Pluripotent stem cells for Parkinson's disease: progress and challenges

Parkinson's disease (PD) is a common debilitating neurodegenerative disease. The motor symptoms of PD are caused mainly by a progressive loss of dopaminergic neurons from the substania nigra, resulting in a loss of dopamine production. Current therapies are palliative and, in the long term, ineffective. In addition, some can result in significant clinical side effects. The relatively localized pathology of PD makes it an ideal candidate for cell replacement therapy. Initial efforts focused on fetal cell transplantation, and significant clinical benefit lasting more than 10 years has been reported in some cases. However, the approach is controversial and results have been inconsistent. Inherent limitations of this approach for widespread use are the limited availability and variability of transplant material. In contrast, the self-renewal and differentiation potential of human pluripotent stem cells (hPSCs) make them a promising alternative cell source for cell replacement therapy for PD. Efforts in the past decade have demonstrated that hPSCs can be induced to differentiate in culture to functional dopaminergic neurons. Studies in delivering these cells into PD animal models have demonstrated survival, engraftment, and behavioral deficit improvements. Several groups are developing these cells with clinical trials in mind. Here, we review the state of the technology and consider the suitability of current manufacturing processes, cell purity, and tumorgenicity for clinical testing.


Cell therapy for Parkinson's disease
Th e lack of an eff ective long-term curative pharmaceutical or surgical therapy for PD has led to eff orts over the past three decades to develop a cell replacement approach. Although lower brain stem and cortical areas may also be aff ected in PD, the largely localized loss of the relatively small population of DA neurons of the substantia nigra makes targeted delivery of dopamineproducing replacement cells appealing. After a decade of diff erentiation methodology development and animal studies, human pluripotent stem cell (hPSC)-derived DA neurons have emerged as a promising approach and appear headed for clinical trials.
Evidence supporting the rationale for developing an hPSC-derived DA cell replacement therapy is provided by animal studies that since the early 1980s have evaluated the eff ects of transplantation of fetal mesencephalic Abstract Parkinson's disease (PD) is a common debilitating neurodegenerative disease. The motor symptoms of PD are caused mainly by a progressive loss of dopaminergic neurons from the substania nigra, resulting in a loss of dopamine production. Current therapies are palliative and, in the long term, ineff ective. In addition, some can result in signifi cant clinical side eff ects. The relatively localized pathology of PD makes it an ideal candidate for cell replacement therapy. Initial eff orts focused on fetal cell transplantation, and signifi cant clinical benefi t lasting more than 10 years has been reported in some cases. However, the approach is controversial and results have been inconsistent. Inherent limitations of this approach for widespread use are the limited availability and variability of transplant material. In contrast, the self-renewal and diff erentiation potential of human pluripotent stem cells (hPSCs) make them a promising alternative cell source for cell replacement therapy for PD. Eff orts in the past decade have demonstrated that hPSCs can be induced to diff erentiate in culture to functional dopaminergic neurons. Studies in delivering these cells into PD animal models have demonstrated survival, engraftment, and behavioral defi cit improvements. Several groups are developing these cells with clinical trials in mind. Here, we review the state of the technology and consider the suitability of current manufacturing processes, cell purity, and tumorgenicity for clinical testing. tissue containing nigral DA cells into the striatum of rodents and non-human primates [4][5][6][7][8][9]. Th e majority of these experimental studies demonstrated that this approach is capable of reversing behavioral parkinsonism despite minimal survival and integration of engrafted cells in the host brain, providing the basis for subsequent clinical trials in which fetal mesencephalic tissue was transplanted in patients with PD [10][11][12][13]. Although evidence of signifi cant clinical benefi t has been reported [10,11], reports concerning the effi cacy of this type of treat ment [14] and the occurrence of dyskinesias (reviewed in Isacson and Kordower [15] in 2008), which might be due to contaminating serotonergic neurons in the cell graft (as reported by Politis and colleagues [16] in 2010) have varied, underscoring the need to obtain cells through a controllable manufacturing processes that mini mizes or eliminates undesirable contaminating cell types.
Although surviving DA neurons have been observed after more than 14 years following fetal cell transplants, Lewy bodies, a hallmark of PD, have been observed in some of the transplanted cells in a subpopulation of patients, raising the concern that transplanted/donor cells might undergo progressive neurodegeneration. Whether this is a reaction to the surrounding pathological tissue or the transmission of an adventitious agent from the host remains unclear (reviewed by Braak and Del Tredici [17] in 2008).
