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Stem cell-derived dopamine neurons for brain repair in Parkinson’s disease One of the prospects for a curative treatment for Parkinson’s disease is to replace the lost dopaminergic neurons. Preclinical and clinical trials have demonstrated that dissected fetal dopaminergic neurons have the potential to markedly improve motor function in animal models and Parkinson’s disease patients. However, this source of cells will never be sufficient to use as a widespread therapy. Over the last 20 years, scientists have been searching for other reliable sources of midbrain dopamine neurons, and stem cells appear to be strong candidates. This article reviews the potential of different types of stem cells, from embryonic to adult to induced pluripotent stem cells, to see how well the cells can be differentiated into fully functional dopamine neurons, which cells might be the best candidates and how much more research is required before stem cell technology might be translated to a clinical therapy for Parkinson’s disease. KEYWORDS: animal models n bone marrow stem cells n differentiation n dopamine neuron n embryonic stem cells n induced pluripotent stem cells n neural stem cells n Parkinson’s disease n review n transplant

Parkinson’s disease (PD) is a widespread neuro degenerative disorder, affecting 1% of those over the age of 60 years. The symptoms of akinesia (reduced movement), rigidity, gait and resting tremor are normally apparent only after the destruction of over 80% of dopaminergic projections from the substantia nigra (SN) to striatum within the basal ganglia (once many of the dopaminergic neurons are lost). This, coupled with the lack of clear causative factors for over 95% of idiopathic PD sufferers, means that to date, no curative therapies have been developed to prevent disease onset or protect remaining neurons, and drug or surgical therapies to reduce and alleviate symptoms remain the treatment of choice. At present, cell-replacement therapies may provide the most promising curative treatment for PD. If cells can be placed in the brain to produce suitable, controlled levels of dopamine release in the striatum, then there is hope of attenuating the disease process and restoring motor function in patients. Since the 1970s, scientists have been assessing the feasibility of cell replacement, using dissected fetal dopamine neurons. Animal studies demonstrated the efficacy of such an approach, and since the late 1980s numerous clinical trials have been conducted, with over 300 patients transplanted worldwide (for a review see [1]). These trials have met with some success, although the use of fetal tissues remains controversial, both in the ethics of procuring large amounts of suitable tissues

and in the variability of positive outcomes (from no apparent benefit, to patients being able to reduce drug medication and return to a reasonably normal life). Dopamine neurons remain the cell of choice for transplantation in PD, as the ability to interconnect host and transplant neuronal circuitry allows for controlled dopamine release and therefore the restoration of normal movements. In order to realize the full potential of cell replacement, alternative sources for dopamine neurons need to be identified. These cells should be plentiful – to enable many more patients to receive treatment – and also provide a safe and reliable source of dopamine neurons. The focus in the last two decades has been on stem cells. This article reviews the potential of different types of stem cells in their ability to produce functional dopamine neurons and their promise for brain repair in PD.

10.2217/RME.10.3 © 2010 Future Medicine Ltd

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Rosemary A FrickerGates† & Monte A Gates Author for correspondence: Keele University, Keele, Staffordshire, UK Tel.: +44 178 273 3937 Fax: +44 178 273 3516 r.a.fricker-gates@ hfac.keele.ac.uk †

Fetal neural stem cells The most obvious place to look for stem cells that can make dopamine neurons is the developing midbrain (ventral mesencephalon [VM]), the origin of the SN dopamine neurons lost in PD. These cells are most likely to be similar in phenotype, and thus should have a more straightforward differentiation pathway to form mature dopamine neurons. Since the late 1970s, methods have been developed to isolate, maintain and expand cells from various regions of the developing nervous ISSN 1746-0751

267

[113,118,121,122]

[105–107]

Yes (nonspecific ? effect?) Yes? (one Stepping and paw reaching? report [121] ) (one report [121] ) Yes? Variable Adult bone marrow

Yes?

Appear immature

Immature markers only Yes

?

? Yes

?: Not yet reported; DA: Dopamine; PD: Parkinson’s disease; TH: Tyrosine hydroxylase.

