| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TRANSLATIONAL AND CLINICAL RESEARCH |
aDivision of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA;
bMedical Research and Surgery Services, Veterans Affairs Medical Center, Durham, North Carolina, USA;
cStem Cell Unit, Laboratory of Neurosciences, National Institute for Aging, NIH, Baltimore, Maryland, USA;
dInvitrogen, Corporate Research Laboratories, Carlsbad, California, USA
Key Words. Adult neurogenesis • Doublecortin • Neural stem/progenitor cells • Stem/progenitor cell transplantation • Stem cell aging Stem cell differentiation
Correspondence: Ashok K. Shetty, M.Sc., Ph.D., Division of Neurosurgery, DUMC Box 3807, Duke University Medical Center, Durham, NC 27710, USA. Telephone: (919) 286-0411, ext. 7096; Fax: (919) 286-4662; e-mail: Ashok.Shetty{at}Duke.Edu
Received on November 8, 2006;
accepted for publication on May 4, 2007.
First published online in STEM CELLS EXPRESS May 17, 2007.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Disclosure of potential conflicts of interest is found at the end of this article.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Investigation of discrete regulatory steps of DG neurogenesis in rats insinuates that aging does not lower either the extents of neuronal differentiation and survival of newly born cells [10] or the number of putative neural stem/progenitor cells (NSCs) in the subgranular zone (SGZ) of the DG [22]. However, the number of proliferating NSCs in the SGZ decreases greatly at middle age [22, 23]. This is considered to be due to age-related changes in the DG milieu, because studies imply that various factors that are known to stimulate the proliferation of NSCs exhibit considerable decline at middle age. These comprise fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), vascular-endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neuropeptide Y, and phosphorylated cyclic-AMP response-element-binding protein [24–26]. Moreover, the concentration of glucocorticoids, one of the negative regulators of neurogenesis, intensifies at middle age [27], suggesting that the microenvironment of the DG becomes nonconducive for greater levels of neurogenesis at middle age. Hence, strategies that alter the DG milieu and promote enhanced proliferation of NSCs may increase neurogenesis in the aging hippocampus.
We hypothesize that grafting of fresh glial-restricted progenitors (GRPs) or NSCs into the aging hippocampus may enrich the DG microenvironment and thereby stimulate an increased production of new granule cells from endogenous NSCs. We characterized DG neurogenesis in the hippocampi of middle-aged F344 rats after grafting of GRPs from embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase (AP) gene or NSCs from the embryonic day 9 caudal neural tubes of Sox-2:EGFP transgenic mice. At 3 weeks postgrafting, the survival and differentiation of grafted cells were analyzed. Additionally, new cells/neurons born in the SGZ after grafting were rigorously characterized. Dentate neurogenesis was similarly quantified from middle-aged rats receiving sham-grafting surgery and naive middle-aged rats for comparison. We chose middle-aged hippocampus for studying the effects of GRP and NSC grafting because the window of major decline in neurogenesis during aging occurs between young age and middle age [23]. Our results confirm the hypothesis and suggest that, although NSCs can generate neurons in the hippocampus, their predominant effect is to mobilize endogenous stem cells.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
dshbwww), anti-NG2 (1:500; Chemicon), anti-FGF-2 (1:200, Chemicon), anti-VEGF (1:200, Chemicon), anti-IGF (1:200, Chemicon), and anti-green florescent protein (anti-GFP, 1:200; Chemicon). The polyclonal primary antibodies included anti-BDNF (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-Olig-2 (rabbit, 1:500; Chemicon), anti-S-100ß (rabbit, 1:1,000; Chemicon), anti-ß-III tublin-1 (anti-TuJ-1, rabbit, 1:1,000; Covance, Princeton, NJ, http://www.covance.com), anti-GFAP (rabbit, 1:1,000; Dako, Glostrup, Denmark, http://www.dako.com), and anti-doublecortin (anti-DCX, 1:250; Santa Cruz Biotechnology). The secondary antibodies included biotinylated anti-mouse, anti-rat, and anti-rabbit IgG from Vector Laboratories (1:100; Burlingame, CA, http://www.vectorlabs.com); anti-mouse, anti-rat, and anti-rabbit IgG conjugated to Alexa Fluor (488 and 594); and streptavidin fluorescein/Texas Red from Molecular Probes (Eugene, OR, http://probes.invitrogen.com).
