Grafting of neural progenitors has been shown to reverse a wide variety of neurobehavioral defects. While their role of replacing injured cells and restoring damaged circuitries has been shown, it is widely accepted that this cannot be the only mechanism, as therapy can occur even when an insufficient number of transplanted cells are found. We hypothesized that one major mechanism by which transplanted neural progenitors exert their therapeutic effect is by enhancing endogenous cells production. Consequently, in an allographic model of transplantation, prenatally heroin-exposed genetically heterogeneous (HS) mice were made defective in their hippocampal neurobehavioral function by exposing their mothers to heroin (10 mg kg−1 heroin on gestation days 9–18). Hippocampal damage was confirmed by deficient performance in the Morris maze (P<0.009), and decreased production of endogenous cells in the dentate gyrus by 39% was observed. On postnatal day 35, they received an HS-derived neural progenitors transplant followed by repeated bromodeoxyuridine injections. The transplant returned endogenous cells production to normal levels (P<0.006) and reversed the behavioral defects (P<0.03), despite the fact that only 0.0334% of the transplanted neural progenitors survived and that they differentiated mainly to astrocytes. An immunological study demonstrated the presence of macrophages and T cells as a possible explanation for the paucity of the transplanted cells. This study suggests one mechanism for the therapeutic action of neural progenitors, the enhancement of the production of endogenous cells, pointing to future clinical applications in this direction by use of neural progenitors or by analogous cell-inducing techniques.
Neural precursor cell transplantation has been proposed as a mean of cell replacement therapy. Presumably, transplanted neural progenitors migrate to the damaged area, replacing defective cells and restoring damaged circuitries.1, 2 Indeed, grafting of neural progenitors into the hippocampus was shown to reverse various neurobehavioral defects in the impaired animals.3, 4, 5, 6, 7 Recently, it has been also suggested that transplanted neural precursors have trophic properties that induce endogenous regenerative processes in the host brain.8 Obviously, once grafted cells have integrated in the host circuitry, it is practically impossible to differentiate between their effects as cell replacement and their trophic effects.
We hypothesized that one specific mechanism by which neural progenitors exert their trophic therapy on the host brain and behavior is via the induction of endogenous neural precursor production. The mechanism by which neural progenitors might induce such an effect is not known. One possibility is the induction of cytokines by neural progenitors,8, 9, 10 which enhance proliferation of endogenous neural cells.11, 12, 13, 14, 15
Here, we aimed to examine our hypothesis by employing the prenatally heroin-damaged brain model. The heroin model was chosen because, among other effects, it induces deficits that can be related to specific synaptic functions. Accordingly, administration of heroin to pregnant mice induced deficits in hippocampus-related offspring behavior and concomitant cholinergic-related abolishment of protein kinase C (PKC) activation.16, 17, 18, 19, 20, 21
Consequently, in the present study we employed our previously established allographic mouse model for the reversal of neurobehavioral defects by transplantation of neural progenitors toward testing the present question. Consistent with our previous studies, genetically heterogeneous HS/Ibg mice22 were made deficient in the hippocampus-related Morris maze behavior and in its supposed mechanism, PKC function, by exposing them to heroin transplacentally. They were grafted with neural progenitors derived from the same strain to reverse the neurobehavioral deficits. An extensive study was designed to demonstrate the expected low rates of survival and differentiation to neurons, immune evaluation was carried out to ascertain the possible role of rejection and, most importantly, our hypothesis—that the transplanted neural progenitors, before disappearing, induced an increase in endogenous proliferation of cells, restoring damaged circuitries and resulting in neurofunctional recovery—was tested.
Materials and methods
Heterogeneous stock (HS/lbg) mice were used in this study since this strain is very prolific, even under drug exposure.23, 24 Four female and one male were housed in each mating cage and maintained under standard conditions of 24° C and a 12-h light–dark cycle. Females were checked daily at 0800. Those that had mated, as evidenced by the existence of a vaginal plug (date was termed as gestational day 1, GD1), were then housed with the other pregnant females. Heroin was administered on GD9–GD18. On GD18, every female was housed individually. The pups were weaned at postnatal day 25 (PN25), segregated by sex and housed in groups of five. On PN35, they were transplanted with neural precursor cells or with Dulbecco's modified Eagle's medium (DMEM) media and divided into three groups for the following three experiments (see summary in Table 1).
