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Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, Florida, USA
Key Words. Green fluorescent protein • Irradiation • Multipotent astrocytic stem cell • Neurosphere • Neuroblast • Neural stem cell • Subependymal zone
Correspondence: Gregory P. Marshall II, Ph.D., 1600 SW Archer Road, Gainesville, Florida 32611, USA. Telephone: 352-392-0231; Fax: 352-392-0025; e-mail: gpm2{at}ufl.edu
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ABSTRACT |
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INTRODUCTION |
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NSs are capable of increasing in number after repeated passages in culture while retaining both their multipotency (self-renewal) and the ability to integrate into host neural tissue upon transplantation [18], leading many to believe that the NS-forming cell is the in vitro correlate of the in vivo NSC. Multipotent astrocytic stem cells (MASCs) isolated from the SEZ of neonatal mice have also been identified as an in vitro correlate of the NSC, displaying the potential not only to generate multipotent NSs but also to integrate into host neural tissue upon transplantation in much the same way as the NS [21, 22].
Transplantation of NSs or MASCs derived from animals transgenic for the reporter gene encoding green fluorescent protein (gfp+) into the LV of normal adult C57BL/6 mice results in minimal engraftment, with few donor-derived neuroblasts present in the RMS or OB. Transplantation into neonatal mice, however, results in relatively robust levels of engraftment with comparatively high numbers of donor-derived neuroblasts and interneurons present in the OB weeks after transplantation. This presents a quandary in the research of adult NSCs because neonatal and adult brains are dramatically different with respect to graft receptivity. A method for enhancing the engraftment of donor cells into adult neurogenic regions is crucial to a better understanding of the differentiation and integration potential of grafted cell types.
Perhaps the most well-characterized adult stem cell is the hematopoietic stem cell (HSC), which, in addition to the aforementioned stem cell characteristics of pluripotency and self-renewal, is also capable of long-term reconstitution of the entire hematopoietic system of a myeloablated animal. In fact, ablation of endogenous HSCs by exposure to high doses of ionizing radiation is critical for facilitating maximum engraftment of transplanted HSCs, which cannot normally compete with the native HSCs for access to the stem cell niche [23–25].
Depletion of the NSC niche has been previously achieved with antimitotic agents [26] and both ionizing and x-irradiation [27–32], yielding transient and long-term depletion of neurogenesis in the hippocampus and SEZ. Neurogenesis in the hippocampus can be attenuated by exposure to varying levels of x-irradiation, as seen by a decrease in the number of migrating granular neurons out of the subgranular layer [28–30, 32]. Previous studies investigating the effects of focused exposure of x-irradiation to the brain of adult rats on SEZ neurogenesis have reported ablation of NSCs in the SEZ immediately after irradiation [27, 31]. A dose-dependent recovery of NSCs occurred within 2 months of exposure, with recovery never reaching basal levels in all but the lowest doses. Little has been done to characterize the effects of a single, whole-body lethal or sublethal dose of ionizing radiation on SEZ neurogenesis and subsequent engraftment of transplanted in vitro–derived NSCs in the adult mouse.
We show here that a single, lethal dose of ionizing radiation significantly depletes the SEZ, RMS, and OB of migrating neuroblasts and that this depletion persists at 3 months after irradiation. The SEZ of lethally irradiated (LI) mice contains fewer neurosphere-forming cells than untreated, age-matched mice, indicating that the stem cell pool of the SEZ has been adversely affected by the radiation. LI renders the SEZ unreceptive to gfp+ MASCs transplanted into the LV, as no donor-derived cells were observed in the OB after transplantation, in contrast to engraftment seen in control mice.
Exposure to milder levels of radiation resulted in a transient decrease in mitotic SEZ neuroblasts, with donor-derived MASC engraftment into the OB at significantly higher levels than seen in controls. These results offer a potential model system for the analysis of NSC candidates in the adult animal, allowing researchers to more easily determine the engraftment potential of candidate NSCs.