Preparation of AP+ GRPs and Sox-2:EGFP Neural Stem Cells for Grafting
The donor GRPs isolated from the spinal cords of embryonic day 13.5 (E13.5) transgenic F344 rats that express the marker gene human placental AP [28, 29] and the NSCs isolated from E9 caudal neural tubes of Sox-2:EGFP transgenic mice brains [30–32] were expanded in vitro using standard methods [29, 30, 32]. The cultured GRPs were dissociated, washed, and resuspended in a fresh culture medium. The NSCs were labeled with BrdU in vitro, dissociated, washed, and resuspended in a fresh medium. The labeling procedure mainly comprised overnight incubation of NSCs in a medium containing 0.5 µM BrdU. Prior to grafting, the cells were rinsed thoroughly to remove excess BrdU. For preparation of both donor cells for grafting, the dissociated cells were centrifuged and cells were counted, and the viability of cells was assessed using trypan blue exclusion test in a hemocytometer. The live and dead cells were counted and the percentage of live cells relative to the total number of cells was calculated. The final volume was adjusted to a cell density of 0.55 x 105 viable cells per microliter of the culture medium for GRPs and 1.3 x 105 viable cells per microliter for NSCs, aliquoted into centrifuge tubes and stored on ice. A few aliquots were cultured on poly-L-lysine (0.05 mg/ml) coated 35-mm tissue culture plates for up to 6 days to determine their differentiation potential at the time of grafting. Cultures were maintained in a differentiation medium comprising Neurobasal (96.4 ml/100 ml; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), B-27 nutrient medium with retinoic acid (2 ml/100 ml; Invitrogen), L-glutamine (1.25 ml/100 ml; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), penicillin (40 U/ml), streptomycin (0.1 µg/ml), and Fungizone (0.004%). The presence of neurons, astrocytes, and oligodendrocytes in GRP+ and NSC cultures was ascertained using immunofluorescence for TuJ-1, GFAP, and rip [33]. The BrdU labeling index of NSC suspension was ascertained using BrdU immunostaining of one-hour cultures of NSCs [34]. This revealed that 98% of cells (mean ± SEM = 98.0 ± 1.5, n = 4) in the suspension were immunopositive for BrdU. We chose Sox-2:EGFP mice for collecting NSCs because Sox-2:EGFP expression in NSCs allowed collection of a purified population of NSCs as donor cells in this study. Additionally, because Sox-2:EGFP expression disappears once these cells differentiate into neuronal or glial phenotypes, the use of Sox-2:EGFP cells labeled with BrdU was helpful for determining whether most NSCs differentiate or remain undifferentiated following grafting into the host brain.
Analyses of Neurotrophic Factors Within AP+ GRPs and Sox-2:EGFP NSCs In Vitro
To determine whether AP+ GRPs and Sox-2:EGFP NSCs synthesize neurotrophic factors that are considered positive regulators of dentate neurogenesis, we performed extensive immunocytochemical analyses of these cells using antibodies specific for various neurotrophic factors. We plated both GRPs and Sox-2:EGFP cells on poly-L-lysine coated dishes using Dulbecco's modified Eagle's medium (DMEM) alone. Cultures were terminated using 2% paraformaldehyde solution either at 1 hour or at 24 hours after plating and processed for immunostaining. The cultures were washed in phosphate-buffered saline (PBS), blocked using appropriate serum (10%), and incubated overnight in primary antibody solutions of BDNF, IGF-1, FGF-2, or VEGF (1:200). Following this, cultures were rinsed thrice in PBS, incubated in either goat anti-mouse IgG or anti-rabbit IgG conjugated to Alexa Fluor 488/594 for 1 hour, washed thoroughly in PBS, and counterstained using 4,6-diamidino-2-phenylindole. Negative control cultures (where primary antibody step was omitted) were processed in parallel to rule out any nonspecific immunoreaction. The cultures were washed again and covered with a slow-fade mounting medium (Molecular Probes) and observed under a florescent microscope for the expression of various neurotrophic factors.
Transplantation Procedure
The grafting surgery was done in four sessions (n = 3 rats per session; total = 12 rats; six for grafting GRPs and six for grafting NSCs). The host middle-aged rats were anesthetized and fixed into a stereotaxic apparatus. The grafting procedure is described in earlier reports [35, 36]. One microliter of the cell suspension (containing 55,000 GRPs or 130,000 Sox-2:EGFP NSCs) was injected into each of the three sites in the hippocampus bilaterally. We chose to transplant differing amounts of GRPs and NSCs per graft because our preliminary studies suggested that GRPs survive grafting into the adult brain very well, whereas NSCs are more vulnerable to grafting-related trauma. The following stereotaxic coordinates were employed for implantation of the grafts into the hippocampus: (a) anteroposterior = 3.1 mm from bregma, lateral (L) = 1.5 mm from midline, ventral (V) = 3.5 mm from the surface of brain; (b) anteroposterior = 4.1 mm, L = 3.0 mm, V = 3.5 mm; and (c) anteroposterior = 4.8 mm, L = 4.0 mm, V = 4.0 mm. Grafting was performed within 3–4 hours after the preparation of cell suspension. Rats receiving mouse NSC grafts were immunosuppressed using daily subcutaneous injections of the cyclosporine A (12 mg/kg body weight). To determine the effects of grafting surgery on neurogenesis, a group of middle-aged rats (n = 6) received sham-grafting surgery, which involved 1.0-µl injections of the tissue culture medium at the above stereotaxic coordinates. In addition, to ascertain the changes in the extent of neurogenesis in grafted and sham-grafted animals, age-matched naive control rats (n = 6) were also included in this study.
