Transplantation of neural cells derived from retinoic acid-treated cynomolgus monkey embryonic stem cells successfully improved motor function of hemiplegic mice with experimental brain injury

We induced neural cells by treating cynomolgus monkey embryonic stem (ES) cells with retinoic acid. The treated cells mainly expressed betaIIItubulin. They further differentiated into neurons expressing neurofilament middle chain (NFM) in elongated axons. Half of the cells differentiated into Islet1+ motoneurons in vitro. The monkey ES-derived neural cells were transplanted to hemiplegic mice with experimental brain injury mimicking stroke. The neural cells that had grafted into periventricular area of the mice distributed extensively over the injured cortex. Some of the transplanted cells expressed the neural stem/progenitor marker nestin 2 days after transplantation. The cells expressed markers characteristic of mature motoneurons 28 days after transplantation. Mice with the neural cell graft gradually recovered motor function, whereas control animals remained hemiplegic. This is the first demonstration that neural cells derived from nonhuman primate ES cells have the ability to restore motor function in an animal model of brain injury.     

Intraventricular transplantation of embryonic stem cell-derived neural stem cells in intracerebral hemorrhage rats

In the present study, we attempted to explore cell transplantation therapy for intracerebral hemorrhage (ICH) using embryonic stem (ES) cells. Collagenase-induced ICH rats were used as model animals. Mouse ES cells were differentiated into nestin-positive neural stem cells in vitro by alltrans retinoic acid (ATRA). ATRA-treated ES cells (10(5)) were transplanted into the lateral ventricle in the hemisphere contralateral to the hemorrhage 7 days after collagenase infusion. Twenty-eight days after transplantation, ES-derived neurons and astrocytes were observed around the hematoma cavities of the brain in all of the ten rats receiving grafts. Graft-derived neurons were found in the subependymal area of the lateral ventricle as cellular nodules. Although one of the ten rats receiving grafts showed uncontrolled growth of astroglia derived from the ES cells, intraventricular transplantation of ATRA-treated ES cells is an effective delivery system of neuronal lineage-committed progenitor cells toward the site of ICH.     

Double lox targeting for neural cell transgenesis

ES cells differentiated along the neural lineage in vitro are an attractive model system. Here we have developed ES cell lines that are suitable for inserting transgenes at a single chromosomal site. ES cell line CE1 (for Cassette Exchange) contains one "acceptor" module (CE1) that allows for efficient double lox targeting. The site is also permissive for gene expression in neural progenitor cells, as well as differentiated neurons and glia. Line CE2 was derived by swapping a puromycin resistance cassette into CE1. Neural progenitors derived from this line are puromycin-resistant. A beta-actin/GFP expression cassette was inserted into the CE1 site to create CE3. The CE3 cell line was differentiated into neural cells and displayed strong EGFP expression in neural progenitors, differentiated neurons and glia. Differentiated CE3 ES cells (4-/4+ RA) were transplanted into the injured rat somatosensory cortex where many of the transplanted cells survived and differentiated into neurons expressing GFP. This strategy for creating sets of transgenic lines with multiple cassettes inserted into a single chromosomal site provides a powerful tool for studying development and function of ES cell-derived neural cells. Many of these will be useful in transplantation research.     

Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures

Embryonic stem (ES) cells can generate neural progenitors and neurons in vitro and incorporate into the adult central nervous system (CNS) following transplantation, suggesting their therapeutic potential for treating neurological disorders. However, our understanding of the conditions that direct ES-derived neural progenitor (ESNP) migration and differentiation within different regions of the adult CNS is incomplete. Rodents treated with the chemoconvulsant kainic acid (KA) experience seizures and display hippocampal sclerosis, as well as enhanced hippocampal neurogenesis, similar to pathological findings in patients with temporal lobe epilepsy (TLE). To examine the potential for ESNPs to incorporate into the adult hippocampus and differentiate into hippocampal neurons or glia following seizure-induced damage, we compared the fates of ESNPs after they were transplanted into the CA3 region or fimbria 1 week following KA-induced seizures. After 4-8 weeks, ESNPs grafted into the CA3 region had migrated to the dentate gyrus (DG), where a small subset adopted neural stem cell fates and continued to proliferate, based on bromodeoxyuridine uptake. Others differentiated into neuroblasts or dentate granule neurons. In contrast, most ESNPs transplanted into the fimbria migrated extensively along existing fiber tracts and differentiated into oligodendrocytes or astrocytes. Hippocampal grafts in mice not subjected to seizures displayed a marked tendency to form tumors, and this effect was more pronounced in the DG than in the fimbria. Taken together, these data suggest that seizures induce molecular changes in the CA3 region and DG that promote region-specific neural differentiation and suppress tumor formation.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

