Rescue of radiation-induced cognitive impairmentthrough cranial transplantation of humanembryonic stem cellsMunjal M. Acharyaa, Lori-Ann Christieb, Mary L. Lana, Peter J. Donovanc, Carl W. Cotmanb, John R. Fiked,and Charles L. Limolia,1

aDepartment of Radiation Oncology, University of California, Irvine, CA 92697-2695; bInstitute for Memory Impairments and Neurological Disorders,University of California, Irvine, CA 92697-4540; cDepartments of Biological Chemistry and of Developmental and Cell Biology, and the Sue and Bill GrossStem Cell Research Center, University of California, Irvine, CA 92697-3940; and dDepartment of Neurological Surgery, University of California, San Francisco,CA 94110

Edited by James E. Cleaver, Room N431, University of California, San Francisco, CA, and approved September 30, 2009 (received for review August 17, 2009)

Cranial irradiation remains a frontline treatment for the control oftumor growth, and individuals surviving such treatments often man-ifest various degrees of cognitive dysfunction. Radiation-induceddepletion of stem/precursor cell pools in the brain, particularly thoseresiding in the neurogenic region of the hippocampus, is believed, inpart, to be responsible for these often-unavoidable cognitive deficits.To explore the possibility of ameliorating radiation-induced cognitiveimpairment, athymic nude rats subjected to head only irradiation (10Gy) were transplanted 2 days afterward with human embryonic stemcells (hESC) into the hippocampal formation and analyzed for stemcell survival, differentiation, and cognitive function. Animals receiv-ing hESC transplantation exhibited superior performance on a hip-pocampal-dependent cognitive task 4 months postirradiation, com-pared to their irradiated surgical counterparts that did not receivehESCs. Significant stem cell survival was found at 1 and 4 monthspostirradiation, and transplanted cells showed robust migration tothe subgranular zone throughout the dentate gyrus, exhibiting signsof neuron morphology within this neurogenic niche. These resultsdemonstrate the capability to ameliorate radiation-induced normaltissue injury using hESCs, and suggest that such strategies mayprovide useful interventions for reducing the adverse effects ofirradiation on cognition.

cognition � stem cells � radiotherapy

Exposure of the brain to ionizing radiation occurs under avariety of clinical situations, and is an important adjuvant

treatment for primary and metastatic brain tumors, head andneck cancers, and central nervous system leukemias/lymphomas.While radiotherapy is the single most effective treatment aftersurgical resection (1), the dose that can be safely administeredis limited by the tolerance of normal brain tissue. Typical dosesfor whole or partial brain irradiation can exceed 50 Gy (2, 3),while more spatially constrained procedures (e.g., LINAC-basedor gamma knife radiosurgery) can deposit single doses well inexcess of 15 Gy to normal tissue at the tumor margins (4). Whilerecent advances in dose delivery have minimized overt tissueinjury, less severe morphologic injury can occur at these andmuch lower doses, resulting in variable degrees of cognitivedysfunction in both pediatric and adult patients (5–8). Whileradiation-induced cognitive deficits have a varied character, theyoften include significant impairments in hippocampus-dependent function such as learning, memory, and spatialinformation processing (9–11).

The hippocampus is one of two active sites of neurogenesis inthe mammalian brain (12), and stem/precursor cell proliferationin the subgranular zone (SGZ) of the dentate gyrus generatescells that migrate to the granule cell layer (GCL) and differen-tiate into mature neuronal and glial phenotypes (13). Significantwork has now demonstrated that these newly born neuronsdevelop granule cell morphology and express mature neuronal

markers (14), project connections to their normal CA3 targetregion and develop action potentials and synaptic inputs (15, 16).Further evidence supporting the functionality of these newlyborn neurons include data showing that changes in neurogenesisare positively correlated with changes in cognition (17–19).Insults such as irradiation (20), inflammation (21, 22), and stress(23) inhibit neurogenesis and elicit cognitive declines, whilemanipulations of the redox microenvironment (24), the use ofanti-inflammatory agents (25) or environmental enrichment(26) upregulate neurogenesis and can ameliorate, at least in part,these cognitive dysfunctions. While the extent and precisemechanisms by which new neurons functionally impacthippocampal-dependent processes remains uncertain, it seemslikely that strategies capable of augmenting neuronal cell num-bers in the hippocampus may provide an avenue for rescuingcertain cognitive deficits brought on by the inhibition of neuro-genesis and/or attrition of granule cell neurons.

