Stem Cells http://www.epitomics.com
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baier, P. C.
Right arrow Articles by Trenkwalder, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baier, P. C.
Right arrow Articles by Trenkwalder, C.

Stem Cells 2004;22:396-404 www.StemCells.com
© 2004 AlphaMed Press

Behavioral Changes in Unilaterally 6-Hydroxy-Dopamine Lesioned Rats After Transplantation of Differentiated Mouse Embryonic Stem Cells Without Morphological Integration

Paul Christian Baiera,*, Jan Schindehütteb,*, Keneuoe Thinyanea, Gabriele Flüggec, Eberhard Fuchsc, Ahmed Mansourib, Walter Paulusa, Peter Grussb, Claudia Trenkwaldera

a Department of Clinical Neurophysiology, Georg-August University Göttingen, Göttingen, Germany;
b Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany;
c German Primate Center, Göttingen, Germany

Key Words. Embryonic stem cells • Transplantation • 6-hydroxy-dopamine lesion • Parkinson’s disease

Paul Christian Baier, M.D., Department of Clinical Neurophysiology, University Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. Telephone: 49-551-39-8453; Fax: 49-551-39-8126; e-mail: pbaier{at}gwdg.de


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective. Transplantation of fetal mesencephalic cells into the striatum has been performed in about 350 patients with Parkinson’s disease and has been intensively studied in rat models of Parkinson’s disease. Limited access to this material has shifted the focus toward embryonic stem (ES) cells. The grafting of undifferentiated ES cells to 6-hydroxy-dopamine (6-OHDA)-lesioned rats leads to behavioral improvements but may induce teratoma-like structures. This risk might be avoided by using more differentiated ES cells. In this study, we aimed to investigate differentiated mouse ES cells regarding their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced after transplantation.

Methods. Mouse ES cells were differentiated on PA6 feeder cells for 14 days before grafting. Twenty to twenty-five percent of the neurons obtained were positive for tyrosine-hydroxylase (TH). PKH26-labeled cells were transplanted in the striata of unilaterally 6-OHDA-lesioned rats.

Results. Direct PKH26 fluorescence visualization and TH staining proved the existence of cell deposits in the striata of all grafted animals, indicating cell survival for at least 5 weeks posttransplantation. There was no evidence of tumor formation. Immunocytochemical staining showed glial immunoreactivity surrounding the grafted cell deposits, probably inhibiting axonal outgrowth into the surrounding host tissue. There was a significant reduction in amphetamine-induced rotational behavior seen in grafted animals, which was not observed in sham-operated animals.

Conclusions. The findings of this study suggest that the amphetamine-induced rotational behavioral test without histological confirmation is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Parkinson’s disease (PD) is a common neurologic disorder caused by a progressive degeneration of dopamine (DA)-producing midbrain neurons. The resulting dopaminergic deficit at the striatal dopamine receptors causes the characteristic symptoms of akinesia, rigidity, and/or tremor [1]. Pharmacological treatment of the disorder with levodopa or DA agonists initially alleviates motor symptoms, but is frequently limited in advanced stages by the occurrence of both motor and psychiatric complications [2]. Therefore, there is a strong need for alternative therapeutic approaches, such as deep-brain stimulation, either in the globus pallidum or the subthalamic nucleus [3, 4], or cell replacement therapies. More than 20 years ago, the first experiments transplanting neural tissue to reduce Parkinsonian symptoms were performed in a rat model of PD [5]. Since then, transplantation of dopaminergic fetal mesencephalic cells into the striatum has been intensively studied in rats [6] and nonhuman primates [7]. More than 350 PD patients all over the world have received fetal transplants using various protocols and techniques with partially positive results [8–11]. However, the practical and ethical limitations of using human fetal cells led to a search for new sources of dopaminergic cells that are readily available and not limited in number. Embryonic stem (ES)-cell-derived neurons may serve as a possible source of grafts, although their potential for generating tumors may limit their use. Recently, undifferentiated murine ES cell grafts, functionally integrated in the unilaterally 6-hydroxy-dopamine (6-OHDA)-lesioned rat striatum, led to behavioral improvements, but 26% of those rats developed teratoma-like structures [12]. In a further study, mouse ES cells were efficiently differentiated into dopaminergic neurons and grafted in the 6-OHDA rat model [13]. Neurophysiological and histological studies revealed the dopaminergic fate and functional integration of grafted cells in the rat striatum. Grafted animals showed significant improvements in amphetamine-induced rotational behavior and no tumor formation.

