HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Files All Versions of this Article: 2005-0393v1 24/6/1433    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brederlau, A. Articles by Li, J.-Y. Search for Related Content PubMed PubMed Citation Articles by Brederlau, A. Articles by Li, J.-Y.

EMBRYONIC STEM CELLS

Transplantation of Human Embryonic Stem Cell-Derived Cells to a Rat Model of Parkinson’s Disease: Effect of In Vitro Differentiation on Graft Survival and Teratoma Formation

^a Institute of Anatomy and Cell Biology, Göteborg University, Gothenburg, Sweden;
^b Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden;
^c Department of Neurosurgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan;
^d Institute of Physiology and Pharmacology, Department of Pharmacology, Göteborg University, Gothenburg, Sweden;
^e RIKEN Center for Developmental Biology, Chuo, Kobe, Japan;
^f The Arvid Carlsson Institute for Neuroscience, Institute of Clinical Neuroscience, Sahlgrenska University Hospital, Göteborg University, Gothenburg, Sweden

Key Words. Differentiation • Teratoma formation • Dopaminergic neurons • Parkinson’s disease • Neural transplantation • Human embryonic stem cells

Correspondence: Jia-Yi Li, M.D., Ph.D., Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, BMC A10, 221 84 Lund, Sweden. Telephone: +46-46-222 05 25; Fax: +46-46-222 05 31; e-mail: jia-yi.li{at}med.lu.se or Prof. Peter S. Eriksson, M.D., Ph.D., Institute of Clinical Neuroscience, Göteborg University, Sahlgrenska University Hospital, 413145, Göteborg, Sweden. Telephone: +46-31-7733433; Fax: +46-773401; e-mail: peter.eriksson{at}neuro.gu.se

Received August 16, 2005; accepted for publication March 14, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinson’s disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC-derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion-induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
Human embryonic stem cells (hESCs) can potentially be used for cell replacement therapy in Parkinson’s disease (PD) [1, 2]. Differentiation into dopamine (DA) neurons is necessary prior to their use for transplantation. The optimal in vitro differentiation protocol should produce a sufficient number of committed DA progenitors and eliminate pluripotent cells that can give rise to teratomas.

Several culture conditions direct ESCs toward differentiation into DA neurons [3–6]. Coculturing with the PA6 stromal cell line rapidly generates high numbers of DA neurons from mouse and monkey ESCs by an unknown mechanism named stromal-derived inducing activity (SDIA) [3, 7]. These DA neurons can survive transplantation, integrate into the host striatum, and reduce functional deficits in animal models of PD [3, 7–11]. The same protocol induces DA cell fate in cultures from hESCs, but the efficacy varies between different hESC cell lines [12, 13]. Furthermore, the survival of hESC-derived DA neurons after transplantation is low and no functional benefit from these grafts has been reported [12, 13].

Results concerning teratoma formation from grafted SDIA-derived ESCs are controversial. Thinyane et al. reported the development of teratomas 5 weeks after transplantation of mouse ESCs first differentiated on PA6 cells in vitro [9]. In contrast, no teratoma formation was detected in studies performed on grafted hESCs that had been predifferentiated on PA6 cells [12, 13].

In the current study, we characterized the differentiation of hESCs (line SA002.5) into DA neurons when cocultured with PA6 cells in vitro and examined their survival after grafting into the striatum of a rat model of PD. The hESCs were transplanted after 16, 20, or 23 days of differentiation in coculture. Changes in lesion-induced behavioral deficits were monitored. Survival, neuronal differentiation, and teratoma formation were assessed 2 and 13 weeks after transplantation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
SDIA-Mediated Differentiation of hESCs into DA Neurons
Undifferentiated hESCs of line SA002.5 (Cellartis, Gothenburg, Sweden, http://www.cellartis.com) were maintained as previously described [14]. PA6 cells were cultured as described elsewhere [7]. For differentiation experiments, PA6 cells were plated on type I collagen-coated chamber slides or 0.1% gelatin-coated Petri dishes (diameter, 6 cm) at confluency 1 day before introducing hESCs into the coculture. The hESCs were dissociated after 15–30 minutes of incubation in type IV collagenase (200 U/ml; Sigma, St. Louis, http://www.sigmaaldrich.com) and plated on PA6 cells at the density of 2 x 10³ cells per cm² in ESC differentiation medium (ESCDM) that consisted of Glasgow’s modified Eagle’s medium (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 8% Knockout Serum Replacement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1 mM pyruvate (Sigma), 0.1 mM nonessential amino acids (Gibco), and 0.1 mM 2-mercapto-ethanol (Gibco). From day 6 onward, half of the medium was changed every 3rd day. Two weeks after plating, colonies were separated from the feeder using a papain dissociation kit (Worthington Biochemical Corporation, Lakewood, NJ, http://www.worthington-biochem.com) and plated on poly-DL-ornithin (PORN)/laminin-coated eight-well glass chamber slides (Falcon, Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) containing ESCDM.