Regenerative medicine holds great promise for the treatmentof a variety of ailments, but to date, the only routine applicationfor stem cell therapy involves hematopoietic stem cell transplan-tation for the treatment of leukemias and lymphomas (27). Bonemarrow-derived stem cells that are capable of reconstituting theimmune system after irradiation (27), have also been found tofacilitate the repair of brain injury in rodent models (28). Anumber of stem and progenitor cell types have been proposed forthe treatment of neurological diseases including neural, bonemarrow, umbilical cord, and embryonic stem cells (29, 30). Manyof these approaches have been used successfully in animalmodels to ameliorate degenerative conditions such as Alzhei-mer’s (31, 32), Parkinson’s (33, 34), and Huntington’s (35)disease, as well as other disorders such as epilepsy (36–38),excitotoxic brain damage (39), and traumatic brain injury (40).While a recent laboratory study has demonstrated the efficacy oftransplanting salivary stem cells to alleviate radiation-inducedxerostomia (41), with the exception of the bone marrow, theapplication of stem cell therapies to reduce radiation-inducednormal tissue damage is still in its infancy. Ultimately, thesuccess (or failure) of such approaches will depend on thefunctional engraftment of transplanted cells into the irradiatedtissue bed. In the CNS, irradiation triggers a complex andmultifactorial response involving persistent increases in oxida-tive species and inflammatory cytokines that actively participate

Author contributions: C.L.L. designed research; M.M.A. and M.L.L. performed research;P.J.D. contributed new reagents/analytic tools; M.M.A., L.-A.C., C.W.C., and C.L.L. analyzeddata; and M.M.A., L.-A.C., J.R.F., and C.L.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail: climoli@uci.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0909293106/DCSupplemental.

19150–19155 � PNAS � November 10, 2009 � vol. 106 � no. 45 www.pnas.org�cgi�doi�10.1073�pnas.0909293106

Dow

nloa

ded

by g

uest

on

June

7, 2

020

in the remodeling of the irradiated microenvironment (42, 43).The protracted nature of the CNS radiation response contributesto the inhibition of endogenous neurogenesis (21, 44) andimpairs the differentiation of neural precursor cells when trans-planted into the irradiated brain (45). Such changes are nothowever, irreversible (44), and here we report that humanembryonic stem cells (hESCs) transplanted into the hippocam-pus can rescue cognitive impairment caused by irradiation,thereby providing evidence that these pluripotent cells can beused to improve radiation-induced normal tissue injury.

ResultsIn Vitro and in Vivo Immunostaining of hESCs. Before transplanta-tion, hESCs were maintained on matrigel in an undifferentiatedstate. Pluripotency of hESCs was verified by immunostaining forOct-4 (transcription factor Octamer-4) and SSEA-4 (glycolipidsurface - Stage Specific Embryonic Antigen-4), and under ourculture conditions, cells showed abundant expression of thesemarkers (Fig. 1 A–D). For the studies presented, hESCs wereused at passages 42–49.

To trace hESCs after grafting, hESCs were labeled in vitrowith BrdU before transplantation. In vitro controls verified thecolabeling of hESCs with BrdU and a human specific nuclear

antigen (Fig. 1E). These labels were also used to verify thepresence of the BrdU positive human cells in tissue sections ofanimals engrafted with hESCs (Fig. 1F). The correspondencebetween cells colabeled with BrdU and human specific markersindicated that grafted cells did not undergo significant prolifer-ation postgrafting, and validates these markers for tracking ofgrafted hESCs in the host tissue. A schematic representation ofour study is illustrated in Fig. 1G.

Transplanted hESCs Rescue Cognitive Deficits. Four months post-grafting, rats were habituated and trained on a novel placerecognition task (NPR). The NPR task uses the innate tendencyof rats to explore novelty. First, rats are exposed to two identicalobjects in specific spatial locations within a test arena. Followinga retention interval, one of the objects is moved to a new spatiallocation and rats are placed in the test arena again. Rats thatremember the previous spatial arrangement of the objects willspend more time exploring the object that has been moved to thenovel spatial location. Successful performance of the task hasbeen shown to rely on intact hippocampal function (46, 47).

