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Meeting Reports

Proceedings: Cell Therapies for Parkinson’s DiseaseFrom Discovery to Clinic

ROSA CANET-AVILES, GEOFFREY P. LOMAX, ELLEN G. FEIGAL, CATHERINE PRIEST

California Institute for Regenerative Medicine, San Francisco, California, USA

Key Words. Parkinson’s disease x Cell therapies x Clinical trials x Experimental models

ABSTRACT

In March 2013, the California Institute for Regenerative Medicine, in collaboration with the NIHCenter for RegenerativeMedicine, held a 2-day workshop on cell therapies for Parkinson’s disease(PD), with the goals of reviewing the state of stem cell research for the treatment of PD and dis-cussing and refining the approach and the appropriate patient populations in which to plan andconduct new clinical trials using stemcell-based therapies for PD.Workshopparticipants identifiedpriorities for research, development, and funding; discussed existing resources and initiatives; andoutlined a path to the clinic for a stem cell-based therapy for PD. A consensus emerged among par-ticipants that the development of cell replacement therapies for PD using stem cell-derived prod-ucts could potentially offer substantial benefits to patients. Aswith all stem cell-based therapeuticapproaches, however, there are many issues yet to be resolved regarding the safety, efficacy, andmethodology of transplanting cell therapies into patients. Workshop participants agreed that de-signing an effective stem cell-based therapy for PDwill require further research and developmentin several key areas. This paper summarizes the meeting. STEMCELLS TRANSLATIONALMEDICINE

2014;3:979–991

INTRODUCTION

InMarch2013, theCalifornia Institute forRegener-ative Medicine (CIRM), in collaboration with theCenter for RegenerativeMedicine of the NIH, helda 2-day workshop on cell therapies for Parkinson’sdisease (PD), with the goals of reviewing the stateof stemcell research for the treatment of PD anddiscussing and refining the approach and theappropriate patient populations in which toplan and conduct new clinical trials using stemcell-based therapies for PD.

Thegroupcomprisedapproximately50scien-tists, clinicians, cell manufacturers, clinical trialand regulatory experts aswell asmembersof bio-technology and pharmaceutical industries andfunding agencies and patient advocates (a listof attendees is shown in supplemental onlineTable 1). Pioneers and global leaders in the fieldpresented their work and discussed the chal-lenges and opportunities they have encounteredin bringing experimental cell therapies for PD tothe clinic.

Workshop participants identified prioritiesfor research, development, and funding; dis-cussed existing resources and initiatives; andoutlined a path to the clinic for a stem cell-based therapy for PD. A consensus emergedamong participants that the development ofcell-replacement therapies for PD using stem

cell-derived products could potentially offersubstantial benefits to patients. As with all stemcell-based therapeutic approaches, however,many issues have yet to be resolved regardingthe safety, efficacy, and methodology of trans-planting cells as therapies for patients. Work-shop participants agreed that designing aneffective stem cell-based therapy for PD will re-quire further research and development in thefollowing areas:

c Identifying thebest cell types touseas sourcesfor the cellular therapeutic in future clinicaltrials (e.g., human induced pluripotent stem[hiPS] cells, human embryonic stem cells[hESCs], adult stem cells) and the degree ofcell differentiation and purification requiredbefore transplantation;

c Optimizing conditions for engraftment, sur-vival, and integration with existing neural cir-cuitry in vivo;

c Understanding the ongoing glial and neuroin-flammatory effects of progressive PD on the lo-cal environmentand theeffectson transplantedneurons and discussing the optimal immuno-therapy needed to support the grafted cells;

c Standardizing the protocols and processes foroptimal cell manufacturing;

c Developing standard surgical methods forcell transplantation that accurately and

CIRM was established in Novem-ber 2004 with the passage ofProposition 71, the CaliforniaStem Cell Research and Cures Act.The statewide ballot measure,which provided $3 billion in fund-ing for stem cell research at Cali-fornia universities and researchinstitutions, was overwhelminglyapproved by voters, and called forthe establishment of an entity tomake grants and provide loans forstem cell research.

The Proceedings of the CaliforniaStem Cell Agency is a monthlyseries of commentaries, articles,interviews, webinars, forums, andconcise reviews on a wide range oftopics in regenerative medicine.

Correspondence: Rosa Canet-Aviles, Ph.D., Neuroscience,Research Partnerships, Foundationfor the National Institutes ofHealth, 9650 Rockville Pike,Bethesda, Maryland 20814, USA.Telephone: 301-402-5346; E-Mail:[email protected]

Received July 24, 2014; acceptedfor publication July 28, 2014; firstpublished online in SCTM EXPRESSAugust 22, 2014.

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http://dx.doi.org/10.5966/sctm.2014-0146

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effectively distribute the cellswhile causingminimal damage tothe target region of the brain;

c Developing biological assays to assess the survival and efficacyof transplanted cells in vivo;

c Designing new functional clinical assessments to evaluate thesuccess of potential interventions; and

c Identifying the most relevant considerations for the design ofsuccessful clinical trials (e.g., patient population, endpoints,monitoring, length of follow-up).