In the present study, we aimed to investigate the utility of mouse ES cells differentiated on PA6 feeder cells [14] with regard to their in vivo development and fate after transplantation in the striatum in the 6-OHDA rat model and the behavioral changes induced 4 weeks after transplantation. Autologous grafting would be more elegant, but technical problems in creating unilateral 6-OHDA lesions and in doing behavioral tests in mice limit investigations in this species. As all attempts to establish a rat ES cell line so far have failed, autologous transplantation in a rat model is not yet possible. Although most studies apply cyclosporin A (CsA) for xenotransplantations, we omitted immunosuppression because CsA may interact with the locomotor effects observed after neural transplantation [15] and it does not necessarily improve the survival rate of grafts [16].


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ES Cell Differentiation
Induction of neuronal differentiation was performed as previously described by Kawasaki et al. [14]. Undifferentiated murine ES cells (MPI I) were maintained and expanded on gelatin-coated cell culture dishes in Glasgow minimal essential medium (G-MEM; GIBCO-Invitrogen; Karlsruhe, Germany; http://www.invitrogen.com) containing 1% fetal calf serum (KSR, GIBCO), 10% knockout serum replacement (GIBCO), 2 mM glutamine (GIBCO), 0.1 mM nonessential amino acids (GIBCO), 1 mM sodium pyruvate (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich; Munich, Germany; http://www.sigmaaldrich.com), and 2,000 U/ml leukemia inhibitory factor (GIBCO). For derivation of dopaminergic neurons, colonies of undifferentiated ES cells were trypsinized, resuspended in differentiation medium, and replated as a single-cell suspension on mitomycin-C-inactivated PA6 feeder cells (Riken cell bank; Ibarak, Japan; http://www.brc.riken.jp/lab/cell). Cells were plated at a density of approximately 400 cells/cm2. The differentiation medium was composed of G-MEM supplemented with 10% KSR, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol. ES cells were cultured on PA6 feeder cells in differentiation medium for 8 days before it was replaced with induction medium and cultured for an additional 6 days. The induction medium consisted of G-MEM including N-2 supplement (GIBCO), 100 µM tetrahydrobiopterin (Sigma), 200 µM ascorbic acid, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 0.1 mM 2-mercaptoethanol. After 4 days of differentiation, the medium was changed daily. On day 14 of the protocol, cells were either analyzed by immunocytochemistry or dissociated with AccutaseTM (PAA Laboratories; Pasching, Austria; http//www.paa.at) for transplantation and quantification.

Quantification of Dopaminergic Neurons
To make a rough estimation of the occurrence of neurons, a total of 200 randomly chosen colonies was screened for Tuj1, a marker for class III ß-tubulin, and tyrosine-hydroxylase (TH)-positive staining. Colonies containing a substantial number of cells double-positive for Tuj1 and TH were counted as positive. Percentages of positive colonies were calculated. In order to determine cell survival after dissociation and viability before transplantation, differentiated cells were washed with phosphate-buffered saline (PBS) and exposed to AccutaseTM for 20 minutes at 37°C. Then, the enzyme was diluted with PBS and the cells were mechanically dissociated by pipetting gently up and down. Single cells were replated on gelatin-coated flask slides and cultured in induction medium for 24 hours before the attached cells were stained by immunocytochemistry with Tuj1 (Babco; Princeton, NJ; http://www.covance.com) and TH (anti-TH; Chemicon; Temecula, CA; http://www.chemicon.com). Using an Olympus BX60 microscope (http://www.olympus.com) and the imaging software Analysis® (Soft Imaging System; Münster, Germany; http://www.soft-imaging.de), Tuj1-positive and TH-positive cells were counted in 10 randomly chosen microscopic view fields, and the percentage of double-positive cells was calculated.