Electrophysiological Recordings
Three to 4 weeks after plating hESCs on PA6 cells, the cultures were placed on an upright Nikon E600FN microscope (Nikon Corporation, Tokyo, http://www.nikon.com) and continuously superfused with a solution (bubbled with 95% O₂–5% CO₂ [pH ~7.4]) containing 124 mM NaCl, 3 mM KCl, 1.6 mM CaCl₂, 2.4 mM MgCl₂, 26 mM NaHCO₃, 1.25 mM NaH₂PO₄, and 10 mM D-glucose. Whole-cell patch-clamp recordings were performed on 61 hESCs (from six different cultures) identified using infrared-differential interference contrast video microscopy. Patch pipette resistances were 2–8 M, and series resistance varied between 5 and 25 M. Recordings were made in current-clamp or voltage-clamp mode using an EPC-9 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany, http://www.heka.com). Data were sampled at 20 kHz and filtered at 3 kHz. A series of 10 hyperpolarizing and depolarizing current pulses (40–140 pA, 500-ms duration) was applied when the membrane potential was held at –60 and –80 mV.

Electromicroscopy
Cultures differentiated for 3 weeks were fixed in 0.1 M PBS with 2.5% glutaraldehyde, 2% paraformaldehyde (PFA), 0.05% sodium azide, and 0.05 M sodium cacodylate. Cells were post-fixed in OsO₄ and dehydrated in ethanol before embedding in epoxy resin. Ultrathin sections (60–70 nm) were cut on a Reichert Ultramicrotome (Reichert Microscope Services, Depew, NY, http://www.reichertms.com), contrasted with lead citrate and uranyl acetate, and examined in a LEO 912AB transmission electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany, http://www.leo-em.com) equipped with a Proscan and a Megaview III camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany, http://www.soft-imaging.net).

6-Hydroxydopamine Lesions and Transplantation of hESC-Derived DA Neurons
Female Sprague-Dawley rats (B&K Universal Ltd., Sollentuna, Sweden, http://www.bku.com) weighing 180–200 g received two stereotaxic injections of 6-hydroxydopamine (6-OHDA) into the right nigra-striatal pathway [15].

Differentiated hESCs that had been grown on PA6 cells for 16, 20, or 23 days were separated from the PA6 cells using the Worthington Papain Dissociation System. (See supplemental online Fig. 1 for the detailed experimental scheme.) They were then dissociated into a single-cell suspension using a pipette after incubation in Accutase cell detachment solution (In novative Cell Technologies, Inc., San Diego, http://www.innovativecelltech.com). The cell suspension contained 50,000 viable cells per µl (assessed by trypan blue) in Hanks’ balanced saline solution with 0.05% DNase. The right striatum of each rat was stereotactically implanted using a 10-µl Hamilton micro-syringe (Hamilton Company, Reno, NV, http://www.hamiltoncomp.com) with 100,000 viable hESC-derived cells (2 µl of cell suspension) cultured for 16 (group 1, n = 22), 20 (group 2, n = 8), or 23 days (group 3, n = 8) on PA6 cells. Injection coordinates (in mm) were anterior = +1.0; lateral = +3.0; ventral = –4.5 and –5.0 [16]. All rats were immunosuppressed with intraperitoneal injections of 15 mg/kg of Cyclosporine A given 1 day prior to transplantation, daily for 2 weeks after transplantation, and at 10 mg/kg per day thereafter. Cells that were not implanted were seeded on PORN/laminin-coated eight-well chamber slides (14,000 cells/well) and fixed 24 hours later. To assess ongoing cell proliferation, each rat was injected with 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, i.p.) that incorporates into the DNA in the S phase of the cell cycle. To label most of the dividing cells in the grafts, BrdU was injected three times at 8-hour intervals 24 hours prior to sacrifice.