Designed to confirm, in the present experimental groups, our previous findings25 on the effects of prenatal heroin exposure and transplantation of neural precursor on cognitive ability. Neural precursors and medium-transplanted mice were tested in the Morris maze on PN90–PN95. A sample group was perfused, and the brains were taken for immunocytochemical assessment of the graft survival and differentiation.
Neural precursors and medium-transplanted mice were stained for T cell and macrophage markers 20 days after transplantation to assess the possible immunological response to the graft.
Beginning 2 h after transplantation, and continuing for the following 4 days, mice received two daily injections of the mitotic marker bromodeoxyuridine (BrdU), for a total of 10 injections, a dose which has been shown to be a valid marker of neural progenitors in the brain and which avoids nonproliferating cells labeling artifacts.26 Four hours after the last injection, the mice were perfused and brains were taken for immunocytochemical evaluation of the newly born cells in the dentate gyrus.
Prenatal heroin administration
Heroin administration paradigm was based on our previous studies showing neurobehavioral deficits after its prenatal administration.17, 23 Pregnant female mice received a daily subcutaneous injection of 10 mg kg−1 of heroin or saline on GD9–GD18, the period when most brain structures develop.24 The drug was not given after that period in order to prevent neonatal withdrawal, which would adversely affect offspring–maternal interaction.27
Isolation of neural precursors and growth of neurospheres
This procedure is based on the studies of Ben-Hur et al.28, 29 For the derivation of neural progenitors, newborn (PN1–PN2) HS/Ibg mice were used. Cerebral cortices were minced, digested in 0.025% trypsin for 20 min and dissociated with a 5 ml pipette into a single-cell suspension. Debris was removed by centrifuging after addition of 4% bovine serum albumin. Then the pallet was suspended in serum-free F12/DMEM medium supplemented with 10 mg% human apo-transferrin, 1 mM sodium-pyruvate, 0.05% bovine serum albumin, 10 ng ml−1 D-biotin, 30 nM sodium selenite, 20 nM progesterone, 60 μM putrescine, 25 μg ml−1 bovine insulin, 2 mM L-glutamine and 25 μg ml−1 gentamycin (all from Sigma, St Louis, MO, USA). The viability of the cells was monitored throughout the incubation using Trypan blue. The cells were plated 10 × 106 cells/T-75 uncoated flask and incubated in a CO2 air jacket incubator for 6–8 days until grafting. The cells were supplemented daily with 10 ng ml−1 of basic fibroblast growth factor 2 (R&D, Minneapolis, MN, USA) and 20 ng ml−1 of epidermal growth factor 2 (R&D). Under these conditions, most cells died and approximately 0.2% of cells proliferated into clusters of small round cells that grew into floating spheres. After 3 days of incubation, the cells were centrifuged at 800 r.p.m. for 8 min and were resuspended in half of the original volume and incubated for 3 more days. As already described, these spheres consisted mainly of PSA-NCAM+, nestin+ and NG2(−) cells that generated GFAP+ astrocytes, GalC+ oligodendrocytes and few neurofilament+ neurons upon differentiation.28, 29
On PN35, the mice treated prenatally with heroin/saline where transplanted with mouse neurospheres. Animals were anesthetized with an intraperitoneal injection of 80 mg kg−1 pentobarbital and grafted on a stereotaxic apparatus with 2500 neurospheres (100–200 cells per neurosphere) at a volume of 2–5 μl using a 10 μl Hamilton syringe into each hippocampus at the following coordinates: 1.8 mm posterior to bregma, ±1.5 mm lateral from the midline and 1.7 mm below calvarium.17, 30 DMEM media was chosen as the control solution. The issue of an appropriate control has been coped with since the days of grafting of embryonic differentiated cells, as opposed to neural progenitors. Transplantation of dead cells or tissues destined to be metabolized in the brain appeared to have an averse effect on the control group. Nevertheless, such a control may be called for in a future extensive experiment with several control groups so that all variables may be controlled for.