Labeling of Dissociated Cells for Transplantation
After dissociation, cells were labeled with PKH26 (PKH26 Fluorescent Cell Linker Kit; Sigma) and resuspended in G-MEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (both from GIBCO).

Animal Preparation
The experiments were performed on 15 young adult male Wistar rats with body weights of approximately 300 g at the beginning of the experiment. The animals were housed in pairs in ventilated sound-attenuated rooms under a 12-hour-light/12-hour-dark schedule at an ambient temperature of 21°C-23°C with food and water available ad libitum. Animal experiments were conducted in accordance with the European Council Directive of November 24, 1986 (86/EEC) and were approved by the Government of Lower Saxony, Germany. The minimum number of animals required to obtain consistent data was employed.

Before anesthesia, animals were pretreated with desipramine hydrochloride (DMI; 25 mg/kg i.p.; 12.5 mg/ml; 0.2 ml/100 g of animal weight). Surgery was performed under deep i.p. ketamin/xylazin anesthesia (at doses of 40 mg/kg and 5 mg/kg body weight for the two drugs, respectively). Each animal was positioned in a stereotaxic operation frame (TSE-Systems). Through a burr hole (position: anterior +2.8 mm and lateral +2.0 mm relative to the bregma), a metal canula (diameter [{Phi}]: 0.47 mm; deep +8.6 mm relative to the dura) was introduced close to the middle forebrain bundle. Thirty to 45 minutes after pretreatment with DMI, 6-OHDA (4 µl of 3.75 mg/ml 6-OHDA [Sigma] dissolved in 0.1% ascorbic acid) was instilled at a rate of 0.5 µl/minute. The needle was left in situ for a further 4 minutes and was then slowly retracted.

Behavioral Testing
The 6-OHDA lesion was evaluated with amphetamine- (AMP, 1 mg/kg i.p. [Sigma]) and apomorphine- (APO, 0.25 mg/kg s.c. [Sigma]) induced rotational testing on day 23 and day 25 postlesioning, respectively. Animals were placed in an automated rotameter bowl, and left and right full-body turns were counted. AMP net rotations over a period of 60 minutes, starting 30 minutes after injection, and APO net rotations over a period of 30 minutes, starting 5 minutes after injection, were determined, and the mean number of rotations per minute was calculated. Animals showing less than two full turns/minute in the pretransplantation AMP rotational test were excluded from the experiment. A lateralized grip strength (LGS) test [17] was performed. Grip strength was measured on each forelimb separately (Grip Strength Meter; TSE-Systems; Bad Homburg, Germany; http://www.tse-systems.de), and the grip strength ratio (LGS ratio) was calculated as the ratio between the mean strength out of 10 pulls of the side contralateral to the lesion and the mean strength of the ipsilateral side. Furthermore, for all animals, the ratios of grip strength post- and pretransplantation (baseline) were calculated for each forepaw, and changes were displayed as a percentage of the baseline value. All behavioral tests were repeated posttransplantation (AMP day 27, APO day 29, and LGS day 31 posttransplantation).

Cell Transplantation
One animal was excluded due to insufficient lesion. Nine animals received cell grafts; of these, three were later excluded from statistical analyses as histology could not be performed due to technical reasons. Five animals received sham surgery (Table 1Go). Surgery was performed under i.p. ketamin/xylazin anesthesia (40 mg/kg and 5 mg/kg body weight, respectively) on day 28 postlesioning. Three burr holes were drilled (position of burr hole A: A –1.8 mm and L +2.2 mm; burr hole B: A –0.6 mm and L +4.0 mm; and burr hole C: A +0.6 mm and L +5.2 mm relative to the bregma, with the incisor bar set to –2.5 mm below the interaural line). Through a metal canula ({Phi}: 0.47 mm), a suspension of dissociated cells (2 µl per site, 5 x 104 cells/µl) or vehicle (G-MEM with 100 U/ml penicillin and 100 µg/ml streptomycin) was injected at a rate of 0.5 µl/minute at four transplantation sites (burr hole A: 5.0 mm and 6.0 mm; burr hole B: 6.0 mm; and burr hole C: 5.0 mm below the bregma) with a 10-µl glass microsyringe. The needle was left in situ for a further 2 minutes to allow the cells to diffuse. Seven animals were grafted and five animals received sham surgery.


