Experimental Neurology 177, 40–49 (2002)doi:10.1006/exnr.2002.7965

Lipid-Mediated Glial Cell Line-Derived Neurotrophic Factor GeneTransfer to Cultured Porcine Ventral Mesencephalic Tissue

Matthias Bauer,*,†,‡ Morten Meyer,‡ Thomas Brevig,‡ Thomas Gasser,† Hans Rudolf Widmer,§Jens Zimmer,‡ and Marius Ueffing*,1

*GSF-National Research Center for Environment and Health, Institute for Human Genetics, Ingolstadter Landstrasse 1, 85764Neuherberg, Germany; †Department of Neurology, Klinikum Großhadern, University of Munich, Marchioninistrasse 15, 81366Munich, Germany; ‡Department of Anatomy and Neurobiology, SDU-Odense University, Winsløwparken 21, 5000 Odense C,

Denmark; and §Department of Neurosurgery, Inselspital, University of Bern, Freiburgstrasse, CH-3010 Bern, Switzerland

Received December 27, 2001; accepted May 10, 2002

Transplantation of dopaminergic ventral mesence-phalic (VM) tissue into the basal ganglia of patientswith Parkinson’s disease (PD) shows at best moderatesymptomatic relief in some of the treated cases. Exper-imental animal studies and clinical trials with allo-genic and xenogenic pig-derived VM tissue grafts toPD patients indicate that one reason for the poor out-come of neural transplantation is the low survival anddifferentiation of grafted dopaminergic neurons. Toimprove dopaminergic cell survival through a gene-therapeutic approach we have established and reporthere results of lipid-mediated transfer of the gene forhuman glial cell line-derived neurotrophic factor(GDNF) to embryonic (E27/28) porcine VM tissue keptas organotypic explant cultures. Treatment of the de-veloping VM with two mitogens, basic fibroblastgrowth factor and epidermal growth factor, prior totransfection significantly increased transfection yields.Expression of human GDNF via an episomal vectorcould be detected by in situ hybridization and by themeasuring of GDNF protein secreted into the culturemedium. When compared to mock-transfected con-trols, VM tissue expressing recombinant GDNF con-tained significantly higher numbers of tyrosine hy-droxylase-positive neurons in the cultured VM tissue.We conclude that lipid-mediated gene transfer em-ployed on embryonic pig VM explant cultures is a safeand effective method to improve survival of dopami-nergic neurons and may become a valuable tool toimprove allo- and xenotransplantation treatment inParkinson’s disease. © 2002 Elsevier Science (USA)

Key Words: transfection; plasmid vector; porcineventral mesencephalic tissue; dopaminergic neurons;GDNF.

1 To whom correspondence should be addressed. Fax: �49 89 31873297. E-mail: marius.ueffing@gsf.de.

400014-4886/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

INTRODUCTION

Parkinson’s disease (PD) is a common neurodegen-erative disease affecting 1 to 2% of the elderly popula-tion (8). Transplantation of dopaminergic neurons intothe dopamine-depleted basal ganglia may be a thera-peutic option for some of these patients. Strategiesusing human embryonic ventral mesencephalic (VM)tissue have shown modest, although significant, im-provements of motor symptoms in a subset of youngerpatients (34, 14), primarily depending on sufficientsurvival and differentiation of dopaminergic neuronswithin the grafts. Unexplained low yield of dopaminer-gic neurons, requiring more donors per PD patient, andethical concerns with regard to the use of human fetaltissue in general have thus turned the focus towarddopaminergic cell survival and differentiation and to-ward alternative donor sources.

Transplantation of porcine dopaminergic neuronsovercomes some of the drawbacks associated with theuse of human embryonic tissue. Results from a clinicalsafety study using porcine VM tissue for intrastriataltransplantation in Parkinson’s patients were pub-lished previously (9, 29). A limited clinical improve-ment in some of the patients was accompanied withvery low survival of dopaminergic neurons, as observedin one patient who died from unrelated causes (9).Insufficient immunosuppression with rejection will ofcourse decrease the survival of grafted cells (6). No signof major damage within the porcine transplant was,however, seen in the autopsy material mentionedabove. Together with the general observations in hu-man and animal allograft cases, this again points toother reasons, including deficient neurotrophic sup-port, for the low survival of grafted dopaminergic neu-rons. Thus, to further improve the efficacy of cell trans-plantation approaches it is crucial to develop strategiesfor delivering neurotrophic factors with the aim of im-

proving both survival and integration of the cells fol-lowing transplantation.

Several neurotrophic factors, most notably glial cellline-derived neurotrophic factor (GDNF), have beentested to support the survival, differentiation, and in-tegration of transplanted dopaminergic neurons (27).Continuous delivery of GDNF within the first weeksposttransplantation has been shown to be most effec-tive (3). For local delivery, encapsulated, GDNF-pro-ducing BHK-cells have been placed next to grafts (28),just as GDNF-producing HiB5 cells have been mixedwith VM cell suspensions for cotransplantation in a ratmodel of Parkinson’s disease (23). For this purposeorganotypic explant cultures of rat VM tissue haverecently been modified by lipid-mediated gene transferof human GDNF, followed by subsequent grafting to arat model of PD (4).

Lipid-mediated gene transfer (“transfection”) to eu-karyotic cells was introduced by Felgner and Ringold(12). Since then a constant improvement of reagentshas been achieved, lowering toxicity and increasing thetransfection efficiency (for review, see Ref. 20). In con-sequence, the range of target cells has broadened enor-mously over recent years, facilitating nonviral genetransfer not only to rapidly proliferating cell lines (36),but also to primary tissue (4). In addition, plasmidvectors allowing strong and long-lasting transgene ex-pression have been developed. For example, insertionof EBNA1 and oriP sequences from Epstein–Barr virus(EBV) into the vector backbone results in persistenceand replication of the plasmid episome in the nucleus(2, 32). The combined use of improved transfectionreagents and nonviral vector constructs, featuringsome of the functional advances of viral vectors, hasaccordingly made these protocols a safe alternativecompared to viral transduction.