Yes Modest numbers

20–40 (patient derived) Low proportion Induced pluripotent Adult neural

?

system using mitogens such as EGF and basic FGF [2–4] . The cell aggregates possessed the ability to produce the three main cell types of the CNS: neurons, astrocytes and oligodendrocytes. Working in the early 1990s Reynolds, Tetzlaff and Weiss dubbed their free-floating cell aggregates ‘neurospheres’ [5] and demonstrated that cells in these spheres could undergo unlimited proliferation, a hallmark property of neural stem cells. We now know that neurospheres actually contain a heterogeneous mix of cell types, including stem cells, and more restricted neural progenitors or precursors. The neurosphere technique has allowed researchers to use rodent, primate and human fetal VM tissues to generate neural stem cell/progenitor cultures and to attempt to define conditions where these proliferating cells could be induced to become dopamine neurons. Success in producing dopamine neurons from fetal neural progenitors has been varied and, at best, modest. Techniques tried include: the addition of cytokines and neurotrophic factors such as interleukins, angiotensin II and glial-derived neurotrophic factor [6–8] , or adapting the culture environment to mimic the developing ventral mesencephalon, including the use of antioxidants (e.g., ascorbic acid) and lowered oxygen conditions [9–14] . A further method has been to insert genes that normally induce a dopaminergic phenotype in the developing midbrain, such as Nurr1, Shh, Wnt-5a and Lmx1a [15–18] . Yields of dopaminergic neurons from mesencephalic precursors have also been increased by refining the cells that are expanded, either through selecting the precise donor age of the cells [19] or eliminating serotonergic cells in the cultures [20] . Expanded neural precursors and dopamine neurons derived from these cells have been transplanted into animal models of PD. Again results have been variable and early research showed limited survival and function of human-derived neural precursors in the 6‑hydroxydopamine (6‑OHDA) rat model [21,22] . Techniques such as altering the in vitro conditions or gene expression of neural precursors have enabled researchers to increase the numbers of dopamine neurons prior to transplantation [23] . Thus, transplants of expanded rodent neural cells have been shown to restore motor function, the main effects measured as a reduction in amphetamine-induced rotational asymmetry [24–26] . Similar results have been shown for expanded human neural precursors, where predifferentiation with cocktails of neurotrophic factors yielded more dopamine neurons in the transplants and greater motor

Very low numbers, poor survival Variable

[91,95,98,99,100]

[38,48,54,57,68,80]

Yes

Yes 11–90 (mouse) 10–80 (human) Embryonic

Yes (K+ evoked)

Large numbers (but transfected cells)

Yes

Generally poor. Some evidence in primate model Limited: Rotarod, paw reaching and stepping (transfected cells) ? Yes Limited numbers Yes

Yes Yes (transfected cells) 15–20 Fetal neural

TH-positive Reduction in Amelioration of skilled neuronal survival drug-induced motor function in PD models rotation in vivo Electro DA release physiological in vitro function in vitro Stem cell source % of neurons Expression of expressing TH midbrain and in vitro dopaminergic markers

Table 1. Potential of stem cells from different sources to differentiate into functional dopaminergic neurons. 268

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Stem cell-derived dopamine neurons for brain repair in Parkinson’s disease

recovery [27] . More recently, human-derived neural precursors have been successfully transplanted to a primate model of PD, yielding surviving dopamine neurons and improving parkinsonian behaviors [28] . Two problems yet to be tackled with in vivo studies are: evidence to suggest that predifferentiated dopamine neurons preferentially die posttransplantation [29] ; and the ability of transplants to improve more subtle motor impairments (e.g., forelimb use and contralateral neglect). In essence, neural stem cell/progenitor transplants have yet to prove their ability to recover these more refined motor problems, with crucial evidence required to prove their usefulness before proceeding to the clinic. More importantly, the main issue preventing greater success with expanded neural stem cells/ precursors appears to be retaining a dopaminergic phenotype in neural cells cultured for long periods. Typically, over time, neural precursor cultures lose the ability to differentiate into dopamine neurons [Bagga V, Fricker-Gates RA, Unpublished Data] , functional neurons [30] , and indeed any type of neuron at all [31] . Many of the transplant studies showing successful dopaminergic neuron differentiation have used cells that have been proliferated for less than 2 weeks. Although this culture period yields a 7–10-fold increase in cells, dopamine neurons differentiated from these cells do not survive well following transplantation [32,33] . Thus, at present, expanded neural precursors cannot yet provide large numbers of dopamine neurons for cell-replacement therapies. Most preclinical studies to date report little or no cell division by transplanted fetal neural precursors. However, long-term studies in animal models are required to ensure the safety and efficacy of fetal neural stem cells.