Breakout sessionsprovideda forumforparticipants todiscussall these points and to suggest additional opportunities for whichsynergy of efforts could improve the probability of success inbringing stem cell therapies to the clinic. Although participantsagreed that collaboration is crucial for success in the field, theyalso indicated that supporting multiple approaches might in-crease the chance that a stem cell therapy for PD will translateto the clinic. The regulatory approval of a safe and effective prod-uct must first advance down the development pathway througha series of clinical trials. The workshop identified eight critical ini-tial steps to facilitate development of a phase I/II clinical trial forstem cell therapy.

First, define the cell source. Either hiPS cells or hESCs couldbe used in the first stem cell trial. hESCs provide a more ben-eficial commercial model because a single hESC line can bebanked in large quantities to treat multiple patients. hiPS cellscan be autologous or allogeneic. Ideally, pluripotent stem cellbanks should be matched to patients to minimize immune ef-fects. Experiments to evaluate the different cell sources areunder way, and several approaches are under investigation. Cur-rently the effort is focused on prioritizing the different ap-proaches and moving forward into clinical trials with thehighest priority.

Second, choose a differentiation protocol that can producefunctional authentic nigral A9 dopaminergic (DA) neurons. Itwill be critical to select a differentiation protocol that producesthe right type of DA neuron both in vitro and in vivo in animalmodels. The method of differentiation is currently being opti-mized in different laboratories. Specifically, during characteriza-tion of cells through differentiation stages, there is a need toshow robust expression of midbrain DA (mDA) neuron lineagemarkers (TH, Nurr1, Foxa2, Lmx1a, Pitx3, Engrailed1, Engrailed2)as well as mature neuronal markers in the final product, includingTuj1, synapsin, dopamine transporter (DAT), and G-protein cou-pled, inwardly rectifying potassium channel (GirK2). An importantobservation is that TH expression is shared among all catechol-aminergic lineages and is not specific to midbrain. Other neuronaland non-neuronal fates should also be ruled out through markerexpression.

Third, develop biological assays that predict in vivo func-tional activity and biological action. The likelihood of an effec-tive approach for clinical application will increase when thefunctional features of authentic nigral A9 DAneurons are shownin the cell candidate. One such feature is an appropriate neuro-physiological profile. Autonomous pacemaking or hyperpolariz-ing activated currents areprimarily observed inA9mDAneuronsand represent a very important functional assay to be included inthe in vitro characterization of the candidates in development.Another such feature is DA production and release in response

to physiological stimuli. Measuring production and release ofDA as well as its metabolites (DOPAC and HVA) with specificand sensitive techniques such as HPLC is necessary as part ofthe functional characterization assays of the candidate cell fortransplantation. Finally, animal models should be used to opti-mize conditions for cell survival and to show integration (signifi-cant fiber outgrowth with synapse formation) and functionalbenefits. Long-term engraftment (minimum of 6 months) in anappropriate rodent model of PD (e.g., 6-hydroxydopamine [6-OHDA]-lesioned rat) and complete restoration of amphetamine-induced rotation behavior as well as significant improvements inat least twoothermotor performancebehaviors need tobe shownwith the candidate cells. Further research is needed to define ad-ditional biomarkers and to develop potency assays that providecorrelations between cell survival and predictive functionaloutcome.

Fourth, develop purification strategies. Purification of DAcells from other cells is also necessary to further enrich for DAneurons and obtain fully uniform populations at an optimizedstage for transplantation. This will allow for better controlof the ratio of serotoninergic (SER) to DA neurons in graftpreparations and will avoid dyskinesias and preclude tumorformation.

Fifth, determine the minimum effective dose and the maxi-mum feasible dose in large animal models. Preclinical experi-ments in large animal models should be conducted on theselected cell to optimize integration with existing neural circuitryand restoration of function. Although the number of cells re-quired to replace the nigral cell loss is unclear, other cell therapyprojects and fetal transplant experiments indicate that it is likelyto be around 100,000 DA neurons, assuming they have the sameinnervation potential as fetal nigral DA cells.

Sixth, develop a consistent transplantation protocol thatshows low risk of hemorrhage and no tumor formation. The firsttrial should involve bilateral transplantation into the putamen.The physician conducting the transplant would select the optimalmethod of transplantation, which may include the use of newtransplantation devices, but the device for cell delivery is notthought to be a rate-limiting step for the phase I/II trial.

Seventh, select target patients for the phase I/II trial. Giventhe results from the fetal transplants trials, it seems clear thatthe target patients should ideally be younger patients who areearly in their disease course and responsive to oral DA replace-ment therapies.

Eighth, select outcomes and follow-up studies for the phase I/IItrial.Most participants agreed that the patientsmust be followedfor at least 3 years after transplantation. Outcome parametersshould include a variety of clinical measures along with magneticresonance imaging (MRI) and positron emission tomography(PET).