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MATERIALS AND METHODS |
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Irradiation and Bone Marrow Reconstitution
Animals were placed in a plexiglass container for irradiation. LI was induced by exposure to a Cs137 source in a Gamma Cell 40 irradiator until 850 rad had been obtained. This amount of radiation is sufficient to deplete the bone marrow of viable cells while not inducing immediate death (unpublished observations). Immediately after irradiation, isofluorane-anesthetized mice were administered a rescue dose of 1 x 106 syngeneic whole bone marrow (WBM) cells in 150 µl of phosphate-buffered saline (PBS) via retro-orbital sinus injection.
WBM was isolated from the femurs of a euthanized littermate, washed in 10 ml of PBS, and resuspended in an appropriate volume of PBS after cell quantification with a hemacytometer. Animals were allowed to recover before being returned to conventional animal housing. For sublethal irradiation (SLI) studies, the animals were exposed to 450 rad of ionizing radiation as described above. No rescue dose of WBM is required for survival of the animal at this exposure, and animals were immediately returned to conventional housing.
Tissue Immunohistochemistry
Control, LI, and SLI animals were anesthetized with a lethal dose of Avertin and perfused through the left ventricle with 4% paraformaldehyde (PFA) in PBS. Brains were removed, postfixed overnight in 4% PFA at 4°C, and then serially sectioned through either the coronal or sagittal plane at 40 µm using a vibratome (model VT-1000-S) (Leica, Heerbrugg, Switzerland, http://www.leica.com) equipped with a sapphire blade. Tissue was prepared for immunohistochemistry by blocking at room temperature (RT) for 1 hour in PBS containing 10% fetal bovine serum (FBS), 5% dry milk, and 0.01% Triton X-100. Primary antibodies (polyclonal anti–ß-III tubulin: PRB-435P, 1:5,000 [Covance, Princeton, NJ, http://www.covance.com]; monoclonal anti–PSA-NCAM: MAB5324, 1:100 [Chemicon, Temecula, CA, http://www.chemicon.com]; monoclonal anti-BrdU[Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA]; 1:30) were applied to the sections overnight with moderate agitation at 4°C.
After three 5-minute washes (PBS plus 0.01% Triton X-100), secondary antibodies (rhodamine Red-X, goatanti-mouse IgG, R-6393,1:500[Molecular Probes, Eugene, OR, http://probes.invitrogen.com]; rhodamine Red-X, goat anti-rabbit IgG, R-6394, 1:500 [Molecular Probes]; fluorescein anti-rabbit IgG, FI-1000, 1:500 [Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com]; Oregon Green 514 goat anti-mouse IgG, O-6383, 1:500 [Molecular Probes]) were applied at RT for 50 minutes. Finally, sections were washed in PBS three times for 5 minutes, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, 12-550-15 [Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com]), and allowed to dry for 15 minutes at 37°C before being cover-slipped in Vectashield (H-1000; Vector Laboratories) mounting medium. Sections were analyzed and photographed using epifluorescence and confocal microscopy.
Identification of Proliferative Cells by BrdU Labeling
Three days, 3 weeks, or 3 months after irradiation, age-matched control and LI mice (n = 4 per time point) received intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU) (B-5002; Sigma, St. Louis, http://www.sigmaaldrich.com) three times a day at 2-hour intervals for 3 days (0.1 mg BrdU per gram of body weight in 300 µl saline). For SLI studies, age-matched animals (n = 4 per time point) were injected twice with BrdU 6, 24, and 48 hours and 3 and 6 weeks after irradiation. The brains were fixed, removed, and sectioned as above. Tissue sections were processed for BrdU immunohistochemistry by first incubating in x2 standard saline citrate (SSC)/formamide solution (1:1) for 2 hours at 65°C. After washing in x2 SSC for 5 minutes at RT, sections were incubated in 2 N HCl for 30 minutes at 37°C. Finally, sections were washed in 0.1 M borate buffer for 10 minutes at RT and then processed for double-immunolabeling with monoclonal anti-BrdU antibody and polyclonal anti–ß-III tubulin as described above. BrdU+ cells in the SEZ were quantified from serial coronal sections using a blind study format (sections coded and scored by separate investigators). The region of the SEZ analyzed encompassed an area extending from the inferior tip of the LV, superiorly along the lateral wall of the LV (extending approximately five cell bodies deep to the ependymal cell layer), to a point extending laterally approximately 700 µm from the dorsal most extent of the LV (see Fig. 1
for a representation of this area). The defined region was carefully analyzed at a magnification of x40 at the top focal plane. To maintain consistency between sections, BrdU-labeled cells were scored as positive regardless of the intensity of the antibody fluorescence. Three adjacent sections per tissue were scored using the location of the anterior commissure as a consistent landmark between animals. The cell numbers were collected, averaged, and placed into a graphical format using an MS Excel spreadsheet (Microsoft, Redmond, WA, http://www.microsoft.com), with both LI and SLI yields presented as percentages of their respective controls. Statistical significance of values was determined by Student’s t-test analysis; p < .05 was deemed significant.