In a recent study, genetically modified rat VM tissueshowed markedly higher amounts of dopaminergicneurons after 1 week in culture and a faster functionalrecovery when grafted in a rat model of PD (4). The aimof the present study was to expand and further im-prove this method for porcine brain tissue. We nowshow that pRep7-GDNF8 can effectively be transferredinto cultured explants of embryonic pig VM tissue,resulting in significantly improved survival and differ-entiation of the dopaminergic neurons.

MATERIAL AND METHODS

Tissue Preparation and Tissue Culture

Ventral mesencephalon from embryonic day 27/28(E27/28) pig embryos (Danish Landrace X Yorkshire)was isolated as previously described (23). The tissuewas divided into eight equal-sized pieces (each approx.0.5 mm3) and placed in a conical plastic tube with serum-containing culture medium (88.5% RPMI (Gibco), 1.5%

glucose, 10% fetal calf serum (Gibco)). The tubes werethen placed in a roller-drum at 37°C (15), and thetissue was maintained as organotypic “free-floatingroller tube” cultures as described by Spenger and co-workers (30).

For transfection, epidermal growth factor (EGF;R&D Systems), basic fibroblast growth factor (bFGF;R&D Systems), or a combination of both factors wereadded to the medium shortly after dissection (at “day 0in vitro,” termed DIV0). Equal amounts (20 ng/ml) ofEGF and bFGF were used.

Vector Constructs

For optimization of the transfection procedure, a vec-tor coding for �-galactosidase (�-Gal) driven by a RousSarcoma Virus (RSV) promotor (pRSV-lacZ; Clonetech)was used. This vector construct has been shown toresult in strong transgene expression in brain tissue(4).

For expression of glial cell-derived neurotrophic fac-tor, an EBV-based vector construct on the basis ofpRep7 (Invitrogen) was used. The pRep7 backbone vec-tor construct contains a RSV promoter, a truncatedEBNA1, and oriP sequences. To generate a vector con-struct coding for GDNF, the coding sequence of GDNFwas amplified from HEK293 cDNA with the XhoI-tailed primers GDs.F (5�-ATGGCCGCCTCGAGATGA-AGTTATGGGATGTCGTGG-3�) and GDx.R (5�-GCCG-CACTCTCGAGTCAGATACATCCACACCTTTTA-3�)using AmpliTaqGold (Perkin–Elmer) according to themanufacturer’s recommendations. The following PCRprotocol was used: initial denaturation for 10 min at94°C, followed by 32 cycles of 1 min at 94°C and 1 minat 60°C, and a last extension step at 72°C for 10 min.The PCR product was cloned into pGEM-T (Promega)and cut with XhoI. This fragment was then cloned intopREP7 (Invitrogen) and the resulting vector constructwas termed pREP7-GDNF8. Transgene expression ca-pabilities and suitability of this vector in primary braintissue has been assessed previously (4, 16).

Transfection Procedure

For transfection, four VM culture pieces (togethercorresponding to one half VM) were pooled in oneroller-tube. Culture medium containing DNA–Effect-ene solution was added as described previously (4). TheVM explants were exposed to different amounts ofplasmid DNA (2 �g to 8 �g pRSV-lacZ), purified withthe EndoFree Kit (Qiagen), in the presence of 20 �l to100 �l Effectene (Qiagen), using lipid–DNA ratios of10:1 to 50:1. The transfection mixture was left in placefor 18 h, and each piece of VM explant was transferredto one roller-tube with fresh culture medium and fac-ultatively supplemented with EGF and/or bFGF asdescribed above. Cell counts of �-Gal-positive cellswere performed on every single explant culture, taking

41GENETIC MODIFICATION OF XENOGRAFTS IN PARKINSON’S DISEASE

one culture as one treatment unit. Cell numbers arepresented as counts per volume.

GDNF mRNA in Situ Hybridization

Culture sections were in situ hybridized using analkaline–phosphatase (AP)-labeled oligodeoxynucle-otide probe specifically recognizing human GDNF(oligo-sequence 5�CTACTTTGTCACTCACCACCCTT-CTATTTC 3�). Prior to hybridization, the sections wereair-dried at room temperature, heated to 55°C for 10min, and then treated with 96% ethanol at 4°C over-night. The sections were then air-dried and incubatedin hybridization medium overnight at 39°C (13, 35).Posthybridization treatment consisted of rinses in 1�standard saline citrate (0.15 M NaCl � 0.015 M Na–citrate; 3� 30 min) at 55°C, followed by rinses in Tris–HCl, pH 9.5 (2� 10 min), at room temperature prior toapplication of the AP developer. AP developer was pre-pared immediately before use and contained nitrobluetetrazoleum (Sigma), 5-bromo 4-chloro 3-indolyl phos-phate (Sigma), and Tris–HCl–MgCl2 buffer, pH 9.5(13). AP development took place in the dark at roomtemperature for 48 h. The color reaction was stoppedby rinsing the sections in distilled water. The sectionswere dehydrated in graded acetone, cleared in xylene,and cover-slipped using Depex. Control reactions con-sisted of (1) pretreatment of the sections with RNase A(50 �g/ml; Pharmacia, Sweden) prior to hybridization,(2) hybridization of the sections with an excess (100�)of unlabeled probe, and (3) hybridization of the sectionswith hybridization buffer alone.