Fate of pre-differentiated mouse embryonic stem cells transplanted in unilaterally 6-hydroxydopamine lesioned rats: histological characterization of the grafted cells

We transplanted mouse embryonic stem (ES) cells pre-differentiated on a PA6 feeder cell layer into the striatum of 6-hydroxydopamine hemi-lesioned adult rats and studied the fate of the grafted cells 1 and 5 weeks post-grafting. At both time points, ES cell grafts contained tyrosine hydroxylase positive (TH+) and 5-HT immunoreactive cells. Between 1 and 5 weeks, there was an enlargement of the grafts and an increase in number of TH+ cells although the differences between the two time points were not significant. The mean number of TH+ neurons per striatum was 330 +/- 73 after 1 week and 1220 +/- 400 after 5 weeks. Over the same time period, mean soma profile area of the TH+ neurons increased significantly by 25.2%. Neurites were longer after 5 weeks (by 24.9%), but the difference to 1 week post-grafting was not reliable. The percentage of TH+ somata without neurites increased from 6.7% after 1 week to 38.3% after 5 weeks (not significant). After 5 weeks, two out of fifteen graft recipients had tumors indicating that pre-differentiation of mouse embryonic stem cells using this differentiation protocol is not sufficient to prevent tumor formation.     

Survival and early functional integration of dopaminergic progenitor cells following transplantation in a rat model of Parkinson's disease

Dopaminergic (DA) grafts in rat models of Parkinson's disease (PD) have previously been derived from embryonic day (E) 14 grafts. Because there is an increasing interest in the restorative capacity of DA stem and progenitor cells, in the present study we examined the survival and early and late functional behavioral effects of DA progenitor cells derived from E12, E13, E14, and E15 grafts transplanted into rats with unilateral 6‐hydroxydopamin lesions. DA transplant–induced functional recovery was already observed in postural balancing reactions after 10 days and in stepping behavior after 13 days, that is, in spontaneous complex behaviors, and later, after 16 days, in the amphetamine‐induced rotation test. Three distinct patterns of functional recovery could be observed at 6–9 weeks posttransplantation. First, behavioral improvements in drug‐induced rotational asymmetry, stepping, and skilled forelimb behavior were directly related to DA neuron survival and TH‐positive fiber reinnervation. Second, recovery in postural balancing reactions was closely related to a specific developmental time window of donor age, for example, only seen in E13 and E14 grafts. Finally, no functional graft effects were seen in the table lift test. Interestingly, DA neuron graft survival, TH‐positive fiber outgrowth, and graft volume were significantly influenced by the developmental time window in which the DA progenitor cells were dissected from the ventral mesencephalon, that is, from E12, E13, E14, or E15 rat embryos. These data highlight the complexity of graft–host interactions and provide novel insights into the dynamics of DA progenitor graft‐mediated functional recovery in animal models of Parkinson's disease. © 2009 Wiley‐Liss, Inc.     

Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy

Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that the new cells functionally integrate into epileptic hippocampal circuitry.     