Participants proposed that CIRMandNIHprioritize their fund-ing efforts in the area of PD to support projects that will advancethese research goals, especially around the generation of suffi-ciently large numbers of authentic DA nigral neurons such thatstemcell-based therapies canbedevelopedmore rapidly as treat-ment options for PD.

Many researchers provided inputduring thisworkshop.Read-ers seeking more information about particular details and con-tacting researchers in certain areas may access that informationby contacting the author.

980 Cell Therapies for Parkinson’s Disease

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BACKGROUND

Overview of the Incidence, Anatomy, andSymptomatology of PD

PD is the second most common neurodegenerative disorder inthe world, affecting 1%–2% of the population aged older than65 years and reaching a prevalence of almost 4% in those aged85 years or older. It is characterized by progressive motor dys-function expressed as a classic “resting tremor,” slowness ofmovements (bradykinesia), muscle rigidity, and difficulties withgait and balance [1]. PD patients also exhibit a variety of nonmo-tor features, including mood disturbances (e.g., depression,anxiety), dementia, pain, and sleep disturbances. Autonomicproblems, including bladder or bowel dysfunction, sexual dys-function, sweating abnormalities, and gastrointestinal abnor-malities are also common. These nonmotor aspects arise atany stage of the disease and often present before motor fea-tures develop [2].

The primary signs of PD arise from damage to neuronal cir-cuits in the basal ganglia involved in the precise control ofmotor function [3–5] (Fig. 1). The basal ganglia are a collectionof nuclei composed of the striatum, globus pallidus, subthalamicnucleus, and substantia nigra. DA neurons of the substantianigra pars compacta (SNc), the neurons progressively lost inPD, project to striatal medium spiny neurons and influencethe activity of motor circuits running through the basal gangliato the thalamus and cortex. Degeneration of DA neurons leadsto a disruption of the delicate balance of excitatory and inhibi-tory feedback that is necessary for appropriate motor function(Fig. 1).

Clinically, the severity in PD is measured using the modifiedUnified Parkinson’s Disease Rating Scale (UPDRS), which isused to follow the longitudinal course of PD. This scale is the

most commonly used in the clinical study of PD and tracks bothmotor and nonmotor features of the disease [6, 7]. The modifiedUPDRS consists of four broad scales that evaluate nonmotorexperiences with daily living, motor experiences with daily living,motor features, and motor complications. Further researchon the current clinical outcome measures is needed because dif-ficulty interpreting the results of studies in recent years hasbeen attributed to problems with the chosen outcome, giventhe natural variability in the scores that arise from UPDRSassessments [8].

The PD disease process in the brain can be staged postmor-tem using the Braak classification [9] by analyzing fixed tissuesections for the accumulation and regional distribution ofa-synuclein and Lewy bodies (LBs), the pathological hallmark ofPD. LBs are large, insoluble protein aggregates that result fromthe accumulation and aggregation of misfolded proteins, partic-ularly a-synuclein [10]. The Braak system has been proposed asa method to stage the severity of PD based on observations ofthe regional distribution of LBs in the central nervous system[9, 11, 12].

A relationship between Braak staging and clinical severity hasbeen difficult to establish, particularly given the fact that LBs arenotuniversally observed in all formsof PD [13] andmight not be inthe right distribution for this classification.

Pathogenesis of PD: CellularMechanisms, Genetics, andImmune Effects

Formany decades, PDwas thought to be an idiopathic, late-adult-onset disease resulting from unknown environmental factors[14]. However, the identification in 1997 ofMendelianmutationsin the SNCA gene [15], which codes for the a-synuclein proteinthat is deposited in the classic pathological lesion of PD, the LB

Figure 1. Coronal section of the human brain showing the relevant regions (left) and circuits (right) underlying the direct and indirect motorpathways under normal physiologic conditions. Neurons in the primarymotor cortex synapse project tomedium spiny neurons in the striatum,which, in turn, regulate motor activity via two mutually antagonistic circuits known as the direct and indirect pathways (reviewed in [3, 4]).Abbreviations: GPe, external globus pallidus; GPi, internal globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars retic-ulata; STN, subthalamic nucleus; STR, striatum.

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[10], was key to uncovering the role of genetics in the disease.Since then, a number of other loci have been implicated. Severalgenes have been found to underlie dominant or recessive formsof PD. These include recessivemutations in thePINK1, Parkin, andDJ-1 genes, which cause early onset forms of PD, and the LRRK2gene, which is an autosomal-dominant risk factor for PD [14]. Al-though these genetic forms of PD are rare, composing approxi-mately 3%–5% of cases [16], and can lack LB pathology, newand powerful genomewide association studies have mappedmany new gene variants that alter the risk for PD, and geneticistspredict that more may be discovered [16–19]. Consequently, ge-netic risk factors contribute to the etiology of even typical formsof PD [16].