GDNF Enzyme-Linked Immunosorbent Assay(ELISA)

Five hundred microliters of culture medium was col-lected at 4 days (DIV6) and 12 days (DIV14) posttrans-fection from each roller-tube containing one eighth ofan embryonic pig VM. New medium had been added 2days prior to collection. Medium from four culturescorresponding to one half pig VM was pooled and mon-itored for GDNF expression using the GDNF Emax Im-munoassay System (Life Technologies).

Tissue Processing and Immunohistochemistry

Cultures were fixed for 60 min in 0.1 M phosphatebuffer (PB) containing 4% paraformaldehyde (pH 7.4).After equilibration in 20% sucrose–PB for 24 h, cul-tures were frozen and cut in parallel series at 20 �m,mounting three sections on each slide. Slides with sec-tions were washed in 0.1 M phosphate-buffered saline(PBS) for 30 min, incubated for 60 min in PBS contain-ing 0.3% Triton X-100 and 10% horse serum (Gibco),and then incubated with polyclonal rabbit anti-�-Galantibody (1:1000, Biotrend), monoclonal anti-nestin (1:1000; Pharmingen; 1:100, BD Biosciences; 1:100,

Chemicon), monoclonal anti-MAP-2 (1:2000, Sigma),monoclonal anti-NF70 (1:500, Chemicon), and mono-clonal anti-GFAP (1:800, Boehringer), overnight at4°C. After washing three times, the sections were in-cubated for 2 h at room temperature either with cy3-conjugated donkey anti-rabbit IgG or cy2-conjugateddonkey anti-mouse IgG (1:100, Jackson ImmunoRe-search Lab.), respectively. After washing, sectionswere cover-slipped in 25% glycerol containing PBS.Immunoreactive cells were observed by fluorescencemicroscopy (Zeiss).

For X-Gal histochemistry, fixed cultures were ex-posed to the X-Gal dye (0.01% Na–Deoxycholat, 0.02%NP-40, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mMK4Fe(CN), 0.05% X-Gal in 0.1 M PBS (reagents pro-vided by Sigma)). To avoid background, the pH of thedye solution was raised to 7.6 according to a protocol ofRosenberg and co-workers (26). After incubation, thesections were washed with 0.1 M PBS and cover-slipped for microscopical investigation.

For tyrosine hydroxylase (TH) immunocytochemis-try the mounted sections were first washed three timesin PB and incubated in 0.3% Triton X-100–PB solutioncontaining 10% horse serum (Gibco) for 60 min. Afterwashing, sections were incubated with anti-TH anti-bodies (1:500, Roche) diluted in 0.1% Triton X-100–PBS containing 2.5% horse serum at 4°C for 12 h. Thesections were washed again in PBS and then incubatedfor 2 h with a biotinylated anti-mouse-antibody (1:200,Vector Lab.) and an avidin-biotinylated horseradishperoxidase complex according to the supplier’s in-structions (Vector Lab.). Finally, TH-immunoreactive(TH-ir) cells were visualized by incubation with 0.1%DAB (Pierce) in 0.1 M PB, and the sections were dehy-drated in increasing alcohol concentrations, cleaned inxylene, and cover-slipped. Specificity of immunostain-ing was determined by omission of primary or second-ary antibodies.

Histological Analysis of Sections, Cell Counts, andSoma Size Estimation

For estimation of transfection efficiencies, �-Gal-pos-itive cells were counted by fluorescence microscopy(Zeiss) in every third section. To be counted, a �-Gal-positive cell had to display (1) dense staining and (2) aclearly defined cell body. Estimations of cell countswere done as described below; counts are displayed as�-Gal-positive cells per volume.

Counts of TH-ir cells and area measurements wereperformed in bright-field microscopy using a three-dimensional neuron tracing system (Eutectic). To becounted, a TH-ir cell had to display (1) dense staining,(2) a well-preserved cellular structure, and (3) a dis-tinct nucleus. Cell numbers were corrected accordingto Abercrombie’s formula (1). Estimations of TH-ir cellsin the culture pieces were done as described previously

42 BAUER ET AL.

(4, 22). In brief, cell counts were performed in threesections (thickness 20 �m) covering a 360-�m-widesegment of the central part of the tissue explants. Thevolume of the culture was approximated assuming thatthe culture blocks have a spherical shape. One individ-ual culture was assumed as one treatment group. To beincluded in the analysis, cultures had to contain morethan 10 TH-ir cells in at least one of the three sections.Counts are presented as number of cells per volume.

Soma size estimation was done using the OlympusC.A.S.T.-Grid system (Olympus, Denmark), composedof an Olympus microscope, an X-Y step motor stage runby an IBM PC computer, and a microcator (Heiden-heim, ND 281) connected to the stage. Three sectionsfrom the central part of each VM culture were delin-eated, and a 1675-�m2 counting frame was moved sys-tematically through each delineated area (step length:150 �m, X axis and 150 �m, Y axis). For inclusion inthe analysis, TH-positive neurons had to demonstratea clearly defined nucleus and be located within a pre-defined depth (4 to 12 �m with a section thickness of 20�m) of the section using the optical dissector. In anaverage area of approx. 2.1 mio. �m2, a minimum of 25TH-positive neurons (25 to 106 cells) were sampledfrom each individual culture. The data were pooled andpresented as mean soma size (�m3 volume) in sham-and pRep7-GDNF8-transfected VM tissue. Both cellcounts and soma size estimations were done in a blindfashion.