Distinct efficacy of pre-differentiated versus intact fetal mesencephalon-derived human neural progenitor cells in alleviating rat model of Parkinson’s disease

Neural progenitor cells have shown the effectiveness in the treatment of Parkinson's disease, but the therapeutic efficacy remains variable. One of important factors that determine the efficacy is the necessity of pre-differentiation of progenitor cells into dopaminergic neurons before transplantation. This study therefore investigated the therapeutic efficacy of mesencephalon-derived human neural progenitor cells with or without the pre-differentiation in alleviating a rat model of Parkinson's disease. We found that a combination of 50 ng/ml fibroblast growth factor 8, 10 ng/ml glial cell line-derived neurotrophic factor and 10 microM forskolin facilitated the differentiation of human fetal mesencephalic progenitor cells into dopaminergic neurons in vitro. More importantly, after transplanted into the striatum of parkinsonian rats, only pre-differentiated grafts resulted in an elevated production of dopamine in the transplanted site and the amelioration of behavioral impairments of the parkinsonian rats. Unlike pre-differentiated progenitors, grafted intact progenitors rarely differentiated into dopaminergic neurons in vivo and emigrated actively away from the transplanted site. These data demonstrates the importance of pre-differentiation of human progenitor cells before transplantation in enhancing therapeutic potency for Parkinson's disease.     

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.     

Generation of graftable dopaminergic neuron progenitors from mouse ES cells by a combination of coculture and neurosphere methods

Parkinson's disease is characterized by a loss of midbrain dopamine (DA) neurons and is generally viewed as a potential target for stem cell therapy. Although several studies have reported the generation of postmitotic DA neurons from embryonic stem (ES) cells, it is unknown whether the proliferative progenitors of DA neurons can be isolated in vitro. To investigate this possibility, we have developed a combined approach in which ES cells are cocultured with PA6 stromal cells to expose them to stromal cell-derived inducing activity (SDIA) and are then cultured as neurospheres. Mouse ES cell colonies were detached from PA6 feeder cells after 8 days of SDIA treatment and then expanded as spheres for another 4 days in serum-free medium supplemented with fibroblast growth factor-2. The spheres exhibited neural stem cell characteristics and contained few DA neurons at this stage of culture. After being induced to differentiate on polyornithine/laminin-coated dishes for 7 days, these spheres generated DA neurons in vitro at a relatively low frequency. Intriguingly, addition of PA6 cell conditioned medium to the sphere culture medium significantly increased the percentage of DA neurons to 25-30% of the total number of neurons. Transplantation of conditioned medium-treated day 4 spheres, which contained DA neuron progenitors, into the mouse striatum resulted in the generation of a significant number of graft-derived DA neurons. These findings suggest that progenitors of DA neurons are generated and can proliferate in ES cell-derived neurospheres induced by serial SDIA and PA6 conditioned medium treatment.     

Zonisamide promotes survival of human-induced pluripotent stem cell-derived dopaminergic neurons in the striatum of female rats

The transplantation of dopaminergic (DA) progenitors derived from pluripotent stem cells improves the behavior of Parkinson's disease model animals. However, the survival of DA progenitors is low, and the final yield of DA neurons is only approximately 0.3%–2% the number of transplanted cells. Zonisamide (ZNS) increases the number of survived DA neurons upon the transplantation of mouse-induced pluripotent stem (iPS) cell-derived DA progenitors in the rat striatum. In this study, we induced DA progenitors from human iPS cells and transplanted them into the striatum of female rats with daily administration of ZNS. The number of survived DA neurons was evaluated 1 and 4 months after transplantation by immunohistochemistry, which revealed that the number of survived DA neurons was significantly increased with the administration of ZNS. To assess the mechanism of action of ZNS, we performed a gene expression analysis to compare the gene expression profiles in striatum treated with or without ZNS. The analysis revealed that the expression of SLIT-and NTRK-like protein 6 (SLITRK6) was upregulated in rat striatum treated with ZNS. In conclusion, ZNS promotes the survival of DA neurons after the transplantation of human-iPS cell-derived DA progenitors in the rat striatum. SLITRK6 is suggested to be involved in this supportive effect of ZNS by modulating the environment of the host brain.     

