Improving the Survival of Human CNSPrecursor-Derived Neurons AfterTransplantation

J.E. Le Belle,1* M.A. Caldwell,1 and C.N. Svendsen2

1Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom2The Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin

We have examined the effects of predifferentiation andenergy substrate deprivation on long-term expandedhuman neural precursor cells (HNPCs). The pre-differentiation of HNPC cultures produced large numbersof neurons (�60%) and mature glial cells capable ofgenerating glycogen stores that protected the neuronalpopulation from experimental metabolic stress. Whenpredifferentiated HNPCs were transplanted into intactadult rat hippocampus, fewer cells survived compared toundifferentiated HNPC transplants. This cell death wascompletely attenuated, however, when predifferentiatedHNPC cultures were pretreated to boost glial energystores and resulted in greatly increased neuronal survivalin vivo. The transplanted cells primarily engrafted withinthe granular layer of the dentate gyrus, where a largeproportion of the predifferentiated HNPCs co-expressedneuronal markers whereas most HNPCs outside of theneuronal layer did not, indicating that the predifferenti-ated cells remained capable of responding to local cuesin the adult brain. Undifferentiated HNPCs migrated morewidely in the brain after grafting than did the prediffer-entiated cells, which generally remained within thehippocampus. © 2004 Wiley-Liss, Inc.

Key words: human; neural; precursor; transplant; sur-vival

Cell replacement therapy is a promising treatmentfor many intractable neurodegenerative diseases such asParkinson’s and Huntington’s disease, and injuries such asstroke and traumatic brain and spinal cord injury. Formany years now clinical trials of fetal tissue transplantationin Parkinson’s disease have demonstrated the valuable clin-ical benefit of this type of treatment. Despite some successwith fetal tissue transplantation, however, if treatment is tobecome widely available to many patients who may ben-efit from such therapy, alternative cell sources are requiredfor practical reasons, such as limited availability of fetaltissue and the ethical unacceptability of its use in somecountries. Additionally, recent clinical studies have dem-onstrated potential complications such as increased dyski-nesias with fetal tissue transplants, which may be related tothe relatively variable cell composition of each graft com-

pared to more purified populations from lab-derived cul-tures. One such alternative source of lab-derived cells fortransplantation are cultures of human neural precursor cells(HNPCs), which can be expanded for long periods inculture to generate stocks of cells to graft many patientsfrom a single source of tissue. To date, multipotentHNPCs have been cultured for extensive periods of time(Svendsen et al., 1998; Wright et al., 2003). Several stud-ies, however, have shown that the cells produce fewneurons away from the graft core and differentiate primar-ily into astrocytes, which migrate extensively when trans-planted into non-neurogenic regions of the adult rodentcentral nervous system (CNS) (Lundberg et al., 1997;Svendsen et al., 1997; Winkler et al., 1998; Herrera et al.,1999; Ostenfeld et al., 2000; Cao et al., 2001). A potentialsolution to this problem is to predifferentiate the cells intohigh neuronal-yield cultures before transplantation. Cald-well et al. (2001) have produced large numbers of neurons(�60% of total cells) from long-term expanded HNPCcultures using a cell-cell contact model of differentiation incombination with neurotrophic factor treatment. Despitethe advantage of having many differentiated neurons fortransplantation, it is not known if the survival of thesemore mature cells will be reduced after grafting into theadult brain. We have therefore transplanted both predif-ferentiated and undifferentiated HNPCs into intact adulthippocampus to determine whether neuronal or overallcell survival has been affected by the differentiation of thecells.

Even undifferentiated HNPCs and fetal tissue graftshave a low survival rate (5–10% of transplanted cells) aftertransplantation. Despite the small numbers of cells surviv-ing, clinical studies have shown a functional clinical ben-efit to the treatment in most patients (Herman et al., 1994;

Contract grant sponsor: Glaxo SmithKline.