Embryonic stem cells In the last decade, a major candidate in the running to provide dopamine neurons for PD has been the embryonic stem cell (ESC). ESCs are derived from the inner cell mass of the preimplantation blastocyst, and are therefore very primitive pluripotent cells with the potential to form any cell within the body. Since their discovery in the early 1980s [34,35] , ESCs have been manipulated to differentiate into many cell types, with the prospect of treating diseases such as diabetes, heart failure and neurodegenerative disorders. They can be grown for extensive periods in culture, maintaining pluripotency and self-renewal either by coculture on mitotically inactivated fibroblast feeder layers or with leukemia inhibitory factor. future science group

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The challenge in providing cells to treat PD lies both in manipulating ESCs to form neurons and, thereafter, a specific dopaminergic phenotype. Similar to expanded neural precursors, studies have used neurotrophic factors and overexpression of midbrain-specific genes to induce a dopaminergic phenotype in mouse ESC-derived neurons. Shh, FGF‑8, ascorbic acid, TGF‑b and cyclic AMP all increase the differentiation of dopaminergic cells in culture, shown by their expression of the rate-limiting enzyme tyrosine hydroxylase (TH) [36,37] . One of the most effective inducers of a dopaminergic fate comes from stromal-derived inducing activity from coculture with stromal cell lines such as PA6 and MS5 [38] . Interestingly, a recent study demonstrated that dopaminergic neurons may be specified from ESCs either before or during their early commitment to a neural fate [39] . Reports from different research groups suggest that the percentage of neurons adopting a dopaminergic phenotype when differentiated from ESCs varies from 11 to 90% of total neurons in vitro [40–45] . In addition, many studies do not identify the proportion of ESCs that differentiate into neurons, and those studies that do, demonstrate that the conversion rate is less than 10% [43] , suggesting that the majority of ESCs do not differentiate into a useful cell type using current differentiation methods. In order to overcome this inefficiency, researchers have overexpressed genes and transcription factors known to promote the development of midbrain dopamine neurons, Nurr1, Pitx3 and Lmx1a [46–53] , resulting in much higher yields of dopamine neurons in vitro and in vivo. To confirm whether ESC-derived neurons carry features of the ventral midbrain dopaminergic SN neurons, most groups have used reverse transcriptase PCR to identify specific midbrain genes, such as En1, Pitx3, Nurr1, Lmx1b and Girk2, and immunohistochemistry or reverse transcriptase PCR to detect dopaminergic markers, such as the dopamine transporter, vesicular monoamine transporter and the enzyme aromatic amino acid decarboxylase [48,49,51] . An additional and vital assessment is how these ESC-derived neurons function. In vitro studies have assessed the ESC-derived neurons’ ability to release dopamine on stimulation with potassium [41,44] and their electrophysiological properties (i.e., firing action potentials, forming synapses and generating postsynaptic currents) [48,51] . So, how do ESCs perform in vivo? A number of key studies suggest that transplanted mouse ESCs, or their neuronal derivatives, can form www.futuremedicine.com

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dopamine neurons with a midbrain phenotype and can ameliorate at least some of the motor symptoms in rodent models of PD [48,50,54,55] . Most of these in vivo studies have used intrastriatal transplants and have measured reduction of whole-body rotation induced by amphetamine or apomorphine to indicate dopamine release from grafted cells and normalization of dopamine receptors in the host striatum, respectively. In a small number of studies, other more complex movement tasks, such as the cylinder, stepping and rotarod tests, were shown to be improved by the presence of a transplant, although to a limited extent [48,56] . These data suggest that ESC transplants are functioning at a more superior level than transplants derived from neural stem cells, most probably due to the much higher numbers (up to thousands) of dopamine neurons present in the grafts [48,49,54] . However, a note of caution is needed here; many of the studies showing high numbers of dopamine neurons and more effective behavioral recovery have used cells transfected with dopamine-inducing genes or pretagged with fluorescent marker genes and enriched by FACS [42,48] . Clearly, this transgenic approach cannot be taken directly to the clinic until the safety of such manipulations can be proved. Original studies using mouse ESCs have been replicated using primate ESCs [57–60] ; and with the derivation of human ESCs in 1998 [61] , there has been a push to develop human ESCs for PD repair. Human ESCs show inherent differences to mouse ESCs in terms of their requirements for proliferation and differentiation, including for some cell lines the necessity to grow cells on a fibroblast feeder layer. That being said, many studies have effectively adapted the embryoid body [47,62–65] , five-stage [66] or stromal-derived inducing activity methods [67–74] to differentiate human ESCs. Data available show a great variation using these methods, with a range of 10 to 80% of stem cell-derived neurons in the cultures expressing markers for midbrain dopaminergic phenotypes. More recently, great strides have been made to define chemical conditions that enhance differentiation, in an attempt to dispose with feeder layers or cocultures that might contaminate any transplant suspension [75,76] . A newly published study has gone a step further by attempting to identify the specific active properties of stromal cells that induce dopaminergic neurons (the stromal-derived inducing activity method) and has identified four factors that when added to human ESCs greatly enhance production of both neurons and those that are a dopaminergic phenotype [77] . 270

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Similar to their mouse counterparts, humanderived dopaminergic neurons also possess the ability to release dopamine in vitro, as well as show specific gene and protein expression, and the electrophysiological properties of midbrain SN dopamine neurons [47,62–64,67,71–83] . These cells have been transplanted in numerous studies, to rodent models of PD, to assess their function in vivo. Early in vivo studies demonstrated survival of transplanted ESC-derived neurons, expressing dopaminergic markers such as the enzyme TH. However, survival was fairly low and many of the early transplants showed evidence of proliferating ESCs and teratoma formation [66,71,72] . More recently, cells differentiated with more refined protocols or for extended periods have been shown to produce larger numbers of dopamine neurons in transplants, and these have a midbrain phenotype [79,80] . Furthermore, increased differentiation prior to transplantation appears to eliminate ES-like cells in the grafts, although dividing cells are often present in the core of grafts and appear to be most commonly of neural lineage [76,84,85] . Human ESC-derived transplants have been demonstrated to ameliorate at least some motor impairments in the 6‑OHDA rat model of PD, including reduction in amphetamine- and apomorphine-induced whole-body rotation [47,68,75,79,84] . Other more complex motor functions measured by stepping and placing tasks have proven more difficult to compensate with human ESC-derived transplants, with some studies showing no recovery [80] , and others observing some improvement in function [84,86] . Clearly, although promising, there remain a number of scientific and safety issues to address before these cells can be seen as safe and useful to treat PD patients.

Adult stem cells Embryonic stem cells show great promise for dopamine replacement in PD, but the ethical issues surrounding the use of human embryos for research and clinical therapies have led to the search for alternative, less controversial cell sources that can be utilized for brain repair. Ideally, these cells should be adult in origin, and preferentially patient-derived, to avoid problems with immunocompatibility. A number of candidates have been investigated for their functional efficacy in animal models of PD. Induced pluripotent stem cells In 2006, a seminal publication from Yamanaka and colleagues first demonstrated that four factors could be used to reprogram embryonic and future science group


adult fibroblasts to produce cell lines with very similar properties to ESCs [87] . This group and others induced pluripotency in somatic cells using various combinations of the transcription factors Oct4, Sox2, Klf4 and c‑Myc, with the resulting cell lines showing long-term proliferation, expression of pluripotent markers, and the ability to make adult chimeras and to undergo germline transmission [88–90] . In 2007, human induced pluripotent stem (iPS) cells were generated using the same factors, introducing these with retroviruses or lentiviruses [91–94] . To date, only a small number of research groups have investigated iPS cells for their ability to differentiate into dopamine neurons. Aasen et al. showed efficient dopamine neuron differentiation in vitro from iPS cells generated from human keratinocytes [95] , and Huangfu et al. were able to generate TH-positive neurons from human fibroblast-derived iPS cells [96] , although the extent of differentiation was not given. A more recent study used two inhibitors of SMAD signaling to increase neuronal differentiation of human iPS cells, and demonstrated that it is possible to induce a dopamine neuron phenotype [97] . Two recent transplant studies have demonstrated that predifferentiated iPS cells can survive and develop into mature dopaminergic neurons in the 6‑OHDA rat model of PD [98,99] . Interestingly, in the study by Wernig et al., four out of five transplanted rats showed good recovery of amphetamine-induced rotational bias, with associated presence of over 20,000 TH-positive cells, whereas an animal with only 1500 TH-positive cells showed no recovery [98] . This suggests that, in this study at least, dopamine neurons derived from iPS cells are not as efficient as neuronal transplants of dissected primary VM tissue, the current gold standard. In addition, teratoma formation was observed in both studies, with both tumor-like cells and nestin-positive neural progenitors within transplants [99] . Tumor formation could be prevented by presorting cells by fluorescence-activated cell sorting to remove pluripotent progenitors based on their expression of the cell surface marker SSEA‑1 [98] . Our experiences with iPS cell-derived mouse neural progenitors transplanted to a 6‑OHDA lesion mouse model of PD have shown similar findings, where transplanted cells express TH and a neuronal phenotype (Figure 1) . iPS cells differentiate more readily compared with ESCderived progenitors, with ESC-derived teratomas forming much more rapidly [Fricker-G ates et al ., future science group

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Unpublished Data] .

However, iPS cell transplants tend to contain many dividing neural progenitors at 4 weeks post-transplantation (shown as nestinpositive cells in Figure 1), and numbers in the range of 1500 to 2400 surviving TH-positive neurons are too few to elicit reduction in amphetamineinduced rotation, suggesting their inappropriate lineage or immaturity. The holy grail of any stem cell treatment for PD is to be able to generate autologous stem cells from patients that could then be converted to dopamine neurons. iPS technology should enable this, and a recent report suggests that it is possible to generate iPS cells from PD patients [100] . This study also achieves two further goals: the efficient differentiation of 20–40% of iPSderived neurons into a dopaminergic phenotype; and a further step towards increasing the safety of iPS cells, by using cre-recombinase excisable viral vectors to induce pluripotency in the cells, allowing the removal of these vectors prior to any cell transplantation. Further advances are being made to increase the safety of iPS cells, including using proteins rather than viral vectors to reprogram cells, although this technique currently yields very low numbers of true stem cells [101,102] . Clearly, there is a lot more research to be carried out with iPS cells. However, early signs are very promising that this source could provide a more ethically acceptable and patient-specific alternative to ESCs. Adult neural stem cells An alternative stem cell source that could be tapped into for patient-specific cell-replacement therapy for PD are neural stem cells, which TH

Nestin/Oct4/DAPI

Figure 1. Transplants of induced pluripotent stem-derived neural progenitors to the adult 6‑hydroxydopamine-lesioned mouse striatum at 4 weeks survival. (A) TH-positive transplant cells with neuronal morphologies at the periphery of the transplant. Dotted line indicates graft/host border. Scale bar: 100 μm. (B) Double-labeling reveals distinct regions containing mitotic cells with a stem cell phenotype (Oct 4, green), or neural progenitor phenotype (nestin, red). Graft and host cells are counterstained with DAPI (nuclei, blue). Scale bar: 500 μm. DAPI: 4’,6-diamidino-2-phenylindole; TH: Tyrosine hydroxylase.

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are resident in neurogenic regions of the adult brain. Adult neural stem cells can be isolated from the anterior subventricular zone (SVZa) and the granule cell layer of the dentate gyrus in the hippocampus [103,104] . These adult stem cells function to continually repopulate neurons in the olfactory bulb and dentate gyrus, respectively, and SVZa neural stem cells are able to generate new dopaminergic neurons within the periglomerular layer of the olfactory bulb. In addition, the SVZa region lies adjacent to the striatum; therefore, it is plausible that SVZa stem cells could be stimulated to divide and produce dopaminergic neurons that migrate laterally and release dopamine in the striatum, where it is required for the alleviation of PD motor symptoms. Attempts to develop dopaminergic neurons from adult neural stem cells/progenitors have only been moderately successful, both in vitro and in vivo. In culture, adult progenitors from the SVZa or hippocampus have produced only small numbers of TH-positive neurons, and one report indicates that these neurons are phenotypically and electrophysiologically immature [105] . Similarly, when transplanted in vivo, studies suggest that any behavioral recovery in rodent models of PD is less due to the presence of stem cell-derived dopamine neurons in transplants, but more likely a general neuroprotective effect of the non-neuronal grafted cells [106,107] . An alternative to generation of adult neural stem cells ex vivo is to stimulate the endogenous neural stem cells or progenitors in situ, to differentiate these into dopaminergic neurons. Attempts to stimulate the development of new neurons in the striatum of the 6‑OHDA lesioned rat have yet to prove efficacious. One study using stimulation with TGF‑a led to the production of many new cells that migrated laterally into the striatum; however, none of these cells were neuronal or a dopaminergic phenotype, and there was no behavioral recovery in treated animals [108] . Ideally, stimulation of neurogenesis in the SN would offer a better means to reinstate dopamine neurons in their normal environment in order to potentially reconnect the normal basal ganglia circuitry and, therefore, increase functional recovery in PD. At present, there is much controversy as to whether neurogenesis occurs in the adult SN. Some studies suggest evidence that new neurons can be generated in the adult SN in the normal brain and that the number of new dopamine neurons is increased following 1‑methyl-4-phyenyl‑1,2,3,6-tetrahydropyridine 272