Morris water maze test
Morris maze testing began on PN90 and continued for 5 days. Both the apparatus and the test procedure that were originally developed for rats31 were adapted to enable testing with mice, as previously described.32 The test was performed in a tank with a diameter of 87 cm filled with opaque white water (by adding powdered milk) at 24° C. A clear platform, 10 × 8 × 9 cm high, was submerged 1 cm under water. In the place test, the mice were given two blocks of four trials on each day of the test. In all trials, the mouse was given 60 s to swim, find the platform and climb onto it. The mouse stayed on the platform for 20 s until placed in the water for another trial. Mouse that failed to find the platform in 60 s was manually placed on the platform until the next trial. The time to reach the platform (latency) was recorded. On the fifth day, the mice were given only one block of four trials of the place test followed by one block of four trials of the spatial probe test. In the latter test, the platform was removed and the extinction of the learned behavior was evaluated as the sequential decline in the proportion of the distance swum in the quadrant of the missing platform, over the four 60-s trials. To control for the possible effect of swimming ability on the results, the swimming speed during each of the trials was calculated.
Immunocytochemical evaluation of the transplanted cells in the host brain
A sample group of four animals, which were behaviorally tested, were immunocytochemically evaluated to determine the fate of the transplanted cells. In this group, 25 μg ml−1 BrdU was added to the growth media 72 and 48 h prior to transplantation, so that the transplanted cells could be identified in the host brain. After behavioral testing, the mice were anesthetized with an overdose of pentobarbital and perfused transcardially with 4% paraformaldehyde (Gadot, Netanya, Israel). The brains were then removed and postfixed overnight at 4° C in 4% paraformaldehyde, cryoprotected in 30% sucrose for another 24 h, and finally deep frozen in liquid nitrogen and stored in −70° C. The whole hippocampal area was cut into coronal 10 μm frozen sections. Every sixth section was mounted. Sections were fixed in acetone for 10 min in −20° C, dried, and incubated in 0.3% H2O2 in methanol for 15 min. After phosphate-buffered saline washes, the sections were incubated with 0.1 mg ml−1 proteinase K for 15 min. The sections were treated with 4 N HCl for 10 min and washed to neutralize the pH. Sections were incubated with anti-BrdU (clone Bu20a, 1:20 dilution; Dako, Glostrup, Denmark) overnight at 4° C. A goat anti-mouse immunoglobulin G secondary antibody, conjugated to Alexa 488 (Molecular Probes-Invitrogen, Carlsbad, CA, USA; 1:100), was added for 50 min at room temperature. Counterstaining was carried out with 4,6-diamidino-2-phenylindole. For double labeling, rabbit anti-GFAP (Dako, 1:100) or rabbit anti-neurofilament (Chemicon, Temecula, CA, USA; 1:100) was added and detected by Cy5-conjugated goat anti-rabbit antibody (Jackson, West Grove, PA, USA; 1:100). Mouse anti-neuronal nuclei was also used for double labeling (Chemicon; 1:200) together with a rat anti-BrdU antibody (Axyll, Westbury, NY, USA; 1:200) and detected with fluorescein isothiocyanate-conjugated goat anti-rat antibody and Cy5-conjugated goat anti-mouse antibody (both from Jackson; 1:100).
Coronal sections (10 μm) were cut spanning the entire rostral/caudal width of the hippocampus. Every sixth section was stained for BrdU and counted. The total number of BrdU+ cells was extrapolated for the entire hippocampus. For immunofluorescent analysis, BrdU-positive cells were first examined with fluorescent microscopy (Olympus, Melville, NY, USA) for double labeling with neurofilament or GFAP. Confirmation of double labeling was performed on a confocal microscope (Leica, Wetzlard, Germany).
Detection of macrophages and T cells in the host hippocampus
Twenty days after transplantation, control and neural-precursor-transplanted mice were anesthetized and perfused as described above. Coronal sections (10 μm) from the hippocampal area were further fixed in 4% paraformaldehyde for 20 min. After washing the sections in phosphate-buffered saline, they were blocked in 10% normal goat serum for 1 h at room temperature and incubated with rat anti CD-3 or rat anti-MAC-2 antibodies (1:100; Serotec, Oxford, UK) overnight at 4° C. A goat anti-rat immunoglobulin G secondary antibody, conjugated to Alexa 488 (Molecular Probes-Invitrogen; 1:100), was added for 50 min at room temperature. Counterstaining was performed with 4,6-diamidino-2-phenylindole.