*Correspondence to: J.E. Le Belle, Department of Pharmacology, UCLASchool of Medicine, Room 1246 CIMI, 700 Westwood Plaza, Los Angeles,CA 90095. E-mail: jlebelle@mednet.ucla.edu

Received 8 September 2003; Revised 10 October 2003; Accepted 5November 2003

Published online 19 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.20035

Journal of Neuroscience Research 76:174–183 (2004)

© 2004 Wiley-Liss, Inc.

Lindvall et al., 1994; Olanow et al., 1996; Kordower et al.,1998; Lindvall, 1999; Freed et al., 2001). It has beenestimated that only one-fifth of the normal complement ofdopaminergic neurons in the human substantia nigra arerequired to survive transplantation to confer a clinicalbenefit, despite the limitations and potential complicationsof the ectopic graft location in PD patients (Bjorklund andLindvall, 2000). Although there are improvements in theirclinical condition, most patients are not asymptomatic andrecovery is not complete. Improving cell survival aftertransplantation, therefore, has the potential to reap furtherfunctional benefits for this therapy. There are several rea-sons that cells may not survive after grafting, includingoxidative stress, lack of trophic support, and energy sub-strate deprivation until integration with the host CNStissue is complete. Many studies have demonstrated thebenefits of antioxidant and neurotrophic treatment ongrafted tissue and cells (Knusel et al., 1990; Grasbon-Frodlet al., 1996; Bjorklund et al., 1997; Clarkson et al., 1997,2001; Karlsson et al., 1999; Brundin et al., 2000; Hanssonet al., 2000; Akerud et al., 2001). The contribution ofenergy substrate deprivation, however, has not been ex-plored to the same extent. There is indirect evidence thatthis metabolic stress may contribute to cell death in trans-plants. For example, it is known that substrate deprivationin neural cultures can induce both necrotic and apoptoticcell death (Gwag et al., 1995; Ouyang et al., 2000; Cava-liere et al., 2001). In addition, overall cell survival has beenshown to be much higher (20% of grafted cells) for trans-plants extending into the ventricle compared to thoselocated entirely within the hippocampus (5–10% of graftedcells), which has been attributed to cerebrospinal fluid(CSF) acting as a nutritive medium for the cells (Rosen-stein and Brightman, 1978; Freed, 1985; Shetty et al.,1991).

Predifferentiation of HNPCs, the focus of this study,may have further advantages in addition to the greatlyincreased number of neurons, such as exploitation of themetabolism of the mature cell phenotypes before trans-plantation to address the issue of substrate deprivation. Thelargest energy reserve in the nervous system is astrocyte-derived glycogen (Lowry et al., 1964). Several studies havedemonstrated in rodent cell cultures that astrocytic glyco-gen can act as an energy store for astrocytes themselves orfor neighboring neurons, oligodendrocytes, and microgliaby releasing glycogen-derived lactate that can then bemetabolized by other cells (Dringen et al., 1993; Magis-tretti et al., 1993; Pellerin et al., 1997). Mature astrocytesare the only CNS cell type to contain the required en-zymes, such as glycogen phosphorylase, to breakdownglycogen for metabolism (Reinhart et al., 1990) and theyhave been shown to protect cultured neurons from celldeath induced by anoxia, hypoxia, and hypoglycemia(Pauwels et al., 1985; Vibulsreth et al., 1987; Swanson andChoi, 1993). We therefore investigated in the presentstudy whether boosting astrocyte glycogen stores in ma-ture, predifferentiated HNPC cultures may improve neu-

ronal survival after transplantation by reducing the energycrisis experienced by the cells.