Statistical Analysis

For comparison of two different treatment groups weused t test (hGDNF protein concentrations, TH-posi-tive cell counts) or the Mann–Whitney rank sum test(transfection yields, soma size comparison). For com-parison of multiple treatment groups we used one-wayanalysis of variance (ANOVA) followed by the Tukeytest (transfection yields and mitogen treatment). Allvalues are expressed as mean � SE.

RESULTS

Optimization of Transfection

A vector construct coding for �-Gal, pRSV-lacZ, wasused to optimize the transfection procedure. Up to 100�l (20 to 100 �l) of the cationic lipid (Effectene; Qiagen)was complexed with up to 8 �g (2 to 8 �g) plasmid DNAusing different lipid–DNA ratios (10:1 to 50:1). Trans-fections were performed 2 days postdissection (“2 daysin vitro,” DIV2). Using different lipid–DNA ratios(10:1, 25:1, and 50:1), transfection efficiencies in termsof �-Gal-expressing cells were significantly lower incultures exposed to transfection mixtures with 2 �gplasmid DNA (mean 24 cells/mm3 � 10, n � 12) com-pared to mixtures with 4 �g (mean 240 cells/mm3 � 50,

n � 8) (P � 0.001; Mann–Whitney rank sum test). Afurther increase in the amount of plasmid DNA to 8 �gdid not result in any increase or cause significantchanges of transfection efficiency (mean 134 cells/mm3 � 67, n � 4). With a given DNA concentrationthere were no statistically significant differences intransfection yields in treatment groups due to changesin the lipid–DNA ratio. Prolongation of the cultureperiod from 2 to 4 days prior to transfection did notresult in a significant change of transfection yields. Insummary, optimal transfection yields were obtainedwhen 4 �g vector DNA was complexed with 40 �l lipidsuspension and the transfection was performed 2 dayspostdissection (DIV2). The following experiments wereperformed under these conditions.

To further improve the transfection yields, cultureswere exposed to the mitogens EGF and bFGF fromculture start and during transfection. Such treatmentof pig VM explant cultures with EGF, bFGF, or acombination of EGF and bFGF resulted in a 1.6-foldincrease of transfection yields for EGF (423 cells/mm3 � 124, n � 8), a 2.1-fold increase for bFGF (553cells/mm3 � 138, n � 8), and a 2.3-fold increase forEGF plus bFGF (615 cells/mm3 � 91, n � 14) com-pared to nontreated controls (268 cells/mm3 � 42, n �12) (P � 0.01; ANOVA) (Fig. 1).

Histological investigation of transfected culturestreated with EGF/bFGF showed �-Gal-positive cell

FIG. 1. Mitogen treatment increases transfection yields in pigVM tissue. VM pig tissue was transfected with pRSV-lacZ after 2days in vitro (DIV2). The transfection mixture consisted of 4 �gplasmid DNA and 40 �l lipid–solution (Effectene; Qiagen) and wasleft in place for 18 h. Analyses of transfection yields in terms ofcounts of �-Gal-positive cells were performed at DIV4. EGF, bFGF,or the combination of both mitogens were supplemented to the cul-ture medium each in a concentration of 20 ng/ml. Mitogens wereadded to the culture medium at the start of the culture period(DIV0), directly to the transfection mixture at DIV2, and to themedium after removal of the transfection mixture at DIV3. Trans-fected cultures not exposed to the mitogens were used as controls(nc). Combined EGF/bFGF treatment of the cultures resulted inmarkedly higher transfection yields compared to untreated controls(*P � 0.05 vs nc., ANOVA, Tukey test; bars represent SE).

43GENETIC MODIFICATION OF XENOGRAFTS IN PARKINSON’S DISEASE

bodies up to 100 �m beneath the surface of the VMtissue block (Fig. 2b). Targeted cells typically displayedspherical cell bodies and multiple dendritic-like pro-cesses oriented tangentially below the surface of theculture (Fig. 3). �-Gal-positive immunofluorescent cellsdid not show a corresponding immunoreactivity to an-tibodies raised against the astroglial marker GFAP orthe neuronal markers NF70, MAP2, or TH (data notshown).

GDNF Gene Transfer

For enhanced trophic support of dopaminergic (TH-positive) neurons, a vector coding for the neurotrophicfactor hGDNF, pRep7-GDNF8, was transferred to theembryonic pig VM explants. The transfection mixtureconsisted of 4 �g plasmid DNA complexed with 40 �llipid suspension and the tissue was exposed to bFGF/EGF from start of the culturing and during transfec-tion performed at DIV2.

hGDNF mRNA was detected by in situ hybridization2 days posttransfection (DIV4) in cultures transfected

at DIV2 (Fig. 4). Labeled cell bodies were detected nearthe surface of the cultures. The distribution of thehybridization signal in cultures transfected with

FIG. 3. Morphological diversity of transfected cells in E27/28 pigVM tissue transfected with pRSV-lacZ after 2 days in vitro (4 �gplasmid DNA complexed with 40 �l lipid–solution, incubation time18 h, EGF/bFGF mitogen treatment) and fixed 2 days posttransfec-tion for analysis by �-Gal immunofluorescence. (a) Spherical-shapedtransfected cells at the surface of the culture. (b) Cell with a processapprox. 150 �m in length. (c) Transfected cell showing at least fourprocesses; scale bar, 50 �m.

FIG. 2. Embryonic porcine VM explant cultures transfected withpRSV-lacZ at 2 days in vitro and processed for histology 2 days later.Histochemical detection of �-Gal-positive cells in sections of a controlculture (no mitogen treatment) (a) and a culture exposed to EGF/bFGF (b); scale bar, 200 �m.