Zuckerkandl's organ improves long-term survival and function of neural stem cell derived dopaminergic neurons in Parkinsonian rats

Transplantation of neural stem cells (NSC) derived dopamine (DA) neurons has emerged as an alternative approach to fetal neural cell transplantation in Parkinson's disease (PD). However, similar to fetal neural cell, survival of these neurons following transplantation is also limited due to limited striatal reinnervation (graft with dense neuronal core), limited host-graft interaction, poor axonal outgrowth, lack of continuous neurotrophic factors supply and principally an absence of cell adhesion molecules mediated appropriate developmental cues. In the present study, an attempt has been made to increase survival and function of NSC derived DA neurons, by co-grafting with Zuckerkandl's organ (a paraneural organ that expresses neurotrophic factors as well as cell adhesion molecules); to provide continuous NTF support and developmental cues to transplanted DA neurons in the rat model of PD. 24 weeks post transplantation, a significant number of surviving functional NSC derived DA neurons were observed in the co-transplanted group as evident by an increase in the number of tyrosine hydroxylase immunoreactive (TH-IR) neurons, TH-IR fiber density, TH-mRNA expression and TH-protein level at the transplantation site (striatum). Significant behavioral recovery (amphetamine induced stereotypy and locomotor activity) and neurochemical recovery (DA-D2 receptor binding and DA and DOPAC levels at the transplant site) were also observed in the NSC+ZKO co-transplanted group as compared to the NSC or ZKO alone transplanted group. In vivo results were further substantiated by in vitro studies, which suggest that ZKO increases the NSC derived DA neuronal survival, differentiation, DA release and neurite outgrowth as well as protects against 6-OHDA toxicity in co-culture condition. The present study suggests that long-term and continuous NTF support provided by ZKO to the transplanted NSC derived DA neurons, helped in their better survival, axonal arborization and integration with host cells, leading to long-term functional restoration in the rat model of PD.     

Neuromodulation for Parkinson's disease]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of midbrain dopaminergic (DA) neurons and a subsequent reduction in striatal dopamine. As a treatment for advanced Parkinson's disease, deep brain stimulation (DBS) of the thalamus was introduced in 1987 to treat tremor, and was applied in 1993 to the subthalamic nucleus. Now high-frequency stimulation of the subthalamic nucleus has become a surgical therapy of choice. Another surgical treatment is a cell replacement therapy. Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief, however, the technical and ethical difficulties in obtaining sufficient and appropriate donor fetal brain tissue have limited the application of this therapy. Then, neural precursor cells and embryonic stem (ES) cells are expected to be candidates of potential donor cells for transplantation. We induced DA neurons from monkey ES cells, and analyzed the effect of transplantation of the DA neurons into MPTP-treated monkeys as a primate model of Parkinson's disease. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced neurological symptoms. DA neurons have also been generated from several human ES cell lines. Furthermore, functional recovery of rat PD models after transplantation was observed. One of the major problems in ES cell transplantation is tumor formation, which is caused by a small fraction of undifferentiated ES cells in the graft. So, it is essential for undifferentiated ES cells to be eliminated from the graft in order for transplantation to be feasible. These efforts will lead to clinical application of ES cell transplantation to the patients with PD.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

Cocaine differentially alters behavior and neurochemistry in periadolescent versus adult rats

The expansion and differentiation of neural progenitor cells in vitro provides an approach to study the development and differentiation of neurons. The ventral mesencephalic area of the brain is an important source of neural progenitor cells and the differentiated neural progenitor cell has paramount potential for use in transplant therapies such as those used in the treatment of neurodegenerative diseases. Here, the controlled conversion of human foetal progenitor cells derived from ventral mesencephalon into dopaminergic neurons is reported. The immunoreactivity to tyrosine hydroxylase (TH) and levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), secreted into culture medium, were used to assess dopaminergic neuronal phenotype. Expansion of the neural progenitor cells for 3 weeks in the presence of basic fibroblast growth factor (2 ng/ml) followed by its withdrawal resulted in approximately 60% of cells staining positive for TH, when challenged in concert with brain-derived neurotrophic factor (50 ng/ml), DA (10 microM) and forskolin (10 microM) for a further 3 weeks. A corresponding 41-fold increase in DA and DOPAC was measured in the incubation medium by HPLC. Therefore, the successful conversion of human foetal progenitor cells in vitro resulting in the desired dopaminergic neuronal phenotype, could provide a solution to the problem of limited availability of human foetuses for clinical surgical transplantation therapies, which are currently in progress for the treatment of neurodegenerative diseases such as Parkinson's disease.     

Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity

A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported. When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression. In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days). SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum. The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation. This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions. When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells. In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo.