Following initial work by Thomson et al. reporting a method for establishing primate ES cells (18
), Suemori et al. (19
) recently devised a similar scheme for generating M. fascicularis
ES cells. Lee et al. recently reported a 5-step method to induce DA neurons from ES cells through the induction of neural progenitor cells from embryoid bodies (20
). Transplantation of induced DA neurons derived from mouse ES cells improves the neurological symptoms of rats with a Parkinson-like syndrome induced by treatment with 6-hydroxydopamine (6-OHDA) (21
). We have also reported a method of inducing DA neurons based on SDIA resulting from coculture of ES cells on a PA6 stromal feeder layer. By the use of this method, mouse ES cells are diverted to a neuronal fate with TH-positive DA neurons composing 30% of total TuJ1-positive neurons (10
). Furthermore, this method produced similar results with M. fascicularis
ES cells (11
). In this study, we produced a highly enriched population of proliferating neural progenitors derived from SDIA-treated monkey ES cells. Furthermore, treatment of these cells with a combination of FGF2 and FGF20 induced the generation of a large population of DA neurons from ES cell–derived neural progenitors. Using MPTP-treated monkeys as a primate model for PD, we analyzed the effect of administration of DA neurons generated from monkey ES cells in vivo. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons, attenuating the MPTP-induced symptoms.
In our preparation of graftable cells, we made two major modifications to our previously published protocol (10
). First, we induced the formation of neurospheres, expecting an enrichment of neuronal progenitor cells. We detached the ES cells from the feeder layer on day 14 and cultured them on noncoated dishes. Under these conditions, the cells formed floating spheres composed of neural precursor cells. Since the serum-free culture medium was suitable for neural cell growth, any contaminating nonneural and PA6 cells were likely eliminated as a result of a low proliferation rate and/or adherence to the bottom of the dish. Our previous report (22
) demonstrated that, when grafted into the brain, fully maturated TH-positive neurons survived less efficiently than DA neuron progenitors induced by SDIA, probably due to their susceptibility to mechanical stress. This result is consistent with the fact that transplantation of mesencephalon tissues from early gestation stage embryos undergoing neurogenesis of DA neurons resulted in good survival of TH-positive cells (1300–18,000) and increased dopamine concentrations in the caudate nucleus of MPTP-treated monkeys, whereas these effects are not observed when mesencephalon tissues from later stages are transplanted (23
). Thus, ES cell–derived neuronal progenitors competent to generate DA neurons appear to be more suitable for transplantation than DA neurons matured in vitro.
Another important modification to our previous protocol is the use of FGF2 and FGF20 treatment to enhance the generation of DA neurons. The percentage of TH-positive neurons generated from neurospheres was 5.4 ± 1.8% of TuJ1, much lower than that generated by culturing on PA6 cells (35%; ref. 10
). This discrepancy most likely results from mechanical damage caused by detaching the cells or inappropriate culture conditions for spheres. To increase the number of DA neurons differentiated from neurospheres, we examined the effects of various additional growth factors. Ascorbic acid and Sonic hedgehog, which were used in the 5-step method (20
), did not increase the proportion of TH-positive cells (data not shown). In contrast, FGF20 treatment in combination with FGF2 was able to efficiently increase the proportion of TH-positive cells. FGF20 is a secreted protein that is preferentially expressed in the substantia nigra pars compacta of the rat brain (13
). The expression profile of FGF20 is quite different from that of other FGF family members, which suggests that FGF20 plays a unique role in the brain. Furthermore, recombinant FGF20 enhances the survival of primary DA neurons (13
). FGF receptor–1c, the receptor through which FGF20 activates the mitogen-activated protein kinase pathway, is also preferentially expressed in the substantia nigra pars compacta (24
). Our results raise the possibility that FGF20 in combination with FGF2 may support the survival or promote the proliferation of progenitors of DA neurons, resulting in the enrichment of DA progenitor cells in spheres. The mechanism by which this combined stimulation of FGF2 and FGF20 facilitates the production of DA neurons remains to be clarified.
FGF2 and EGF are reported to play different roles in the differentiation of neural precursors. Although FGF2 and EGF promote proliferation of neural precursor cells, the former promotes neuronal differentiation, while the latter induces glial differentiation (12
). They also have different effects on the differentiation of embryoid bodies derived from human ES cells (27
). In the present study using neural precursors derived from primate ES cells, FGF2 increased differentiation of ES cells into DA neurons, while EGF suppressed this process even in the presence of FGF2 and FGF20. It is possible that EGF interferes with the differentiation of SDIA-treated spheres into DA neurons directly or indirectly by promoting astroglial induction. Alternatively, EGF may stimulate proliferation or differentiation of a different cell population than that stimulated by FGF2. The differential effects of growth factors present an intriguing topic for future investigation.