44 BAUER ET AL.

pRep7-GDNF8 resembled that of �-Gal-positive cells incultures transfected with pRSV-lacZ. pRep7-trans-fected cultures served as controls and did not show anypositive signal.

Pooled medium from four explant cultures, corre-sponding to one half pig VM, was collected at 4 (DIV6)and 12 (DIV14) days posttransfection and analyzed byELISA for GDNF protein. At DIV6, GDNF protein wasdetected in all samples of pRep7-GDNF8-transfectedcultures (65 � 11 pg/ml, n � 8), whereas only two ofeight samples of pRep7-transfected control culturesexpressed detectable levels of GDNF (9 � 6 pg/ml, n �

8) (P � 0.001, t test). At DIV14, GDNF concentrationsin the medium of pRep7-GDNF8-transfected culturesdropped to 32% (P � 0.01, t test) of the initial DIV6baseline level, but considerable amounts of GDNF pro-tein were still detectable in the medium (mean 21 � 6pg/ml, n � 7). GDNF concentrations in control culturesat DIV14 were 4 � 4 pg/ml (n � 7) (Fig. 5).

Effects on TH-Positive Cell Numbers and Soma Size

The effect of ectopic hGDNF expression was assessedby counts of TH-positive neurons after 2 weeks in cul-ture (DIV14; 12 days posttransfection). The number ofTH-positive neurons in cultures transfected withpRep7-GDNF8 (mean 5384 � 588 cells/mm3, n � 12)was 1.8-fold higher (P � 0.01, t test) than in thepRep7-transfected control cultures (3069 � 502 cells/mm3) (Figs. 6 and 7). The volume of the explantcultures did not differ significantly (P � 0.14) be-tween pRep7-GDNF8- and pRep7-transfected cul-tures (0.53 � 0.06 mm3 (n � 12) and 0.70 � 0.1 mm3

(n � 11)), respectively. Soma size of TH-positive neu-rons within sham-transfected and pRep7-GDNF8-transfected cultures did not show any significant dif-ferences (223 � 10 �m3, n � 270 vs 193 � 7 �m3, n �374, respectively).

FIG. 5. GDNF production by transfected embryonic porcine VMexplant cultures. The explants were transfected under optimizedconditions (see text) with pRep7-GDNF8 and pRep7 (control) atDIV2. Medium from four corresponding cultures (four times 500 �l,conditioned for 2 days), collected at DIV6 and DIV14, was pooled forGDNF protein measurements. At both culture ages, significantlyhigher amounts of GDNF were detected in the medium of pRep7-GDNF8-transfected cultures compared to the medium of pRep7-transfected control cultures (***P � 0.001, t test vs pRep7, “4 daysposttransfection”; *P � 0.05 vs pRep7, “12 days posttransfection”).Measurements revealed up to 100 pg GDNF per milliliter mediumcorresponding to 200 pg GDNF produced by half a pig VM. Concen-tration of GDNF in the medium of pRep7-GDNF8-transfected cul-tures decreased significantly with time (##P � 0.01 t test vs pRep7-GDNF8, “12 days posttransfection”). Bars represent SE.

FIG. 4. Detection of hGDNF mRNA in transfected explant cul-tures derived from E27/28 pig VM. The VM tissue was exposed to atransfection mixture consisting of 4 �g pRep7-GDNF8 plasmid vec-tor and 40 �l cationic lipid for 18 h after 2 days in vitro. To enhancetransfection efficiency cultures were treated with 20 ng/ml of EGFand bFGF from start of culture, during transfection, and until 2 daysposttransfection. In situ hybridization for hGDNF mRNA was per-formed after 4 (a) and 14 (b) days in vitro. Sham- (pRep7) transfectedcultures served as controls and did not show any positive signal. Thearrow indicates the surface of the culture; scale bar, 25 �m.

45GENETIC MODIFICATION OF XENOGRAFTS IN PARKINSON’S DISEASE

DISCUSSION

We have previously established a lipid-mediatedgene transfer method to introduce a plasmid vectorcoding for hGDNF into rat VM tissue (4). EctopichGDNF expression resulted in higher numbers of TH-positive neurons within the transfected tissue, andtransplantation of transfected VM cultures in a ratmodel of Parkinson’s disease caused a faster recoveryfrom experimentally induced movement deficits com-pared to animals receiving nontransfected transplants.The findings suggest that transplantation treatment inPD can be improved by nonviral gene transfer asshown in the rat to rat allotransplantation situation.To switch to a cellular system of a larger animal, thepig, which may become more relevant for a clinicalapplication (9, 29), lipid-mediated gene transfer wasadapted and optimized to introduce a vector constructencoding hGDNF into embryonic pig VM explant cul-tures.

For the optimization of the gene transfer method,pRSV-lacZ, a vector construct coding for the Rous Sar-

FIG. 6. Increased numbers of TH-positive neurons in transfectedembryonic porcine VM explants. Mitogen-treated (EGF/bFGF) cul-tures were transfected (DIV2) with pRep7 and pRep7-GDNF8, re-spectively. Counts of TH-positive neurons at DIV14 revealed signif-icantly higher numbers in cultures transfected with pRep7-GDNF8(**P � 0.01, t test).

FIG. 7. Sections of TH-immunostained embryonic pig explants transfected with pRep7 (control; a and b) and pRep7-GDNF8 (c and d)(scale bar, 50 �m).