View larger version (44K): [in this window] [in a new window]   Figure 1. In vitro differentiation of human embryonic stem cells (hESCs) by the SDIA method. (A): Quantification of ß-III-tubulin- or TH-positive colonies (A) or cells (B) at different time points. Values represent means ± SD. (C–H): Morphology of stromal-derived inducing activity (SDIA)-treated hESCs after 2.5 weeks. ß-III-Tubulin-positive neurons (green, [C] and [F]), TH-positive neurons (red, [D] and [G]), and merged images plus Hoechst 33,258 nuclei staining. (E) displays a merge of (C) and (D); (H) displays a merge of (F) and (G). (C–E): Images of cell cluster. (F–H): Images of cells in monolayer. Scale bar = 50 µm. Voltage responses to hyperpolarizing and depolarizing current injections from two representative cells showing mature (I) and immature (J) action potentials. (K): Electron micrographs showing synapses at different levels of sectioning. Scale bar = 1 µm. (L): Electrophysiological recording of spontaneous synaptic currents in one cell. Inset shows the averaged current. (M): high-performance liquid chromatography chromatogram for DA release from hESCs cocultured with PA6 cells for 20 days (blue line). Pink line represents DA standard. Abbreviations: DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; TH, tyrosine hydroxylase.

Immunohistochemistry and Cell Quantification
Immunostained cell cultures prepared for the study of SDIA-mediated differentiation of hESCs into DA neurons were visualized by a Zeiss fluorescent microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) attached to a Nikon digital camera. The total number of colonies and the number of ß-III-tubulin- and tyrosine hydroxylase (TH)-positive colonies were counted. A colony was defined as "immunoreactive" if more than approximately 10% of the cells were stained by the marker in question. In some randomly chosen colonies, the proportions of TH/ß-III-tubulin-immunoreactive cells were evaluated (60–200 cells/colony, approximately 1,800 cells/group).

Rats were perfused with 4% PFA. The brains were dissected and post-fixed with the same fixative for 24 hours, and 40-µm thick coronal sections were cut using a microtome. Immunostaining was performed on free-floating sections. (See supplemental online Table 1 for information of antibodies used.) Immunostained brain sections and cell cultures were analyzed under a fluorescent microscope (Olympus BX60; Olympus, Tokyo, http://www.olympus-global.com). Double-stained cells were visualized by confocal microscopy (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany, http://www.leica.com). Cell numbers were assessed using an Olympus CAST-Grid system (Olympus). Surface areas in sections covered by grafted human nuclei (HNuc)-positive cells were delineated. Using a systematic random sampling technique, the numbers of HNuc- and BrdU- or neuronal nuclei (NeuN)- or TH-positive cells were assessed in 1% of the areas. The total numbers of cells within the grafts were estimated by the equation N = n x x x y/A(f) x 10, where N is total number of cells, n is number of cells in one series, x is x-step length, y is y-step length, and A(f) is frame area.

Statistical Analysis
Effects of time in culture on hESC differentiation into DA neurons was examined using one-way analysis of variance and Scheffé post hoc test. In the transplantation studies (i.e., survival, proliferation, and neuronal differentiation), statistical analyses were performed using the nonparametric Kruskal-Wallis test. Post hoc pairwise analysis between two groups at a same time point or between two time points within the same group were performed using the nonparametric Mann-Whitney U test.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
In Vitro Differentiation of hESCs
The hESCs were cultured on PA6 cells for 2 weeks and then transferred to PORN/laminin-coated chamber slides for another 0.5, 2, and 4 weeks in vitro. At 2.5 weeks in culture, 42% ± 20% (mean ± SD) of the colonies expressed ß-III-tubulin, and 38% ± 22% were positive for TH (Fig. 1A). The percentages of ß-III-tubulin- and TH-positive colonies, as well as the percentages of ß-III-tubulin- and TH-positive cells within each colony (Fig. 1B), did not change significantly over time. After 6 weeks in culture, only 1.0% ± 2.0% of TH-positive cells were immunoreactive for peripherin, a marker for neurons in the peripheral nervous system, indicating that the vast majority of the generated TH-positive neurons had a central nervous system phenotype (supplemental online Fig. 2).