Th is variation likely results from diff erences in cell preparation, cell dose, and specifi c delivery site. In spite of this, evidence of signifi cant clinical benefi t has been reported [10,11]. Results of these trials, however, have raised a number of concerns from ethical, practical, and clinical standpoints. Furthermore, tissue availability (multiple fetuses are needed for each transplantation procedure) is not compatible with large-scale application of fetal cell transplants. Finally, the clinical benefi ts of fetal cell transplantation in patients with PD have been inconsistent [12], emphasizing this procedure's potential pitfalls that may derive from the variable cell composition (DA neurons represent only a small proportion of mesencephalic tissue), donor age, and methods of preparation of the transplants [10][11][12].
Ex vivo expansion of a neuronal population from adult or fetal neural stem cells (NSCs) might address some of the limitations of the fetal transplant approach but has not been reported and, owing to the limited capacity of these cells to proliferate, may not be possible. Despite the limitations of this approach, the important conclusion after three decades of animal and human studies is that a cell replacement approach to PD can provide signifi cant long-term clinical benefi t. Th e demon strated proof-of-concept, along with the limitations of this approach, has driven the eff ort to identify an alternative cell source.

Pluripotent stem cells as a cell replacement therapy
Human embryonic stem cells (hESCs) and adult stem cell sources such as mesenchymal stem cells have been shown to be capable of generating DA neurons in cell culture models. Whereas both adult and embryonic PSC sources have shown promise, hESC sources have made the most progress.
It has been less than 15 years since the fi rst report of the successful isolation and culture of hESCs [18]. Although undiff erentiated pluripotent hESCs and human induced pluripotent stem cells (hiPSCs) are not likely to be used directly as clinical products, they do provide a starting cell source for many diff erentiated cell types. Th e unlimited potential for self-renewal of these hPSCs and their capacity to be directed to diff erentiate by manipulation of their cell culture environment make them promising alternatives to adult or fetal tissue in many regenerative, medical applications. Th e ability to reproducibly generate large batches of diff erentiated cells allows for the standardization, consis tency, and detailed characterization that will facilitate animal studies and that will be required prior to testing in human studies.
Th e clear clinical potential of these cells has led to signifi cant eff orts to optimize methods to direct their diff erentiation into a variety of specifi c precursor or terminally diff erentiated cell types that could have considerable benefi t in the treatment of a broad range of human disease and genetic disorders, including PD. A critical advancement toward the therapeutic use of hPSCs in PD has been the demonstration that authentic DA neurons can be generated from hPSCs [19,20] and that grafts can release dopa mine and ameliorate behavioral defi cits in rodent PD models [21][22][23][24][25][26][27]. Although these studies are encouraging, development of these cells for clinical study requires the establishment of a scalable process that produces a suffi cient yield and purity of DA neurons that will be compatible with US Food and Drug Administration require ments for safe and well-characterized cell products.

Methods of generating dopaminergic neurons
During the past decade, there has been an explosion in developing methods to direct DA neuronal diff erentiation of hESCs, and many groups have reported the generation of tyrosine hydroxylase (TH)-positive DA neurons from hESCs [21,22,[27][28][29][30][31][32]. Most of these methods were initially developed by using mouse ESCs (mESCs) and subsequently adapted for use with hESCs. Th e approaches used to generate DA neurons from ESCs have used information from developmental biology. In general, ESCs are systematically exposed to factors that induce diff erentiation in a stepwise fashion, thereby directing the cells toward specifi c cell fates. While these methods vary in the process of neuronal diff erentiation, effi ciency of DA neuron generation, and the degree of DA neuron characterization, they defi ne two fundamental approaches: (a) via NSCs by embryoid body (EB) formation followed by exposure to various growth factors and (b) by coculture with stromal cells or astrocytes. Both approaches were fi rst developed in mESC systems and later were adapted with modifi cations to hESCs. Variations of both protocols include direct diff erentiation of hESCs into a neural lineage without going through the EB stage, using conditioned media from mouse stromal cells, and a defi ned media cocktail based in part on factors secreted by stromal cells and work done in transgenic mouse models [33].