MATERIALS AND METHODS

Human Neural Precursor Cell Culture

Human embryonic tissue (8 weeks post conception) wascollected after routine terminations of pregnancy. Methods ofhuman tissue collection conformed with the arrangements rec-ommended by the Polkinghorne Committee for the collectionof human tissues and to the guidelines set out by the UnitedKingdom Department of Health. The cortex was isolated fromthe human fetal samples and the tissue was treated with trypsin(0.1% for 20 min), washed in Dulbecco’s modified Eagle me-dium (DMEM) and DNase I and dissociated into a single-cellsuspension. Cells were seeded initially at a density of 500,000cells/ml in T75 flasks containing 20 ml of a defined, serum-freemedium (DMEM:Ham’s-F12 at 3:1) supplemented with B27(1% vol/vol), epidermal growth factor (EGF, 20 ng/ml), andfibroblast growth factor (FGF2, 20 ng/ml) with heparin(5 �g/ml). The cells formed neurosphere cell aggregates by 2–5days of growth and were cultured as neurospheres for 16 weeksaccording to the methods of Svendsen et al. (1998). Briefly, thehuman cells were passaged every 14 days by sectioning thespheres into 350-�m sections and re-seeding into fresh growthmedium. After 4 weeks of growth, cultures were switched fromB27 to N2 supplements (1% v/v) and EGF only.

Predifferentiation of Human Neurosphere Cultures

Neurospheres from the long-term expanded human cul-tures were predifferentiated for 7 days to obtain approximately60% neurons according to the methods of Caldwell et al. (2001).Briefly, whole neurospheres were plated directly onto poly-D-lysine/laminin-coated flasks in serum-free (DMEM:Ham’s-F12)containing N2 supplement and neurotrophin-4 (NT4,20 ng/ml) but without mitogens. Over 7 days, cells migratedaway from the intact neurospheres and formed a monolayer ofdifferentiating neuronal and glial cells. A group of the predif-ferentiated cells were also treated by incubation in a high-glucose medium (20 mM) 24 hr before transplantation or ex-perimental substrate deprivation to increase glial glycogen storesaccording to the methods of Swanson et al. (1989a) (Fig. 1).

In Vitro Experiments

Predifferentiated neurosphere cultures were grown onglass chamber slides and incubated for 24 hr in either normalmedia or high-glucose media to boost glycogen energy stores inthe differentiated astrocytes (n � six 8-well chamber slides pertreatment group). These cultures were then exposed to substratedeprivation for 6 and 24 hr (n � 3 per time point) using aserum-free, defined culture media deficient in glucose andamino acids (Gibco, UK). The culture media was analyzed forthe release of lactate dehydrogenase (LDH) as an indicator of celldeath. The chamber slides were also fixed using 4% paraformal-dehyde in phosphate-buffered saline (PBS) for 20 min andimmunocytochemistry was carried out using standard protocols.Fixed cell cultures were blocked in 3% goat serum with 0.3%Triton X-100 and incubated with primary antibodies to�-tubulin III (monoclonal, 1:500; Sigma, UK) and glial fibrillary

Improving HNPC Survival 175

acidic protein (GFAP, polyclonal, 1:1,000; DAKO). Cultureswere then incubated in either biotinylated goat anti-mouse orfluorescein-conjugated goat anti-rabbit secondary antibodies.Biotinylated cultures were visualized with a streptavidin-rhodamine conjugate, and Hoechst 33258 was used as a nuclearstain. Quantification of cells was achieved by viewing the cellsunder a fluorescence microscope (40� objective) and countingHoechst-stained nuclei along with labeled neurons in threeindependent fields per well of the chamber slide with five slidesper group/time point.

LDH Assay

LDH release into the culture media after 6 and 24 hr ofsubstrate deprivation was determined according to the methodsof Juurlink and Hertz (1993) using an assay kit from Sigma.Briefly, 200 �l of culture media was taken from predifferentiatedcell cultures at 6 and 24 hr of incubation in the followingconditions: (1) control (normal media), no substrate deprivation;(2) normal media 24 hr before substrate-deprivation media; and(3) high-glucose media (glycogen-boosting) 24 hr beforesubstrate-deprivation media. The colorimetric assay is based onthe reduction of NAD by the action of LDH and the resultingNADH is utilized in the stoichiometric conversion of a tetra-zolium dye, which is then measured spectrophotometrically(wavelength 490 nm). LDH content during substrate depriva-tion in the normal media and glycogen-boosted pretreatmentgroups is expressed as a percent of control (non-substrate-deprived) levels of the enzyme.