Characterization of Transplant Survival and Differentiation
Three weeks after grafting or sham-grafting surgery, rats were perfused transcardially with 4% paraformaldehyde. Brains were cryoprotected in sucrose and sliced coronally (30-µm-thick sections) through the hippocampus using a cryocut and collected serially in phosphate buffer. Every 10th section was stained for Nissl, and the sections were scanned to identify the location of transplants in relation to the different hippocampal cell layers. In all animals, transplants were located either predominantly inside the hippocampus or partly inside the hippocampus. When transplants were located only partly inside the hippocampus, they projected into the corpus callosum above and/or into the lateral ventricle below. For further analyses of transplants, we chose transplants that were predominantly intrahippocampal. In animals receiving GRP cell grafts, every 10th section through each chosen transplant was processed for AP immunostaining, whereas, in animals receiving grafts of NSCs, every 10th section through each chosen transplant was processed for BrdU immunostaining using the avidin-biotin-complex (ABC) method, as detailed in our recent reports [10, 37]. These BrdU immunostained sections were later used for quantification of the yield of surviving cells per graft using the optical fractionator method.
To identify the presence of neurons, astrocytes, and oligodendrocytes among surviving grafted cells in rats receiving GRP cell transplants, representative sections through transplants were processed for dual immunofluorescence for AP and markers of neurons (NeuN, Tuj-1), astrocytes (GFAP, S-100ß), and oligodendrocytes (NG2, olig-2, rip). In addition, to ascertain proliferating grafted cells among cells derived from transplanted GRP, BrdU, and AP, dual immunostaining was performed. In animals receiving grafts of BrdU-labeled Sox-2:EGFP NSCs, dual immunofluorescence staining was performed for BrdU and the above-mentioned markers of neurons and glia. In addition, to identify undifferentiated Sox-2:EGFP NSCs in the transplant, we stained representative sections using an antibody to enhanced green fluorescent protein (EGFP). For visualizing AP and markers of neurons or glia using dual immunofluorescence, free-floating sections containing grafts were washed in PBS, blocked in 3% normal horse serum, incubated overnight in a monoclonal antibody against AP, washed in PBS, and treated with goat anti-mouse IgG conjugated to Alexa Fluor 594. Following this, sections were rinsed in PBS, incubated overnight in antibodies against markers of neurons or glia, washed with PBS, treated with a biotinylated secondary antibody, washed in PBS, and incubated in streptavidin fluorescein solution, rinsed again in PBS, and mounted on clean slides using a slow-fade mounting medium (Molecular Probes). For demonstrating proliferating cells among grafted GRPs, sections were first processed for AP staining as mentioned above and then proceeded for BrdU pretreatment protocols [37] and incubated overnight in rat anti-BrdU and developed using goat anti-rat IgG conjugated to Alexa Fluor 488. To demonstrate BrdU and markers of neurons or glia in cells derived from NSC grafts, free-floating sections containing grafts were first washed in PBS, subjected to BrdU pretreatment protocols [37], rinsed in PBS, blocked in 3% normal goat serum, and incubated overnight in rat anti-BrdU solution. The sections were then washed in PBS, treated with goat anti-rat IgG conjugated to Alexa Fluor 594, and washed again in PBS. Following this, sections were blocked in normal serum, incubated overnight in antibodies against neuronal or glial markers (1:100–1:1,000), washed in PBS, and treated with an appropriate secondary antibody (biotinylated goat anti-mouse IgG/goat anti-rabbit IgG). The sections were then washed in PBS, incubated in streptavidin fluorescein solution, rinsed in PBS, and mounted on clean slides using a slow-fade mounting medium (Molecular Probes). Dual-labeled cells in all sections were analyzed via Z-section analyses in a confocal laser-scanning microscope (LSM-410; Carl Zeiss, Jena, Germany, http://www.zeiss.com; [10]). To identify undifferentiated Sox-2:EGFP cells in NSC grafts, representative sections were incubated overnight in mouse anti-GFP (1:200) and then treated with goat anti-mouse Alexa Fluor 488 (1:200) for 60 minutes.