Th e classic method for generating DA neurons from mESC lines is by EB formation followed by selection for neural precursors followed by expansion and then induction of neuronal diff erentiation by a combination of several growth factors [34]. Th e procedure generates a small number of DA neurons (about 7% of TuJ1-positive neurons, which comprises about 72% of the total surviving cell number; that is, about 5% of the fi nal cell population were TH-positive neurons), but the percentage of TH-positive cells increases to 33% of the total number of neurons, or about 20% of total cells, when Shh, FGF8, and ascorbic acid are added. DA neuron cell yields can be further enhanced by using genetically modifi ed mESCs by overexpression of transcription factor nuclear receptor related 1 (Nurr1) [9].
Initial eff orts to adapt mESC diff erentiation procedures to hESC cultures demonstrated that human neural precursors and neurons could be similarly generated via EB formation [35,36] in the early 2000s. Th is method, however, did not produce mesencephalic post-mitotic neurons, such as midbrain DA neurons, at a high frequency. More effi cient generation of DA neurons through EB formation was reported a few years later [31,32]. Schulz and colleagues [31] reported the successful diff erentiation of hESCs to form neurons expressing markers of the midbrain DA lineage in a serum-free suspension culture system in the absence of added neuron-inducing agents or growth factors. Large networks of TH-positive neurons were generated in the aggregates (in suspension) that co-expressed a panel of markers of the midbrain DA lineage. In another study, DA subtype-specifi c neurons were effi ciently generated by applying Shh and FGF8 in a specifi c sequence [32]. Th e authors suggested that early exposure of FGF8 to neural precursors prior to the expression of Sox1 was required for effi cient generation of midbrain DA neurons but that treatment with FGF8 and Shh in Sox1-expressing neural precursors resulted in the effi cient production of forebrain DA neurons. Both groups reported that hESC-derived DA neurons were electrophysiologically active and could release dopamine upon depolarization with KCl. Although these results showed that it was technically feasible to generate authentic, electrically mature neurons that had the biochemical and functional phenotype of a striatal DA neuron, the overall effi ciency of DA diff erentiation with these methods was relatively low, resulting in high levels of contaminating cells. In addition, these methods employed a process of diff erentiation from hESCs to DA neurons which in general takes 8 weeks, resulting in a low yield of DA neurons from the starting hESC culture. Signifi cant improvements in DA cell yields from diff erentiation via EB formation have recently been made. One of these improvements took advantage of being able to generate and store cells at an intermediate stage of the diff eren tiation process, the NSC stage (with nearly 100% purity) [26]. Th e homogeneous NSCs can then be diff erentiated into DA neurons, resulting in greater than 40% of the fi nal cell product expressing TH after only 3 to 4 weeks of diff erentiation [26].
DA neurons can be generated effi ciently from hESCs by co-culture methods. Generation of DA neurons by coculturing with stromal cells (for example, PA6 and MS5) was fi rst reported by Kawasaki and colleagues [37] with mESCs. Th e method is effi cient and rapid, as evidenced by the observations that more than 90% of cells became neural cell adhesion molecule-positive (NCAM + ) when cultured on the mouse stromal cell lines, PA6 or MS5, for a week and that about 30% of the TuJ1-positive neurons (about 52% of total cells) were TH-positive after 12 days of co-culture. Th e same group later successfully used the co-culture method to produce DA neurons from nonhuman primate ESCs [38]. Th e yield of DA neurons by this simple co-culture method is comparable to the more complex method of overexpression of Nurr1 combined with multiple growth factors via EB formation described in the previous section.
Several laboratories reported the adaptation of the mESC stromal cell co-culture method of generation of DA neurons to hESCs in 2004. Perrier and colleagues [21] reported that hESCs diff erentiated into midbrain DA neurons after co-culture with the mouse stromal cell line MS5 for 4 weeks followed by 2 to 3 weeks of culture in the presence of Shh and FGF8 without stromal feeder cells. Th e neurons produced by this procedure had midbrain DA characteristics at a high effi ciency, and 79% of the neurons were TH-positive. DA neurons could also be generated from hESCs by co-culturing hESCs with mouse PA6 stromal cells in the presence of glial cell linederived neurotrophic factor (GDNF) [30]. Zeng and colleagues [22] described the effi cient generation of DA neurons by co-culture with PA6 cells without the addition of growth factors. After 3 weeks of co-culture, about 80% of the colonies contained TH-positive cells that coexpressed many midbrain and DA markers. In addition, DA neurons generated by co-culture with stromal cells produced a signifi cant amount of dopamine in response to KCl stimulation and displayed neuronal electrophysiological properties. Th ese methods were further optimized by a combination of growth factors and by use of media conditioned on stromal cells at various stages of neural diff erentiation [33,39]. Co-culture with other cell types such as astrocytes was also reported to be effi cient in generating functional DA neurons from hESCs [27].
