Progress on stem cell research towards the treatment of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive accumulation of Lewy body inclusions along with selective destruction of dopaminergic (DA) neurons in the nigrostriatal tract of the brain. Genetic studies have revealed much about the pathophysiology of PD, enabling the identification of both biomarkers for diagnosis and genetic targets for therapeutic treatment, which are evolved in tandem with the development of stem cell technologies. The discovery of induced pluripotent stem (iPS) cells facilitates the derivation of stem cells from adult somatic cells for personalized treatment and thus overcomes not only the limited availability of human embryonic stem cells but also ethical concerns surrounding their use. Non-viral, non-integration, or non-DNA-mediated reprogramming technologies are being developed. Protocols for generating midbrain DA neurons are undergoing constant refinement. The iPS cell-derived DA neurons provide cellular models for investigating disease progression in vitro and for screening molecules of novel therapeutic potential and have beneficial effects on improving the behavior of parkinsonian animals. Further progress in the development of safer non-viral/non-biased reprogramming strategies and the subsequent generation of homogenous midbrain DA neurons shall pave the way for clinical trials. A combined approach of drugs, cell replacement, and gene therapy to stop disease progression and to improve treatment may soon be within our reach.

however, we have begun to understand why the A9 subtype is more vulnerable to degeneration. Guzman and colleagues [5] showed that A9 (not A10) DA neurons engaged plasma membrane L-type calcium channels throughout the pacemaking cycle. Knocking out DJ-1 (PARK7) down regulates the expression of uncoupling proteins, com pro mises calcium-induced uncoupling, and increases oxida tion of matrix proteins specifi cally in A9 neurons. Th ere fore, A9 neurons are dying of high oxidative stress due to high calcium fl uxes [5]. As PD is associated with the destruction of the A9 neurons located in the nigrostriatal tract, a straightforward approach to cure the disease may be to generate A9 DA neurons to reconstruct and provide reinnervation to the striatum.

Stem cell grafts in patients with Parkinson's disease and in animal models
Although early clinical trials were limited in size and number, they did highlight the therapeutic potential of stem cells for neurodegenerative diseases. In 1995, Kordower and colleagues [6] grafted fetal mesencephalic tissue harvested from a total of seven human embryos (at ages of 6.5 to 9 weeks) into the post-commissural putamen of a patient with PD. Up to 18 months after the procedure, not only did these bilateral grafts survive and remain viable but also there was marked DA reinnervation in the striatum. It was observed, through a series of positron emission tomography (PET) scans, that fl uoro dopa uptake increased markedly after 6 and 12 months, respectively, and this would refl ect improved neuronal function in the region surrounding the transplanted tissue. Th e transplant recipient exhibited motor abilities and considerable improvement in the Unifi ed Parkinson's Disease Rating Scale (UPDRS) test [6]. A similar trial reported further clinical benefi t and provided the opportunity for complete withdrawal of L-DOPA (L-3,4-dihydroxyphenylalanine) treatment [7]. However, some other studies had poorer clinical responses [8,9], and a smaller number of grafted DA neurons or severe stages of the disease or both were considered to be the causes [10]. More recently, an improvement was ascertained in subpopulations of these same patients upon post hoc analysis [11].
Th us, the long-term eff ects of stem cell transplantation therapy have been diffi cult to evaluate because of a combi nation of limited clinical trials, the typically late onset of the disease, and the fact that clinical follow-ups have been conducted mostly within an 18-month period [11]. Piccini and colleagues [7] found that, 10 years after transplantation, there was continuous benefi t, and the patient had no rigidity but only 'minor hypokinesia' . However, Kordower and colleagues [11] analyzed the longest-surviving transplant patient, 14 years after the operation, and observed a low UPDRS in the fi rst 10 years, but the patient experienced gait problems, diffi cult balancing, and falls from 11 years after transplantation. On post-mortem analysis, the grafts were found to have Lewy body-like structures, staining positively for α-synuclein and ubiquitin, strongly suggestive of a PD progression in the patient after transplantation [11]. Th ese fi ndings raise the possibility that transplanted grafts are not invincible to damage by PD progression. Side eff ects were also associated with fetal mesencephalic grafts, and dyskinesia was a particular problem [8,9]. Additionally, fetal grafts have not been able to fully reconstruct the nigrostriatal tract [10], highlighting the need for diff erentiated A9 DA neurons (rather than neural stem cells from fetal brain) for the therapy of PD [4].