What are themechanisms responsible for the highly selectivedeath of the SNc DA neurons in PD patients? Postmortem evalu-ation and, more recently, genetic and molecular studies havehelped uncover the evidence for many possible mechanisms toexplain the cellular pathogenesis of sporadic PD, which is likelyto be complex, involving alteredmetabolism and possible spreadof a-synuclein, lysosomal dysfunction, mitochondrial dysfunc-tion, and possibly a dysregulated inflammatory response [20].In terms of mitochondrial defects, these were found to be asso-ciated with the occurrence of classic PD symptoms that devel-oped in young adults after self-exposure to the neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Later,mechanistic studies revealed that MPP1 (the actively toxic me-tabolite of MPTP) inhibits mitochondrial metabolism [21]. Sub-stantial progress toward understanding the role of mitochondriain the disease process has been made by the identification andcharacterization of genes causing familial variants of PD. Studiesof the function and dysfunction of these genes have revealed thatvarious aspects of mitochondrial biology appear to be affected inPD, including mitochondrial biogenesis, bioenergetics, dynamics,transport, and the fidelity of the mitochondrial quality controlsystems [22].

Some genetic forms of PD directly impair mitochondrial func-tion and thereby cause bioenergetic failure. Studies publishedover the last 7 years have established a model for the PINK1/Parkin pathway of mitochondria quality control [23]. Further-more, data from iPS cell-derived neural cells from PD patientswith LRRK2/PINK1 mutations have shown altered bioenergeticprofiles and mitochondrial dynamics [24]; however, the precisemechanisms linking mitochondrial dysfunction to neuronaldeath in PD remain unclear. As described above, affected neuronsin PD tend to accumulate large amounts of a-synuclein in theform of LBs [10]. LBs are one of several known types of insolubleprotein aggregates that form in a number of neurodegenerativedisorders, the composition of which varies according to specificdisease (i.e., b-amyloid and tau in Alzheimer’s disease, TDP-43in amyotrophic lateral sclerosis [ALS], and mutant huntingtin inHuntington’s disease [HD]; reviewed in [25]). Whether LBs actu-ally cause disease or are a secondary coping response to diseasepathogenesis is amatter of intensedebate [13]. Early hypotheses,based on postmortem observations of PD brains, suggestedthat LBs within the neurons themselves might be linked to DAcell death [26]. Recent evidence, however, suggests that LBsmight instead be essential for sequesteringmisfolded or dysfunc-tional proteins that otherwise cause cellular damage, and thusLBs might be protective [27]; similar observations have beenmade in HD models [28]. Other cellular mechanisms designed tomanage the accumulation of such proteins, such as proteosomal

degradation and autophagy, have been shown tobe dysregulatedin PD [24, 29, 30]. In this last area, the recognition thatGBAmuta-tions are quite common in sporadic PD has suggested that lyso-somal dysfunction may be a much more important pathway fordisease than previously thought.

DA neurons are also disproportionately vulnerable to oxida-tive damage and inflammatory effects. A distinguishing featureof nigrostriatal DA neurons is their increased reliance on intracel-lular oxidative processes related to the synthesis of dopamine[31], which makes them particularly susceptible to oxidativestress. This aspect was directly revealed in studies with hiPScell-derived neurons from PD patients with PINK1/LRRK2 muta-tions, whereby patient-derived DA neurons were more vulner-able to pharmacologically induced oxidative stress than DAneurons from healthy controls [24, 32]. DA neurons also haveincreased iron content [33] and reduced levels of the antioxi-dant glutathione [34], which increases their susceptibility to ox-idative stress [35], as well as specific calcium channels that mayin some way link to disease [36]. In addition, there are moremicroglia, the resident macrophages of the central nervous sys-tem, in the substantia nigra relative to other regions of thebrain.Because activated microglia produce increased levels of inflam-matory cytokines and chemokines and generate reactive oxygenspecies that harm DA neurons [35], immune activation in theSNc may influence the onset and progression of the disease,leading to accelerated DA neuron damage and death in PDpatients.

Treatment for PD: Current Standard of Care

Presently, there is no cure for PD. The current competition in theglobal PD therapeuticsmarket isweak becausedrugs available fortreatment provide only symptomatic relief and do not act to re-verse disease progression. There is, therefore, an unmet medicalneed to develop treatments that address the underlying biolog-ical causes of the disorder with diseasemodifying capability ther-apies to control disease progression.

Early stage PD is typically treated pharmacologically withtherapies that replace the dopamine normally secreted by DAneurons in the SNc. Levodopa (L-DOPA), a dopamine precursorthat is transformed into dopamine in the brain, is always givenwith carbidopa, a peripheral dopamine decarboxylase inhibitorthat inhibits the metabolism of L-DOPA outside the central ner-vous system, thereby enhancing its delivery to the brain. Combi-nation carbidopa-levodopa therapies have proven particularlyeffective at reducing disease symptoms while minimizing sideeffects of the drugs in the short term; however, these drugsare associated with severe adverse effects with long-term use.Other therapies for early stage PD include dopamine agonists,which mimic the effect of L-DOPA by binding directly to striataldopamine receptors. Dopamine agonists are often used as first-line therapies, and their early use delays the appearance ofmotorcomplications and other dopamine-related symptoms; however,recent reports link them to a variety of behavioral problems [37].Other adjunctive therapies, such as monoamine oxidase-B inhib-itors, which are used to prolong dopamine signaling by blockingdopamine metabolism; catechol O-methyltransferase inhibitors,which work in a similar way; and anticholinergic agents, whichhave classically been used in younger patients to treat tremor,can provide added relief [38, 39].