View larger version (13K): [in this window] [in a new window]   Figure 2. In vivo functionality of human embryonic stem cell (hESC)-derived dopamine neurons transplanted into the lesioned striatum of the 6-hydroxydopamine-lesion rat model of Parkinson’s disease. The graph represents the mean number of net ipsilateral turns after 90 minutes at pregrafting (point 0, group 1, n = 22; group 2, n = 8; group 3; n = 8) and at 2 (group 1, n = 22; group 2, n = 8; group 3; n = 8), 4 (group 1, n = 18; group 2, n = 4; group 3, n = 4), 8 (group 1, n = 10; group 2, n = 4; group 3, n = 4), and 13 (group 2, n = 3; group 3, n = 4) weeks after implantation of hESC-derived cells. The net ipsilateral turns are calculated subtracting the contralateral turns to the ipsilateral turns. There is no clear recovery of rotation rates in any of the three groups. Mean ± SEM within each group is represented.

Transplantation of SDIA-Mediated Differentiated hESCs

Functionality of DA Neurons Derived by the SDIA Method.   Release of DA was detected by HPLC in supernatant from PA6/hESC cocultures grown in vitro for 20 and 23 days, but not from those grown for only 16 days (Fig. 1M). Hemiparkinsonian rats with 6-OHDA lesions, transplanted with differentiated hESCs, were subjected to the amphetamine-induced rotation test. No significant changes in rotation scores were seen in any of the three groups of transplanted rats at 2, 4, 8, and 13 weeks after transplantation (Fig. 2), indicating that the grafts were not functional.

Survival, Proliferation, and Tumor Formation.   Supernumerary hESC-derived cells harvested before transplantation surgery were replated and grown in vitro. Some of them expressed both ß-III-tubulin and TH, indicating that they had survived the dissociation procedure. The proportion of TH-immunopositive cells increased over the time between 16 and 23 days of coculturing with PA6 cells (Table 1).

In the grafts, hESC-derived cells were detected at 2 and 13 weeks after transplantation by immunostaining for HNuc (Fig. 3A). At 2 weeks after transplantation, four rats from each group were sacrificed. Stereological analysis of HNuc-positive cells revealed that cells differentiated for 23 days did not survive the grafting procedure as well as the cells cultured for 16 days (p < .05). Some grafts contained cell numbers (in the range of 250–800 x 10³) by far exceeding the number of implanted cells, suggesting proliferation after grafting. All rats of group 1 and two out of four rats of group 2 had been lost because of teratoma formation at week 13 after transplantation.

View larger version (73K): [in this window] [in a new window]   Figure 3. Survival and proliferation of human embryonic stem cell (hESC)-derived cells after grafting and teratoma formation. (A): Number of surviving cells, identified by HNuc-immunoreactivity, in each transplanted rat perfused at 2 and 13 weeks after transplantation. The short horizontal lines represent the median values within each group at the two different time points after transplantation. (B): Grafted HNuc-positive cells. (C): A transplant with a very low number of surviving HNuc-positive cells. (D): Percentage of proliferating cells, identified by BrdU-immunoreactivity, within the total number of HNuc-positive cells in each transplanted rat perfused at 2 and 13 weeks after transplantation. (E–G): Confocal images of proliferating cells double-positive for BrdU and HNuc within a graft, (E) HNuc-positive cells, (F) BrdU-positive cells, and (G) merged image. (H): Scheme representing teratoma formation per group over time. (I): Macroscopic image of a brain from group 1 showing a teratoma in the transplantation site. (J): Microscopic image of a teratoma in a brain from group 1 showing different tissue structures. (K–M): Confocal images of hESC-derived endodermal cells double-positive for -fetoprotein and HNuc within a graft, (K) HNuc-positive cells, (L) -fetoprotein-positive cells, and (M) merged image. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; HNuc, human nuclei.

The tendency to form teratomas differed between the three groups (Fig. 3H). Between 6 and 11 weeks after transplantation, all rats in group 1 developed teratomas with classic morphological features (Fig. 3I, 3J) and had to be sacrificed. In group 2, two rats were lost due to teratoma formation after 12–13 weeks. No teratoma was found in rats from group 3. In line with this, Oct-3/4-positive cells were found in grafts from groups 1 and 2 at 2 weeks after transplantation, but not in grafts from group 3 (supplemental online Fig. 3A–3C). In contrast, grafts in some rats in all three groups still expressed the endodermal marker -fetoprotein (Fig. 3K, 3L).