Th e limited availability of human embryonic tissue for transplantation has driven researchers to investigate alternative sources of stem cells. For example, adult mesen chymal stem cells were exploited in an MPTP mouse model of PD [12]. Five weeks after transplantation, 5-bromo-2'-deoxyuridine (BrdU)-labeled mesenchymal transplants were reported to express tyrosine hydroxylase (TH), the rate-limiting enzyme of DA synthesis. Mice had signifi cantly improved performance on the rotarod test. Uncommitted neural stem cells from the subventricular zone of adult brain were extracted and investigated for TH neuronal diff erentiation [13]. Th is line of research may provide information for promoting endogenous neurogenesis but may not be realistic for supplying donor cells for cell replacement therapy.
Establishment of human embryonic stem (hES) cells, with their unlimited diff erentiation potential, off ers unequivocal prospects for regenerative medicine [14]. Before hES cells can be considered clinically, we must demonstrate that they provide long-term improvements in motor function and mobility in addition to alleviating symptoms of drug resistance in animal models. Th e most commonly used PD models in animal trials are generated by using 6-hydroxydopamine (6-OHDA), a neurotoxin that selectively induces exten sive degeneration of striatal DA neurons via apoptotic and necrotic pathways in rodents [15][16][17][18][19][20][21][22]. Th e success of the transplantation experiments is measured by behav ioral improvements in the amphetamine-or amorphine-induced rotation behavioral test, adjusting step test, the cylinder test, and the paw-reaching test, along with immunohistochemical evidence for the survival and integration of grafts within host brains [15,16,20].
Th e therapeutic potential of ES cells has been refl ected in numerous animal trials, which are largely encouraging. Björklund and colleagues [23] demonstrated that ES cells injected stereotactically into the striatum of rat models of PD diff erentiated into DA neurons spontaneously, along with the development of some 5-HT + neurons that were shown to increase synaptic DA release [24]. PET and magnetic resonance imaging, in addition to histological examination at the endpoint of animal trials, revealed the integration of the ES cell-derived neurons. Th e resulting regenera tion of DA neural networks in the striatum corre lated with an improvement of the rat models in behavioral tests [23]. In a sham-controlled trial, Kim and colleagues [20] (2002) reported that rats grafted with ES cell-derived DA neurons showed signifi cant improvements in several behavioral tests and that the cells exhibited electrophysiological properties typical of midbrain DA neurons. Similar results were elicited when hES cell-derived DA neurons were enriched by co-culture with immortalized midbrain astrocytes and grafted into 6-OHDA-lesioned rats [15]. Transplantation of grafts composed mainly of the A9 subtype DA neurons (GIRK2 + ) led to substantial functional improvement [15]. In contrast, Brederlau and colleagues [17] (2006) observed no improvement in the motor symptoms of 6-OHDA-lesioned rats grafted with hES cell-derived cells. Th is was interpreted as being caused by the lack of suffi cient TH + cells from a stromal cell-derived inducing activity (SDIA) protocol [17]. Th e number of DA neurons is indeed reported to correlate directly with the outcome of behavior [16]. However, concerns surrounding immune rejection of the grafts and ethical issues together with a shortage of supply have severely curtailed investigation of hES cells for clinical applications.
A groundbreaking technology has recently emerged in the fi eld of regenerative medicine. Takahashi and colleagues showed that fi broblasts harvested from either mice [25] or humans [26] can be converted into induced pluri potent stem (iPS) cells in culture via viral transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc. Th ese iPS cells make it possible to bypass hES cells, to treat patients with their own somatic cell-derived stem cells (Figure 1), and (in theory) to avoid immune rejection caused by patient-donor cell incompatibility [18]. iPS cells have been shown to display properties similar to those of ES cells [22,25]. For example, Swistowski and colleagues [22] compared iPS cells with hES cells and concluded that the two had similar genomic stability, transcription profi les, pluri potency, and DA neuron diff erentiation capacity. Several groups have succeeded in generating DA neurons from iPS cells independently [18,21,22,27]. iPS cell-derived DA neurons were shown to integrate into the striatum of parkinsonian rats with behavioral improve ments comparable to those observed using ES cell-derived DA neurons [18,21,22].