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Unfortunately, the effectiveness of carbidopa-levodopa-basedtherapies decreases as the disease progresses, typically within5 years of treatment initiation [40, 41]. Fluctuations in the re-sponse to medication as well as alterations in the productionof endogenous L-DOPA result in “on-off” states in which themedication produces alternating phases of good response andno response and/or the development of involuntarymovementsresulting from dysregulation of dopamine balance. These invol-untarymovements, dyskinesias, worsen over time, requiring ad-ditional therapies to control them, such as amantadine or moreaggressive enteral or surgical treatments [39–41]. Deep brainstimulation (DBS) is a relatively new therapy that is increasinglyused by physicians. In DBS, electrodes are surgically implantedinto one of two specific regions of the basal ganglia, the subtha-lamic nucleus or the internal globus pallidus [40]. An impulsegenerator delivers electrical stimuli to the cells and fibers lo-cated closest to the implanted electrode, thereby modulatingthe firing rate and patterns of neurons within the basal ganglia,which in turn influences thalamocortical circuits.DBS is a surgicaltechnique typically used in patients with moderately advancedPD who have disabling on-off fluctuations, dyskinesias, andtremor and who remain responsive to L-DOPA [39, 40]. DBShas been shown tobe very effective at reducing themotor symp-toms of PD in patients, and numerous clinical trials have demon-strated significant relief [39, 40, 42].

The mechanisms by which DBS relieves PD symptomsremain unclear [40, 43], and the procedure carries risks that con-cern physicians and patients. Foremost is the potential for infec-tion or intracranial hemorrhage, either of which often requiredevice removal and an extended waiting period before consid-eration of further implantation surgery. Hardware-related com-plications, including electrode lead fractures, also occur withhigh frequency. Finding the optimal DBS stimulation patternsfor each patient is also a challenging process of trial and error[43].

A number of other neurological and neuropsychiatric issuescan occur with DBS, including cognitive impairment, speech dis-turbances (particularly with respect to verbal fluency), memoryloss, sensory disturbances, and mood disturbances [40, 44, 45].Although some of these effects may be corrected by adjustingthe location of the implant and electrical stimulation, othersare permanent or relieved only by cessation of the therapy. Fi-nally, DBS treatment carries significant ongoing expense beyondthe initial surgery, owing to the cost of device andbattery replace-ments and ongoing physician visits to monitor stimulationefficacy [43]. In summary, although dopamine-replacement ther-apies and DBS can provide relief from motor symptoms forpatients with PD, they present some important disadvantages,have been received with mixed patient acceptance, and do nottreat the underlying causes of the disease.

In addition to the degradation of motor function, PD patientsoften display a variety of cognitive and psychiatric symptoms, in-cluding dementia, hallucinations, anxiety, and depression; distur-bances in sleep, bowel or bladder, and sexual functions; and pain[39, 41]. These symptoms significantly diminish patient quality oflife [4, 41]. Despite their impact, few clinical trials have directlyaddressed these issues, and current management of thesesymptoms in late-stage PD is often poor [39, 40]. Although datafrom fetal cell transplants suggest that replacing DA neuronslikely will not affect these nonmotor symptoms [46], cell

replacement may be a clinically competitive option that couldlead to significant improvements in the quality of life forpatients [47].

Because PD progresses slowly over a period of years to dec-ades, patients often require extensive medical and home care,thereby placing enormous burdens on patients, their families,and society that have been estimated at $25 billion annually inthe U.S. alone [48]. Although a variety of therapies treat the mo-tor features of PD, these treatments diminish in efficacy overtime. Furthermore, they neither reverse the underlying biologicaldefect nor modify disease progression. Stem cell-based replace-ment therapies could represent a revolutionary approach thatmay slow, halt, or reverse many of the motor aspects of PD.In the long term, such an approach might be the best path toa disease-modifying therapy for this devastating neurodegenera-tive disorder.

Where Have We Been? History of Cell Transplantationin PD

The potential of cell-replacement therapies for significant long-term relief of PD symptomswithout the need for continued phar-macological or surgical therapy provided the early rationale forconducting transplantation trials using fetal ventral mesence-phalic (VM) tissue [49, 50]. The results from these studies willbe critical for informing the design of clinical trials to assess theeffectiveness of stem cell-based transplantation for PD.