View this table:
[in this window]
[in a new window]
  		Table 1. Individual results of behavioral testing
 
Histology and Immunocytochemistry
Thirty-four days after transplantation, the animals were transcardially perfused under deep ketamin anesthesia (80 mg/kg body weight) with 100 ml saline for 2 minutes followed by 200 ml of paraformaldehyde (4% in PBS) for 8 minutes. The brains were postfixed in the same solution, cryoprotected in 30% sucrose, and frozen. Coronal sections (30 µm) were cut on a cryostat, collected in PBS, and divided into series. One series of sections was mounted on gelatin-coated slides for direct PKH26 visualization. After incubation in 3% hydrogen peroxide in PBS and blocking with 3% normal goat serum, separate sections were incubated overnight in the primary antibody. The following primary antibodies were used: polyclonal rabbit anti-TH (TH; 1:500; Biotrend; Cologne, Germany; http://www.biotrend.com), monoclonal mouse anti-rat ED1 (ED1; 1:300; Serotec; Oxford, UK; http://www.serotec.co.uk), and glial fibrillary acidic protein (GFAP; 1:5,000; Sigma). Sections were then incubated in either biotinylated secondary antibody goat anti-rabbit or goat anti-mouse (1:100; Vector Laboratories; Burlingame, CA: http://www.vectorlabs.com), and all were subsequently incubated in avidin-biotin complex (Vectastain ABC kit ELITE; Vector). The peroxidase was visualized with 3,3'-diaminobenzidine (DAB kit; Vector) used according to the manufacturer’s instructions. Immunostained sections were mounted onto gelatin-coated slides, coverslipped, and analyzed with normal light and differential interference contrast microscopy using a Zeiss Axioplan microscope.

Statistical Analysis
Statistical analyses were performed using the analysis program Sigma Stat 2.03. A two-factor analysis of variance was applied to the results of the behavioral tests, with treatment (cell versus sham) and condition (pre- versus posttransplantation) as experimental factors. A Tukey test for pairwise multiple comparisons was performed, when appropriate. Differences in post- and pretransplantation LGS ratios for the intact and lesioned sides were analyzed with an unpaired Student’s t-test between grafted and sham-operated animals.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differentiation of ES In Vitro
Cocultivation of murine ES cells with PA6 feeder cells was efficient for the generation of dopaminergic neurons. Starting from single ES cells, complex colonies with cells of neuronal morphology developed. After 14 days of differentiation 96% ± 2% of the generated colonies contained Tuj1-positive cells, and 32% ± 6% of those colonies were also positive for TH. Exemplary colonies are shown in Figures 1AGo–1CGo. Of the Tuj1-positive cells (mean number calculated per view field was 106 ± 23), 28% ± 6% were TH positive cells, detectable after dissociation with AccutaseTM. An example is shown in Figure 2Go.



View larger version (132K):
[in this window]
[in a new window]
  		Figure 1. ES-cell-derived Tuj1- and TH-positive colonies. After 14 days of differentiation 96% ± 2% of all colonies were positive for class III ß-tubulin, as detected by the Tuj1 antibody. Tuj1-positive colonies are shown (A, D, G) at different magnifications (1:4, 1:10, 1:60). Of the Tuj1-positive colonies, 32% ± 6% of contained a substantial number of TH-positive cells (B, E, H). Examples of counted colonies are marked with arrows (A, B). Signals from the Tuj1 and TH staining, in addition to nuclear Hoechst staining, were merged (C, F, I).