Analyses of Newly Born Cells Using BrdU in Animals Receiving GRP Cell Grafts
In order to quantify newly born cells and neurons following GRP cell grafting, one intraperitoneal injection of BrdU (Sigma) at a dose of 100 mg/kg of body weight was given daily to animals in this group (n = 6) for 12 consecutive days, commencing from the day of grafting. For comparison, animals receiving sham-grafting surgery (n = 6) and naïve control animals (n = 6) were also given similar BrdU injections. Animals in all groups were perfused with 4% paraformaldehyde at 10 days after the last BrdU injection. In animals receiving GRP cell grafts and sham-grafting surgery, this time point is equivalent to 3 weeks after grafting surgery, as described above for characterization of transplant survival and differentiation. Every 15th 30-µm-thick section through the entire hippocampus was selected in each of the animals belonging to the above three groups and processed for single BrdU immunostaining using a monoclonal antibody to BrdU. The BrdU immunostaining was performed using the ABC method (Elite ABC kit; Vector) with diaminobenzidine as the chromogen. The detailed methods for visualization of BrdU in newly born cells are described in our earlier report [37]. The immunostained sections were mounted on gelatin-coated slides, air-dried, counter-stained with hematoxylin, dehydrated, cleared, and coverslipped. The BrdU immunostained sections were used for quantification of the total number of newly born cells added to the SGZ/granule cell layer (GCL) over a period of 12 days following grafting of GRP cells or sham-grafting surgery using the optical fractionator method. For quantification of the effects of GRP cell grafts on the number of newly born cells added to the SGZ/GCL, we chose hippocampi where GRP cell grafts were predominantly intrahippocampal. Additional series of sections (6–10 per animal) in all groups were processed for BrdU and NeuN dual immunofluorescence, and dual-labeled cells (i.e., cells positive for both BrdU and NeuN) in the SGZ/GCL were analyzed via Z-section analyses in a confocal laser scanning microscope (LSM-410; [10]).
Assessment of Neurogenesis Using DCX Immunostaining
In all animals belonging to different groups (animals receiving GRP cell grafts, NSC grafts or sham-grafting surgery, and naïve control animals), we analyzed neurogenesis using DCX immunostaining of serial (every 15th) sections through the entire hippocampus. The choice of DCX as a marker of new neurons in the DG is based on our earlier finding that neurons visualized with DCX immunostaining in the adult rat DG are new neurons that are predominantly born during the 12 days prior to euthanasia [37]. Sections were processed for DCX immunostaining using a polyclonal antibody to DCX and the ABC method as described in an earlier report [38]. The peroxidase reaction was visualized using vector gray as the chromogen, and the sections were counterstained using nuclear red staining. These sections were employed for assessment of the status of dentate neurogenesis via quantification of the total number of DCX+ neurons in the SGZ and GCL using the optical fractionator method.
Quantification of BrdU+ Cells and DCX+ Neurons Using the Optical Fractionator Method
The BrdU and DCX positive cells in the SGZ (two-cell-thick region from the inner margin of the dentate GCL) and GCL were counted in the following groups: hippocampus receiving GRP cell grafts, hippocampus receiving sham-grafting surgery, and naïve control hippocampus (n = 5 per group). In hippocampus receiving NSC grafts (n = 5), DCX+ new neurons in the SGZ and GCL and BrdU+ grafted cells dispersed in the entire hippocampus were measured. All cell counts were performed using the StereoInvestigator system (MicroBrightField Inc., Williston, VT, http://www.mbfbioscience.com). These measurements utilized every 15th section through the entire septotemporal extent of the hippocampus for counting cells in the SGZ and GCL and every 10th section through the entire extent of transplants for counting the dispersed BrdU+ cells from NSC grafts. The StereoInvestigator system consisted of a color digital video camera (Optronics Inc., Muskogee, OK, http://www.optronicsinc.com) interfaced with a Nikon E600 microscope. In each animal, cells were counted from 50–200 randomly and systematically selected frames (each measuring 40 x 40 µm, 0.0016 mm2 area) in every selected section using the 100x oil immersion objective lens. The numbers and densities of frames were determined by entering the parameter grid size in the optical fractionator component of the StereoInvestigator system [37].