As in the case of ES cells, teratoma may be formed if iPS grafts are not fully diff erentiated [18]. Brederlau and colleagues [17] (2006) found that the time spent during in vitro pre-diff erentiation via the SDIA method could make a noteworthy impact on teratoma formation: rats developed severe tumors with hES cell grafts prediff erentiated for 16 days; extending this length of time to approximately 20 to 23 days in culture resulted in teratoma-free rats mostly. When SSEA1 + (a marker for pluripotent stem cells) cells were removed from mouse iPS cell-derived neurons by cell sorting, no teratoma was formed in any of the rats 8 weeks after transplantation [18]. Th erefore, the safety issue can be alleviated if homogenously post-mitotic cells are made prior to transplantation.

Protocols used to generate midbrain dopaminergic neurons
In light of the fact that hES cell diff erentiation favors a telencephalic fate, protocols have been designed to direct stem cell diff erentiation toward a mesencephalic fate [27]. Currently, there are two main methods to generate midbrain DA neurons: using stromal cell-derived feeder cells or defi ned culture media. A stromal cell-derived feeder line, PA6, from mouse skull bone marrow was found to promote DA neuron generation from hES cells [28]. However, the molecular nature of the SDIA is still unknown [29]. SDIA directs stem cells to become neural precursors, which then go through regional specifi cation with fl oor plate-derived sonic hedgehog (SHH) and fi broblast growth factor 8 (FGF8). Wernig and colleagues [18] (2008) found that, once these factors were withdrawn, most cells diff erentiated into Tuj + neurons but that only a small fraction became TH + DA neurons. However, the proportion of TH neurons generated was increased with the length of the time they spent in culture [19].
Perrier and colleagues [30] described a protocol to produce approximately 24% to 40% of TH + neurons from ES cells in 6 weeks by culturing clusters of rosettes in stromal feeder conditions with SHH, FGF8, glial cell linederived neurotrophic factor (GDNF), dibutyryl cAMP, and transforming growth factor beta 3 (TGFβ3). Vazin and colleagues [31] shortened the protocol to 1 month and the outcome was similar. Th ey co-cultured hES cells with PA6 cells for 12 days and further diff erentiated them for 18 days with SHH, FGF8, and GDNF, and 34% of cells became TH + . However, more effi cient homogenous DA neuron production is desired. Stromal feeder cells are of animal origin and may retain xenogenic factors such as mouse antigens or pathogens or both, and these concerns prevent their use in clinical applications.
Effi cient protocols have been developed for the derivation of DA neurons from hES cells by using defi ned culture media. For example, Cho and colleagues [19] developed a feeder-free method, with which 67% of hES cells became TH + DA neurons. Th is protocol involves a number of steps. After the formation of embryoid bodies (EBs) through the culture of hES cells in a non-adherent culture dish for 7 days, the EBs are transferred to a Matrigel-coated dish and cultured with 0.5% N 2 supplement for 5 days to select for neural precursors. At this point, basic fi broblast growth factor is added to the culture for 14 days to promote the formation of spherical neural masses, which are transferred to a Matrigel-coated dish and incubated in defi ned diff erentiation media. Growth factors SHH and FGF8 are added to the medium for 10 days to promote neuronal induction and subsequently the cells are incubated with ascorbic acid for a further 6 days to promote DA maturation. Th is protocol has proven to be quite successful in the generation of DA neurons; 77% of the hES cells became neurons (Tuj + ), and 86% of Tuj + cells became TH + DA neurons [19].
TH is a rate-limiting enzyme in synthesizing dopamine and is an important marker for localizing DA neurons in the brain. However, TH marker alone may not be specifi c enough if A9 specifi c DA neurons are to be generated for the treatment of PD [3] since correct transcription factor expression is essential to the maintenance, diff erentiation, and survival of the DA neurons throughout their development [3]. At the progenitor stage, neural pre cursor cells are known to express Otx2, Lmx1a/b, Engrailed 1/2, Msx1/2, Neurogenin 2, and Mash1 [3]. As they mature, these cells continue to express En1/2 and Lmx1a/b but also start to express nuclear receptor-related 1 protein (NURR1) and pituitary homeobox 3 (PITX3). NURR1 (or NR4A1) is a member of the steroid/thyroid hormone/ retinoid receptor superfamily and critical for DA maintenance, whereas PITX3 is a paired homeodomain transcription factor that is important for TH expression and survival of SNpc A9 DA neurons [3]. It is unknown whether SNpc A9 and VTA A10 progenitors diff er at the progenitor stage. Th e earliest distinction within midbrain DA development appears to be that ventro-lateral DA neurons express PITX3 before TH, whereas dorso-medial ones express TH before PITX3 [3]. Subsequently, A9 neurons also express GIRK2 specifi cally whereas A10 neurons express calbindin-D 28K [15].