As expected, irradiated rats (IRR) showed impaired novelplace recognition at the short, 5-min retention interval. Irradi-ated animals spent significantly less time exploring the novel

Fig. 1. Pluripotency markers in hESCs. Immunocytochemical analysis of hESCs counter-stained with DAPI (A) shows strong expression and colocalization of thepluripotent markers Stage Specific Embryonic Antigen, (SSEA-4, B), and transcription factor Octamer-4, (Oct-4, C), shown as merged image (D). Colabeling of BrdU(red) and human specific nuclear antigen (green) in vitro (E) and in vivo (F) confirm the correspondence between BrdU labeled hESC and the presence of a specifichuman antigen. Orthogonal images (E and F) represent 3-D reconstructions of 1-�m confocal sections, and (F) were derived from a coronal section of a rat braingrafted with hESCs 4 months prior. (G) Schematic illustration of transplantation studies. Two-month-old athymic nude rats given 10 Gy head-only irradiation weregrafted with hESC 2 days post-irradiation. At 4 months following these procedures, animals were subjected to cognitive testing then euthanized for thedetermination of transplanted cell survival and differentiation. A cohort of animals that did not undergo cognitive testing was also euthanized at 1 monthpost-transplant for survival and differentiation studies. (Scale bars, 200 �m in A–D; 2 �m in E; and 4 �m in F).

Acharya et al. PNAS � November 10, 2009 � vol. 106 � no. 45 � 19151

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

June

7, 2

020

place compared to control animals (Fig. 2A). In contrast, afterthe 5-min retention interval, irradiated, hESC-transplanted rats(IRR�hESC) did not differ from control animals and spentmore time than expected by chance (indicated by the dashed linein Fig. 2 A) exploring the novel spatial location (Fig. 2 A). Similartrends were observed in terms of total time spent exploring the

novel spatial location (Fig. 2B), and in frequency of visits to thenovel spatial location and latency to explore the novel spatiallocation. These results suggested that hESC transplantationattenuated post-irradiation cognitive impairments and pre-served short-term memory for spatial information in a hip-pocampal-dependent task.

At the 24-h retention interval, IRR rats again showed im-paired NPR compared to control animals (Fig. 2C). By contrast,IRR�hESC rats did not differ significantly from control ani-mals. However, IRR�hESC animals did not spend more timethan expected by chance exploring the novel spatial location.Again, we observed similar trends when we analyzed totalexploration of the novel spatial location (Fig. 2D), as well asfrequency of visits and latency to first explore the novel location.The results suggest that hESC transplantation may also improvelong-term retention of spatial information, although the treat-ment appeared to be more effective at the shorter, 5-minretention interval. During the 24-h test phase only, controlanimals receiving hESCs spent significantly less time exploringthe novel place compared to controls receiving vehicle injections(Fig. 2C). This result suggests that hESC implantation in controlanimals disrupts long-term but not short-term recognition mem-ory, although further behavioral testing is necessary to confirmthis finding. These data suggest that in the intact hippocampus,hESC transplantation may have a distinct dose-response (i.e.,cell number) or may actually disrupt hippocampal function ascompared to an irradiated microenvironment that might be moreconducive to integration of grafted hESCs.

To better understand why IRR animals showed impairedshort- and long-term NPR abilities, we analyzed total time spentexploring both objects during the familiarization phase givenbefore the retention intervals. We found a significant differencebetween the groups; IRR animals tended to spend less timeexploring during the familiarization phase compared to both thecontrol and IRR�hESC animals (Fig. 2E). This result suggestedthat the impairments seen in the IRR animals at 5min and 24 hpost-familiarization might be related to lower exploration duringinitial exposure to the objects.

Location and Migration of hESCs in the Irradiated Rat Hippocampus.At 4 months posttransplant and after cognitive testing, animalsin each group were euthanized for immunocytochemical anal-yses. An additional cohort of animals not subjected to cognitivetests was analyzed at 1 month post-transplant, to assess earlypatterns of incorporation and differentiation of the graftedhESCs. An examination of BrdU immunostained sections fromanimals that received transplants revealed that the majority ofgrafted cells were located within the hippocampal formation andadjacent to the CA1 subfield and corpus callosum (Fig. 3).Representative sections showed the intrahippocampal locationof grafted hESCs at 1 (Fig. 3 A–C) and 4 months post-transplant(Fig. 3 D–F). Similar localization and migration patterns ofhESCs were found at both post-transplant times in all animalsreceiving grafts. Moreover, the data show that grafted hESCsmigrated extensively into the dentate gyrus (DG), hilus (DH),and hippocampal CA1 subfield, (Fig. 3). In both irradiated andunirradiated animals, grafted hESC were also found to exhibitsignificant homing to the SGZ, populating this neurogenic nichethroughout the septo-temporal axis of the host hippocampus.