 


View larger version (54K):
[in this window]
[in a new window]
  		Figure 2. For quantification, differentiated cells were dissociated and replated as single-cell suspensions on gelatin-coated flask slides and stained for class III ß-tubulin and TH. Of the attached Tuj1-positive cells, 28% ± 6% were also positive for TH. A) TH staining. B) Tuj1 staining. C) composite image of A and B.

 
Survival of Transplanted ES Cells
Direct PKH26 fluorescence visualization proved the existence of cell deposits in the striata of all grafted animals close to the transplantation sites, indicating cell survival for at least 5 weeks after transplantation (Fig. 3AGo). There was no evidence of extensive migration (Fig. 3AGo and 3BGo) or axonal outgrowth into the surrounding host tissue (Fig. 3BGo) at 5 weeks posttransplantation. TH-positive cells could be identified in each striatum (Fig. 3BGo and 3CGo). Since the grafted cells formed dense conglomerates that did not allow a definite discrimination of single cells, no quantification of cell survival could be made (Fig. 3CGo). Immunocytochemical staining with ED1 (Fig. 3EGo) and GFAP (Fig. 3FGo) showed glial immunoreactivity surrounding the grafted cell deposits in all grafted animals. No immunoreactivity was seen in any of the striata of sham-transplanted animals (Table 1Go). There was no tumor formation observed in any of the grafted animals.



View larger version (121K):
[in this window]
[in a new window]
  		Figure 3. PKH26 fluorescence visualization proved the existence of cell deposits in the striata of all grafted animals (A). The distribution of PKH26-positive cells indicates that there is no evidence of an extensive migration of the transplanted cells. B) Immunocytochemical detection of TH-positive cells revealed that no axonal outgrowth of the transplanted cells into the surrounding host tissue occurred 5 weeks posttransplantation (TH, overview, same as A). C) TH-positive cells could be identified in each striatum (TH, higher magnification, same as B). Immunocytochemical staining with ED1 (E) and GFAP (F) showed glial immunoreactivity surrounding the grafted cell deposits.

 
Behavioral Parameters
The majority of the grafted animals showed a significant reduction in amphetamine-induced rotational behavior (Table 1Go), represented in the group means (pretransplantation: 4.3 ± 0.5 net rotations/minute; posttransplantation: 3.7 ± 1.9 net rotations/minute). In contrast, all sham-operated animals showed a clear increase in this behavior (Table 1Go; pretransplantation: 4.0 ± 0.3 full body turns/minute; posttransplantation: 6.4 ± 0.4 full body turns/minute; Fig. 4AGo; treatment x condition: F1,19 = 6.537, p = 0.021; the Tukey test revealed that this was due to a difference between treatments within the condition postgrafting, p = 0.006). Results in apomorphine-induced rotational behavior were more heterogeneous in both investigational groups, not leading to any significant changes in the group means. Individual results are shown in Table 1Go (grafted animals: pregrafting 6.7 ± 1.0 full body turns/minute, postgrafting 7.7 ± 2.0 full body turns/minute; sham-operated animals: pregrafting 7.3 ± 1.3 full body turns/minute, postgrafting 6.5 ± 1.2 full body turns/minute; treatment x condition: F1,19 = 1.294, not significant; Fig. 4BGo).



View larger version (10K):
[in this window]
[in a new window]
  		Figure 4. A) Amphetamine-induced (1 mg/kg i.p.) rotations showed a significant difference posttransplantation between grafted and sham-operated animals, with a reduction in the grafted group (n = 6) and an increase in the sham-operated animals (n = 5) (F1, 19 = 6.537, p < 0.05; Tukey-test p = 0.006 for treatment within postgrafting). B) There was no change in apomorphine-induced (0.25 mg/kg s.c.) rotation behavior (F1,19 = 1.294, not significant). Error bars represent the standard error of the mean.