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treatment to induce a PD-like state in adult mice [109,110] . Other groups have disputed the presence of new neurons in the normal adult and PD-like SN [111] , or have suggested that neural progenitors from this region normally only develop into mature glial cells, although they have a neurogenic potential once placed in a permissive environment for neurogenesis [112] . Clearly, any potential neurogenesis in the SN of the PD brain is insufficient to combat dopamine neuron loss during the disease process; and even if substantial neurogenesis could take place in the SN, there is still the obstacle of the cells regrowing the nigrostriatal circuit. However, strategies to stimulate the production of dopamine neurons within the SN provide an exciting prospect to repopulate this region with new neurons. Adult bone marrow stem cells Around the turn of the millennium, a new adult stem cell was being proposed as an alternative source of neural cell – the bone marrow stem cell (BMSC). In vitro data suggested that these BMSCs could ‘transdifferentiate’ into a neuronal lineage in vitro under specific conditions, such as in coculture with fetal midbrain cells [113] . Studies using intravascular delivery of BMSCs showed their migration into the CNS and expression of neuronal antigens, such as NeuN, neuron-specific enolase and neurofilament [114,115] . However, evidence that these cells are biological neurons is weak, and it is well accepted that artificial induction methods can produce cells with some evidence of neuronal properties without being bona fide neurons [46] . Moreover, the transdifferentiation effect demonstrateed by these groups and others has been challenged by reports that BMSCs and stem cells adopt alternative phenotypes by fusion with other cell types rather than a switch in lineage identity [116,117] . A recent study has demonstrated that, after coculture with astrocytes, mouse adult BMSCs can adopt the morphology, phenotype and electrophysiological properties of midbrain dopamine neurons in vitro [118] . However, this work has yet to be replicated elsewhere. Other methods have been used to induce human BMSCs to become dopaminergic neurons, including cocktails of neurotrophic factors used with ESC cultures, sonic hedgehog, FGF‑8 and basic FGF. The immature progenitor-like cells expressed midbrain dopamine-specific genes, were electrically active and secreted dopamine, and could be further matured by the addition of brain-derived neurotrophic factor [119,120] . future science group


Alternative methods of generating dopamine neurons have included transfection of rodent and human BMSCs with the intracellular domain of the Notch signaling protein followed by exposure to neurotrophic factors, such as glial-derived neurotrophic factor [121] . Neurons differentiated in this way were transplanted to 6‑OHDA lesioned rats, and transplants were shown to contain cells expressing the dopaminergic markers TH and dopamine transporter, and to elicit some recovery in apomorphine-induced rotational bias, and stepping and paw-reaching deficits. The results published from this study are controversial owing to the inability of other groups to replicate the findings. Conversely, other studies have shown limited dopaminergic differentiation and only shortterm survival of transplanted neurons derived from BMSCs using neurosphere culture methods [122] . Therefore, at present, it is unclear whether BMSCs show potential as a source of dopamine-producing cells for transplantation in PD. To date, there is little evidence that these cells form mature functional dopaminergic neurons either in vitro or in vivo, reconnect with host circuitry or ameliorate motor deficits.