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Blind Analysis of the Effects of LI and SLI on NS Yield
To determine the effects of irradiation on NS generation, we used a blind paradigm to enable unbiased preparation and examination of the cultures from three control and three LI mice (2 months after lethal irradiation, age-matched) and from four control and four SLI mice (3 weeks after sublethal irradiation, age-matched). Briefly, the animals were euthanized and their brains removed by investigator A, who gave each brain an identifying number (1 through 8). Investigator B removed the SEZ (as described above) from each brain in an identical fashion. The isolated SEZ tissue was recoded with a letter (A through H) by investigator C, who remained the only individual to know both the letter and number code. The tissue was then returned to investigator A for culture (as described above) and quantification. At 21 days in vitro, NSs were collected, pelleted, and resuspended in 2 ml of media. To determine NS yield, four 50-µl aliquots from each culture were placed in a 12-well tissue culture plate. The aliquots were analyzed with a Nikon inverted-phase microscope (Nikon, Melville, NY, http://www.nikonusa.com) at both x4 and x10 magnifications, with NS diameter determined by use of the SPOT program (Diagnostic Instruments, Sterling Heights, MI, http://www.diaginc.com). NSs below 40 µm in diameter were excluded to avoid counting hypertrophied cells. Additionally, spherical aggregates that did not display continuous, phase-bright perimeters characteristic of NSs were omitted. The total number of spheres per aliquot was determined, and the total yield and percent yield of each culture were then calculated from these numbers. Statistical significance of values was determined by Student’s t-test analysis; p < .05 was deemed significant. At the conclusion of the analysis, the code was broken and the identity of the cultures was revealed.
Isolation and Culture of MASCs
Primary SEZ tissue was isolated from postnatal-day-2 mice transgenic for green fluorescent protein (gfp+) and dissociated to a single-cell suspension in the same manner as above. Cells were plated onto tissue culture flasks (TRP, 90076) at high density in growth medium consisting of DMEM/F-12, 5% FBS, L-glutamine (25030-081; Gibco), and N-2 supplement (17502-048; Gibco). Three days after the initial plating, nonadherent cells were removed and fresh medium was applied. Cultures were passaged after generation of a confluent monolayer and were deemed suitable for transplantation at the third passage, when contaminating neurons from the primary dissociation were no longer detectable.
Transplantation of gfp+ MASCs into the LVs of Control, LI, and SLI C57BL/6 Mice
Passage-three gfp+ MASCs were collected via trypsinization and resuspended in 1 ml of growth medium (see above). Cells were resuspended in growth media at 5 x 104 cells per µl. Host mice were anesthetized with Avertin (see above), and 2 µl of cell suspension was stereotaxically injected into the LV at the following coordinates: A-P, –0.2; M-L, –1.2; H-D, –2.5. Transplanted animals were allowed to recover and placed back in general housing.
Analysis of Engrafted gfp+ MASCs into the OB of Control, LI, and SLI C57BL/6 Mice
Three weeks after transplantation, host brains were processed for immunohistochemistry as described above. Each transplanted hemisphere was sectioned through the sagittal plane at 40 µm, and every section containing the OB was collected for analysis, allowing for serial reconstruction of the transplanted hemisphere. Resulting sections were incubated with antibodies against ß-III tubulin as described above and mounted on glass slides for analysis. Gfp+ migratory cells present in the OB proper (the region rostral to the descending limb of the RMS) were scored as engrafted, with sections analyzed at x20 magnification. The total number of gfp+ neuroblasts per transplanted hemisphere was calculated for each condition (n = 14 for both nonirradiated control and for SLI).