BrdU administration and identification of newly born cells in the dentate gyrus
The third group of mice, which were transplanted with neural precursors on PN35, received two daily intraperitoneal injections of 50 mg kg−1 BrdU (Amersham, Buckinghamshire, UK) beginning on the day of grafting and continuing throughout the next 4 days. The commonly applied dose of BrdU (50 mg kg−1 in one daily injection for up to 12 days) has shown no physiological side effects such as weight loss or behavioral changes.26 In our case, 60 mg kg−1 day−1 was administered as two daily injections of 30 mg kg−1 for 5 days. Four hours after the last injection, the mice were perfused and the brains sectioned and stained with an anti-BrdU antibody, similar to the procedure described above. BrdU-positive cells were counted through the rostral/caudal extent of the dentate gyrus (granular cell layer+hilus). All BrdU-positive cells were quantified as described above for the immunocytochemical evaluation of the transplanted cells.
Data are presented as means and s.e., with differences between treatments established by multivariate analysis of variances, followed by Tukey test for post hoc comparisons between groups where appropriate. It should be noted that similar significance levels were shown with a two-tailed t-test for all major findings. Sex differences were considered in the analysis and in agreement with earlier work with prenatal heroin treatment,16, 17 the initial analysis of variance did not identify any significant interaction between treatment and sex; accordingly results from males and females are presented together. Significance for all tests was assumed at the level P<0.05.
Morris water maze performance
In line with our previous work, mice exposed prenatally to heroin displayed impaired Morris water maze performance, requiring more time than controls to reach the platform (Figure 1a; P<0.009); the gap between the control and the treated offspring remained unchanged throughout the entire test period (no interaction of treatment × day). Groups of mice were transplanted with neural precursor cell spheres into the hippocampus bilaterally. Neural sphere transplantation in normal mice had no effect on Morris water maze performance. However, in mice that were exposed prenatally to heroin, the neural progenitors totally reversed the deficits evoked by prenatal heroin exposure. Differences in Morris maze performance did not reflect alterations in swimming ability, as we did not find any differences in the total swimming speed when the platform was removed for the spatial probe test (Table 2). We also did not detect significant differences among groups for extinction in the spatial probe test (Figure 1b): across all groups, there was a progressive reduction in the time spent in swimming in the quadrant in which the platform had been located (P<0.008), but the trial-to-trial variability precluded finding significant intergroup differences in the extinction rate.
The transplant survival rate and fate
At 45 days post-transplantation, upon completion of behavioral testing, the mice were killed for histopathological assessment of grafted cells. Identification of grafted cells was enabled by labeling them prior to transplantation with BrdU. Quantification of surviving BrdU-labeled transplanted cells showed a mean number of 334±49 cells per brain. Considering that each animal was grafted with approximately 106 cells (5000 neurospheres divided between the two hippocampi, each containing 200 cells), this indicates a 0.0334% survival rate of the initial amount of transplanted cells. These were mostly localized within the hippocampal formation (Figure 2a), although a few cells could still be found along the corpus callosum and needle tract. A further study of these surviving cells revealed that 21.6% (72±0.5) had differentiated to astrocytes, as shown by GFAP labeling (Figure 2b), whereas no neuronal marker neurofilament or neuronal nuclei positive grafted could be found indicating a total absence of graft-derived neurons in the host dentate gyrus.
Macrophages and T cells in the transplanted hippocampus
To examine whether the loss of transplanted cells was due to graft rejection, immunofluorescent stains were performed for CD-3T cells and for MAC-2 macrophages to identify the immune response toward the graft. Twenty days after transplantation of neural progenitors, all animals showed a very eminent presence of CD-3-positive (Figures 3a and b) and MAC-2-positive (a galactose-specific lectin—Figures 3c and d) cells around the hippocampal area (especially the CA1 area), the corpus callosum and the needle tract, indicating the presence of T-cell phenotypes33 and inflammatory and mature murine macrophages,34 respectively. No immune cells were observed in the sham-transplanted control groups (Figures 3e and f).
Enhancement of endogenous cell proliferation in the dentate gyrus via neural progenitors transplantation to heroin-impaired offspring
The performance in the Morris water maze memory task is related to the rate of neurogenesis in the dentate gyrus of the hippocampus. Therefore, we counted the number of host brain proliferating neural progenitors in the dentate gyrus, as indicated by uptake of BrdU by the cells.