 
The LGS test showed inconsistent results pretransplantation. The expected increase in strength contralateral to the lesion, as a correlate to rigidity, and represented by an LGS ratio >1 was observed in seven animals only. Those animals showed a difference in the LGS ratio posttransplantation between grafted and sham-operated animals. Whereas the grafted animals (n = 2) had a decrease in LGS ratio to values below 1 after transplantation (pregrafting: 1.4 ± 0.2; postgrafting: 0.90 ± 0.06), the sham-operated animals (n = 5) had an increase (pregrafting: 1.3 ± 0.1; postgrafting: 1.6 ± 0.1). Due to the low number of animals in the grafted group showing LGS ratios >1 pregrafting, a statistical analysis was not performed. The analysis of all animals (including animals with LGS ratios <1 before transplantation), however, revealed a decrease in grip strength on the lesioned side to 93% ± 12% of the pregrafting value in the transplanted animals and an increase to 130% ± 15% in the sham-operated group (t-test, p < 0.05), whereas there was no change in the intact side (grafted animals: 103% ± 20%; sham-operated animals: 106% ± 23%; t-test, p = 0.79).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we could show that dopaminergic neurons, specifically dopaminergic precursor cells derived from murine ES cells, survived and induced behavioral changes 5 weeks after transplantation into 6-OHDA-lesioned rats. The morphologies of these neurons, however, showed that they were not synaptically integrated into the host brain. This suggests that the observed changes in amphetamine-induced rotational behavior do not necessarily indicate a functional and structural integration of grafted cells.

ES Cell Differentiation
Cells were differentiated as described by Kawasaki et al. [14], who found that neuronal induction of ES cells is promoted by cocultivation with a bone marrow stromal cell line (PA6), producing a so-called stromal cell line-derived inducing activity. TH-positive cells derived according to this protocol were shown to produce dopamine, with the highest number of dopaminergic neurons after 20 days [14]. We transplanted cells after 14 days of differentiation to obtain predifferentiated but not fully differentiated cells, because those cells may respond better to microenvironmental factors in the host brain [18]. In addition, a differentiation of 14 days has been shown to be approximately optimal for survival when grafting differentiated murine ES cell colonies into mice [19]. There is evidence that the expression of TH during differentiation of dopaminergic neurons underlies a multistep regulatory mechanism and that committed dopaminergic precursors do not express TH constantly during differentiation [20]. Consequently, a quantification of TH-positive cells before transplantation does not necessarily represent the actual number of committed dopaminergic neurons. However, of the dissociated reattached Tuj1-positive cells in this study, approximately 25%-30% could be expected to become dopaminergic neurons.

Cell Survival/Immune Reponse
Cyclosporin A may influence motor effects [15] and does not necessarily improve the survival rate of grafts [16], although the effect of immunosuppression is debated [21, 22]. Despite the decision not to use CsA in this study, grafted cells still survived after 5 weeks. We observed, however, a dense glial wall surrounding those cells. As the glial wall has not been observed when grafting allogenic, but xenogenic ventral mesencephalic, tissue [23], it was most likely caused by a specific graft rejection of xenogenic murine cells. Duan et al. speculated that the rejection of xenografted fetal mesencephalic cells was due to an inflammation that leads to an accumulation of host defense cells. This, in turn, leads to increased major histocompatibility complex (MHC) expression in and around the grafts [23]. To prove this hypothesis, it would be interesting to measure MHC expression when xenografting ES cells. The fact that only dense TH-positive conglomerates remained close to the transplantation site is in accordance with the finding that axons are more vulnerable to immunological rejection than neural somata [24, 25]. One may speculate that the immunological reaction inhibits further differentiation of transplanted immature cells into dopaminergic neurons. This suggests that immunosuppression should be applied in the xenotransplantation of embryonic cells, regardless of the privileged immunologic features of the central nervous system [26]. No tumor formation occurred, although ES cell-derived grafts, in contrast to the study by Kawasaki et al. [14], were not treated with antimitogenic drugs. The reason for the absence of tumors is most likely xenotransplantation without immunosuppression. In previously published data, where immunosuppression was applied, teratoma-like structures occurred in approximately 26% of grafted animals in spite of xenografting [12].