Glycogen Measurement

Predifferentiated, control and high-glucose-treated cellcultures (n � 3 T25 flasks/group) were analyzed for glycogencontent as described previously (Swanson and Choi, 1993).Briefly, cells were dissociated with trypsin and DNase followed

by the addition of 1 ml ice-cold 85% ethanol/15% 30 mM HClto instantly stop glycogen metabolism, and shaken gently for2 hr. Shaking the cells in the ethanol/acid solution removed allfree glucose so that the glucose measured in the cells reflectedonly hydrolyzed glycogen. Cells were transferred to 0.3 ml of30 mM HCl and sonicated. The glycogen content was thendetermined in the sample by the amyloglucosidase hydrolysis ofglycogen to glucose followed by quantification of the glucosecontent by glucose-6-phosphate dehydrogenase/NADP reac-tion (Passonneau and Lauderdale, 1974). Standards were pre-pared from glucose and glucose content was determined spec-trophotometrically and expressed per milligram of cell proteindetermined using the method of Lowry (1951).

In Vivo Grafting Experiments

Predifferentiated neurosphere cultures were incubated for24 hr before grafting in either normal or high-glucose media toincrease astrocyte glycogen stores (Swanson et al., 1989a). Onthe day of surgical transplantation, all cells were dissociated withtrypsin and DNase and grafted at a density of 500,000 cells intoone site in the intact hippocampus in 2 �l of normal media (n �8 per group: predifferentiated normal media, predifferentiatedhigh-glucose media, and undifferentiated normal media). Afurther three animals received sham grafts of dead, freeze-thawed cells. Injections were made into the left hippocampus atthe following coordinates: AP � �3.6 mm; L � �2.0 mm frombregma, and V � �2.7 mm from dura. All transplanted cellsamples were assessed for cell viability at the end of the surgicaltransplantation session by observation and counting of trypanblue dye exclusion. All animals received immunosuppressionwith daily cyclosporin-A (10 mg/kg, intraperitoneally; Sandoz)for 3 days before grafting and for the remainder of the experi-ment. At 8 weeks after grafting the animals were anesthetizedand their brains fixed by transcardial perfusion with 0.1 M PBSfollowed by 4% paraformaldehyde in 0.1 M phosphate buffer.Fixed brains were equilibrated in 25% sucrose for 48 hr and thencut into 50-�m coronal sections on a freezing microtome.

Immunohistochemistry

Immunofluorescence staining. Double immunoflu-orescence staining for human nuclei antibody (HuNu) and theneuronal markers, neuronal nuclei (NeuN) and �-tubulin III,were used to detect co-labeling of transplanted cells. HuNu is ahuman-specific nuclear protein used to identify the humanxenografts in the rat brain. Free-floating sections were incubatedin 2 N HCl for 30 min at 37°C and then rinsed three times inTris A (0.1 M Tris-HCl and 0.1% Triton X-100) and Tris B(0.1 M Tris-HCl, 0.1% Triton X-100, and 0.05% bovine serumalbumin [BSA]). The sections were then blocked in Tris B with10% normal donkey serum for 1 hr at room temperature. HuNuantibody was applied at 1:50 in Tris B and 0.1% sodium azide atroom temperature for 48 hr. After rinsing in Tris A and B,anti-mouse Alexa 648 in Tris B (1:400; Molecular Probes) wasapplied for 2 hr. After rinsing in Tris A and B, antibodies toNeuN (monoclonal, 1:1,000; Chemicon) or �-tubulin III(monoclonal, 1:200; Sigma) in 0.1 M Tris-HCl, 0.1% Triton,and 0.1% BSA were then applied overnight at room tempera-ture, followed by anti-mouse or anti-rabbit Alexa 488 (1:400;

Fig. 1. HNPCs were grown for 28 weeks prior to being divided into3 experimental groups for transplantation. Seven days before graftingtwo groups of cells were plated down on poly-D-lysine (PDL) andlaminin-coated tissue culture flasks and allowed to differentiate in thepresence of Neurotrophin 4 (NT4) for 7 days. One of these pre-differentiated groups was incubated in a high-glucose medium for thefinal 24 hr before transplantation. All groups were transplanted assingle-cell suspensions following treatment with trypsin and DNaseI.Figure can be viewed in color online via www.interscience.wiley.com.