Neural Differentiation of Transplanted hESCs. To analyze the differ-entiation of engrafted hESCs, dual immunofluorescence and Z-section analyses using confocal microscopy were performed onrepresentative sections at 1 and 4 month post-transplant. Analysisof BrdU colabeling with NeuN or GFAP demonstrated that aportion of cells showed neural and astrocytic differentiation at bothposttransplant times in all animals that received hESC grafting.Grafted cells positive for both BrdU and NeuN displayed mature

Fig. 2. Human embryonic stem cell implantation improves radiation-induced impairments in novel place recognition (NPR). Rats were first famil-iarized with two identical objects in specific spatial locations in an open field,and total time spent exploring was assessed. Following a 5-min retentioninterval, they were re-presented with the same objects with one moved to anovel spatial location. (A) Irradiated animals (IRR) spent a significantly lowerproportion of time exploring the novel place [ANOVA, P � 0.031, FPLSD, P �0.015 vs. controls (Con) and vs. hESC-implanted controls (Con�hESC)]. Incontrast, irradiated animals that received hESC injections (IRR�hESC) did notdiffer from either control group (P �0.25), and spent more time exploring thenovel place than expected by chance (dashed line at 50%) although the latterwas not found to be statistically significant (one sample t-test, P � 0.364).Nonetheless, the fact that the IRR�hESC group did not differ from controlssuggests that performance was partially restored following hESC grafting.Twenty-four hours after the initial familiarization phase, rats were againpresented with the same two objects, with one moved to a new spatiallocation. After the 24-h retention interval, (C) animals in the IRR group spenta significantly lower proportion of time exploring the novel place (ANOVA,P � 0.042, FPLSD, P � 0.007 vs. Con). In contrast, IRR�hESC animals did notdiffer from controls (P � 0.208). The results suggest that hESC transplantationpartially rescued radiation-induced deficits on hippocampal dependent NPRtask. Similar trends were observed when total time spent exploring the novelplace was assessed after both the 5-min (B) and 24-h (D) retention intervals.Analysis of time spent exploring both objects during the initial familiarizationphase revealed that IRR animals tended to spend less time engaged in explo-ration than both the Con and IRR�hESC group (E, ANOVA, P � 0.026, FPLSD,P � 0.068 vs. Con and P � 0.004 vs. IRR�hESC), which may partially explain whythey showed impairments in NPR following the retention intervals. Data arepresented as means � 1 S.E.M.

19152 � www.pnas.org�cgi�doi�10.1073�pnas.0909293106 Acharya et al.

Dow

nloa

ded

by g

uest

on

June

7, 2

020

neuronal morphology (Fig. 4 A, B, E, and F). Grafted cellsexpressing these neuronal markers were predominately found at theSGZ, as opposed to other hippocampal subfields. Assessment ofastrocytic differentiation via BrdU and GFAP dual immunofluo-rescence revealed the presence of GFAP positive astrocytes at eachposttransplant time analyzed (Fig. 4 C, D, G, and H). Grafted cellsexpressing astrocytic phenotypes were uniformly distributedthroughout the hippocampus, with no apparent preference for aparticular hippocampal region.

DiscussionThe data presented here show that transplantation of hESCs intothe hippocampal formation can impact the development ofradiation-induced cognitive impairment. While some data existshowing that such impairments can be ameliorated in part byvoluntary running (26), this report shows that hESC transplan-tation can be effectively used to reduce a serious complicationof cranial irradiation. The mechanism of this cell-mediatedeffect is not yet clear, and whether it depends on cell replacementper se, as opposed to trophic support provided by the trans-planted cells, remains to be determined. In the present study,we were able to show that at both 1 and 4 months post-transplantation, hESC cells expressed neuronal and astrocyticmarkers. Significant survival and migration of the transplantedcells to the SGZ were observed at 1 and 4 months post-irradiation, with greater survival found at 1 month. There was agreater likelihood for the fraction of cells migrating to the SGZto develop mature neuronal phenotypes compared to those cellsresiding in non-neurogenic regions. Whether those cells wereactually integrated into hippocampal circuits is not yet known.Regarding trophic support, studies of ischemic injury foundtransplanted neural stem/precursor cells to be neuroprotectivevia glial cell line-derived neurotrophic factor (GDNF) (48, 49).Other work using a transgenic model of Alzheimer diseaseprovided evidence that brain-derived neurotrophic factor(BDNF) from transplanted rodent neural stem cells (mNSC)played a significant role in restoring certain cognitive deficitswhen assessed 1-month posttransplantation (31). Regardless ofthe manner in which the transplanted pluripotent cells func-

tioned in this study, their mere presence seemed to amelioratethe adverse effects of ionizing irradiation on cognition.