Behavioral Parameters
Results of the amphetamine-induced rotational behavior measurements pretransplantation indicate lesions of 75%-100% of the substantia nigra pars compacta (SNpc) neurons [27]. Although no morphological integration was observed, a reduction in amphetamine-induced rotations postgrafting, similar to previous studies transplanting rat ventral mesencephalic cells [28], porcine xenografts [29], undifferentiated murine ES cells [12], or murine differentiated ES cells [13], was observed. As no spontaneous functional recovery or reinervation occurs in animals with lesions of 75%-100% of the SNpc neurons [27], this effect—only seen in grafted animals—might be explained by an amphetamine-induced release of dopamine from transplanted cells. It, therefore, implies that without histological confirmation, this behavioral test is not proof of morphological integration with axonal outgrowth within the first 4 weeks posttransplantation. A synaptic integration of transplanted cells may be necessary for grafts to have an effect on apomorphine-induced rotational behavior [30]. Fetal mesencephalic grafts may become effective at reducing apomorphine-induced rotation only after several weeks posttransplantation [28, 30]. The reduction in grip strength postgrafting on the lesioned side, not observed after sham surgery, would then have to be interpreted as an unspecific effect of the immunological processes, rather than an improvement in rigidity. However, the results of the grip strength test have to be interpreted very cautiously, as rigidity was not reliably produced in all animals pregrafting.

Future long-term studies using immunosuppression may confirm the continuity of dopamine release combined with functional integration of these dopamine precursor cells in the rat striatum.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by a grant of the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung—01GN0102). The authors thank Christine Crozier for a critical reading of the manuscript and Katharina Schneider and Sharif Mahsur for technical assistance.


   FOOTNOTES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
* These authors contributed equally. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gibb WR, Lees AJ. The relevance of the Lewy body to the pathogenesis of idiopathic Parkinson’s disease. J Neurol Neurosurg Psychiatry 1988;51:745–752.[Abstract]

  2. Clarke CE, Speller JM. Pergolide for levodopa-induced complications in Parkinson’s disease. Cochrane Database Syst Rev 2000;(2):CD000235.

  3. Krause M, Fogel W, Heck A et al. Deep brain stimulation for the treatment of Parkinson’s disease: subthalamic nucleus versus globus pallidus internus. J Neurol Neurosurg Psychiatry 2001;70:464–470.[Abstract/Free Full Text]

  4. Olanow CW, Brin MF, Obeso JA. The role of deep brain stimulation as a surgical treatment for Parkinson’s disease. Neurology 2000;55:S60–S66.

  5. Perlow MJ, Freed WJ, Hoffer BJ et al. Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979;204:643–647.[Abstract/Free Full Text]

  6. Brundin P, Karlsson J, Emgard M et al. Improving the survival of grafted dopaminergic neurons: a review over current approaches. Cell Transplant 2000;9:179–195.[Medline]

  7. Taylor JR, Elsworth JD, Sladek JR Jr et al. Sham surgery does not ameliorate MPTP-induced behavioral deficits in monkeys. Cell Transplant 1995;4:13–26.[CrossRef][Medline]

  8. Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci 2000;3:537–544.[CrossRef][Medline]

  9. Freed CR, Greene PE, Breeze RE et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719.[Abstract/Free Full Text]

  10. Olanow C. Transplantation for Parkinson’s disease: pros, cons, and where do we go from here? Mov Dis 2002;17:S15.

  11. Olanow CW, Kordower JH, Freeman TB. Fetal nigral transplantation as a therapy for Parkinson’s disease. Trends Neurosci 1996;19:102–109.[CrossRef][Medline]

  12. Bjorklund LM, Sanchez-Pernaute R, Chung S et al. Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 2002;99:2344–2349.[Abstract/Free Full Text]

  13. Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002;418:50–56.[CrossRef][Medline]

  14. Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.[CrossRef][Medline]

  15. Borlongan CV, Freeman TB, Hauser RA et al. Cyclosporine-A increases locomotor activity in rats with 6-hydroxydopamine-induced hemiparkinsonism: relevance to neural transplantation. Surg Neurol 1996;46:384–388.[CrossRef][Medline]

  16. Castilho RF, Hansson O, Brundin P. FK506 and cyclosporin A enhance the survival of cultured and grafted rat embryonic dopamine neurons. Exp Neurol 2000;164:94–101.[CrossRef][Medline]