46 BAUER ET AL.

coma Virus promoter, and the marker gene �-galacto-sidase were used. RSV-driven vector constructs havebeen shown to result in strong transient transgeneexpression in central nervous system (CNS) tissue invarious species including rat (4) and human (5). Underoptimized conditions a mean of 113 cells per culture,corresponding to 904 cells per VM, showed transgeneexpression. The transfection efficiency was about 25%lower in the porcine tissue than in the transfected ratVM tissue (4). Several factors may contribute to thisfinding, including lower accessibility of lipoplexes tothe porcine target cells, different charge ratio, andlower baseline mitotic index of target cells in embry-onic porcine tissue (7). There appears to be a correla-tion between the mitotic activity and the transfectionefficiency using cationic lipid–DNA complexes (10, 24).Although transfer of lipid–DNA complexes into thenucleus is dependent on several variables, includingthe size of the complexes and the existence of nuclearlocalization signals within the sequence of the plasmid,it is suggested that lipofection in general preferentiallytargets cycling cells and plasmid entry into the nucleusis facilitated by nuclear membrane break down (forreview see Ref. 20). In support of this hypothesis, wefound in our study that addition of the mitogens EGFand/or bFGF to the pig explant culture medium prior totransfection resulted in a marked increase of trans-gene-expressing cells. Under this condition BrdU label-ing of cultured porcine VM tissue showed clusters ofmitotic cells within the culture, revealing a tendencyfor larger clusters and more intense staining of cellswithin the cluster in mitogen-treated cultures (datanot shown). Due to the inhomogeneity of the explants itwas, however, not possible to clearly quantify and thusstatistically compare the number of BrdU-labeled cellsin untreated versus mitogen-treated pig tissue.

Identification of transfected porcine cells by immu-nohistochemical stains using antibodies raised againstmarkers expressed in mature astroglia or neuronsfailed to identify the targeted cells. In previous studiesimmature nestin-expressing neuroepithelial precursorcells were targeted by lipid-mediated gene transfer inVM tissue derived from embryonic day 14 (E14) ratembryos (4), whereas transfected VM cells from 6 to12-week-old human embryos did not show colocaliza-tion with nestin (5). Despite applying several differentantibodies against nestin, E27/28 porcine VM tissuedid not stain for this marker. Since to our knowledgethere are no data published on nestin expression in VMporcine tissue, whether the antibodies used did notdetect porcine nestin homologues or whether nestin isnot yet expressed in E27/28 porcine VM tissue remainsunclear. However, transfected cells in bFGF/EGF-treated cultures showed some morphological featuresseen in neuroepithelial precursor cells (18), includinglong curly processes, whereas transfected cells in un-treated control cultures preferentially showed a spher-

ical shape. With the lack of appropriate antibodies forpig-specific immature CNS cells to characterize thetransfected cells in mitogen-treated and untreated cul-tures, whether different cell populations were targetedin cultures treated in a different way is a matter ofspeculation.

Despite a limited number of cells being transfected,pRep7-GDNF8-transfected porcine cells produced con-siderable amounts of hGDNF protein 4 days posttrans-fection. Average medium levels of 65 pg protein permilliliter supernatant were detected, which is in aphysiological range in terms of receptor saturation(30–100 pg/ml) and survival-promoting effects in a pri-mary neuronal test system (half maximal effect: 30–40pg/ml) (17, 19). After 2 weeks in culture, hGDNF levelshad decreased significantly but remained easily detect-able. Cell counts in pRep7-GDNF8-transfected ex-plants cultured for 2 weeks as free-floating roller-tubecultures showed significantly better survival of TH-positive neurons than mock-transfected control cul-tures. The survival-promoting effect must be attrib-uted to the ectopically produced hGDNF since pRep7-transfected control cultures were treated with bFGF/EGF in the same way as the cultures of the treatmentgroup (11). The effects seen in the present study arecomparable with those seen in a previous study onporcine VM brain slices cocultured with GDNF-produc-ing HiB5 cells or supplemented with hGDNF protein(10 ng/ml) to the medium (23).

For hGDNF transgene delivery we used an Epstein–Barr virus-based vector construct (pRep7-GDNF8) thatalready proved to be effective in rat VM tissue (4).Vector constructs based on pRep7 express a 3� trun-cated form of EBNA1. EBNA1 is a multifunctionalviral protein of EBV, which contributes to a stablemaintenance of the episome in the nucleus and to aproper replication of the EBV circular episome in hu-man, monkey, and dog cell lines (for review see Ref.33). EBNA1 binds to the viral origin of replication ofEBV, oriP, also present on the pRep7 plasmid, allowingreplication of the viral genome during viral latency(25). EBV-based elements, however, may differ in theirfunction in cells derived from different species (21, 32)and even among different cell populations within onespecies (31). In porcine VM tissue transfected with anEBV-based plasmid, strong transgene expressionwithin the first days posttransfection was followed by amarked decline of ectopic gene expression after 2weeks posttransfection as determined by hGDNF pro-tein measurements and in situ hybridization. Severalpossibilities may apply for the decline of transgeneexpression, including viral promoter shutdown, partialincompatibility of plasmid-derived EBV elements withporcine target structures, or reduction of the cells car-rying the plasmid. Ectopic gene expression of a fractionof cells in a given tissue for gene-therapeutic purposeswill suffice only for expression of those gene products,

47GENETIC MODIFICATION OF XENOGRAFTS IN PARKINSON’S DISEASE

of which the beneficial effects are distributed to non-transfected cells. This applies to our neuroprotectiveapproach. In contrast, gene replacement, gene correc-tion, or antisense expression requires the majority oftarget cells to be successfully transfected. Therefore,future studies have to determine mechanisms and con-ditions to further improve yields and stability of lipid-mediated gene transfer to primary VM tissue for thoseapplications, where expression of the transgene is re-quired in the majority of the treated cells. In parallel,improved vector constructs seem favorable, facilitatinga more robust and long-lasting transgene expression.This could be combined with the principle of exog-enously controlled transgene expression—for exampleby tetracycline—to improve safety and delivery of fac-tors at an optimal time point.