We elected to use the novel place recognition (NPR) task toassess the effects of hippocampal hESC transplantation, as suc-cessful performance on the task has been shown to depend on intacthippocampal function (46, 47). Our behavioral studies have cor-roborated a number of past reports linking radiation-inducedalterations in cognition to impaired hippocampal neurogenesis (9,10), and have extended them by showing that irradiation causessignificant short- and long-term memory impairments on the NPRtask in rats. Irradiated animals that received vehicle injectionsexhibited significantly impaired NPR compared to unirradiatedcontrols � hESC, suggesting their short- (5-min) and long-term(24-h) memory for the former spatial arrangement of the objectswas impaired (Fig. 2). Transplantation of hESC into irradiatedanimals (IRR�hESC) was found to reduce postirradiation cogni-tive impairments. At short (5-min) and long (24-h) retentionintervals, animals that received IRR�hESC did not differ signifi-cantly from unirradiated controls � hESC, suggesting that hESCrestored NPR to at least levels intermediate between the controland IRR groups. Animals that received IRR�hESC showed atrend to spend more time exploring the novel spatial location thanexpected by chance (5min test), suggesting preservation of short-term memory. Further, in the irradiated groups, the duration andfrequency of exploration of the novel place was significantly higherin animals transplanted with hESC versus vehicle (t-tests; P �0.05).However, more rigorous testing using additional hippocampal-dependent tasks such as the Morris water maze (50, 51) orcontextual fear conditioning (52) may provide more informationabout the robustness of our observed effect, and possibly about itsunderlying cause.

The cells used for transplantation were pluripotent, at leastbased on marker expression (Oct4, SSEA4, Fig. 1) in vitro, andthe BrdU labeling indices of those cells was approximately 98%.Additionally, there was a one-to-one correspondence betweencells staining positive for the BrdU label and the human specificnuclear antigen (Fig. 1). While the survival of the transplantedcells was qualitatively greater at 1 month than at 4 months aftertransplantation, there were still large numbers of BrdU� cells at

Fig. 3. Survival and intrahippocampal location of transplanted hESCs. At 1 month postgrafting, hESCs are shown to incorporate extensively throughout thehost-hippocampus (magnification, �5–20 in A–C). Qualitatively similar patterns of migration were observed at 4 months post-grafting, albeit lower hESCnumbers were evident (magnification, �5–20 in D–F). Grafted cells were detected by BrdU immunostaining (dark brown nuclei) and counterstained withhaematoxylin. Transplanted hESCs migrated extensively from the site of injection throughout the hippocampal formation (dentate gyrus, DG, dentate hilus, DH,granule cell layer, GCL, and CA1) and partially in the corpus callosum. Images shown were derived from irradiated animals. (Scale bars, 200 �m in A and D; 100�m in B and E; and 50 �m in C and F.

Acharya et al. PNAS � November 10, 2009 � vol. 106 � no. 45 � 19153

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

June

7, 2

020

the later time point (Fig. 3). Further, those cells had migratedpervasively throughout the hippocampus and seemed to bepredominantly located in or near the dentate SGZ (Figs. 3 and4). It was of particular interest that the transplanted cells near theSGZ showed morphologic changes consistent with mature neu-

rons while those at the site of transplantation were more glial innature. This suggests that the neurogenic niche had someinfluence on the morphologic character of the cells, althoughexactly how is not known. It is also worth noting that no overtadverse cognitive sequelae (e.g., ataxia) were observed in thecohort of animals receiving hESC grafting (Con/IRR�hESC),suggesting that intracranial teratogenesis was not problematic,although this was not tested for directly.

While transplantation of stem cells for the therapeuticmanagement of disease and degenerative conditions may oneday be realized, this approach has to be considered in thecontext of a number of potential limitations, including immu-norejection (53), teratogenesis and tumorigenesis (54), andethical debates (55). Recent advances in generating inducedpluripotent stem cells (iPS) may alleviate some of theseconcerns (56), but considerable work is still needed to deter-mine if a transplantation effect, such as that shown here, is aviable option for use in humans. Regardless, the data shownhere constitutes a finding that transplanted hESCs can survive,differentiate along neuronal lineages, and ameliorate cogni-tive impairments after cranial irradiation. These data areencouraging and offer the promise of tailoring other stem cellstrategies for minimizing the adverse side effects associatedwith exposure to ionizing radiation.

Materials and MethodsAnimals and Irradiation. Young (2-month-old) athymic nude rats acquiredfrom National Cancer Institute (strain 02N01 Cr:NIH-rnu) were used in thisstudy. All animal procedures were performed in strict accordance with theNational Institutes of Health and Institutional IACUC guidelines. Anesthetizedrats were subjected to head only �-irradiation (10 Gy) using a 137Cs irradiator(J.L. Shepard and Associates Mark I) at the dose rate of 2.07 Gy/min. Growthand characterization of hESC and the processing of all tissue sections followedstandard procedures (see SI Methods for further details).