In heroin-exposed offspring there were 31% less BrdU-positive cells in the dentate gyrus as compared to control animals (370.5 cells mean heroin+sham, as opposed to 537.43 cells mean control). However, when transplanted with neural progenitors, the number of BrdU-positive cells in the dentate gyrus of heroin-exposed offspring returned to normal (79% increase as compared to sham-grafted heroin-exposed) and even showed some, albeit not statistically significant, overshoot compared to control levels (P<0.02, for the prenatal exposure × transplantation, interaction, analysis of variance). Control animals (not exposed to heroin prenatally) grafted with media or with neural progenitors did not differ in the number of BrdU-positive cells in the dentate gyrus and consequently their results here are pooled. Representative immunocytochemical pictures are shown in Figure 4. The quantitative differences between the neural progenitors and sham-grafted mice can be seen in Figure 5.
The present study attempts to resolve the apparent paradox of the successful reversal of neurobehavioral defects by neural progenitors transplantation on the one hand and, and on the other, the host brain paucity, primarily in xenographic and allographic studies, of visible transplanted cells, particularly neurons; a paucity, which according to the present findings, may be related to an immune reaction. We suggest that the production of new cells in the dentate gyrus is reduced by the insult (prenatal heroin exposure in the present study), and that the transplanted neural progenitors restore production of endogenous neural cells.
Heroin exposure during prenatal development was chosen as the insult that produces the neurobehavioral birth defects for the present model but, as was shown in models of similar teratogens, it does not produce gross morphological aberrations.35, 36 Although heroin is a major ‘hard’ drug of abuse in the US as well as in many other countries,37, 38 it was not chosen for its significance as a teratogen but rather for the fact that prenatal heroin has been shown, in our model, to produce specific defects in the mouse septohippocampal cholinergic innervation-related behaviors and the specific mechanism of this defect has been identified and extensively described. In particular, heroin-induced deficits in eight arm and Morris maze behaviors.16, 17, 18, 19, 20, 21, 39, 40, 41 Both of these behavioral tests are affected by the cholinergic septohippocampal projection.42, 43, 44 On the biochemical/molecular level, prenatal heroin induced both pre- and postsynaptic hyperactivity in the hippocampal cholinergic innervation,16, 17, 23, 45 converging on a total abolishment of the specific cholinergic-induced activation of PKCγ.16, 21, 41, 46 This appears to be a major mechanism of these behavioral defects.
The reversal of the PKCγ inactivation and behavioral deficits with neural progenitors was recently demonstrated41 and the behavioral deficits and their reversal were confirmed in the present study. Although PKC analysis could not be replicated as the brains were needed for immunocytochemical evaluations, based on previous studies,41 the behavioral results suggest that PKC inactivation occurred and was reversed by neural progenitors transplantation. Here we took the heroin model a step further, revealing another mechanism by which this drug exerts its teratogenic effect—inhibiting endogenous production of cells in the dentate gyrus. A central enigma in stem cell research is the possibility of a successful restoration of normalcy in the face of lower than expected visible transplanted cells in the host brain. Although this phenomenon was mostly shown in allographic and xenographic transplantation and/or grafting including our own,41, 47, 48 it was reproduced even with autographic neural progenitors.3 It was shown not only in a Parkinson's model, where a small number of surviving cells are expected and sufficient to produce reversal,47 but even in other models such as hippocampal neurofunctional deficits.48 Thus, to enable survival in allographic and xenographic models, immunosuppression became a routine procedure.9
In the present study we were able to demonstrate clearly this effect by employing an allographic model of neural progenitor transplantation to enhance rejection. HS/Ibg is a HS mouse stock made by crosses of eight inbred strains22 and was since kept heterozygous via a maximum outbreeding program, creating a strain very suitable for allographic transplantation. As already shown in a previous study,41 the survival rate of the grafted neural precursor cells was low and the prelabeled-transplanted cells differentiated mainly into glia. Because morphological defects are expected to be small, we could preclude any transplant-derived neuromorphological regenerative element in the beneficial effect of the graft.