Neuronal Differentiation in the Graft.   Two weeks after implantation, the proportions of HNuc-positive cells that expressed the neuronal marker NeuN did not differ between the three grafted groups (Fig. 4A). Disregarding rats that developed teratomas, the proportion of grafted cells that expressed NeuN showed similar numbers at all time points after surgery (Fig. 4A). Only 10 to 50 TH-positive cells were found in each graft (Fig. 4E– 4J), and these numbers did not differ systematically between the three groups. Furthermore, we examined sections from grafted brains to see whether the hESC-derived cells stained for other neural markers. None of the examined transplanted cells were immunolabeled for NG2 (oligodendrocytes; Fig. 4K), GAD (GABAergic neurons), 5-HT (serotonergic neurons), and ChAT (cholinergic neurons; Fig. 4L), whereas some host cells were immunoreactive for these markers.

View larger version (54K): [in this window] [in a new window]   Figure 4. Neuronal phenotype of grafted human embryonic stem cell (hESC)-derived cells. (A): Percentage of neuronal cells, identified by NeuN immunoreactivity, within the total number of HNuc-positive cells in each transplanted rat perfused at 2 and 13 weeks after transplantation. The short horizontal lines represent the median values within each group at the two different time points after transplantation. (B–D): Confocal picture of grafted neurons double-positive for NeuN and HNuc within a graft, (B) HNuc-positive cells, (C) NeuN-positive cells, and (D) merged image. (E–J): Confocal image of a grafted dopamine neuron double-positive for tyrosine hydroxylase (TH) and HNuc, (E, H) HNuc-positive cells, (F, I) TH-positive cells, and (G, J) merged images. (K, K'): Double immunostaining for NG2 (green) and HNuc (red) for identification of hESC-derived oligodendrocytes where no double-positive cells were seen. (L, L'): Double immunostaining for choline acetyltransferase (green) and HNuc (red) for identification of hESC-derived cholinergic neurons where no double-positive cells were seen. Abbreviations: HNuc, human nuclei; NeuN, neuronal nuclei.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
The SDIA method efficiently induces neuronal and DA cell fate in hESCs. The risk of teratoma formation after transplantation is significantly decreased with differentiation time in vitro. The fact that no tumors, but a substantial number of neurons, were detected in animals transplanted with hESCs differentiated for 23 days is appealing. This prompts additional protocol optimizations in future studies focusing on in vitro predifferentiation for either direct transplantation of hESC-derived neural cells or for the generation of neuronal progenitors. Furthermore, reasons for the low frequency of TH-positive cells in the grafts need to be understood and counteracted. Importantly, our data support the statement that safety issues in hESC application for transplantational therapy of PD (such as a risk for teratoma formation) should be fully apprehended before approaching the stage of actual clinical trials.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 
A.B. and A.S.C. are regarded as joint first authors. This study was supported by the United States Army Medical Research Acquisition Activity (award W81XWH-04-1-0366), National Institutes of Health (grant 1 R21 NS043717-01A1), Swedish Research Council (project nos. K2002-99SX-14472-01A and K2004-33X-15072-01A and grant 12535), Research Foundation of the Swedish Parkinsons Disease Association, and Fundação para a Ciência e a Tecnologia from the Portuguese government (reference SFRH/BD/11804/2003 to A.S.C.). We thank Birgitta Larsson for her help in administration and Britt Lundgren and Birgit Haraldsson for their excellent technical assistance.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures References

 

1. Levy YS, Stroomza M, Melamed E et al. Embryonic and adult stem cells as a source for cell therapy in Parkinson’s disease. J Mol Neurosci 2004;24:353–386.[CrossRef][Medline]

2. Roybon L, Christophersen NS, Brundin P et al. Stem cell therapy for Parkinson’s disease: Where do we stand? Cell Tissue Res 2004;318:261–273.[CrossRef][Medline]

3. Kawasaki H, Suemori H, Mizuseki K et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.[Abstract/Free Full Text]

4. Perrier AL, Tabar V, Barberi T et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 2004;101:12543–12548.[Abstract/Free Full Text]

5. 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]

6. Kim JY, Koh HC, Lee JY et al. Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression. J Neurochem 2003;85:1443–1454.[CrossRef][Medline]

7. 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]

8. Yoshizaki T, Inaji M, Kouike H et al. Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells. Neurosci Lett 2004;363:33–37.[CrossRef][Medline]

9. Thinyane K, Baier PC, Schindehutte J et al. Fate of pre-differentiated mouse embryonic stem cells transplanted in unilaterally 6-hydroxydopamine lesioned rats: Histological characterization of the grafted cells. Brain Res 2005;1045:80–87.[Medline]