We cut 30-µm-thick sections through the hippocampus using a cryostat. Confirmation of the section thickness using the StereoInvestigator system incorporating XYZ stage controller equipped with z-axis position control (Ludl Electronic Products Ltd, Hawthorne, NY, http://www.ludl.com) suggested that the variability among sections is minimal (i.e., ± 1 µm). However, following immunostaining, sections showed significant shrinkage along the z-axis. Measurement of the thickness of sections in different regions of the DG using the StereoInvestigator system described above revealed that, in sections processed for BrdU or DCX immunostaining, the average thickness was reduced to 53% of the initial thickness. Hence, at the time of data collection, the thickness was 16 µm for both BrdU and DCX immunostained sections. For cell counting, in every section, the contour of GCL/SGZ area or transplant area was first delineated using the tracing function. The optical fractionator component was then activated, and the number and location of counting frames and the counting depth for that section were determined by entering parameters such as the grid size, the thickness of top guard zone (4 µm), and the optical dissector height (i.e., 8 µm). The grid size was 75 x 75 µm for all cell counts except for the measurement of BrdU+ cells derived from NSC grafts, where 200 x 200 µm was chosen. A computer-driven motorized stage then allowed the section to be analyzed at each of the counting frame locations. In every counting frame location, the top of the section was set, after which the plane of the focus was moved 4 µm deeper through the section (guard zone) to get rid of the problem of uneven section surface. This plane served as the first point of the counting process. All cells that came into focus in the next 8-µm section thickness were counted if they were entirely within the counting frame or touching the upper or right side of the counting frame. Based on the above parameters and cell counts, the StereoInvestigator program calculated the total number of BrdU/DCX positive cells per SGZ and GCL utilizing the optical fractionator formula [37].
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Expression of Neurotrophic Factors in AP+ GRPs and Sox-2:EGFP NSCs
We analyzed GRPs and Sox-2:EGFP NSCs for the expression of various neurotrophic factors in vitro. We found that cells derived from both GRPs and NSCs (at 60 minutes after plating on poly-L-lysine coated dishes containing just DMEM) were robustly immunoreactive for many of the neurotrophic factors that are considered positive regulators of dentate neurogenesis. These include BDNF, FGF-2, VEGF, and IGF-1 (Fig. 1A1–1D3, 1F1–1I3). As negative control cultures (with omission of primary antibody during immunocytochemistry) displayed no such immunoreactivity (Fig. 1E1–1E3, 1J1–1J3), these observations imply that both GRPs and NSCs have the ability to secrete these neurotrophic factors following grafting. Interestingly, all of these neurotrophic factors were also found in the cytoplasm of cells derived from GRPs and NSCs at 24 hours after plating (data not shown).
|
28% of cells differentiating into rip+ oligodendrocytes. This high percentage of oligodendroglial differentiation likely reflects the culture condition employed in this study. From the above, it is also clear that the two donor cell types chosen in this study are quite distinct; one (i.e., GRPs) is capable of differentiating mostly into astrocytes and oligodendrocytes, whereas the other one (i.e., NSCs) is capable of differentiating into all three CNS phenotypes.
Survival of AP+ GRPs and Sox-2:EGFP NSCs in the Aging Hippocampus
We analyzed the survival of grafted GRPs through AP immunostaining of serial brain sections from animals receiving GRP cell grafts. This revealed the location and distribution of GRP cell grafts. In all animals, grafts were located mostly inside the hippocampus with extensions into the DG (Fig. 2A1). However, portions of grafts also projected either into the corpus callosum above (likely a backflow along the needle track) or into the lateral ventricle below (Fig. 2A1). Characteristically, a vast majority of grafted GRPs remained close to the grafted site as a core with migration of only a few cells away from the core (Fig. 2A2). However, processes emanating from the core invaded the surrounding hippocampal tissue (Fig. 2A3). These processes presumably represent glial processes, as the subsequent phenotypic analyses revealed a large number of cells morphologically resembling astrocytes and expressing immunoreactivity for S-100ß and GFAP were seen in the core of the graft (Fig. 3A1–3B15). As Sox-2+ NSCs were prelabeled with BrdU prior to grafting, we analyzed the survival of these cells through BrdU immunostaining of serial sections through grafts and optical fractionator cell counting. In contrast to GRPs, grafted NSCs migrated profusely into all layers of the DG and hippocampal CA1 and CA3 subfields (Fig. 2B1–2B3). In the DG, fractions of cells were engrafted into the SGZ (Fig. 2B4). When grafts were placed close to the end of the upper blade of GCL, fractions of cells also migrated into the stratum lucidum of the CA3 region (Fig. 2B2). Quantification revealed that the yield of surviving cells from NSC grafts is equivalent to 205% of injected cells (mean ± SEM = 205% ± 29.0%, n = 5), suggesting significant cell division after grafting. Thus, grafts of both GRPs and NSCs exhibit robust survival following grafting into the intact aging hippocampus. Additionally, the GRP cells tend to stay in clusters following grafting into the aging hippocampus, whereas NSCs exhibit widespread migration into all layers of the aging hippocampus.