176 Le Belle et al.

Molecular Probes). Hoechst 33258 (1:5,000) was used as anuclear stain.

Immunoperoxidase staining. Endogenous hydrogenperoxidase activity was quenched by treating sections with 10%hydrogen peroxide in 10% methanol for 5 min. For each pri-mary antibody a one in six series of sections was blocked in 3%goat serum with 0.3% Triton X-100 and subsequently incubatedfor 48 hr (4°C) with antibodies to human nuclei (monoclonal,1:200; Chemicon) and �-tubulin III (monoclonal, 1:250;Sigma). Sections were then incubated in either biotinylated goatanti-mouse (rat pre-absorbed, 1:200; Vector Labs) or biotinyl-ated goat anti-rabbit (1:200; DAKO) followed by transfer to anavidin-biotin complex (Vectastain ABC kit; Vector Labs) inTBS. Antigens were then visualized using diaminobenzidine(DAB) as chromogen.

Quantitative Analysis

Quantification of total cell survival and double-labeledHuNu cells with �-tubulin III was calculated in a one in sixseries of brain sections using a 2D stereological sampling algo-rithm (West et al., 1991) on an Olympus CAST grid stereologysystem (Olympus, Denmark) and subsequently corrected afterAbercrombie (1946). Co-localization of HuNu with the neu-ronal markers NeuN and �-tubulin III was conducted by con-focal microscopy using a Zeiss LSM-510 confocal dual-laser-scanning microscope on an inverted Axiovert microscope usinga 63� oil immersion objective. Double-labeled cells were con-firmed by collecting 1 �m sections through the slice.

Statistical Analysis

The presented values are mean � SEM. Comparisonsbetween groups were made with one-way analysis of variance(ANOVA) followed by Bonferroni post-hoc tests (SigmaStat2.03). P 0.05 was considered statistically significant.

RESULTSIn Vitro Experiments

Cell death in response to 6 and 24 hr of substratedeprivation was measured by LDH release in the culturemedia of predifferentiated HNPC cultures after pre-

Fig. 2. A: LDH release measured in pre-differentiated HNPC culturesexposed to 6- and 24-hr of substrate deprivation after pre-incubation ineither normal media or high-glucose media to boost astrocyte glycogenstores. Media LDH is expressed as a percentage of control LDHmeasured in sister cultures not exposed to substrate deprivation.B: Neuronal survival in pre-differentiated HNPC cultures exposed to6- and 24-hr of substrate deprivation after pre-incubation in eithernormal or high-glucose media. The data is presented as a percentage ofthe control cultures that did not undergo substrate deprivation.

Fig. 3. A: The total HuNu-positive cells surviving 8 weeks post-grafting expressed as a percentage of the total number of cells originallygrafted (500,000). B: Total double-labeled cells (HuNu � �-tubulin)within the hippocampus were significantly lower in the un-differentiated (*P � 0.0005) and normal media (**P � 0.004) groupscompared to the glycogen-boosted group .

Improving HNPC Survival 177

incubation in either normal media or high-glucose media,which elevates astrocyte glycogen stores by 59% (normalmedia � 12 � 3; high-glucose media � 29 � 5 �g/mgprotein). The glycogen-boosted cultures had 32% lessLDH release (cell death) at 6 hr of substrate deprivation(P � 0.002) and 34% more �-tubulin III-positive neuronswere seen to survive in the glycogen-boosted cultures at6 hr (P � 0.003, Fig. 2). Furthermore, there was nosignificant difference in neuronal numbers between thecontrol group that received no substrate deprivation andthe glycogen-boosted group (P � 0.11), indicating thatthe pretreatment with high glucose completely attenuatedthe neuronal death induced by 6 hr of substrate depriva-tion. No significant differences, however, were detectedbetween groups after 24 hr of deprivation.