Early open-label trials in Europe and theU.S. showed substan-tial clinical improvement in some patients receiving fetal celltransplants grafted into the basal ganglia. Studies reportedimproved motor symptoms [51–55], improved 18F-fluorodopa(18F-DOPA) uptake [53–58] and robust long-term graft survivaland reinnervation of the grafted site in these patients [56, 59].However, two placebo-controlled trials conducted in the U.S.,known as the Colorado-Colombia (CC) and Tampa/Mount Sinai/Rush (TMR) trials, showed only modest benefit, if that [60, 61].Of concernwas that a significant proportion of patients appearedto develop graft-induced dyskinesias (GIDs) that persisted evenin the absence of levodopa medication [60–62]. These resultsled to a halt of all cell therapy trials for PD.

Subsequent long-term evaluation of secondary endpoints byPET, 18F-DOPA, and UPDRS scoring, combined with stratificationof patients by age and severity, showed a statistically significantbenefit of fetal tissue transplantation for certain patient popula-tions. Specifically, younger or less severely affected patients whohad been responsive to dopamine-replacement therapy at thetime of transplantation showed marked improvement starting2–4 years after transplantation, in line with reports from theopen-label trials [56, 63–66]. Details such as methods for tissuepreparation, surgical technique, and immunosuppressive therapyseemed to have particular impact on the success of the interven-tion, as did the nature of the primary endpoint chosen for use inthe study.

In the end, the CC and TMR trials revealed the importance ofpatient selection, trial design, and rigorous follow-up in clinicaltrials using cell-replacement therapies. In addition, the clinicalendpoints chosen, particularly in the CC trial, were later recog-nized to beproblematic and revealed the importance of definingobjective, quantifiable measures as primary endpoints thatare assessed some time after surgery when the graft hashad a chance to mature and integrate into the host brain.




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Furthermore, we now understand the importance of controllingthe transplantation procedure itself, including surgical trans-plantation methods and immunotherapy, to ensure successfuloutcomes. Finally, studies revealing that patients continued toimprove years and even decades after transplantation of fetalVM tissue [63, 67, 68] underscores the importance of includinglong-term, rigorous follow-up of patients to monitor ongoingchanges and improvements in motor symptoms.

To reflect on these findings, the workshop discussed the cur-rent efforts to optimize clinical trial design for cell transplanta-tion in PD. A European consortium, TRANSEURO (http://www.transeuro.org.uk), will conduct the first fetal cell transplantationstudy since the CC and TMR trials. The principal objective ofTRANSEURO is to develop a safe, long-lasting, and efficacious ap-proach to treating PD patients using fetal cell-based therapeuticsthat can serve as a template for future clinical trials, includingthose for other stem cell-based therapies. This clinical trial is be-ing designed using a very tightly controlled approach tominimizeprocedural variables. It includes specific criteria for selection ofthe patients (defined age range, stage, and type of PD); tissuepreparation (specified number of cells grafted, standardizedmethods for tissue collection to ensure a high percentage ofDA neuroblasts and to limit the number of serotoninergicneurons); and defined methods for tissue placement, graft sup-port, and improved trial design (number of patients, follow-uptime, and endpoints). Very careful consideration of the discussionsand analysis that have led to this new clinical trial has beenreviewed previously [63]. According to TRANSEURO, the primaryendpoints for the clinical study are safety and motor effects, butthe intent is to evaluate the effects of “tissue preparation anddelivery, patient selection and immunosuppressive treatment”and to show that consistency and efficacy of DA cell replacementin PD can be improved by careful attention to those parameters.This study, informedbypreviouschallengesandsetbacks,will serveas a valuable resource to inform future stem-cell transplantationstudies.

CHALLENGES AND OPPORTUNITIES IDENTIFIED IN THEWORKSHOP

Therapeutic Candidate Profile for Stem Cell Therapies

Stem cell-based replacement therapies could potentially providelifetime reversal of many of the symptoms of PD. Given the avail-ability of other therapeutic options for PD patients (L-DOPA, en-zyme inhibitors, dopamine agonists, DBS) and the risks incurredby surgery, to be clinically competitive, a DA cell therapy shouldprovide long-lasting, major improvement (.60%–70%) of motorsymptoms and suppression of dyskinesias.

Participants in the workshop proposed a therapeutic profilethat should be expected for an ideal candidate for cellular therapy(see further detail as described in the Introduction).

Τransplanted cells must have the potential to recreatethe characteristics of neurons lost to the disease. The principalcell type lost in PD is the A9 DA neuron, found selectively inthe SNc. Cell therapy should aim to replace this particulargroup of DA neurons, supported by in vitro and in vivo datain preclinical animal models. Candidate cells must expressthe features of authentic nigral A9DAneurons, such as the righttranscriptional and neurophysiological profile (i.e., pacemakerpotentials), dopamine production and release in response to

physiological stimuli, and significant fiber outgrowth with syn-apse formation.

