http://www.nature.com/nbt/journal/v3.../nbt.2077.html


Nature Biotechnology | News and Views

Dopaminergic neurons for Parkinson's therapy
Olle Lindvall
Nature Biotechnology 30,56–58(2012)doi:10.1038/nbt.2077Published online 09 January 2012



A differentiation protocol guided by developmental principles produces more-authentic dopaminergic neurons for transplantation in patients.



In Parkinson's disease, impaired control of movement stems from the degeneration of dopaminergic neurons in the substantia nigra and the resulting loss of dopamine in the striatum. Clinical studies in which human fetal mesencephalic tissue, rich in dopaminergic neuroblasts, was transplanted into the striatum of individuals with Parkinson's disease have shown in principle that dopaminergic neurons can be replaced and symptoms reduced in some cases1. But the functional outcome of this treatment has proved highly variable, and human fetal tissue is scarce, suggesting that other sources of dopaminergic neurons are needed to develop a clinically useful cell therapy. A recent report in Nature by Studer and colleagues2 describes the conversion of human embryonic stem cells (hESCs) into substantia nigra dopaminergic neurons that ameliorate Parkinson's disease symptoms in animal models without forming tumors. From the clinical perspective, this new differentiation protocol, which generates large numbers of transplantable dopaminergic neurons of the correct phenotype, represents a major advance toward the first application of hESC-derived dopaminergic neurons for grafting in patients.

Previous studies by my group and others have shown that dopaminergic neurons in fetal grafts can reinnervate the striatum and restore dopamine release in the brain of Parkinson's disease patients1. Some of our patients improved to the extent that they have managed for several years without treatment with L-dopa (a chemical that crosses the blood-brain barrier and after decarboxylation increases brain dopamine levels)3. These clinical programs came to an end after troublesome graft-induced involuntary movements, or dyskinesias, developed in ~15% of patients1. This problem now seems to be solved as the graft-induced dyskinesias in three of our patients were found to be caused by graft-derived serotonergic hyperinnervation of the striatum and could be abolished by pharmacological blockade of serotonin signaling3. In other studies, a small fraction of fetal graft–derived dopaminergic neurons was shown to contain Lewy bodies (the hallmark of Parkinson's disease) many years after transplantation1, 4. However, the progression of pathology to grafted neurons was slow, and they were still functional after a decade, with patients showing long-term improvement.

Stem cells could become a source of virtually unlimited numbers of dopaminergic neuroblasts for transplantation. To induce substantial clinical benefit, stem cell–derived dopaminergic neurons must be of human origin and must exhibit the specific properties of substantia nigra neurons. Promising results have been obtained using hESCs5, 6, 7, but several problems of fundamental importance for clinical translation remain8. The identity of the cells as substantia nigra dopaminergic neurons, their ability to extensively reinnervate the striatum in a larger brain and their capacity to markedly improve deficits resembling the symptoms experienced by Parkinson's disease patients—none of these prerequisites has been convincingly demonstrated yet. Moreover, hESC-derived grafts are associated with a risk of tumor formation5, and some protocols for generating dopaminergic neurons involve ex vivo gene delivery7, which may hinder clinical application.

Studer and colleagues2 provide solutions to several of these problems (Fig. 1). Compared to previous protocols for generating dopaminergic neurons from hESCs, the most important change in the new method is that the cells are passed through a developmental stage at which they exhibit the characteristics of floor plate tissue. The floor plate is a signaling center during neural development, located along the ventral midline of the neural tube, and the midbrain floor plate is neurogenic and a source of dopaminergic neurons. Studer and colleagues2 derived floor plate cells from hESCs in vitro using dual inhibition of SMAD signaling and high levels of sonic hedgehog9, 10. Midbrain floor plate identity was induced by activation of WNT signaling, and cells were exposed to BDNF, ascorbic acid, GDNF, cAMP, TGFβ3 and DAPT. Differentiation to dopaminergic neurons with extensive fiber outgrowth occurred with high efficiency. The cells expressed phenotypic markers typical for substantia nigra dopaminergic neurons and expressed genes that define these neurons during development. They also exhibited electrophysiological properties of nigral neurons and released large amounts of dopamine in vitro.

Figure 1: Generation of functional dopaminergic substantia nigra neurons for transplantation in Parkinson's disease.


Floor plate cells are derived in vitro from hESCs by dual inhibition of SMAD signaling and high levels of sonic hedgehog. Midbrain floor plate identity is induced by activation of WNT signaling, and dopaminergic neuron precursors are generated in the presence of trophic factors, ascorbic acid, cAMP and DAPT. After intrastriatal transplantation in mice, rats or non-human primates, these cells give rise to large numbers of dopaminergic neurons with a substantia nigra phenotype, which reinnervate the striatum and ameliorate behavioral deficits resembling the symptoms in Parkinson's disease patients.
Full size image (242 KB)

Notably, high numbers of dopaminergic neurons with a substantia nigra phenotype survived for a long time after intrastriatal transplantation of the hESC-derived dopaminergic precursors both in mice (~5,000 cells per graft at 4.5 months) and rats (~15,000 cells per graft at 5 months)2. These quantitative data suggest that the cells could be clinically useful because successful fetal transplants in Parkinson's disease patients require ≥100,000 dopaminergic neurons per putamen. In a nonhuman primate model, the grafted dopaminergic neurons extended their axons up to 3 mm into the striatum already at 1 month2. This is important because the axonal outgrowth from human stem cell–derived dopaminergic neurons generated so far has been poor8. The new hESC-derived dopaminergic neurons seemed to be able to reinnervate a major portion of the denervated striatum also in a larger brain, which will be necessary for clinical efficacy. Finally, the dopaminergic cell grafts not only completely reversed drug-induced rotational asymmetry but also improved clinically relevant behavioral deficits resembling symptoms in Parkinson's disease patients.

These findings are supportive of a future hESC-based therapy for Parkinson's disease, but several issues remain to be addressed. A critical issue for clinical translation is safety. The protocol for generating dopaminergic neurons should be fully chemically defined, and the components of animal origin eliminated. The potential for graft-induced dyskinesias after transplantation should be assessed in appropriate animal models. Notably, serotonergic neurons were rare in the grafts in rodents2, suggesting that the risk of dyskinesias in the clinical setting is probably low. The authors observed no tumors after transplantation, and the percentage of proliferating cells was low. However, before the cells are used clinically, their tumorigenicity must be assessed more rigorously. As part of this evaluation, it will be important to determine the identity of all cell types in the implants. Use of cell sorting to eliminate tumor-forming cells or of regulatable suicide genes may be necessary to improve safety.



Another question concerns the functional efficacy of the hESC-derived dopaminergic neurons. To be competitive with alternative therapies, grafts must bring about major recovery of motor function. Motor symptoms in Parkinson's disease patients can already be treated rather well with L-dopa, dopamine agonists, enzyme inhibitors and deep brain stimulation. Studer and colleagues2 found that the hESC-derived grafts contained large numbers of dopaminergic neurons and were efficacious in rodents. A cell potency assay comparing hESC-derived neurons with fetal dopaminergic neurons would be useful to estimate the number of cells needed for humans. Also, the growth capacity of the dopaminergic cells must be carefully analyzed to determine the number and distribution of implants required to reinnervate most of the human striatum. Finally, it should be noted that restoration of striatal dopamine transmission by cell therapy is unlikely to improve nonmotor problems in Parkinson's disease, which are due to the degeneration of other dopaminergic and nondopaminergic neuron systems.

Although several challenges lie ahead for stem cell therapy in Parkinson's disease, the findings of Studer and colleagues2 are cause for optimism. If implantation of their dopaminergic cells is found to enable robust, long-lasting improvement of motor function and to allow withdrawal of dopaminergic medication, as observed in some patients with fetal grafts3, this study would represent a breakthrough in the field that could make cell therapy available to large numbers of patients.

Another important study, by Olle Lindvall:

"Clinical application of stem cell therapy in Parkinson's disease"

http://www.biomedcentral.com/1741-7015/10/1