In Vivo ExperimentsThe total predifferentiated HNPCs surviving 8

weeks after grafting into the intact hippocampus of adultrats was significantly lower than the undifferentiated cells(P 0.001). When glycogen stores were boosted in thecells before grafting, however, the overall cell survival wasincreased significantly (P � 0.005, Fig. 3) to levels similar

to that seen in the undifferentiated cells. Control trans-plants that had been killed by freeze-thawing before graft-ing did not contain any detectable positive staining for theHuNu antibody. Most transplanted cells in all groupsincorporated themselves into the granular layer of thedentate gyrus (Fig. 4). Although many cells were seenmigrating across the midline of the subcortical white mat-ter tracts to the contralateral hemisphere, the cells did notenter the overlying cortex or underlying hippocampus.Significantly more cells migrated away from the transplantsite in the undifferentiated group compared to both pred-ifferentiated groups (P � 0.001, Fig. 5).

The percentage of neurons surviving 8 weeks post-graft were very similar in both of the predifferentiatedgroups (glycogen � 49 � 4% and normal media � 50 �4%, P � 0.91) but significantly greater than in the undif-ferentiated grafted cells (6 � 1%, P � 0.0002). Despite asimilar percentage of HNPC-derived neurons in the (pre-differentiated) normal media and glycogen-boosted cells,the total number of neurons was higher in the glycogengroup because a significantly higher overall cell survivalwas achieved with this treatment. The total number of

Fig. 4. A: Diagram of the rat hippocampus. Abbreviations: GL, granular layer; DG, dentate gyrus;HF, hippocampal fissure. B: Transplanted HNPCs populate the granular layer (GL) of the hippocam-pal dentate gyrus (10� magnification). Staining for NeuN (green) and HuNu (red) indicates thelocation of the graft. C,D: HuNu positive cells (red) double-label (yellow) with the neuronal markerNeuN (green) within the GL shown at 20� and 63� magnification. Hoechst 33258 was used as anuclear stain (blue).

178 Le Belle et al.

predifferentiated HNPC-derived neurons was thus ap-proximately 31% higher in the glycogen group comparedto the normal media (P � 0.004) and 81% higher than theundifferentiated cell group (P � 0.0005, Fig. 3). The cellviability assessed by trypan blue dye exclusion at the end ofthe graft surgery indicated a similarly high survival rate atthe time of transplantation in all groups (89 � 10% pre-differentiated � glycogen; 83 � 8% undifferentiated;79 � 14% predifferentiated).

The cells in all groups engrafted in the granular layerof the dentate gyrus were shown to co-express �-tubulinIII and NeuN with the human nuclei marker, whichindicates their differentiation into appropriate neural phe-notypes within the hippocampus (Fig. 4 and 6).

DISCUSSIONThe present study has demonstrated that long-term

expanded HNPC cultures that have been predifferentiatedto obtain high numbers of neurons do retain the ability torespond to in vivo cues in the adult rat brain after trans-plantation and engraft into appropriate sites while main-taining neuronal differentiation. This pretreatment resultsin the presence of almost 81% more neurons in the graftedhippocampus than are present in transplants from undif-ferentiated cultures of the same cells. Although prediffer-entiation does seem to cause the cells to be more suscep-tible to cell death after transplantation, we havedemonstrated that addressing cellular stressors such as en-ergy substrate deprivation can attenuate this negative ef-fect. The combination of boosting cell energy substratestores with other strategies shown to increase cell survivalsuch as antioxidant treatment may improve this survivalrate even further. Finally, the transplantation of undiffer-entiated neural precursor cells is known to result in wide-spread migration of the cells throughout the brain, but wehave found that the predifferentiation of HNPC cultures

results in significantly less migration and more neuronsremaining within the grafted hippocampus. This is advan-tageous in the case of transplants that target a focal brainregion, such as the case with Parkinson’s disease. Thisdecrease in the cells’ migratory nature, however, may notbe a desired characteristic for application to more diffuseneuronal injuries or neurodegeneration.