Once transplanted into the right anatomical location, DAneu-rons must survive, reinnervate the striatum, and functionally in-tegrate into the host’s neural circuitry. Consequently, rigorouslydefining and determining the number of cells required for suc-cessful implantation will be essential to achieving lasting survivaland integration.

Transplanted neurons must provide measurable, biologicaloutcomes in terms of dopamine synthesis and regulated releaseon physiological stimuli. The fieldmust place emphasis on the de-velopment of potency assays to establish correlations betweencell properties in vitro and predictive functional outcomes in vivo.The tools necessary to assess the survival and function of trans-planted DA neurons are being developed, and some of these fea-tures must be inferred by analyzing the activity of the cells inculture and in large animal models.

Safety issues must be addressed, including tumor formation;GIDs, either through the presence of serotoninergic neuronshyperinnervating the striatum or uneven distribution of trans-plantedmaterial in the target region; host immunological reactiv-ity to the graftedmaterial; and inappropriate stem cell migration.

Finally, the therapymust result in significant improvement ofmotor deficits in patients that is robust and sustained over a pe-riod of years to decades.

The following sections highlight avenues of current develop-ment in stem cell research that were presented and discussedduring the workshop and that are directly or indirectly contribut-ing to the development of cells with this therapeutic candidateprofile.

Tools for Modeling PD In Vitro

The ability to create iPS cells from human skin biopsies has revo-lutionized the ability to model numerous neurodegenerative dis-eases in vitro.Models of Alzheimer’s disease [69, 70], HD [71–74],ALS [75, 76], and autism [77, 78], among others, provide an exper-imental platform for researchers to investigate disease-specificphenotypes at cellular and molecular levels. This approach couldprovide invaluable insights for determining the underlying mech-anisms of disease and a cell culture system for screening for newtherapeutic treatments.

Nonetheless, significant challenges accompany this ap-proach, for example, the ability to overcome clonal variabilityand minimize the genomic and phenotypic changes that celllines present over passage and time. One of the proposed solu-tions for monogenic and genetically defined disorders is togenerate isogenic control lines that harbor defined geneticalterations [79] through the use of advanced genome-editingtechnologies, including zinc-finger nucleases, transcriptionactivator-like effector nucleases, and clustered/regularly inter-spaced palindromic repeats. Specific sequences can be insertedinto the cellular genome to introduce defined mutations (ormutation-corrected sequences) for study [77, 80]. By otherwisepreserving all other genetic features of an hiPS cell line, thesetechnologies allow for superior experimental control and canbe used to address the challenges associated with clonalvariability.

In PD, recent studies using neural cell types derived fromhuman LRRK2-G2019S hiPS cells have revealed increased ex-pression of oxidative stress-response genes and a-synuclein




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protein [32], increased susceptibility to proteosomal [81] andoxidative [24] stress, and key genes and signaling pathways in-volved in in vitro pathogenesis [82] of the disease. Someof thesestudies have also shown how isogenic correction of this muta-tion could reverse several of the identified in vitro phenotypes[81, 82].

Adifferent challenge that the field facedwas theneed for a re-pository ofwell-characterized, publicly available, anddiverse hiPScell lines derived from genetic PD patients. In response, the Na-tional Institute of Neurological Disorders and Stroke Parkinson’sDisease iPCCell LineConsortium (http://www.pdips.org) initiatedthe development of such a resource in 2009 [83]. The consortiumhasalreadygenerated iPS cells frompatientswithdisease-causingmutations for analysis of how cells with different PD-associatedgenotypes and phenotypes respond to cellular stressors, suchas oxidative stress. In addition, although it is unclear whetherDA neurons frompatientswith sporadic PD exhibit the same cel-lular perturbations observed in genetic forms of the disease,hiPS cell studies can provide an important framework for study-ing different types of PD. Moreover, hiPS cell models frompatients with a diversity of genetic and idiopathic forms willbe essential for extracting clinically meaningful information re-lated to disease phenotypes and potential treatments. Promis-ing neuroprotective molecules, for example, could be tested inpatient-derived cells to identify a patient’s responsiveness toa given therapy and to understand the mechanisms of disease.In addition, hiPS cell-derived neurons could be used to assessthe efficacy of cell-based treatments in vivo and to developnew targets for drug development. Studying these modelsmight lead to new insights into the cellular defects underlyingthe death of SNc neurons in PD patients. Finally, hiPS cell-based models can be used to optimize neuronal differentiationprotocols (e.g., to generate the A9 SNc neurons needed for stemcell transplantation) and to identify conditions that optimize cellsurvival.

These patient-derived lines can also provide a powerful toolfor drug discovery. By grouping the genetic PD iPS cell linesaccording to shared cellular phenotypes and the clinical featuresof the donor patients, this platform could be used for screening ofsmall molecule libraries to identify candidate drugs that modifyspecific phenotypes, thereby providing a tool to identify patientsand at-risk cohorts that may be amenable to specific treatments.Expanding the resource to include data from idiopathic patienthiPS cell lines may provide a means to extrapolate the findingsto predict drug responsiveness for different cohorts of this spo-radic disorder.