In keeping with the literature,49 this scarcity of transplanted cells is expected to owe itself to immune rejection. MAC-2 (a galactose-specific lectin) is characteristically expressed by inflammatory and mature murine macrophages,34 and CD-3 indicates a presence of T-cell phenotype.33 These cells are the key mediators of graft rejection50 and their presence in the neural progenitors-transplanted brains coupled with their total absence sham-transplanted brains provides evidence of rejection processes resulting from neural progenitors transplantation.
Importantly, despite this rejection, reversal took place on the behavioral and cellular levels, confirming our hypothesis that one important mechanism by which transplanted neural progenitors exert their therapeutic effect is via the enhancement of endogenous cells production, as supported by a related study on bone marrow culture.51 To further substantiate this hypothesis, it should be noted that studies have shown that inflammatory processes have a negative effect on cell proliferation.52, 53 As such, molecular and behavioral recovery can be stimulated despite the fact that, as a result of the inflammatory and rejection processes, many of the transplanted cells are eventually absorbed by the host immune system. Further study ascertaining the fate of the endogenous cells produced as pertaining to the neurobehavioral recovery is still needed. Since in the present study the endogenous cells were only studied soon after their genesis, their fate obviously cannot yet be known. It can only be noted that they are positive for markers like Ki67 (a marker of the cell cycle), doublecortin (a marker for migrating neuronal cells) and nestin (marker of proliferating and migrating cells), all of which are downregulated as the cells start to differentiate.
It is important to note that even if a large proportion of the endogenous cells induced differentiated top glia and not neurons, it will still bear great therapeutic significance.
Whereas the importance of endogenous differentiation to neurons is obvious, there is a growing awareness of the role of glial cells, mainly astrocytes, in the reversal of neurobehavioral deficits.4, 6 Astrocytes are located close to the neural stem cells in the adult dentate gyrus54 and can express several factors that are able to independently increase proliferation.55, 56 Of particular relevance to the present study is the clear connection found between astrocytes, learning and PKCγ.57 Thus, this suggests the mechanism for behavioral reversal, given the well-known connection between PKCγ and Morris maze performance.58, 59, 60, 61, 62, 63 Thus, neural progenitors-induced endogenous differentiation can contribute to the therapeutic effect, even if that differentiation is not neuronal.
The mechanisms by which prenatal heroin exposure causes reduction in the production of endogenous cells are not yet known, though previous studies provide some indications. Muscarinic manipulation implicated the muscarinic signaling cascade, involving G proteins, Ca2+ elevation and PKC, in the proliferation of neural precursors.64 In our model, prenatal heroin exposure induced defects of the cholinergic innervation,16, 17, 23, 45 terminating in PKC inactivation,16, 21, 41, 46 thus serving as a possible explanation for prenatal heroin inhibition of endogenous neural precursors proliferation. It is possible that the muscarinic signaling cascade may be part of, and/or play a synergistic role to, the cytokine system, whose role in neurogenesis has been suggested by other investigators.11, 12, 13, 14, 15 Whether neurogenesis was causative of biochemical recovery, most importantly of PKC, or merely parallel thereof, remains a subject of future investigation.
In conclusion, allographic transplantation of neural progenitors was shown to restore neurobehavioral defects induced by an insult, in the present case, prenatal heroin exposure, despite lower than expected demonstrable transplanted neural progenitors-originating cells in the host brain. To explain this paradox, we have shown that a possible central mechanism for the therapeutic action of the neural progenitors is the induction, directly or indirectly, of endogenous cells production in neurogenic regions, originally depleted by the insult. Further insight can be gained in the future through very extensive studies where proliferation of endogenous cells, as well as the extent of grafted cells, immune markers and cytokines, is evaluated at more temporal points, including at the time of expression of behavioral correction. Additionally, the fate of the endogenous cells thus produced and their therapeutic mechanisms, whether it is by rescuing existing cells (through differentiation to glia) or by replacing lost cells and restoring damaged circuitries (through differentiation to neurons), remains the subject of future investigations.
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This work was supported by USPHS Grant ES13147, the United States-Israel Binational Science Foundation BSF2005003 and the Israeli Anti-Drug Authority.
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Ben-Shaanan, T., Ben-Hur, T. & Yanai, J. Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain. Mol Psychiatry 13, 222–231 (2008). https://doi.org/10.1038/sj.mp.4002084
- endogenous cells
- immunological rejection
- neural progenitors therapy
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