8 \/ d8 [$ o @/ W3 z. [ jc German Primate Center, G?ttingen, Germany , @" }# z& h: T% t, } 9 M0 P* p: }$ O7 v0 hKey Words. Embryonic stem cells ? Transplantation ? 6-hydroxy-dopamine lesion ? Parkinson’s disease % C$ m6 p, E3 h- [ . A" U; a( ~- w% r$ j' }( lPaul Christian Baier, M.D., Department of Clinical Neurophysiology, University G?ttingen, Robert-Koch-Str. 40, 37075 G?ttingen, Germany. Telephone: 49-551-39-8453; Fax: 49-551-39-8126; e-mail: pbaier@gwdg.de / {8 i: H) E2 \$ \* X \ o$ J4 }' B1 ^
ABSTRACT $ {! o; w# G- f- T t- e, v 8 l& e- w; p1 S5 AParkinson’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 . 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 . Therefore, there is a strong need for alternative therapeutic approaches, such as deep-brain stimulation, either in the globus pallidum or the subthalamic nucleus , 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 . Since then, transplantation of dopaminergic fetal mesencephalic cells into the striatum has been intensively studied in rats and nonhuman primates . More than 350 PD patients all over the world have received fetal transplants using various protocols and techniques with partially positive results . 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 . In a further study, mouse ES cells were efficiently differentiated into dopaminergic neurons and grafted in the 6-OHDA rat model . 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. * D: [, k3 G- b$ h5 S8 S- Q, h2 U _0 X: `( ^+ M
In the present study, we aimed to investigate the utility of mouse ES cells differentiated on PA6 feeder cells 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 and it does not necessarily improve the survival rate of grafts .$ q& g5 [5 j- ~" y
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MATERIALS AND METHODS : [" p7 b; Z, A. [- |/ o0 k: i+ @4 o" u
Differentiation of ES In Vitro 2 m1 S2 U# w# M& D/ e- h6 M$ s5 C Y( X. b1 f# U+ M! [
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 1A–1C. 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 2. 8 ?& Y: I) X& w$ I' B: V: n! D# d5 F) Z7 Y7 @
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 ?-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). ' z) t) `# e$ p3 z! [% G% x# e- m0 J" R" R! B
Figure 2. For quantification, differentiated cells were dissociated and replated as single-cell suspensions on gelatin-coated flask slides and stained for class III ?-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.) n; z% ~8 B- ~
( r A7 @3 p, ^: `. E6 VSurvival of Transplanted ES Cells ; v; u0 K; ` g. d7 U+ W( f" N% A " ~0 k4 m! S( K/ o! D" ZDirect 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. 3A). There was no evidence of extensive migration (Fig. 3A and 3B) or axonal outgrowth into the surrounding host tissue (Fig. 3B) at 5 weeks posttransplantation. TH-positive cells could be identified in each striatum (Fig. 3B and 3C). 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. 3C). Immunocytochemical staining with ED1 (Fig. 3E) and GFAP (Fig. 3F) 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 1). There was no tumor formation observed in any of the grafted animals.8 L6 [6 f- G5 s
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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. 7 Q# T" I$ [9 z* _3 e) L. s, n3 m+ G1 `9 m9 p0 ?/ s- q
Behavioral Parameters* k8 r. [8 N" g* u- d. P' o. L
% \% H% r; }- L6 OThe majority of the grafted animals showed a significant reduction in amphetamine-induced rotational behavior (Table 1), 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 1; pretransplantation: 4.0 ± 0.3 full body turns/minute; posttransplantation: 6.4 ± 0.4 full body turns/minute; Fig. 4A; 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 1 (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. 4B).& U4 d$ f6 ?2 {3 o( j1 P5 K- [
( i8 Q2 \' `$ ]$ J/ f0 @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 - J2 V! S! ~/ t- ^/ S
7 K$ Y9 ~/ I2 }* W2 C- g$ [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 9 T" L) Z4 D# w% J$ n0 A& T+ { ; h8 M* Y. j2 z# d+ w) `9 IDISCUSSION4 Z7 j4 x9 Y+ c# s% w }. `
/ D) [6 n2 a$ \: l) F! }. WThis 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. - {. N; f% ]0 r: Q' w' V" s3 o# w7 {) |% [% ^: _
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