|
|
We analyzed cells derived from grafts of NSCs in different regions of the hippocampus using BrdU and NeuN, BrdU and S-100ß, BrdU and olig-2, and BrdU and NG2 dual immunofluorescence methods (Fig. 3F1–3K3). Analyses of NSC-derived cells engrafted into the SGZ/GCL revealed that 4% (mean ± SEM = 4.3% ± 0.6%) of migrated cells differentiated into NeuN+ mature neurons (Fig. 3F1–3F3), 37% (37% ± 0.2.6%) differentiated into S-100ß+ mature astrocytes (Fig. 3G1–3G3), and 36% (36% ± 3.3%) differentiated into olig-2+ cells (presumably oligodendrocytes; Fig. 3I1–3I3). Thus, 23% of cells that migrated into the dentate SGZ exhibited immature characteristics. These cells likely remain as NSCs in the SGZ. In other regions of the hippocampus, none of the cells derived from NSC grafts differentiated into NeuN positive neurons. However, a significant fraction (39.0% ± 9.6%) of NSC-derived cells expressed S-100ß and morphologically resembled astrocytes (Fig. 3H1–3H3). In addition, a fraction of cells (41% ± 7.8%) expressed olig-2 (Fig. 3J1–3J3). We also examined cells derived from NSCs (i.e., BrdU+ cells) for the expression of rip and NG2. Many BrdU+ cells displayed NG2 expression (Fig. 3K1–3K3) but none exhibited rip immunoreactivity, suggesting that differentiation of grafted NSCs into mature oligodendrocytes does not happen at the postgrafting time point examined in this study. Nevertheless, the occurrence of NG2+ cells among BrdU+ cells suggests that some of the grafted NSCs will eventually give rise to mature oligodendrocytes.
Thus, in the aging hippocampus, NSC-derived cells exhibit some neuronal differentiation only when migrated into the neurogenic zone. In non-neurogenic regions of the aging hippocampus, NSC-derived cells differentiate into glia. As most of the grafted NSCs differentiated, EGFP expression was lost in a great majority of BrdU+ grafted cells. However, a small fraction of undifferentiated cells (Sox-2:EGFP positive) was seen in the host hippocampus (Fig. 3L1), and some of the undifferentiated cells migrated into the posterior subventricular zone (Fig. 3L2). These undifferentiated cells retained robust Sox-2:EGFP expression, providing a clear demonstration that stem cells mostly differentiate and do not persist as undifferentiated cells after transplantation except in stem cell niches.
Effects of GRP Cell Grafting on Dentate Neurogenesis in the Aging Hippocampus
We first quantified newly born cells and neurons in middle-aged hippocampus following GRP cell grafting via BrdU immunostaining and BrdU and NeuN dual immunofluorescence assays, and the data were compared with results from naïve control hippocampus and hippocampus receiving sham-grafting surgery (n = 5 per group). In all groups, BrdU immunostaining visualized newly generated cells in the GCL and the SGZ. However, the density of BrdU + cells clearly appeared greater in middle-aged hippocampus receiving sham-grafting surgery or GRP cell grafts in comparison with naïve middle-aged hippocampus (Fig. 4A1–4C2). Quantification of the total number of new cells (i.e., BrdU+ cells) generated over a period of 12 days in the GCL and SGZ using the optical fractionator method revealed that addition of new cells to GCL is increased in hippocampus receiving GRP cell grafts in comparison with both naïve age-matched hippocampus (128% increase; p < .001) and hippocampus receiving sham grafting surgery (23% increase; p < .05; Fig. 4D1).
|
|
We next analyzed newly born neurons in the entire GCL and SGZ through measurement of DCX+ neurons. In all groups, DCX immunostaining visualized newly formed neurons in the SGZ and GCL (Fig. 6A1–6C3). The cell bodies of DCX+ neurons were located in the SGZ and the inner third of the GCL. In naive hippocampus and hippocampus receiving sham-grafting surgery, DCX+ neurons were far fewer in number and morphologically appeared immature with horizontally oriented or basal dendrites (Fig. 6A1–6B3). Additionally, vertically oriented dendrites going through the GCL were infrequent and, when present, they were conspicuously shorter. In contrast, in middle-aged hippocampus receiving GRP cell grafts (Fig. 6C1–6C3), a significant fraction of DCX+ neurons exhibited relatively more mature phenotypes with vertically oriented dendrites extending into the outer two thirds of the dentate molecular layer [40]. The overall dendritic branching also appeared greater in these neurons in comparison with their counterparts in naive control hippocampus and hippocampus receiving sham-grafting surgery. Quantification of the number of DCX+ neurons in the entire DG revealed that addition of new neurons to the GCL is increased in rats receiving GRP cell grafts in comparison with both naïve control hippocampus (87% increase; p < .001) and hippocampus receiving sham-grafting surgery (237% increase; p < .001; Fig. 6D1). Because DCX+ cells in F344 rats represent neurons that are mostly added during the 2 weeks prior to euthanasia [10, 37] and animals were killed at 3 weeks postgrafting in this study, the results obtained with DCX analyses reflect neurogenesis that occurs at 2–3 weeks postgrafting. Thus, both BrdU and DCX assays suggest that neurogenesis in the aging hippocampus can be considerably enhanced through grafting of GRP cells. Additionally, it is clear that the increase in overall neurogenesis is not due to injury inflicted at the time of grafting surgery, as hippocampi receiving sham-grafting surgery do not upregulate neurogenesis.