Cooper and colleagues [27] (2010) reported that another transcription factor, FOXA2, a key marker of fl oor plate development, is required to specify and maintain ventral DA phenotype. Previous protocols were not able to generate FOXA2 + cells. An early exposure to retinoic acid improved regional specifi cation and -in combination with a high activity of SHH, FGF8a, and WNT1 -gave 'robust diff erentiation' of FOXA2 + DA neurons [27]. Fasano and colleagues [32] showed that early high-dose SHH (125 to 500 ng/mL) could also induce FOXA2 expression for success ful midbrain DA neuron derivation from hES cells. Kriks and colleagues [33] used a fl oor plate-based strategy to obtain engraftable midbrain DA neurons that coexpressed TH with FOXA2, PITX3, and NURR1. Th e in vivo survival and function of the neurons were demonstrated in mouse, rat, and monkey PD hosts. Th is shows, for the fi rst time, that hES cell-derived transplants may be feasible.
Additionally, evidence has emerged that post-transcriptional and post-translational modifi cations play a role in DA neuron phenotype. For example, the leucine-rich repeat kinase 2 (LRRK2) gene is frequently mutated in PD, and LRRK2 phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein (4E-BP). 4E-BP is a translation inhibitor and its chronic inactivation by mutant LRRK2 deregulates protein translation, eventually resulting in loss of DA neurons [34]. When the Droso phila homolog of 4E-BP, Th or, is overexpressed, it imposes a limit on DA neuron loss in Parkin and Pink1 mutant fl ies [35]. Pharmacological activation of 4E-BP by rapamycin also prevents parkinsonian DA neuron loss [35].
Micro-RNAs (miRNAs) have also been implicated in DA development. Th ese non-coding 18-to 25-base mRNAs regulate gene expression post-transcriptionally by binding to specifi c mRNA targets, leading to mRNA degradation or translational inhibition. Dicer is an enzyme critical for miRNA biosynthesis from larger transcripts. When Dicer is conditionally knocked out in mice by Wnt1 promoter-driven Cre recombinase, it produces deformities of the midbrain, cerebellum, and mandible and almost complete elimination of midbrain TH neurons together with a lack of miR-9, miR-124, and miR-218 expression [36]. Th is highlights the importance of miRNAs in DA neuron production. Signifi cantly, using quantitative polymerase chain reaction (PCR), Kim and colleagues [37] demonstrated that a particular miRNA, miR-133b, is specifi cally expressed in midbrain DA neurons and is downregulated in the midbrain of patients with PD. Th is causes the loss of nigrostriatal DA neurons because miR-133b normally functions to repress PITX3 expression as part of a feedback loop. Two other miRNAs, miR-7 and miR-153, are involved in maintaining the α-synuclein level [38,39], and accumulation of αsynuclein is the main pathological feature of PD. Th ese miRNAs bind specifi cally to the 3' untranslated region of SNCA mRNA and downregulate production of α-synuclein protein. Th e repression of α-synuclein by miR-7 has been shown to be protective against oxidative stress and apoptosis of DA neurons in the striatum [39]. Th ese studies suggest that regulation at post-trans crip tional and posttranslational levels may represent viable therapeutic approaches for PD. An understanding of miRNA involvement in the maintenance of neurons is critical to the use of stem cell-derived DA neurons as a viable therapy for patients with PD.

