Cluster around Implantation Site in Immunocompetent Mice but Not Immunocompromised Mice
In nude mice, 14 days after the transplantation into the frontal lobe, X-Gal–positive C17.2-Luc NPCs were mainly observed around the implantation site (Fig 2) and in the adjacent subventricular zone. C17.2-Luc NPCs were less numerous in C57 mice and CD-1 mice, which was consistent with the bioluminescent imaging data. In addition, cells that were not X-Gal–positive clustered around the implantation site (Fig 2). Fluorescent staining of the adjacent sections (Fig 3) showed that the clustered cells were CD45-positive cells, which most likely represented infiltrating peripheral leukocytes. The outer rim of the cell cluster consisted of both CD45- and CD11b-positive macrophages and/or microglia cells (22). In contrast, CD45- or CD11b-positive cells were rarely observed infiltrating the transplantation site in nude mice (Fig 3).


Figure 3
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  		Figure 3: Fluorescent micrographs. A, B, Inflammatory cells cluster around the NPC implantation site (arrow) in immunocompetent mice. C, D, In contrast to immunocompromised nude mice, C57 mice have CD45-positive myelomonocytic cells (green) clustering densely near the implantation site (arrowhead). The outer rim of the cell cluster consists of both CD45- and CD11b (red)-positive monocytic cells. DAPI = 4',6-diamidine-2-phenylindole, scale bars = 50 µm.

 
The survival and proliferation of C17.2-Luc NPCs transplanted into the brains of C57 mice are influenced by ischemic episodes.

Baseline imaging was performed 7 days after NPC implantation (ie, day 7). Transient forebrain ischemia or sham surgery was performed at day 9, and imaging was performed immediately before the insult and thereafter. In line with the results of the previous experiment to determine the influence of immune systems on cell survival (Fig 2), all control C57 mice showed decreased luciferase activities at day 14 compared with day 9. However, this was not the case in the ischemia group. In contrast to a steady decline in the control group, a resurgence of the bioluminescent photon counts was observed at day 14 in the ischemia group (Figs 4 and 5). The mean of the normalized percentage of the luciferase photon counts at day 14 in the ischemia group (34 ± 13) was higher than that in the control group (1.9 ± 1.1), which showed marginal nonsignificance (P = .07, Student t test).


Figure 4
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  		Figure 4: Image shows that survival and proliferation of C17.2-Luc NPCs transplanted into brains of C57 mice are affected by ischemic episodes. In this image, false-color luciferase photon emissions are superimposed on gray-scale white-light images. The photon emissions in the representative sham-surgery animal (top row) gradually decrease over time, in contrast to the gradual increase in the animal that received 18 minutes of forebrain ischemia (vertical gray line). Time in days is indicated, with day 0 as the time of NPC implantation. After sham surgery or forebrain ischemia, serial follow-up images are shown (D = day, h = hour).

 

Figure 5
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  		Figure 5: Graph shows influence of ischemia on NPC survival in C57 mice. After imaging at 7 and 9 days after NPC implantation (D7 and D9), C57 mice underwent either 18 minutes of forebrain ischemia (n = 6; gray, right column in each category) or sham surgery (n = 6; white, left column in each category); arrow indicates the time of ischemia or sham surgery. All control mice show decreased luciferase activities (normalized percentage relative to the baseline value at day 7) at day 14 compared with day 9. However, a resurge of bioluminescent photon counts are observed in the ischemia group. Thus, luciferase activities at day 14 in the ischemia group tend to be higher than those in the control group (P = .07).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
Our data demonstrate that although the brain is traditionally considered to be immune-privileged, progenitor cell survival is still substantially dampened by an active immune system.

Transient forebrain ischemia paradoxically caused a temporary reversal of NPC loss in immunocompetent C57 mice (marginally nonsignificant).

Immune System and NPC Survival
NPC were reported to have low immunogenic properties and thus to be relatively inert to immune surveillance (4,5). C17.2-Luc NPC strongly expressed the stem cell marker nestin but no detectable MHC I surface antigens under basal (unstimulated) conditions in vitro. However, findings of our study show that MHC I expression on neural stem cells can be induced by an inflammatory factor (IFN{gamma}) and that active transplant rejection in the central nervous system occurs in immunocompetent animals in vivo. IFN{gamma} has been widely implicated in various acute and chronic brain diseases (23–27), and thus, it is likely that the phenomenon we observed occurs more widely. This supports previous notions that the immunoprivilege of NPCs and the central nervous system may not be absolute (9,28–31).