Intrahippocampal Transplantation of hESCs. Rats received bilateral intrahip-pocampal injections of BrdU labeled hESCs 48 h after cranial or sham irradiation.1.0 � 105 live cells were injected in 1 �L of the cell suspension using a 5-�LHamilton microsyringe (33-gauge) at an injection rate of 0.25 �L/min. Eachhippocampi received four distinct graft injections using the following stereotaxiccoordinates: (i) AP: 3.0 mm from bregma, ML: 1.8 mm from midline, and ventralDV:3.2mmfromthesurfaceof thebrain; (ii)AP:3.6mm,ML:2.5mm,DV:3.2mm;(iii) AP: 4.2 mm, ML: 3.2 mm, DV: 3.2 mm and (iv) AP: 4.8 mm, ML: 4.0 mm, DV: 3.2mm. Thus, the hippocampus of each hemisphere received a total of 4.0 � 105 livehESCs. Control and irradiated rats receiving vehicle (sterile conditioned medium)at the same stereotaxic coordinates served as sham surgery groups.

Behavioral Analysis. At 4 months post-grafting, a blinded observer examined ratsin all groups [control (Con�hESC) and irradiated (IRR�hESC) rats receiving hESCgrafts, control (Con) and irradiated (IRR) rats receiving sham surgery and vehicleinjections], for hippocampal-dependent spatial memory using a novel placerecognition (NPR) task according to a standard protocol (46) (SI Methods).

Measures and Analyses. Exploration ratio, or the percentage of total explo-ration time spent exploring the novel spatial location (tnovel/tnovel�tfamiliar) wasused as the main dependent measure. We chose to analyze the behavior of theanimals during minute 1 of the 3-min, 5-min, and 24-h test phases becauseprevious research has shown that preference for the novel place diminishesafter the first minute, as the spatial locations become equally familiar to theanimals (46). For completeness, repeated measures ANOVA that included alldata from each of the three test sessions were performed. These analysesconfirmed our behavioral findings found during the first minute. We alsoanalyzed the total time, frequency of visits, and latency to first explore thenovel place during the test phases. Additionally, time spent exploring bothfamiliar places was recorded during the familiarization phase.

Exploration ratio data were analyzed using univariate ANOVAs for the5-min and 24-h test phases (PASW Statistics version 17.0, SPSS Inc.). When astatistically significant (i.e., P � 0.05) overall group effect was found, FisherProtected Least Significant Difference (FPLSD) post hoc tests were run tocompare the four groups.

Fig. 4. Differentiation of transplanted hESCs. At 1 month post-grafting(A–D), BrdU (red) positive hESCs were observed to differentiate into matureneurons (A and B) or astrocytes (C and D) as indicated by the colabeling of theneuron-specific nuclear antigen (NeuN, green) or glial fibrillary acidic protein(GFAP, green). Similar phenotypes were found 4 months post-grafting (E–H),where BrdU (red)-positive cells were also found to express markers of matureneurons (NeuN, E and F) and astrocytes (GFAP, G and H). Orthogonal recon-structions of BrdU�/NeuN� colabeled cells (B and F) and BrdU�/GFAP� cola-beled cells (D and H) are shown at each post-grafting time. Images shown werederived from irradiated animals. (Scale bars, 20 �m in A and E; 50 �m in C andG; and 20 �m in B, D, F, H.)

19154 � www.pnas.org�cgi�doi�10.1073�pnas.0909293106 Acharya et al.

Dow

nloa

ded

by g

uest

on

June

7, 2

020

ACKNOWLEDGMENTS. We thank Drs. Aileen J. Anderson and Brian J. Cum-mings for their help in defining stereotactic coordinates and other method-ological details and Christina Tu for her technical assistance in the growth andcharacterization of hESCs. This work was supported by California Institute of

Regenerative Medicine Seed Grant RS1-00413 (to C.L.L.) and National Insti-tutes of Health Grants AG 00538 and AG 16573 (to C.W.C.) and RO1 HD49488and PO1 HD47675 and California Institute of Regenerative Medicine GrantRC1-00110 (to P.J.D.).

1. Walker MD, Strike TA, Sheline GE (1979) An analysis of dose-effect relationship in theradiotherapy of malignant gliomas. Int J Radiat Oncol Biol Phys 5:1725–1731.

2. Sheline GE, Wara WM, Smith V (1980) Therapeutic irradiation and brain injury. Int JRadiat Oncol Biol Phys 6:1215–1228.