Direct reprogramming of dopaminergic neurons from somatic cells
Recent research showed that somatic mouse cells can be converted directly to other cell types (that is, DA neurons) by expressing defi ned transcriptional factors. Using a two-step approach, Pfi sterer and colleagues [40] fi rst converted human embryonic fi broblasts, fetal lung fi broblasts, or post-natal fi broblasts into neurons by overexpression of Mash1 (also known as Ascl1), Brn2, and Myt1l in lentiviral vectors. Th e converted neurons were subsequently directed to become DA neurons with expression of Lmx1a and FoxA2. In addition, Caiazzo and colleagues [41] showed that three transcription factors -Mash1, Nurr1, and Lmx1a -were able to reprogram mouse and human fi broblasts directly into functional DA neurons, which release dopamine and exhibit regular electrical activity. Th is can be done by using prenatal and adult fi broblasts of healthy donors or of patients with PD [41]. Subtype-specifi c induced neurons derived from human somatic cells can be valuable for disease modeling and cell replacement therapy. However, this approach has limitations. Genetic modifi cation is needed to introduce the defi ned set of transcription factors. Th e number of neurons that can be generated is strictly dependent on the number of initial fi broblasts from the donor and the effi ciency of direct conversion. Th e capability of directly converted neurons in ameliorating the phenotype in animal models remains to be seen. Nevertheless, the whole process does not proceed via a pluripotent cell inter mediate, and one may speculate that it may off er a reduced risk of tumor formation in transplantation.

Refi nement of induced pluripotent stem cell technology
Since the publication of the fi rst iPS cell generation in 2006, substantial progress has been made to improve the technology. To reduce multiple chromosomal integration sites associated with the initial four retroviral vectors, a single lentiviral reprogramming vector was developed to fuse them into a single open reading frame via selfcleaving 2A sequences [42]. Continuous expression of transgenes in iPS cells (even at low levels) might induce tumor formation in vivo [43] or alter diff erentiation potential. Soldner and colleagues [44] (2009) then developed a Cre recombinase excisable system to remove transgenes after reprogramming via doxycycline-inducible lentiviral transduction. Non-viral methods have been developed for mouse iPS cell generation. Kaji and colleagues [45] (2009) replaced viral vectors with a single plasmid vector expressing the four reprogramming factors linked with 2A peptides. Surprisingly, many iPS colonies diff erentiated spontaneously after Cre recombinasebased removal of the reprogramming factors. Co-transfection of two piggyBac transposons enhanced stable trans fection effi ciencies of human fi broblasts [45]. Th e problem of leftover sequence residues remains.
Non-integrative approaches were subsequently reported. Okita and colleagues [46] (2008) generated iPS cells at a low effi ciency by repeated transfection of two circular plasmid vectors, and as a result of the approach, most iPS clones were free of plasmid integration. Similarly, Yu and colleagues [47] (2009) took advantage of the Epstein-Barr virus to generate iPS cells free of vector or transgene sequences. OriP/EBNA1 from the Epstein-Barr virus functions as stable extrachromosomal replicon and replicates plasmid once per cell cycle under selection. In the absence of drug selection, the episomes are lost at a rate of approximately 5% per cell genera tion because of defects in plasmid synthesis/partitioning. However, this system required three plasmids carrying seven factors, including SV40 large T antigen. It has not been shown to work with adult fi broblasts yet, and expression of the EBNA1 protein may raise concerns of immune rejection if the vector is retained in the reprogrammed cells [46].
An increased expression of reprogramming factors should improve iPS effi ciency, and removal of plasmid vector sequence could greatly increase transgene expression in mammalian cells. Jia and colleagues [48] (2010) took advantage of the øC31-based intramolecular recombi nation system to produce minicircle DNA expressing OCT4, SOX2, LIN28, and NANOG under a CMV promoter. Th e presence of an inducible phage øC31 integrase gene and attB/attP sites enables the generation of two circular plasmids: a minicircle reprogramming cassette and a plasmid back bone. Th e latter is linearized and degraded in bacteria. Th us, minicircle DNA can be purifi ed and repeatedly transfected into somatic cells. Jia and colleagues [48] (2010) used this to generate iPS cells from human adipose cells, and the overall reprogramming effi ciency was approxi mately 0.005%. Th is is approximately half that of typical viral methods (approximately 0.01%) but signifi cantly higher than that of other plasmid vectors [48].