In primate studies, functional neuroimaging is a useful tool for in vivo assessment of differentiation, survival, and functional integration of grafted cells. PET imaging of presynaptic targeting reagents such as fluoro-dopa, fluoro-metatyrosine, or 2β-carbomethoxy-3β-4-fluorophenyltropane (CFT) determines whether cells implanted in vivo have the molecular machinery necessary for dopamine synthesis and/or storage (28
). In this study, we examined the uptake of fluoro-dopa at 14 weeks after transplantation. The significant increase in the mean Ki value in the putamen of ES cell–transplanted animals indicated that the grafted cells functioned as DA neurons. A postmortem examination of the ES cell–transplanted monkey in Figure C revealed that more TH-positive cells survived within the right putamen. This finding reflects the correlation of the PET results with the survival of DA neurons. For future studies, detailed analyses using additional tracers, including postsynaptic markers such as fluoro-raclopride, should allow for further understanding of the functional aspects of grafted cells. In this study, we detected significant differences in the mean Ki values from the entire putamen between the ES cell–transplanted monkeys and the sham-treated control monkeys 3 months after surgery. Widner et al. (8
) reported that striatal uptake of fluoro-dopa was unchanged 5 to 6 months postoperatively, but increased markedly at 12 to 13 and 22 to 24 months in patients who received fetal mesencephalic grafts. Freed et al. (4
) reported an improved Ki value from the entire putamen 6 months after transplantation. Given the lengthier monitoring periods in these reports, our evaluation of mean Ki values at 3 months may still be premature; further PET studies at later time points may result in even greater changes.
MPTP is a neurotoxin that causes selective destruction of DA neurons in the substantia nigra pars compacta, inducing PD-like symptoms in primates (31
). Following repetitive intravenous injections of MPTP (approximately 17 mg in total per animal), monkeys stably exhibited PD-like symptoms more than 12 weeks before transplantation surgery. With a blind evaluation based on neurological scores, we detected significant behavioral improvements in the ES cell–transplanted monkeys 10 weeks after transplantation. Recently, 2 double-blind placebo-controlled clinical trials of fetal nigral transplantation (4
) demonstrated that younger patients and patients with mild symptoms improved after treatment, with behavioral recovery first observed in the 3 to 4 month period following surgery. In this study, posture and motility were the symptoms showing the most marked improvement. These results are comparable with clinical reports (4
) demonstrating improvements in rigidity and hypokinesia.
PET and immunofluorescence studies demonstrated that a substantial number of the grafted cells survived in the putamen to function as DA neurons. We transplanted 300,000–600,000 cells into each side of the brain in each monkey. The number of surviving cells detected by BrdU staining was approximately 8,000 per side. Thus, the survival rate of the grafted cells was 1.3% to 2.7%, although the actual value could be higher (discussed below). Through TH staining, the number of surviving DA neurons was shown to be approximately 4,300 per brain. In normal brains, there are no DA neuron cell bodies in the striatum, only fibers. Thus, these TH-positive cells were considered to be derived from the grafted ES cells. While the grafted cells were labeled by BrdU prior to transplantation, only 65% of these TH-positive cells were immunoreactive for BrdU. This may have resulted from incomplete labeling of the input cells; as the cells were treated with BrdU while being cultured as spheres, the labeling rate was not 100%, but 68.8%. In addition, grafted cells might proliferate in vivo, reducing the concentration of BrdU in the cells. Furthermore, intrinsic striatal TH-positive neurons may be recruited, as reported previously (34
), which may explain the observation of a few TH-positive neurons even in control monkeys.
According to an earlier clinical report, the number of TH-positive cells in the postmortem brain of a PD patient was approximately 200,000 (35
). In 2 recent double-blind trials, however, the number of surviving TH-positive cells was determined to be 50,000–240,000 (4
). Given that the volume of the monkey putamen is 10% of that of the human putamen (36
), it is likely that the required number of TH-positive cells in the monkey (M. fascicularis
) brain is 5,000–24,000. The results of this study remain in keeping with observations made in human patients, suggesting that ES cells are a promising candidate for a donor source for cell transplantation treatment of PD. It should be noted, however, that the MPTP-treated monkey is a model of acute selective nigral destruction whereas human PD patients also experience progressive deterioration and pathological changes of other neural systems (15
Although the results presented here encourage the development of strategies involving ES cell–derived neurons for treatment of neurological diseases, further studies will be needed to address the long-term efficacy and safety of using these cells. For instance, the low survival rate of the grafted cells or neurons is comparable to that noted in previous reports (40
). To increase the number of viable DA neurons produced by grafts in vivo, we used DA neuron progenitors in the present study. Multiple-target grafting (41
) is also a strategy that should be considered. Notably, we observed a number of GABA-positive cells in the graft, suggesting that other types of neurons and/or glial cells in the graft may contribute to both the differentiation and function of transplanted DA neurons (42
). However, the optimal cellular composition of the graft remains to be determined. In addition, while previous studies with rodents have demonstrated that tumor formation can be associated with ES cell grafts (43
), we did not observe tumor formation or Ki67-positive cells within the first 3 months after transplantation. In the future, however, it will be important to examine late tumor formation as well as the possible long-term effects of ES cell grafts on motor behavior. It will also be necessary to use non–TH-positive cells in control grafts to exclude the possibility that the effects of this treatment are mediated by non-DA cells.
Finally, we would like to emphasize that our system (MRI, surgery, PET, etc.) is applicable to humans. Previous work has shown that monkey ES cells have characteristics similar to those of human ES cells (18
). In addition, it was recently demonstrated that neural precursors induced from human ES cells were able to survive in rodent brains (47
). The SDIA method is applicable to human ES cells, allowing for enrichment of DA progenitors (unpublished data). These results suggest that transplantation using ES cells as a clinical therapy for PD is approaching the point of technical feasibility. Two recent double-blind, sham surgery–controlled trials of embryonic mesencephalic transplants for the treatment of PD, however, showed only modest improvement (4
), suggesting the potential limits of cell transplantation. Many basic issues, especially regarding stem cell therapy, remain to be resolved (38
). Before the clinical application of human ES cell transplantation can be attempted, extensive studies assessing the safety and efficacy of ES cell transplantation in monkey models will be necessary.