In Vitro ExperimentsMeasurements of cell glycogen content, cell death

(LDH release), and neuronal survival (�-tubulin III cellnumbers) were used to demonstrate that pre-incubation ofpredifferentiated HNPC cultures in a high-glucose me-dium can elevate cell glycogen stores and attenuate LDHrelease and neuronal loss in response to total energy sub-strate deprivation. Although rodent astrocytic glycogenstores have been shown to play a neuroprotective roleduring cell energy substrate crises, this is the first time thatthis phenomenon has been demonstrated in human neuralcultures. Furthermore, we have shown that the prediffer-entiation method used on the HNPCs to produce highnumbers of neurons (�60%) for transplantation also gen-erates an adequate number of mature astrocytes capable ofproducing cellular energy stores that protect the neuronsfrom energy failure-induced cell death, whereas cultures ofundifferentiated cells are not capable of increasing glyco-gen stores.

The beneficial effect provided by the glycogenenergy stores seems to be limited to acute time periodsbecause there were no significant differences betweentreatment groups after 24 hr of substrate deprivation.The exploitation of this astrocyte-specific metabolismthat has successfully protected neurons in the culturedish would therefore only be of benefit in transplanta-tion if the substrate deprivation experienced by the cellsin vivo lasts for only short periods of time. One possibleexplanation for why even a small energy reserve mayimprove survival is that cells undergoing metabolicstress down-regulate their metabolic expenditure,which allows extended subsistence on energy reserves(Ames, 1992; Yager et al., 1994). This is evident fromthe culture studies mentioned and the present study,which demonstrates that neural cultures can withstandsubstrate deprivation for several hours by utilizing en-ergy reserves such as glycogen, even though in vivoglycogen reserves are thought to provide only mereminutes of additional energy supplies. Transplantedcells, therefore, may not need to integrate fully withhost blood supply to access some form of endogenousenergy substrate within a short period of time.

The viability of the cells used for grafting was as-sessed at the end of the transplantation surgery by trypanblue dye exclusion and a similarly high survival rate wasdemonstrated in all groups. The protective effect seen onthe cell grafts was, therefore, most likely imparted in vivoand was not merely a result of improved survival duringthe transplant process itself.

Fig. 5. The relative percentage of transplanted cells that remainedwithin the hippocampus and migrated out into the sub-cortical whitematter tracts. The undifferentiated cells had significantly more migrat-ing cells than either pre-differentiated groups (P 0.001), which didnot show any significant difference to each other (P � 0.817).

Improving HNPC Survival 179

In Vivo ExperimentsLong-term expanded HNPCs that are not prediffer-

entiated do not spontaneously produce large numbers ofneurons when transplanted into non-neurogenic areas ofthe adult brain (Lundberg et al., 1997; Winkler et al.,1998; Herrera et al., 1999; Cao et al., 2001; Zhang et al.,2001). Predifferentiation of high-yield neuronal HNPCcultures thus may be useful for overcoming the low neu-ronal numbers in vivo. Unfortunately, we have found that

predifferentiation of the cells results in a decreased overallcell survival. Despite this, the total number of neuronsremains greater than that seen in undifferentiated trans-plants of the same cells. We have demonstrated further thatthe cell loss experienced by the predifferentiated cellscompared to undifferentiated cells can be overcome byelevating glial glycogen stores in predifferentiated cells24 hr before transplantation. The number of neuronssurviving after 8 weeks in vivo is significantly higher after

Fig. 6. Human nuclei positive cells (red) in thehippocampus double-labeled with �-tubulin III(green). A,B: Three-dimensional confocal projec-tions of double-labeled staining. C–H: Confocaloptical sections at 1�m intervals showing a double-labeled neuron (arrow).

180 Le Belle et al.

this simple treatment. This metabolic manipulation beforegrafting is accessible to the predifferentiated cells due tothe maturation of metabolic machinery in the differenti-ated astrocytes present in the culture.

We also know from our in vitro studies that theprotective effect of the energy substrate stores is effectivefor less than 24 hr. The significant outcome of this pre-transplant treatment observed in vivo therefore indicatesthat the critical period for energy substrate crisis in cellgrafts may last only a short time. It is known that theexposure of fetal tissue grafts to the CSF can improve thenumber of cells that survive transplantation by providingenergy substrates to the cells (Rosenstein and Brightman,1978; Freed, 1985; Shetty et al., 1991). In addition, thecurrent study has provided further evidence that cellenergy-substrate deprivation may be a significant, yetacute, factor in cell survival that can be overcome to acertain extent by increasing energy stores before transplan-tation. Using this pre-graft culture treatment in combina-tion with other treatments such as antioxidant protectionmay improve further cell survival.