It has been shown in previous work that CD80/CD86-mediated interaction of IFN{gamma}-stimulated neural stem cells with T-cells can lead to the demise of the neural precursors (32). This mechanism was shown in the context of primary inflammatory lesions in mouse models of multiple sclerosis. We therefore used T-cell–deficient nude mice in our transplantation paradigm to extend these observations to a brain disease that is not primarily inflammatory. Mismatching MHC I–expression can be the first signal for CD8–T-cells to differentiate into cytotoxic T-cells. The second signal can be either CD80/CD86 or the presence of cytokines like IFN{gamma}. We chose to use IFN{gamma} in our in vitro experiments.

Compared with the immunocompetent mice, nude mice did not show histologic evidence suggestive of an inflammatory response. In vivo imaging and histologic results demonstrated that higher numbers of C17.2-Luc NPCs survived in the T-cell–deficient nude mice. Therefore, T-cell immunity appears to have had an important role in determining the fate of the C17.2-Luc NPCs in our experimental settings.

Ischemia and NPC Survival
Ischemia is known to have profound effects on NPC behavior, causing them to migrate and home to sites of disease with subsequent differentiation (14,33,34). The biochemical mediators of these behaviors are as yet unclear, but reelin (35,36), SCF (37), and SDF-1 (38,39) have been implicated in homing behavior. Stem cell numbers are regulated by a second set of mediators, trophic factors such as basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor, all of which are typically required for the maintenance of human NPCs in culture (40).

We observed a remarkable reversal of the decrease in NPCs at day 14 in immunocomptetent animals in response to ischemia. This was the only time point at which ischemic and nonischemic groups approached a significant difference in NPC graft survival. It is interesting to note that previous work with similar models of global ischemia demonstrated peak bromodeoxyuridine incorporation at 4–9 days in the native dentate gyri of mice (41), which suggests a link between ischemic stimulus and NPC proliferation.

Possible Proregenerative Interactions between the Immune System, Ischemic Pathophysiology, and Neural Stem Cells
This transient reverse in NPC number decline in immunocompetent animals subject to ischemia indicates to us that the balance between pro-death and anti-death signals was transiently shifted toward a state favoring the survival of C17.2. This is likely due to a prosurvival microenvironment created by the hypoxic-ischemic disease. Trophic mediators released by ischemic or surrounding tissues likely have caused the exogeneous neural stem cells to proliferate in our experimental setup (42–44). We believe that this interesting observation requires further investigation, especially since most in vivo studies on the interaction of neural stem cells and the neuroinflammatory environment were undertaken with the a priori assumption of a detrimental influence of inflammation on neural stem cells (45,46). This is the first report, to our knowledge, of a possible favorable influence of at least certain phases of neuroinflammation on neural stem cell survival and/or proliferation in vivo.

Limitations
There are several caveats to be considered in the interpretation of this data. First, serial luciferase activities that quantitatively reflect changes of C17.2-Luc NPC counts in the same animal over time could in fact be compared between different strains (nude mice vs C57 or CD-1 mice), because a cranial window had been made to minimize strain differences in terms of light scattering by the skull, hair, and other intervening tissues, thus obviating many potential interspecies differences. The cranial window is also believed to have increased the sensitivity of the luciferase imaging, since very small numbers (less than 50 X-Gal–positive cells per 30-µm brain section) of C17.2-Luc NPCs could be reliably detected at in vivo imaging. However, further studies are required to rule out the possibility that the cranial window might have altered the present results in some way, such as by inducing or aggravating the immuno-inflammatory reaction observed. Second, we used a global ischemic model. Alternative ischemic models, including focal ischemia, might yield different results. Third, small sample sizes may have caused a low specificity or a decreased power of the study. Last, the genetic modification of stem cells—such as the insertion of marker genes—represents an experimental influence that may or may not be important and is difficult to control for.

Conclusion
Immune status and host immunity have an influence on NPC graft survival, and these changes can be noninvasively assessed with bioluminescent imaging in our experimental model.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: FITC = fluorescein-isothiocyanate • IFN{gamma} = interferon {gamma} • MHC = major histocompatibility complex • NPC = neural progenitor cell

2 Current address: Department of Neurology, DongGuk University International Hospital, Goyang City, Gyeonggi-do, Korea Back

3 Current address: Department of Hemato-oncology, National Cancer Center, Goyang City, Gyeonggi-do, Korea Back

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, D.E.K., D.S.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, D.E.K., F.J.M., E.Y.S., D.S.; experimental studies, D.E.K., K.T., Y.R.K., F.J.M., H.S.E., E.H.L., D.S.; statistical analysis, D.E.K., F.J.M., E.Y.S., E.H.L., R.W., D.S.; and manuscript editing, D.E.K., F.J.M., E.Y.S., R.W., D.S.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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