Although the molecular mechanism underlying DA diff erentiation by stromal cells is not clear, factors secreted by PA6 cells seem to be suffi cient for DA neuronal induction and maturation after neural initiation [33,39]. Overall, the stromal cell co-culture method has both advantages and disadvantages in the production of DA neurons as compared with diff erentiation via EB methods. Th e co-culture method is technically simpler, and neural diff erentiation of hESCs is not only effi cient but rapid. However, the development of hPSC-derived DA neurons as clinical therapies requires effi cient manufac turing methods that will be compatible with good manufacturing practices (GMPs). Co-culture with mouse stromal cells raises regulatory concerns and makes the method less feasible compared with the EB method, which requires only growth factors. Import antly, Swis towski and colleagues [26] have demonstrated the effi cient generation of functional DA neurons from hiPSCs by using an improved EB method under fully xeno-free culture conditions. Transplants into a rat PD model with cells grown under these 'GMP-compatible' conditions resulted in behavioral improvements comparable to those reported for cells produced by using non-xeno-free culture conditions.

Dopaminergic neuron cell product purity
Although considerable progress has been made in developing diff erentiation protocols capable of yielding a high percentage of midbrain DA neurons, the cell populations generated from these protocols remain heterogeneous and may contain various percentages of non-DA neurons (for example, motoneurons and GABAergic neurons), neural progenitor cells, non-neuronal cells (for example, astrocytes and oligodendrocytes), and non-neural cells (for example, undiff erentiated cells). While TH is the most common marker of DA neurons, recent reports suggested that additional determinants such as Lmx1a and FoxA2 are important for the quality of midbrain DA neurons. A combination of these markers together with more mature or specifi c DA markers, including DAT, Girk2, and VMAT, may be useful to assess the overall quality of DA neuron preparation, as reported in 2010 by Fasano and colleagues [40] and Cooper and colleagues [41].
While the value or risks of non-DA neuronal cell 'contaminants' in grafts are unknown, the presence of undiff erentiated hPSCs or NSCs in a cell product has been reported to result in proliferative cell growths or teratoma formation [27,[42][43][44][45], suggesting that purifi cation of diff erentiated cell products may be required for clinical use. In one study, purifi cation of the cell product by cytometric sorting to remove SSEA-1 pluripotent cells was eff ective in reducing the teratoma formation observed with non-purifi ed DA cells in a rat model while maintaining behavioral recovery by purifi cation of the cell product by cytometric sorting to remove SSEA-1 pluripotent cells [43]. Along with another study [27], this study not only demonstrates the potential of hPSCderived cell products to retain cells with tumorigenic potential even after a prolonged diff erentiation process but also points to the value of a post-diff erentiation purifi cation strategy. However, the tumorigenic potential of sorted cell populations may be diffi cult to judge in PD animal models in which low cell doses are used. Additional eff orts will be required to demonstrate the tumorigenic potential of cells made by using the optimized diff erentiation process and cell doses that will be required for human trials.
Concerns have also been raised over whether current diff erentiation protocols provide effi cient patterning of DA neurons to the A9 type seen in the substantia nigra. hESC-and iPSC-derived TH-positive DA neurons transplanted into the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats survived for several months and resulted in reproducible and some behavior recovery in one study [19], but whereas 50% of the transplanted THpositive DA neurons co-expressed Girk2, as is typically seen in DA neurons of the substantia nigra, only a few DA neuron axons were seen to innervate the host striatum. Although Girk2 and TH are typically coexpressed on DA neurons of the substantia nigra, this pattern is also seen in a subset of other neurons outside the A9 region [46], suggesting that characterization of hESC-derived DA neuron populations with these markers alone, or by TH alone, is inadequate to confi rm the proper substantia nigra A9 phenotype. When transplanted cells were further analyzed, the authors noted that expression of the transcription factor FoxA2, also typically seen together with TH in the midbrain, was absent. A combination of TH and Girk2 with wellcharacterized midbrain and DA markers such as DAT, AADC, VMAT, Nurr1, Pitx3, and Lmx1b [22], along with more novel markers such as FoxA2 [19], will be useful in the development of diff erentiation methods and to determine the optimal phenotype of neurons to be transplanted.