The cell survival in the undifferentiated and predif-ferentiated glycogen groups (32 � 4% and 28 � 2%,respectively) was considerably higher than the typical5–10% survival reported in the literature. This is due mostlikely to the neurogenic properties of the adult hippocam-pus, which may provide neurotrophic and other factorsthat increase cell survival. Rosser et al. (2000) and Jain etal. (2003) have also observed greater numbers of HNPCssurviving transplantation into the hippocampus comparedto those transplanted into non-neurogenic striatum. It is amystery how grafts survive at all, however, because vas-cularization is known to take several days (Krum andRosenstein, 1988; Broadwell et al., 1991; Geny et al.,1994), the brain has mere minutes of energy reserves(Benzi et al., 1978; Swanson et al., 1989b), and significantneuronal loss can be observed within hours of ischemicinsult (Gideon et al., 1992; Aronowski et al., 1999; Davoliet al., 2002). Our results suggest that a possible answer tothis conundrum is that a critical metabolic period exists forthe grafts immediately after transplantation that is notrelated to vascularization, after which some source ofparenchymal substrate supply may be accessible to the cellsin vivo. Boosting cellular energy stores before transplan-tation is therefore a useful way to attenuate this cell deathfrom acute metabolic stress.

Most grafted cells remained within the granular layer(GL) of the dentate gyrus, which is similar to the patternof migration for undifferentiated HNPCs observed byFricker et al. (1999) and Englund et al. (2002). In thepresent study, however, we have shown that more undif-ferentiated cells migrated outside of the hippocampus thandid those from both predifferentiated groups of cells (Fig.6). Most HuNu-labeled cells in the granular layer wereNeuN- and �-tubulin III-positive. Whereas most HuNu-positive cells outside of the GL did not co-express eithermarker, indicating that transplanted cells populating theGL were able to respond to local cues and appropriately

populate a neuronal layer of the hippocampus. This pat-tern of site-specific differentiation has also been observedin undifferentiated HNPCs (Fricker et al., 1999) and ro-dent precursor cells (Gage, 1995; Vicario-Abejon, 1995;Suhonen et al., 1996) after transplantation into the adultrat hippocampus; however, co-labeling of the human cellswith calbindin, a marker for mature granular cells, wasinconclusive. This may be due to the relatively short timeafter transplantation that we looked for mature neuronalphenotypes (8 weeks). Transplants of primary human cellsare known to take considerably longer to mature than dorodent cells, indicating that the human cells retain anintrinsically slower developmental clock (Grasbon-Frodlet al., 1997). In addition, there are other reports of hip-pocampal transplants of undifferentiated HNPCs that donot express appropriate hippocampal neuronal markers,which also suggest that more time may be needed forHNPCs to mature fully in vivo (Englund et al., 2002; Jainet al., 2003).

Predifferentiation alone provided a significant in-crease in the number of neurons surviving hippocampaltransplantation. By providing a large number of neuronsfor grafting from expanded cultures and protecting thosecells from energy crisis after transplantation, human neuralprecursor cells may be a viable replacement for primaryfetal cell transplants in brain repair. The HNPC culturesare particularly suited to cell replacement therapies in thatthey can be expanded extensively to generate large banksof transplantable cells, they are amenable to predifferen-tiation to generate large numbers of neurons, and they canbe manipulated in vitro to maximize cell survival post-transplant. In conclusion, long-term expanded HNPCshave the capacity for integration within the adult brain andcan be predifferentiated into neurons without losing theirability to respond to extracellular signals in the adult CNS.

ACKNOWLEDGMENTSThis work was supported in part by Glaxo SmithK-

line (postdoctoral fellowship to J.E.L.). We thank Ms. X.He and Dr. R. Burnstein for their generous help with thedaily cyclosporin injections and Dr. S. Ball for her helpwith confocal microscopy.

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