  17. Dunnett SB, Torres EM, Annett LE. A lateralised grip strength test to evaluate unilateral nigrostriatal lesions in rats. Neurosci Lett 1998;246:1–4.[CrossRef][Medline]

  18. Nishino H, Hida H, Takei N et al. Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum. Exp Neurol 2000;164:209–214.[CrossRef][Medline]

  19. Morizane A, Takahashi J, Takagi Y et al. Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity. J Neurosci Res 2002;69:934–939.[CrossRef][Medline]

  20. Matsushita N, Okada H, Yasoshima Y et al. Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development. J Neurochem 2002;82:295–304.[CrossRef][Medline]

  21. Finsen B, Poulsen PH, Zimmer J. Xenografting of fetal mouse hippocampal tissue to the brain of adult rats: effects of cyclosporin A treatment. Exp Brain Res 1988;70:117–133.[Medline]

  22. Schwarz SC, Sauer H, Oertel WH et al. Effects of graft pooling of foetal rat and mouse tissue and immunosuppression in the 6-hydroxydopamine rat model of Parkinson’s disease. Exp Brain Res 1997;115:71–82.[CrossRef][Medline]

  23. Duan WM, Widner H, Brundin P. Temporal pattern of host responses against intrastriatal grafts of syngeneic, allogeneic or xenogeneic embryonic neuronal tissue in rats. Exp Brain Res 1995;104:227–242.[Medline]

  24. Coleman MP, Perry VH. Axon pathology in neurological disease: a neglected therapeutic target. Trends Neurosci 2002;25:532–537.[CrossRef][Medline]

  25. Neumann H. Molecular mechanisms of axonal damage in inflammatory central nervous system disease. Curr Opin Neuol 2003;16:267–273.

  26. Brundin P, Hagell P. The neurobiology of cell transplantation in Parkinson’s disease. Clin Neurosci Res 2001;1:507–520.

  27. Stanic D, Finkelstein DI, Bourke DW et al. Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat. Eur J Neurosci 2003;18:1175–1188.[CrossRef][Medline]

  28. Nikkhah G, Falkenstein G, Rosenthal C. Restorative plasticity of dopamine neuronal transplants depends on the degree of hemispheric dominance. J Neurosci 2001;21:6252–6263.[Abstract/Free Full Text]

  29. Armstrong RJ, Hurelbrink CB, Tyers P et al. The potential for circuit reconstruction by expanded neural precursor cells explored through porcine xenografts in a rat model of Parkinson’s disease. Exp Neurol 2002;175:98–111.[CrossRef][Medline]

  30. Schwarting RK, Huston JP. The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol 1996;50:275–331.[CrossRef][Medline]

Received August 19, 2003; accepted for publication December 4, 2003.



This article has been cited by other articles:


Home page
 Stem CellsHome page
E. Hedlund, J. Pruszak, A. Ferree, A. Vinuela, S. Hong, O. Isacson, and K.-S. Kim
Selection of Embryonic Stem Cell-Derived Enhanced Green Fluorescent Protein-Positive Dopamine Neurons Using the Tyrosine Hydroxylase Promoter Is Confounded by Reporter Gene Expression in Immature Cell Populations
Stem Cells, May 1, 2007; 25(5): 1126 - 1135.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
L. Lin and O. Isacson
Axonal Growth Regulation of Fetal and Embryonic Stem Cell-Derived Dopaminergic Neurons by Netrin-1 and Slits
Stem Cells, November 1, 2006; 24(11): 2504 - 2513.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. L. K. Coles, B. Angenieux, T. Inoue, K. Del Rio-Tsonis, J. R. Spence, R. R. McInnes, Y. Arsenijevic, and D. van der Kooy
Facile isolation and the characterization of human retinal stem cells
PNAS, November 2, 2004; 101(44): 15772 - 15777.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baier, P. C.
Right arrow Articles by Trenkwalder, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baier, P. C.
Right arrow Articles by Trenkwalder, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.epitomics.com