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RESULTS |
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). Two weeks after LI, mice show a marked decrease in the number of PSA-NCAM–positive neuroblasts in the RMS (Fig. 2B). This observation was corroborated after staining of tissue sections from the brains of both wild-type (WT) and LI mice with the pan-neuronal marker ß-III tubulin (data not shown). Neuroblast depletion is variable, with some animals retaining small pockets of cells in the RMS. However, the overall abundance of migrating neuroblasts in the RMS of LI mice is never similar to those seen in untreated mice.
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Analysis and Quantification of Neuroblast Depletion in the SEZ by LI
To quantify the degree of neuroblast depletion induced by LI, we used BrdU to label mitotic cells within the SEZ [8]. The SEZ in WT animals immunolabeled with antibodies against ß-III tubulin and BrdU on serial coronal sections, at the level of the anterior commisure, contains a robust layer of dividing neuroblasts (Fig. 3A). Three weeks after LI, this same region exhibits a significant decrease in newly generated neuroblasts (Fig. 3B), and this depletion persists at 3 months after LI (Fig. 3C). A decrease of approximately 60% in the number of BrdU-positive cells was observed at the 3-week time point compared with the control. Student’s t-test analysis confirms that this decrease is significant (p = .003). There is an 87% decrease in the number of BrdU-positive cells at the 3-month time point compared with control (p = .002). The 68% decrease in the number of BrdU-positive cells between 3 weeks and 3 months after LI was also significant (p = .03) (Fig. 3D).
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). These data suggest that the decrease in migrating neuroblasts in the RMS is reflected in the SEZ and that this depletion is significant and long-term.
Effects of LI on NS Yield in Culture
As it is generally accepted that the NS-forming cell is the in vitro manifestation of the NSC [17], we cultured NSs from both control and LI mice in a blind-study format to determine if the stem cell pool in the SEZ was affected by the LI. NSs cultured from LI brains displayed an average decrease in yield of approximately 77% compared with the wild-type cultures (Fig. 4; p = .02). This decrease closely corresponds to the decreased levels of BrdU-positive neuroblasts in vivo after lethal irradiation, further supporting the validity of this finding. It has been reported that the stem cell population of the SEZ is between approximately 0.02% and 1.0% of the total cells [17, 18, 34], as determined by NSs yield from dissociated SEZ tissue, and the average yield of NSs from the control brains in this study falls within this range (0.15%, data not shown). The average yield of NSs isolated from the LI brains was significantly lower, at 0.03%, indicating that exposure to lethal doses of radiation depletes the number of NSCs in the brain responsible for the generation of NSs.
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It is possible that the lethal dose of ionizing radiation inhibited functional engraftment of the transplanted cells (possibly by inducing irreparable damage to the radiosensitive support cells of the SEZ), so a lower exposure dose of 450 rad was assayed as a milder form of injury for the enhancement of gfp+ MASC engraftment.
Analysis of RMS Neuroblast Migration and Quantification of SEZ Neurogenesis After SLI
Unlike LI, SLI (450 rad) does not completely abolish hematopoiesis in the bone marrow of adult mice, and SLI animals do not require a rescue dose of bone marrow for survival. Low levels of focused gamma irradiation (1–3 Gy) have been shown to result in a transient increase in mitotic cell activity in the SEZ, with levels eventually diminishing in the weeks following in a dose-dependent fashion compared with untreated controls [27, 31]. Contrary to the results after LI, we observed no decrease in the overall number of ß-III tubulin–positive migratory neuroblasts in the RMS of irradiated animals 3 weeks after whole-body exposure to 450 rad (a dose equivalent to 4.5 Gy). BrdU quantification assays revealed that although mitotic cell activity in the SEZ was significantly decreased in the hours immediately after irradiation, the levels returned to near normal at 3 weeks and eventually increased to above control levels at 6 weeks (Fig. 5). Blind analysis of NS yield from SLI brains at 3 weeks after SLI revealed a 30% decrease in the number of NSs compared with nonirradiated controls (Fig. 6; p = .032), with control NSs yield again falling into the aforementioned reported range of 0.2%–1.0% (0.12%, data not shown).