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Pattern of Integration of Grafts of GRPs and NSCs into the Aging Hippocampus
We employed two diverse populations of cells (GRPs and NSCs) to investigate their behavior and fate following grafting into the aging hippocampus. The GRPs were obtained from transgenic animals where all cells expressed AP under a ubiquitous promoter [29], which facilitated visualization of GRP-derived cells after grafting through simple AP immunostaining. The NSCs, in contrast, were harvested from mice where GFP was knocked in to the Sox-2 locus to obtain NSCs that are GFP+ [30, 31]. As these NSCs lose their GFP expression following differentiation into neurons or glia, we used BrdU to track NSCs following grafting. Transplanted GRPs differentiated into astrocytes and oligodendrocytic progenitors. Nonetheless, it appeared that some grafted GRPs remained as progenitors, as these cells expressed GFAP but lacked S-100ß and rip/olig-2. Interestingly, cells derived from GRPs were mostly located in and around the graft sites, in sharp disparity to the widespread migration of GRPs detected along white matter tracts after grafting into the adult spinal cord [39, 41]. The incongruity reflects differences in both age of the host and the site of grafting between the two studies. Nevertheless, the current observations insinuate that the migration of GRPs is restricted in the aging brain, which is likely a consequence of swift differentiation of most GRPs following grafting. The likelihood of decreased cues for migration of GRPs in the aging hippocampus is improbable, since NSCs grafted into the age-matched hippocampus demonstrated extensive migration in this study. Additionally, grafted GRPs did not contribute neurons because no AP+ neurons could be found in the hippocampus of animals receiving GRP cell grafts, suggesting that both transdifferentiation of GRP-derived astrocytes into neurons [42] and dedifferentiation of GRPs into NSCs do not occur after grafting. This is consistent with our earlier studies showing that GRPs harvested from Sox-2:EGFP mice fail to dedifferentiate into Sox-2:EGFP NSCs (M.S. Rao, unpublished observations). Thus, transplanted GRPs display limited migration but readily differentiate into astrocytes and oligodendrocytes following grafting into the aging hippocampus.
Grafted NSCs exhibited pervasive migration, as they invaded the entire hippocampus including the dentate GCL and the hippocampal CA1 and CA3 cell layers. Some of the grafted NSCs also appeared to persist in the stem cell niches, as few EGFP+ cells could be visualized in the dentate gyrus and posterior subventricular zone at 3 weeks after transplantation. However, a great majority of cells from NSC grafts differentiated into astrocytes and oligodendrocytic progenitors and so lost their GFP expression. Some cells derived from NSC grafts also differentiated into neurons when they migrated into the SGZ/GCL but not when they engrafted into the white matter or the non-neurogenic regions of the hippocampus. This is in difference to results obtained earlier with both transplants of neuronal restricted precursors, which differentiate into neurons even when grafted into the white matter [39, 41], and committed postmitotic neurons from the fetal hippocampus, which differentiate into mature neurons even when grafted into the senescent host brain [36, 43]. Collectively, these observations indicate that, although NSCs have the proficiency to differentiate into neurons, the induction of neuronal phenotype from NSCs critically requires exogenous signals that seem to be present in only the neurogenic regions of the brain. Although the specific nature of these signals remains to be identified, studies suggest that the possible candidates that regulate neuronal fate-choice decision of NSCs include bone morphogenetic proteins [44], wnt proteins [45, 46], sonic hedgehog [47–50], and cytokines such as IL-1ß and IL-6 released by astrocytes [51]. However, transplanted NSCs that migrated into the neurogenic SGZ did not differentiate exclusively into neurons, as the SGZ of the host hippocampus also contained glia derived from NSC grafts. This suggests that the behavior of grafted NSCs is analogous to the behavior of endogenous NSCs in the SGZ, where they generate neurons and glia throughout life. Thus, grafted NSCs exhibit robust survival, migrate extensively, and differentiate mostly into glia in all regions of the aging hippocampus. Furthermore, grafted NSCs that migrate into the neurogenic region of the aging hippocampus produce both neurons and glia. However, it remains to be seen whether NSCs that persist in the dentate gyrus would continue to produce new neurons and glia for prolonged periods in the aging hippocampus. Although a recent study suggests that in vitro expanded NSCs are incapable of self-renewal after grafting [52], careful long-term analyses of grafted NSCs are needed in future to resolve this issue.