Because of the oncogenic potential, Klf4 and c-Myc were replaced by Nanog and Lin28 [49]. iPS cells could still be generated from mouse and human fi broblasts without c-Myc but at a severely reduced effi ciency [50]. Furthermore, Kim and colleagues were able to reprogram adult mouse [51] and human fetal [52] cells with Oct4 only. Unfortunately, overexpression of Oct4 [53] and Klf4 [54] could be linked with dysplasia. Th erefore, non-DNA approaches are sought to overcome the hurdles in iPS cell derivation. Supplementation of transcription factors to the culture medium has been tried out [52,55,56]. Transportation of the transcription factors from medium to cytoplasm and to nuclei was essential, and the reprogram ming factors were therefore engineered to fuse with a poly-arginine transduction domain for human proteininduced iPS cells [52,56]. Th is method eliminates the risk of genetic modifi cation but is very time-consuming. However, Rhee and colleagues [57] (2011) were able to generate DA neurons from protein-induced iPS cells in affl uent quantities, and DA neurons were robust in survival compared with virus-derived human iPS cells and also produced prominent behavioral improvements in 6-OHDA-lesioned rats. Meanwhile, substantial progress has been made in improving iPS cell production by using small molecules. A cocktail of compounds, including PD0325901 (MEK inhibitor), CHIR99021 (GSK-3 inhibitor), A83-01 (TGFβ type I receptor ALK-5 inhibitor), and HA-100 (ROCK inhibitor), was shown to increase the reprogramming effi ciency of an episomal vector by a factor of approximately 70 [58]. A kinase inhibitor, kenpaullone, can substitute for Klf4 [59]. E616452 and valproic acid -a TGFβ receptor ALK4/5/7 inhibitor and a histone deacetylase (HDAC) inhibitor, respectively -have also been used eff ectively to replace Sox2 [60]. Excitingly, a single molecule, RG108, a DNA methyl transferase inhibitor, is suffi cient to reprogram mouse myoblasts with 5 days of treatment [61]. Whether human iPS cells can be made by using only small molecules remains a question.

Disease 'in a dish'
One of the advantages of iPS cells and ES cells is that they off er opportunities to visualize disease progression in vitro, which otherwise may be diffi cult to observe, by comparing neurons from healthy donors with those derived from patient cells (Figure 1). It is not well understood how the defective proteins aggregate in PD or indeed how they relate to the oxidative stress and specifi c cell destruction during PD progression [62]. Except in rare forms of defi ned genetic causes that constitute 5% to 10% of the incidence of PD, the etiology of the disease is not fully understood [63]. Genetic alterations can be made in ES/iPS cells by knockout, knocking in, inducible expression, or overexpression to mimic the genetics in patients. Th is will allow investigation of the disease progression of the neurological conditions in vitro.
Derivation of iPS cells from patients with known genetic defects can avoid genetic manipulation of iPS/ES cells, which is time-consuming and sometimes technically challenging. PD can be caused by defects in many loci, such as SNCA encoding α-synuclein, LRRK2 encoding leucine-rich repeat kinase 2, Parkin (or PARK2) encoding the ubiquitin E3 ligase, PINK1 for PTENinduced kinase 1, and DJ-1 encoding a protein of the peptidase C56 family [64]. Availability of iPS cells from these patients will enable researchers to characterize eff ects of individual genetic components and to enhance the current understanding of PD pathophysiology. Th ese iPS cells should incorporate all aspects of the pathophysiology and thus, in theory, should generate disease models that are more accurate than those using 6-OHDA or MPTP to destroy DA neurons.
Heterogeneity of iPS cell lines can be a problem in phenotyping iPS cells. Soldner and colleagues [65] (2011) have sought to fi nd an eff ective way of studying PD progression by using zinc-fi nger nuclease-mediated genome-editing technology to generate ES/iPS cells carrying A53T (G209) or E46K (G188A) point mutations in the SNCA gene. Because of the slow progression of the pathological cellular events and late onset of the disease, the in vitro phenotype may be diffi cult to recognize if it is not carefully compared with closely matched controls. A lack of genetically matched controls (that is, non-aff ected sibling donors) could make it more diffi cult to determine whether the changes are relevant to the prevalent disease phenotypes, background, incomplete penetrance, age of onset, or nature of disease progression [65]. To ensure that the point mutations were the only modifi ed variable in their study, Soldner and colleagues [65] either derived iPS cells from patients carrying the mutations and corrected them genetically as controls or produced the point mutations in wild-type hES cells.