In summary, a nonviral transfection protocol for cul-tured porcine dopaminergic tissue has been developed.Ectopic hGDNF gene expression was maintained for atleast 2 weeks, resulting in an improved survival ofTH-positive neurons in vitro. Moreover, in vivo studiesto elucidate the effects of such nonviral transfection oncell survival and function in dopaminergic tissue graftstransplanted into hemiparkinsonian rats are under-way.

ACKNOWLEDGMENTS

We are grateful to Qiagen GmbH, especially Dr. M. Weber, forvaluable help and discussion concerning transfection technology andfor financial support. We thank D. Lyholmer for excellent technicalassistance, Dr. T. Johansen and H. Andersen, NeuroSearch, Copen-hagen, for doing the GDNF ELISA, Dr. Ursula Olazabal for criticalreading and editing of the manuscript, and Professor G. W.Bornkamm for continuously supporting the project. This researchwas supported by Qiagen, Hilden, the Danish MRC, and the DanishParkinson Foundation.

REFERENCES

1. Abercrombie, M. 1946. Estimation of nuclear population frommicrotome sections. Anat. Rec. 94: 239–247.

2. Ambinder, R. F., M. Mullen, Y. N. Chang, G. S. Hayward, andD. Hayward. 1991. Functional domains of Epstein-Barr virusnuclear antigen EBNA-1. J. Virol 65: 1466–1478.

3. Barker, R. A., S. B. Dunnett, A. Faissner, and J. W. Fawcett.1996. The time course of loss of dopaminergic neurons and thegliotic reaction surrounding grafts of embryonic mesencephalonto the striatum. Exp. Neurol. 141: 79–93.

4. Bauer, M., M. Meyer, L. Grimm, T. Meitinger, J. Zimmer, T.Gasser, M. Ueffing, and H. R. Widmer. 2000. Non-viral GDNFgene transfer enhances survival of cultured dopaminergic neu-rons and improves their function after transplantation in a ratmodel of Parkinson’s disease. Hum. Gene Ther. 11: 1529–1541.

5. Bauer, M., M. Meyer, J. Sautter, T. Gasser, M. Ueffing, andH. R. Widmer. 2001. Liposome-mediated gene transfer to fetalhuman ventral mesencephalic explant cultures. Neurosci. Lett.308: 169–172.

6. Brevig, T., J. Holgersson, and H. Widner. 2000. Xenotransplan-tation for CNS repair: Immunological barriers and strategies toovercome them. Trends Neurosci. 23: 337–344.

7. Chesnoy, S., and L. Huang. 2000. Structure and function oflipid–DNA complexes for gene delivery. Annu. Rev. Biophys.Biomol. Struct. 29: 27–47.

8. de Rijek, M., L. Launer, K. Berger, M. Breteler, J. Dartigues,M. Baldereschi, L. Fratiglioni, A. Lobo, J. Martinez-Lage,C. Trenkwalder, and A. Hofman. 2000. Prevalence of Parkin-son’s disease in Europe: A collaborative study of population-based cohorts. Neurologic Diseases in the Elderly ResearchGroup. Neurology 54: S21–S23.

9. Deacon, T., J. Schumacher, J. Dinsmore, C. Thomas, P. Palmer,and O. Isacson. 1997. Histological evidence of fetal pig neuralcell survival after transplantation into a patient with Parkin-son’s disease. Nat. Med. 3: 350–353.

10. Fasbender, A., J. Zabner, B. G. Zeiher, and M. J. Welsh. 1997.A low rate of cell proliferation and reduced DNA uptake limitcationic lipid-mediated gene transfer to primary cultures ofciliated human airway epithelia. Gene Ther. 4: 1173–1180.

11. Fawcett, J. W., R. A. Barker, and S. B. Dunnett. 1995. Survivalof embryonic mesencephalic dopaminergic neurons in explant,three-dimensional and monolayer cultures, and the effect ofbFGF. Exp. Brain Res. 106: 275–282.

12. Felgner, P. L. and G. M. Ringold. 1989. Cationic liposome-mediated transfection. Nature 337: 387–388.

13. Finsen, B. R., N. Tonder, S. Augood, and J. Zimmer. 1992.Somatostatin and neuropeptide Y in organotypic slice culturesof the rat hippocampus: An immunocytochemical and in situhybridization study. Neuroscience 47: 105–113.

14. Freed, C. R., P. Greene, R. E. Breeze, W. Tsai, W. DuMouchel,R. Kao, S. Dillon, H. Wnfield, S. Culver, J. Trojanowski, D.Eidelberg, and S. Fahn. 2001. Transplantation of embryonicdopamine neurons for severe Parkinson’s disease. N. Engl.J. Med. 344: 710–719.

15. Gahwiler, B. H. 1981. Organotypic monolayer cultures of ner-vous tissue. J. Neurosci. Methods 4: 329–342.

16. Grimm, L., E. Holinski-Feder, J. Teodoridis, B. Scheffer, D.Schindelhauer, T. Meitinger, and M. Ueffing. 1998. Analysis ofthe human GDNF gene reveals an inducible promotor, threeexons, a triplet repeat within the 3�-UTR and alternative spliceproducts. Human Mol. Gen. 7: 1873–1886.

17. Jing, S., D. Wen, Y. Yu, P. L. Holst, Y. Luo, M. Fang, R. Tamir,L. Antonio, Z. Hu, R. Cupples, J.-C. Louis, S. Hu, B. W. Altrock,and G. M. Fox. 1996. GDNF-induced activation of the Retprotein tyrosine kinase is mediated by GDNFR-alpha, a novelreceptor for GDNF. Cell 85: 1113–1124.