10. Takagi Y, Takahashi J, Saiki H et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model. J Clin Invest 2005;115:102–109.[CrossRef][Medline]

11. 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]

12. Zeng X, Cai J, Chen J et al. Dopaminergic differentiation of human embryonic stem cells. STEM CELLS 2004;22:925–940.[Abstract/Free Full Text]

13. Park CH, Minn YK, Lee JY et al. In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons. J Neurochem 2005;92: 1265–1276.[CrossRef][Medline]

14. Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.[Abstract/Free Full Text]

15. Clarke DJ, Brundin P, Strecker RE et al. Human fetal dopamine neurons grafted in a rat model of Parkinson’s disease: Ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry. Exp Brain Res 1988;73:115–126.[CrossRef][Medline]

16. Paxinos G, Watson C, Pennisi M et al. Bregma, lambda and the interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. J Neurosci Methods 1985;13:139–143.[CrossRef][Medline]

17. Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system. Brain Res 1970;24:485–493.[CrossRef][Medline]

18. Spigelman I, Zhang L, Carlen PL. Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: Membrane excitability and K⁺ currents. J Neurophysiol 1992;68:55–69.[Abstract/Free Full Text]

19. Ben-Hur T, Idelson M, Khaner H et al. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. STEM CELLS 2004;22:1246–1255.[Abstract/Free Full Text]

20. Schulz TC, Noggle SA, Palmarini GM et al. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. STEM CELLS 2004;22:1218–1238.[Abstract/Free Full Text]

21. Tabar V, Panagiotakos G, Greenberg ED et al. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol 2005;23:601–606.[CrossRef][Medline]

This article has been cited by other articles:

				X.-J. Li, B.-Y. Hu, S. A. Jones, Y.-S. Zhang, T. LaVaute, Z.-W. Du, and S.-C. Zhang Directed Differentiation of Ventral Spinal Progenitors and Motor Neurons from Human Embryonic Stem Cells by Small Molecules Stem Cells, April 1, 2008; 26(4): 886 - 893. [Abstract] [Full Text] [PDF]

				M. S. Cho, Y.-E. Lee, J. Y. Kim, S. Chung, Y. H. Cho, D.-S. Kim, S.-M. Kang, H. Lee, M.-H. Kim, J.-H. Kim, et al. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells PNAS, March 4, 2008; 105(9): 3392 - 3397. [Abstract] [Full Text] [PDF]

				D. Yang, Z.-J. Zhang, M. Oldenburg, M. Ayala, and S.-C. Zhang Human Embryonic Stem Cell-Derived Dopaminergic Neurons Reverse Functional Deficit in Parkinsonian Rats Stem Cells, January 1, 2008; 26(1): 55 - 63. [Abstract] [Full Text] [PDF]

				A. J. Joannides, D. J. Webber, O. Raineteau, C. Kelly, K.-A. Irvine, C. Watts, A. E. Rosser, P. J. Kemp, W. F. Blakemore, A. Compston, et al. Environmental signals regulate lineage choice and temporal maturation of neural stem cells from human embryonic stem cells Brain, May 1, 2007; 130(5): 1263 - 1275. [Abstract] [Full Text] [PDF]

				J. A. Rodriguez-Gomez, J.-Q. Lu, I. Velasco, S. Rivera, S. S. Zoghbi, J.-S. Liow, J. L. Musachio, F. T. Chin, H. Toyama, J. Seidel, et al. Persistent Dopamine Functions of Neurons Derived from Embryonic Stem Cells in a Rodent Model of Parkinson Disease Stem Cells, April 1, 2007; 25(4): 918 - 928. [Abstract] [Full Text] [PDF]

				K.-C. Sonntag, J. Pruszak, T. Yoshizaki, J. van Arensbergen, R. Sanchez-Pernaute, and O. Isacson Enhanced Yield of Neuroepithelial Precursors and Midbrain-Like Dopaminergic Neurons from Human Embryonic Stem Cells Using the Bone Morphogenic Protein Antagonist Noggin Stem Cells, February 1, 2007; 25(2): 411 - 418. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Files All Versions of this Article: 2005-0393v1 24/6/1433    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brederlau, A. Articles by Li, J.-Y. Search for Related Content PubMed PubMed Citation Articles by Brederlau, A. Articles by Li, J.-Y.