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), a fourfold increase in the number of gfp+ migratory cells in the OB was seen, as compared with nonirradiated controls (n = 14) (Fig. 7A–C). Taken as a whole, the average number of gfp+ migratory cells in the OB of SLI animals was twice the average number seen in nonirradiated controls (significant at p = .014) (Fig. 7G), indicating that SLI significantly enhances the engraftment potential of transplanted gfp+ MASCs, albeit in a variable fashion.
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DISCUSSION |
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The extensive investigation of the HSC has produced a gold-standard definition of a stem cell: a single cell that is capable of producing all cell types of a particular organ for the life of the animal via asymmetrical division in which both an exact duplicate of the stem cell and a lineage-committed progenitor daughter cell is generated. Additionally, the stem cell can reconstitute its native niche after transplantation, and this ability is retained after secondary transplantation, a phenomenon known as serial reconstitution. Functional transplantation of a stem cell and its subsequent reconstitution of the niche is a vital requirement for defining a stem cell, as the isolated cell can now be considered a useful tool for tissue repair. Candidate stem cells from other tissues need to meet the same criteria with the same degree of stringency if they are to be classified as true stem cells.
Of some concern to the NSC community is the lack of stable, long-term engraftment by transplanted NSCs in the host SEZ or RMS. Donor-derived neuroblasts can be detected in the RMS of host animals in the immediate weeks after transplantation, but these eventually vanish, suggesting that the transplant has resulted in only transient engraftment of donor cells. The current tactic in the field of hematopoiesis to maximize engraftment of transplanted HSCs is to first deplete the host bone marrow of HSCs by exposure to lethal doses of radiation, a technique referred to as myeloablation. This renders the HSC niche more receptive to transplanted cells, allowing for stable long-term, multilineage engraftment. We hypothesized that depletion of the native NSC pool by lethal irradiation would render the SEZ more receptive to transplanted cells and subsequently enhance the engraftment efficiency of the transplanted NSCs.
Because a single, high dose of ionizing radiation is sufficient to permanently deplete the bone marrow of viable HSCs in adult mice, we believed that a similar result would be observed in the SEZ, with the pool of NSCs being similarly depleted.
The number of mitotic cells in the SEZ was decreased by approximately 60% 3 weeks after irradiation, and this decrease was significantly greater 3 months later, at 87%, indicating a long-term, if not permanent, depletion. The levels of migrating neuroblasts in the RMS reflected this decrease, with the chains decreasing in number at both 3-week and 3-month time points.
If the NSC pool itself had been diminished by the radiation exposure, the number of NSs cultured from those brains should reflect this decrease in the number of BrdU-positive neuroblasts. In fact, we observed 77% fewer NSs isolated from the brains of LI adult mice that had been irradiated 2 months earlier. Only the yield of NSs seemed to be affected, as the resulting spheres were similar in size to control NSs and preliminary analyses indicate they display a similar level of multipotency (data not shown). In addition, the resulting cultures from WT tissue support earlier findings that the number of NSCs in the SEZ is between 0.02% and 1.0%, with the average NS yield in our cultures being approximately 0.15% of the SEZ tissue cultured. The percent yield of NSs cultured from the SEZ of LI mice was diminished, at 0.03%. These observations lead us to conclude that the SEZ is significantly depleted of NSCs after LI and that this depletion is long-term, if not permanent. The reason for this permanent depletion of neurogenesis is not entirely clear, although recent studies indicate that the neuroinflammatory response to ionizing radiation in the hippocampus inhibits neurogenesis by disruption of normal stem cell function [35].