However, it is encouraging that disease-related phenotypes are recaptured in iPS cell-derived neurons in some cases. For example, Devine and colleagues [66] (2011) generated multiple iPS cell lines from SNCA triplication patients. When these iPS cells were diff erentiated into midbrain DA neurons, patient-derived cells expressed increased α-synuclein with relatively lower levels of paralogous proteins SNCB and SNCG. Th is precisely recapitulated the situation in these individuals [66]. Nguyen and colleagues [67] (2011) generated iPS cells carrying the most common PD-related G2019S mutation of the LRRK2 gene. Neurons from the mutated iPS cells express increased α-synuclein and oxidative stressresponse proteins MAO-B and HSPB1. Th ese neurons are also more susceptible to caspase-3 activation and cell death when exposed to various stress agents (for example, MG132 and 6-OHDA) which are known to induce DA degeneration [67]. Such reprodu cible phenotypes from aff ected iPS cell-derived neurons will off er opportunities to examine disease progression in vitro as well as to use them as cellular models for screening compounds that may reverse the pathological phenotypes.

Limitations and practicalities of induced pluripotent stem cells
An overwhelming number of publications demonstrate that iPS cells are similar to ES cells, and both can be diff erentiated into cell types of three germ layers.
However, some recent studies suggest that there may be subtle diff erences between them. For example, in a comparison study, iPS cells were found to be less fl exible in their diff erentiation capacity: only the blood-derived iPS cells showed potential in hematopoietic diff eren tiation, whereas the fi broblast-derived iPS cells favored the osteogenic route [68]. Feng and colleagues [69] (2010) used an SDIA protocol to compare the growth and diff erentiation of both hES and iPS cells down a hematopoietic lineage. Th e phenotype and morphologies of the iPS cells were largely the same, but the iPS cells were 'extremely limited' in diff erentiation, expansion, and ability to form hematopoietic colonies with a greater tendency toward apoptosis [69]. Another study demonstrated that iPS cells would retain a transient epigenetic memory of their somatic origin and that this retention might limit their diff erentiation fates [70]. Th is is thought to be due to the gene expression of their cell origin and epigenetic mechanisms, including DNA methylation. How to modulate or erase this memory is not yet known [71], but continuous passaging of iPS cells appears to attenuate these diff erences [69]. We speculate that the use of small molecules such as inhibitors of HDAC or of DNA methyltransferase or both may help to erase the epigenetic memory and overcome some of these limitations [60,61].
Immune rejection of iPS cells was one unexpected complication in animal trials. Th e widely held assumption was that autologous cells would be immune-compatible and therefore protected from attack by the recipient's immune system. Zhao and colleagues [72] (2011) used teratoma formation in the same strain of mice for immune-compatibility and showed that teratomas formed by retrovirally generated iPS cells from C57BL/6 mouse fi broblasts were widely immune-rejected by C57BL/6 recipients. Th e study has yet to be replicated, and the causes of the immune rejection are unknown. However, the immune response was less severe when a non-integrative episomal approach was used to create the iPS cells. It was speculated that rejection of autologous cells might be due to abnormal gene expression or mutations during iPS cell generation or both [72]. Nevertheless, the immunogenicity of patient-derived iPSCs should be evaluated before autologous transplantation. Th ere may be other practical issues to be considered before clinical application, such as whether donors shall be given the ability to withdraw their cells from study or whether they can exercise their reach-through rights for control or fi nancial gain [73]. Regardless of these questions, scientists have already begun to collect skin biopsies from people with familial PD mutations for iPS cell generation [22]. Diff erent genes may be altered in diff erent individuals, and the establishment of iPS cells from diff erent patients is the fi rst step toward patienttailored medicine.

Conclusions
It is clear that stem cell research has taken huge leaps in recent years, and the treatment of degenerative diseases such as PD may one day have the ability to slow down, halt, or even reverse functional decline by eff ectively regenerating the damaged cells or tissues. ES and iPS cell-derived DA neurons have had promising outcomes in animal trials, although there will be more challenges to overcome to make their use clinically safer. Th e current protocols are constantly being refi ned, and progress toward generating homogenous midbrain DA neurons in vitro is ongoing. It is likely that iPS cells may overtake the race of hES cells in clinical application. Th e use of patient-specifi c iPS cells in lieu of the current therapies, which simply serve to mask symptoms in many instances, may soon become a reality.