Conclusion Which stem cells have the greatest potential for dopamine neuron replacement in PD? (Table 1) At present it seems that all offer some potential: ESCs and iPS cells show greatest efficiency in generating functional midbrain dopaminergic neurons; adult stem cells and iPS cells would allow for autologous transplants from patientderived neurons and circumvent the need for extensive immunosuppression to prevent graft rejection, which is in itself a challenge for the patient. Neural stem cells appear to be lagging behind in terms of their capability, although any breakthrough with stimulation of endogenous adult neural stem cells would surely shoot them back up the list of contenders. There are still some major hurdles to overcome before stem cells can be considered a realistic therapy for PD. From a scientist’s point of view, we are still some way from producing functional dopaminergic neurons on a large enough scale to generate banks of equivalent cells to offer transplants as a widespread therapy. What stands out from the literature on ESCs and iPS cells is that significantly larger numbers of dopamine neurons are required in transplants derived from these cells to elicit functional motor recovery in animal models than from traditional dopaminergic neurons dissected from the developing future science group

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midbrain. It is likely that either these stem cellderived neurons are less efficient because they lack vital properties of midbrain dopamine neurons, or that our ‘gold standard’ VM transplants contain an environment of more suitable cell types to aid reconnection with host circuitry and optimal neuronal dopamine release. A typical VM transplant contains both dopaminergic and serotonergic TH-positive neurons [123] , as well as other neuronal and glial subtypes. We do not yet know how important these other cells may be in influencing neuronal survival and maturation, and whether aiming for stem cellderived transplants containing 100% midbrain dopamine neurons will be as successful in terms of graft function. The safety aspects must also be considered. It is not ethically acceptable to inject cells to humans where they may retain the potential to divide in vivo; therefore, we will have to generate a cell source free of proliferating cells [124] , or develop methods to eliminate these should they appear in the transplants. More problematic is that most iPS cells to date have been induced using pluripotency genes delivered by viruses – these may have to be removed with 100% efficiency, or other nongenetic and nonviral methods will have to be found to generate iPS cells, such as use of recombinant proteins [125] . In addition, most successful dopaminergic differentiation has relied on introduction of midbrain dopamine-specific genes to stem cells. How safe is it to transplant these transgenic cells into humans? Will we require safer protocols to predifferentiate stem cells prior to transplantation?

Future perspective We are clearly at an exciting point in stem cell research. The last decade has seen huge advances in technology, and the next 10 years hold great promise for the development of stem cells as a realistic source for neuronal repair in PD. In the coming decade it is likely that many of the safety issues surrounding stem cells will be resolved, such as removal of dividing stem cells or reprogramming genes prior to transplantation. However, more key scientific issues may be less readily solved in the next 10 years. We do not yet understand some key areas in basic transplantation studies, such as what makes a successful graft of dissected fetal VM tissue, what is the biology underlying the development of graft-induced dyskinesias and why is there such variability in patient recovery? The other key area where there needs to be a major breakthrough is in driving the efficient production of www.futuremedicine.com

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midbrain-specific SN dopamine neurons from a stem cell source. Current techniques still yield fairly modest results, and only by understanding how the normal brain generates its dopamine neurons during development can we hope to produce large quantities of transplantable cells with the same potential to repair the brain in PD. Clearly, there is a lot more research to be done. Nevertheless, the next chapter in stem cell technology looks to be an exciting prospect.

Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Stem cells show great potential as a source of dopaminergic midbrain neurons to treat Parkinson’s disease (PD). A number of different stem cell types have been investigated for their ability to differentiate into fully functional dopaminergic neurons both in vitro and in transplants to animal models of PD. Fetal neural stem cells can make dopaminergic neurons in vitro and in vivo, but show restricted potential over time in culture. Embryonic stem cells form functional neurons in vitro and in animal models of PD. Technology and gene delivery enhance the production of midbrain phenotypes. Adult stem cells have the appeal of circumventing ethical and immunological transplantation issues: -- Induced pluripotent stem cells can be reprogrammed from adult tissues, show similar potential to embryonic stem cells and have now been generated from PD patients -- Adult neural stem cells have proven more difficult to differentiate into dopaminergic neurons although they may be stimulated to divide locally in the brain -- Bone marrow stem cells’ ability to form dopamine neurons remains controversial Stem cell technologies provide the potential to replace dopaminergic neurons in PD, although, as yet, there is no clear candidate stem cell that can achieve this. For stem cells to be a realistic choice for cell-replacement therapy in PD, there is still much research to be undertaken in order to understand what is required for a safe and fully functional transplant.

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