18. Lendahl, U., L. B. Zimmerman, and R. D. G. McKay. 1990. CNSstem cells express a new class of intermediate filament protein.Cell 60: 585–595.

19. Lin, L. F., D. H. Doherty, J. D. Lile, S. Bektesh, and F. Collins.1993. GDNF: A glial cell line-derived neurotrophic factor formidbrain dopaminergic neurons. Science 260: 1130–1132.

20. Lollo, C. P., M. G. Banaszczyk, and H. C. Chiou. 2000. Obstaclesand advances in non-viral gene delivery. Curr. Opin. Mol. Ther.2: 136–142.

21. Mecsas, J., and B. Sugden, 1987. Replication of plasmids de-rived from bovine papilloma virus type 1 and Epstein-Barrvirus in cells in culture. Annu. Rev. Cell Biol. 3: 87–108.

22. Meyer, M., J. Zimmer, R. W. Seiler, and H. R. Widmer. 1999.GDNF increases the density of cells containing calbindin butnot of cells containing calretinin in cultured rat and humanfetal nigral tissue. Cell Transplant. 1: 1–2.

23. Meyer, M., J. Johansen, J. B. Gramsbergen, T. E. Johansen,and J. Zimmer. 2000. Improved survival of embryonic porcinedopaminergic neurons in coculture with a conditionally immor-talized GDNF-producing hippocampal cell line. Exp. Neurol.164: 82–93.

48 BAUER ET AL.

24. Mortimer, I., P. Tam, I. MacLachlan, R. W. Graham, E. G.Saravolac, and P. B. Joshi. 1999. Cationic lipid-mediated trans-fection of cells in culture requires mitotic activity. Gene Ther. 6:403–411.

25. Reisman, D., J. Yates, and B. Sugden. 1985. A putative origin ofreplication of plasmids derived from Epstein-Barr virus is com-posed of two cis-acting components. Mol. Cell Biol. 5: 1822–1832.

26. Rosenberg, W. S., X. O. Breakfield, C. DeAntonio, and O. Isac-son. 1992. Authentic and artifactual detection of E. coli lacZgene product in the rat brain by histochemical methodes. Mol.Brain Res. 16: 311–315.

27. Rosenblad, C., A. Martinez-Serrano, and A. Bjorklund. 1996.Glial cell line-derived neurotrophic factor increases survival,growth and function of intrastriatal fetal nigral dopaminergicgrafts. Neuroscience 75: 979–985.

28. Sautter, J., J. L. Tseng, D. Braguglia, P. Aebischer, C. Spenger,R. W. Seiler, H. R. Widmer, and A. D. Zurn. 1998. Implants ofpolymer-encapsulated genetically modified cells releasing glialcell line-derived neurotrophic factor improve survival, growth,and function of fetal dopaminergic grafts. Exp. Neurol. 149:230–236.

29. Schumacher, J. M., S. A. Ellias, E. P. Palmer, H. S. Kott, J.Dinsmore, P. K. Dempsey, A. J. Fischman, C. Thomas, R. G.Feldman, S. Kassissieh, R. Raineri, C. Manhart, D. Penney,J. S. Fink, and O. Isacson. 2000. Transplantation of embryonicporcine mesencephalic tissue in patients with PD. Neurology54: 1042–1050.

30. Spenger, C., L. Studer, L. Evtouchenko, M. Egli, J. M. Burgun-der, R. Markwalder, and R. W. Seiler. 1994. Long-term survivalof dopaminergic neurons in free-floating roller tube cultures ofhuman fetal ventral mesencephalon. J. Neurosci. Meth. 54:63–73.

31. Teo, C. G., and B. E. Griffin. 1987. Epstein-Barr virus genomesin lymphoid cells: activation in mitosis and chromosomal loca-tion. Proc. Natl. Acad. Sci. USA 84: 8473–8477.

32. Tomiyasu, K., E. Satoh, Y. Oda, K. Nishizaki, M. Kondo, J.Imanishi, and O. Mazda. 1998. Gene transfer in vitro and invivo with Epstain-Barr virus-based episomal vector results inmarkedly high transient expression in rodent cells. Biochem.Biophys. Res. Commun. 253: 733–738.

33. Van Craenenbroeck, K., P. Vanhoenacker, and G. Haegeman.2000. Episomal vectors for gene expression in mammalian cells.Eur. J. Biochem. 267: 5665–5678.

34. Wenning, G. K., P. Odin, P. Morrish, S. Rehncrona, H. Widner,P. Brundin, J. C. Rothwell, R. Brown, B. Gustavii, P. Hagell, M.Jahanshahi, G. Sawle, A. Bjorklund, D. J. Brooks, C. D. Mars-den, N. P. Quinn, and O. Lindvall. 1997. Short-and long-termsurvival and function of unilateral intrastriatal dopaminergicgrafts in Parkinson’s disease. Ann. Neurol. 42: 95–107.

35. West, M. J., K. Ostergaard, O. A. Andreassen, and B. Finsen.1996. Estimation of the number of somatostatin neurons in thestriatum: An in situ hybridization study using the optical frac-tionator method. J. Comp. Neurol. 370: 11–22.

36. Wiesenhofer, B., and C. Humpel. 2000. Lipid-mediated genetransfer into primary neurons using FuGene: Comparison to C6glioma cells and primary glia. Exp. Neurol. 164: 38–44.

49GENETIC MODIFICATION OF XENOGRAFTS IN PARKINSON’S DISEASE