Induced pluripotent stem cells
In addition to having characteristics similar to hESCs and avoiding the ethical and regulatory dilemmas associated with hESCs, iPSCs off er the potential to provide patientspecifi c cell replacement therapies. Although the requirement for immunosuppression with hESC-derived cell replacement therapy for PD is not clear, patient-specifi c iPSC products may avoid the immunorejection problems commonly associated with allogeneic transplants. In addition, iPSC lines have been reported, in some cases, to retain an epigenetic 'memory' of their cells of origin [47] that may skew their diff erentiation potential toward specifi c fates [48], although the durability of this memory over extended culture periods is not clear. While iPSC memory may be a potential disadvantage for some research applications, a diff erentiation bias of an allogeneic iPSC line to a neural progenitor fate might provide higher yields or greater purity following diff erentiation. Improved diff erentiation effi ciency would facilitate the scale of manufacture that will be required for clinical studies.
Although there may be immunological advantages to the use of autologous iPSC-derived DA neurons, there are also potential pitfalls. Th e time and cost required to produce an autologous iPSC-derived DA product may be prohibitive. In addition, cells derived from patients with familial forms of PD carrying PD mutations might need to be genetically corrected before transplantation back to the patients, and this requires additional regulations, safety testing, and costs. Extensive safety testing of autolo gous iPSC-derived cell products may not be practical and could result in increased risk of adverse events. As we learn more about the mechanism of the PD disease process, we will be able to determine whether this type of cell population is suitable for disease applications.
Eff orts to pursue an iPSC alternative to hESCs for PD are under way. An initial report of motor recovery in a PD rat model after transplantation of diff erentiated cells from a mouse iPSC line [43] was followed by the report of the successful generation of iPSC lines derived from patients with PD [20]. Subsequently, several groups reported on the successful generation of midbrain DA neurons with hiPSC-and PD-derived iPSCs [19,41]. Recent reports describe hiPSC-derived DA neuron cell survival and engraftment as well as behavioral improvements in PD rat models [42,49,50]. Th ese reports demonstrate that midbrain DA neurons can be successfully generated from iPSCs and that these cells can function properly.
Although there are several advantages of iPSCs, there are concerns about chromosomal aberrations and epigenetic modifi cations that have been reported to occur during reprogramming and that may be maintained through diff erentiation [47,51]. It is not clear what the eff ect of these mutations and aberrations will be on the activity or safety of diff erentiated cells or whether they will persist after extended culture of an iPSC line [52], but there is a clear need for a better understanding of these cells before they are used in clinical trials. In addition, the diff erentiation potential of iPSC lines may change over time in culture, requiring careful charac ter ization of diff erentiated cell products to ensure consistency and reproducibility in animal studies [53]. It is clear that a better understanding of these cells will be essential before they are used in clinical trials.

Conclusions
A number of groups have made considerable progress in demonstrating that authentic A9 DA neurons can be produced in vitro with high effi ciency from hESC and iPSC lines, including lines derived from patients with PD. Several of these groups have also shown survival and engraftment of these cells in the 6-OHDA rat model as well as evidence of dopamine release and correction of behavioral defi cits. Th ese studies, combined with the proof-of-concept provided by human trials using fetusderived DA neurons, provide compelling evidence to justify the development of these cells for use in human clinical trials. However, there are outstanding issues to be resolved. In many cases, the behavioral improvements observed in animal studies are modest, and there is currently limited evidence that complex motor defi cits can be corrected with engrafted hPSC-derived DA neurons [27]. Th is may be due to the variable or low number of cells in the grafts, limitations of the rodent PD models, or diff erences in the micro environmental signals between human and rat cells in the brain or a result of infl uences from contaminating non-A9 DA cells in the graft. Better animal model systems would facilitate the development of these cells, and predictive long-term effi cacy studies with non-human primate models such as the MPTP monkey model will be of great value. In addition, other issues need to be addressed, including the low survival rate of DA neurons in grafts, diffi culty in integrating transplanted cells into the host brain's circuitry, defi ning the number of DA neurons needed for a transplant (Freed and colleagues [14] suggested that at least about 20,000 cells are needed for transplantation), site of injection, and graft-induced dyskinesia. Some of these issues, such as cell number or dose, injection site, and cell survival, may not be resolved without information from human clinical studies, but it is certain that addressing these issues will require properly controlled animal and human studies using a wellcharacterized cell product made with defi ned protocols and reagents.