3. Tofilon PJ, Fike JR (2000) The radioresponse of the central nervous system: A dynamicprocess. Radiat Res 153:357–370.

4. Shaw E, et al. (2000) Single dose radiosurgical treatment of recurrent previouslyirradiated primary brain tumors and brain metastases: Final report of RTOG protocol90–05. Int J Radiat Oncol Biol Phys 47:291–298.

5. Butler J, Rapp S, Shaw E (2006) Managing the cognitive effects of brain tumor radiationtherapy. Curr Treat Options Oncol 7:517–523.

6. Meyers CA, Brown PD (2006) Role and relevance of neurocognitive assessment inclinical trials of patients with CNS tumors. J Clin Oncol 24:1305–1309.

7. Roman DD, Sperduto PW (1995) Neuropsychological effects of cranial radiation:Current knowledge and future directions. Int J Radiat Oncol Biol Phys 31:983–998.

8. Surma-aho O, et al. (2001) Adverse long-term effects of brain radiotherapy in adultlow-grade glioma patients. Neurology 56:1285–1290.

9. Raber J, et al. (2004) Radiation-induced cognitive impairments are associated withchanges in indicators of hippocampal neurogenesis. Radiat Res 162:39–47.

10. Rola R, et al. (2004) Radiation-induced impairment of hippocampal neurogenesis isassociated with cognitive deficits in young mice. Exp Neurol 188:316–330.

11. Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S (2006) Inhibition of neuro-genesis interferes with hippocampus-dependent memory function. Hippocampus16:296–304.

12. Gage FH (2000) Mammalian neural stem cells. Science 287:1433–1438.13. Palmer TD, Takahashi J, Gage FH (1997) The adult rat hippocampus contains primordial

neural stem cells. Mol Cell Neurosci 8:389–404.14. Cameron HA, Woolley CS, McEwen BS, Gould E (1993) Differentiation of newly born

neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56:337–344.15. Markakis EA, Gage FH (1999) Adult-generated neurons in the dentate gyrus send

axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol406:449–460.

16. van Praag H, et al. (2002) Functional neurogenesis in the adult hippocampus. Nature415:1030–1034.

17. Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal neurons in adult miceliving in an enriched environment. Nature 386:493–495.

18. Kempermann G, van Praag H, Gage FH (2000) Activity-dependent regulation of neu-ronal plasticity and self repair. Prog Brain Res 127:35–48.

19. Song H, et al. (2005) New neurons in the adult mammalian brain: Synaptogenesis andfunctional integration. J Neurosci 25:10366–10368.

20. Mizumatsu S, et al. (2003) Extreme sensitivity of adult neurogenesis to low doses ofX-irradiation. Cancer Res 63:4021–4027.

21. Fike JR, Rola R, Limoli CL (2007) Radiation response of neural precursor cells. NeurosurgClin N Am 18:115–127.

22. Rola R, et al. (2005) High-LET radiation induces inflammation and persistent changesin markers of hippocampal neurogenesis. Radiat Res 164:556–560.

23. Leuner B, Gould E, Shors TJ (2006) Is there a link between adult neurogenesis andlearning? Hippocampus 16:216–224.

24. Rola R, et al. (2007) Lack of extracellular superoxide dismutase (EC-SOD) in themicroenvironment impacts radiation-induced changes in neurogenesis. Free Radic BiolMed 42:1133–1145.

25. Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade restores adult hippocam-pal neurogenesis. Science 302:1760–1765.

26. Naylor AS, et al. (2008) Voluntary running rescues adult hippocampal neurogenesisafter irradiation of the young mouse brain. Proc Natl Acad Sci USA 105:14632–14637.

27. Greenberger JS, Epperly M (2009) Bone marrow-derived stem cells and radiationresponse. Semin Radiat Oncol 19:133–139.

28. Peister A, et al. (2006) Novel object recognition in Apoe(-/-) mice improved by neonatalimplantation of wild-type multipotential stromal cells. Exp Neurol 201:266–269.

29. Hess DC, Borlongan CV (2008) Stem cells and neurological diseases. Cell Prolif 41:94–114.30. Le Belle JE, Svendsen CN (2002) Stem cells for neurodegenerative disorders: Where can

we go from here? BioDrugs 16:389–401.

31. Blurton-Jones M, et al. (2009) Neural stem cells improve cognition via BDNF in atransgenic model of Alzheimer disease. Proc Natl Acad Sci USA106:13594–13599.

32. Yamasaki TR, et al. (2007) Neural stem cells improve memory in an inducible mousemodel of neuronal loss. J Neurosci 27:11925–11933.