Although LI significantly diminished the levels of neurogenesis in the SEZ, transplantation of gfp+ MASCs into the LV of LI mice 3 weeks after irradiation did not result in the expected increase of donor-derived migratory cells in OB. Nonirradiated controls exhibited normal engraftment levels, yet transplanted LI animals were observed to contain donor-derived only cells in the periventricular walls. Although it is unknown why the transplanted cells did not engraft normally into the neurogenic regions of LI animals, it has been proposed that deleterious effects to the microvasculature occurring in the dentate gyrus after 10 Gy of focused irradiation render the region unreceptive to transplanted cells [28] and a similar phenomenon may occur here.
Because LI was not observed to enhance engraftment of transplanted MASCs, we next hypothesized that moderate injury to the SEZ in the form of SLI (450 rad) would result in enhanced engraftment. Numerous studies in stem cell biology have revealed that injury to the candidate site of transplantation is critical for engraftment of the transplanted stem cell because the recipient niche is normally relatively quiescent in a healthy adult animal and induc-tionofinjuryoftenrendersthenichemorereceptivetotransplanted cells. Indeed, transplanted SLI animals were observed to contain significantly more donor-derived migratory cells in the OB compared with nonirradiated controls. It is important to note that although this increase was statistically significant, it was highly variable, with fully half of the transplanted SLI animals exhibiting levels of engraftment similar to controls. The reason for this variability is not known, although it could easily be attributed to the inherent inconsistency observed to occur in adult transplants. One intriguing explanation for the observed variability may be that variations in circadian rhythm influence the engraftment potential of cells transplanted during different seasons, a phenomenon reported to occur with bone marrow transplants in mice [36]. BrdU incorporation experiments to determine the mitotic cell levels at the time of transplantation revealed that although the numbers of mitotic SEZ neuroblasts were significantly depleted immediately after exposure, the levels returned to near normal by 3 weeks and were found to be significantly higher at 6 weeks, an observation further supported by blind NS culture assays of SLI animals in which the observed yield of NSs cultured from brains exposed to SLI 3 weeks earlier was significantly decreased by 30%.
As was observed in the LI study, the NS yield from nonirradiated controls was again between 0.02% and 1 %(0.12%). This mild injury to the brain may potentially enhance neurogenic activity and allow for increased engraftment by transplanted NSCs.
Candidate NSCs have recently been isolated from the brain using antibodies that bind to unique cell-surface antigens such as CD133 and CD15 [37, 38]. Isolated cells are then subjected to culture conditions that will produce NSs, indicating that the cell population isolated contains NSCs. In the field of hematopoiesis, attempted in vitro manipulation and subsequent expansion of cultured HSCs has been shown to result in decreased pluripotency and engraftment by the HSC, with only hematopoietic progenitor cells lacking the capacity for functional reconstitution being successfully expanded in vitro [39], in theory due to the incomplete reconstruction of the bone marrow microenvironment in vitro. Transplantation of noncultured, primary HSCs isolated from donor bone marrow still yields the most robust, long-term engraftment into the niche, and it is entirely possible that the in vitro manipulation of NSCs will result in a similar loss of multipotency and engraftment ability. Ideally, the NSCs would be isolated directly from primary SEZ tissue before manipulation or transplantation, but for the NSCs to be identified and isolated, a model system for robust engraftment is necessary for the analysis of the functional ability of the NSCs. By using the injury model described here, candidate cell populations isolated according to surface antigen expression could be transplanted to the injury-activated SEZ, followed by later analysis of the RMS for increased, long-term production of donor-derived neuroblasts. The resulting observations would then allow for a definitive conclusion to be made as to whether the isolated cell population expressed the characteristics of a true stem cell.
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ACKNOWLEDGMENTS |
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DISCLOSURES
The authors indicate no potential conflicts of interest.
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REFERENCES |
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This article has been cited by other articles:
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  G. P. Marshall II, E. D. Laywell, T. Zheng, D. A. Steindler, and E. W. Scott In Vitro-Derived "Neural Stem Cells" Function as Neural Progenitors Without the Capacity for Self-Renewal Stem Cells, March 1, 2006; 24(3): 731 - 738. [Abstract] [Full Text] [PDF]
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