Potential Mechanisms and Extent of Enhanced Neurogenesis After Grafting of GRPs and NSCs
The precise mechanisms by which grafts of NSCs or GRPs increase neurogenesis in the aging hippocampus are unknown. Nevertheless, increased neurogenesis after grafting is not due to injury or nonspecific release of neurotrophic factors associated with grafting surgery because animals receiving sham-grafting surgery exhibited decreased neurogenesis compared with naïve control animals. Increased neurogenesis might reflect induction of a more conducive microenvironment for proliferation of endogenous NSCs in the presence of cells derived from GRP and NSC grafts. This is because recent studies have shown that the major reason underlying declined dentate neurogenesis during aging is reduced proliferation of NSCs [10, 23, 53], which is likely due to multiple age-related alterations in the hippocampus. These include reduced FGF-2 activity and FGF-2 receptors in astrocytes, decreased concentrations of multiple NSC proliferation factors, and an increased quiescence of NSCs [22, 24, 25, 54]. The positive changes in the milieu following grafting are likely mediated by fresh astrocytes and oligodendrocytic progenitors permeating the aging hippocampus, as most of the cells derived from both GRP and NSC grafts differentiated into these two cell types. It is plausible that addition of these fresh glia increases the concentration of NSC proliferation factors, which in turn stimulates the proliferation of quiescent endogenous NSCs in the aging hippocampus. Indeed, our in vitro analyses of GRPs and NSCs reveal that these cells do synthesize a variety of neurotrophic factors that are considered positive regulators of dentate neurogenesis. These include BDNF, FGF-2, IGF-1, and VEGF. Thus, the effects of GRP and NSC grafts on dentate neurogenesis are likely mediated by the cell types derived from these grafts.
The overall extent of enhancement in neurogenesis was slightly (16%) greater with grafts of NSCs than with grafts of GRPs, although the differences were not significant statistically. There may be many possibilities for this difference. First, although both GRPs and NSCs seem to influence the proliferation of endogenous NSCs by altering their milieu via contribution of new glia, the GRPs did not give rise to new neurons even when grafted directly into the dentate SGZ and GCL. In contrast, NSCs migrating into the SGZ contributed new neurons to the GCL. Second, the timing and duration of growth factor delivery may be different between the two types of grafts. The GRPs, being lineage committed, likely differentiate rapidly into astrocytes and oligodendrocytic progenitors following grafting, leading to swift effects on the proliferation of endogenous NSCs. However, these early effects may not be adequate for increased proliferation of NSCs for prolonged periods, whereas grafts of NSCs, being noncommitted at the time of grafting, likely proliferate for prolonged periods and produce cohorts of fresh astrocytes and oligodendrocytic progenitors at different time points after grafting, leading to a sustained effect on the proliferation of endogenous NSCs. Third, the overall positive effects on microenvironment are likely greater with NSC grafts than with GRP cell grafts because GRPs exhibit very limited migration, whereas NSCs display pervasive migration throughout the hippocampus. Fourth, it is possible that NSCs provide a different set of trophic molecules than those derived from grafted GRPs, which is consistent with our comparative gene expression studies showing significant differences in growth factor profiles of NSCs and GRPs ([55] and M.S. Rao, unpublished observations). Overall, although both GRPs and NSCs are efficient for enhancing neurogenesis in the aging hippocampus, NSC grafts may be slightly better suited than GRP cell grafts. However, additional long-term studies are needed to dissociate differences in mediating neurogenesis between these two groups. Furthermore, unlike NSCs, GRPs do not differentiate into neurons [29], which may be an advantage for using GRPs as donor cells for enhancing neurogenesis in the aging hippocampus. This is because lack of neuronal differentiation may mitigate some of the potential side effects of neural cell grafting in the intact aging hippocampus, such as the development of inappropriate synaptogenesis or the formation of ectopic foci of neurons. Preventing the formation of ectopic foci of neurons is important, as such foci observed in the DG following status epilepticus or infusions of BDNF have been shown to contribute to epilepsy development [56–60].
|
CONCLUSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. K. Singh, S. Gupta, Y. Jiang, M. Younus, and M. Ramzan In vitro Neurogenesis from Neural Progenitor Cells Isolated from the Hippocampus Region of the Brain of Adult Rats Exposed to Ethanol during Early Development through Their Alcohol-Drinking Mothers Alcohol Alcohol., March 1, 2009; 44(2): 185 - 198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Einstein and T. Ben-Hur The Changing Face of Neural Stem Cell Therapy in Neurologic Diseases Arch Neurol, April 1, 2008; 65(4): 452 - 456. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