33. Meissner KK, Kirkham DL, Doering LC (2005) Transplants of neurosphere cell suspen-sions from aged mice are functional in the mouse model of Parkinson’s. Brain Res1057:105–112.

34. Roy NS, et al. (2006) Functional engraftment of human ES cell-derived dopaminergicneurons enriched by coculture with telomerase-immortalized midbrain astrocytes. NatMed 12:1259–1268.

35. Armstrong RJ, Watts C, Svendsen CN, Dunnett SB, Rosser AE (2000) Survival, neuronaldifferentiation, and fiber outgrowth of propagated human neural precursor grafts inan animal model of Huntington’s disease. Cell Transplant 9:55–64.

36. Acharya MM, Hattiangady B, Shetty AK (2008) Progress in neuroprotective strategiesfor preventing epilepsy. Prog Neurobiol 84:363–404.

37. Boison D (2009) Engineered adenosine-releasing cells for epilepsy therapy: Human mes-enchymal stem cells and human embryonic stem cells. Neurotherapeutics 6:278–283.

38. Maisano X, et al. (2009) Embryonic stem cell-derived neural precursor grafts fortreatment of temporal lobe epilepsy. Neurotherapeutics 6:263–277.

39. Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ (2005) Transplanted humanembryonic germ cell-derived neural stem cells replace neurons and oligodendrocytesin the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res82:592–608.

40. Riess P, et al. (2007) Embryonic stem cell transplantation after experimental traumaticbrain injury dramatically improves neurological outcome, but may cause tumors.J Neurotrauma 24:216–225.

41. Lombaert IM, et al. (2008) Rescue of salivary gland function after stem cell transplan-tation in irradiated glands. PLoS One 3:e2063.

42. Limoli C, et al. (2004) Radiation response of neural precursor cells: Linking cellular sensi-tivity to cell cycle checkpoints, apoptosis and oxidative stress. Radiat Res 161:17–27.

43. Chiang CS, et al. (1997) Delayed molecular responses to brain irradiation. Int J RadiatBiol 72:45–53.

44. Fike JR, Rosi S, Limoli CL (2009) Neural precursor cells and central nervous systemradiation sensitivity. Semin Radiat Oncol 19:122–132.

45. Monje ML, Mizumatsu S, Fike JR, Palmer TD (2002) Irradiation induces neural precursor-cell dysfunction. Nat Med 8:955–962.

46. Mumby DG, Gaskin S, Glenn MJ, Schramek TE, Lehmann H (2002) Hippocampal damageand exploratory preferences in rats: Memory for objects, places, and contexts. LearnMem 9:49–57.

47. Save E, Buhot MC, Foreman N, Thinus-Blanc C (1992) Exploratory activity and responseto a spatial change in rats with hippocampal or posterior parietal cortical lesions. BehavBrain Res 47:113–127.

48. Chen B, et al. (2009) Neuroprotective effect of grafting GDNF gene-modified neuralstem cells on cerebral ischemia in rats. Brain Res 1284:1–11.

49. Kameda M, et al. (2007) Adult neural stem and progenitor cells modified to secreteGDNF can protect, migrate and integrate after intracerebral transplantation in ratswith transient forebrain ischemia. Eur J Neurosci 26:1462–1478.

50. Acevedo SE, McGinnis G, Raber J (2008) Effects of 137Cs gamma irradiation on cogni-tive performance and measures of anxiety in Apoe-/- and wild-type female mice. RadiatRes 170:422–428.

51. Morris R (1984) Developments of a water-maze procedure for studying spatial learningin the rat. J Neurosci Methods 11:47–60.

52. Villasana L, Rosenberg J, Raber J (2009) Sex-dependent effects of (56)Fe irradiation oncontextual fear conditioning in C57BL/6J mice. Hippocampus, 10.1002/hipo.20659.

53. Lui KO, Waldmann H, Fairchild PJ (2009) Embryonic stem cells: Overcoming the immu-nological barriers to cell replacement therapy. Curr Stem Cell Res Ther 4:70–80.

54. Knoepfler PS (2009) Deconstructing stem cell tumorigenicity: A roadmap to saferegenerative medicine. Stem Cells 27:1050–1056.

55. Daley GQ, et al. (2007) Ethics. The ISSCR guidelines for human embryonic stem cellresearch. Science 315:603–604.

56. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell 126:663–676.

Acharya et al. PNAS � November 10, 2009 � vol. 106 � no. 45 � 19155

NEU

ROSC

IEN

CE

Dow

nloa

ded

by g

uest

on

June

7, 2

020