Technologies for Stem Cell Transplantation

Recent advances in stemcell technologies, animalmodels of PD,and clinical tools will greatly facilitate the goal of stem cell-based therapies for PD. Although workshop participants wereoptimistic, highlighting a number of key breakthroughs in theseareas, they also discussed the ongoing issues that must beaddressed to make stem cell-based therapies a viable clinicaloption.

Stem Cell Source

Because the availability of fetal tissue is restricted, a variety ofcell sources suitable for generating mDA neurons has emerged

in the past two decades, including in vitro expanded midbrainneural precursors [84–86] and various neural stem cell (NSC)lines [87–89]. However, all of these cell sources and strate-gies have disadvantages [90]. One is the limited potentialof NSC lines to generate authentic mDA neurons. Thus, thegroup discussed the need for a stem cell source that offersaccess to the earliest stages of embryonic development,which would allow control of regional specification during celldifferentiation.

Human pluripotent stem cells may push the field in a new di-rectionbecause theyprovide analternative sourceof cells for der-ivation of DA neurons for therapeutic application and can readilybypass some of the limitations inherent to NSCs [90, 91]. Impor-tantly, translation of any stem cell-based candidate to the clinicalsetting will require evidence of efficacy, safety, and long termfunctional efficacy in preclinical models of disease. Presently,both hiPS cell- and hESC-derived sources are being developedas candidates for clinical application.

The history of derivation of mDA neurons provides an objectlesson in the challenges inherent in achieving clinical application.Differentiation of this cell type from hESCs was reported nearlya decade ago [22]; however, the field struggled with demonstrat-ing in vivo functional engraftment of these cells. It was not untilthe development of floor plate-based neural differentiation pro-tocols [92–94] that the field made a significant step forward to-ward the use of these cells for potential clinical cell therapies.Transplantation of these neurons results in robust in vivo survivaland therapeutic benefit across rodent and primate PD models,demonstrating both therapeutic efficacy and scalability to largeanimal models [93].

This differentiation strategy is based on the direct conversionof ESCs to floor plate precursors that, on exposure to Shh andWntsignaling agonists, are efficiently converted tomDAneurons.Mo-lecular profiling of these cells confirmed a developmental pro-gression of hESCs consistent with the mDA lineage, wherebythey correctly coexpressed the transcription factors Foxa2 andLmx1a at the floor-plate precursor stage and Pitx3 and Nurr1 atlater stages of differentiation [93].

This approach has been adopted by other laboratories, whichhave introduced variations and refinements thathave led to someinteresting alternatives [92]. Participants felt that work in thisarea suggests that the floor plate protocol may have translationalpotential for the development of candidate therapeutic DA cellsfor transplantation in humans.

Safety

For cell-based therapies to be a viable option for treatment of PD,a number of safety issuesmust be addressed in preclinical rodentand larger animal models. Themajor issues appear to be the riskof tumor formation; GIDs, either through the presence of sero-toninergic neurons hyperinnervating the striatum or unevendistribution of transplanted material in the target region (al-though it is extremely hard to mimic this problem in the labora-tory); host immunological reactivity to the graftedmaterial; andinappropriate stem cell migration. Undifferentiated NSCs havethe capacity to undergo extensive migration from the site oftransplantation to nontarget sites of the brain through whitematter tract. The propensity of stem cells to migrate may resultin a theoretical risk that they could cause seizure-like symp-toms or other brain dysfunction. The purification (and efficient




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differentiation) of cell populations to remove undifferentiatedcells, together with methods that yield differentiated precursorcells with high efficiency, will significantly lower the risk for tu-mor formation and inappropriate migration. Purification toremove serotonergic cells or at least to ensure that their num-bers are low relative to midbrain DA neurons, as well as im-proved surgical methods for distributing cells within theputamen (discussed below), is likely to reduce the incidenceof GIDs in patients. Finally, the immune responses to cell trans-plantation in the brain, particularly the effects of local inflamma-tion on the viability of transplanted cells, are being evaluated [95];however, it remains unclear how and to what degree the immunesystem contributes to the long-term viability of grafted material,a question that will be critical to address in the future. That said,it is known that human fetal allografts can survive long termin the adult PD brain in the absence of continuous long-termimmunosuppression.

Sorting and Purification

To what degree will cells need to be sorted and purified prior totransplantation? The workshop participants discussed this con-sideration in a number of contexts. First, pluripotent stem cell-derived populations may pose a risk of tumor formation aftertransplantationbecause they cancontainundifferentiatedorpro-liferating non-neuronal cells [96, 97]. Furthermore, preclinical ev-idence suggests that sorted allogeneic cell populations displayimproved survival and engraftment with reduced incidence of tu-mor formation [98]. Some procedures are being developed forpurifying DA neurons from mixed cultures [98] to remove undif-ferentiated stem cells and to enrich for cells expressing latedifferentiation-stage markers, but these differentially purifiedcell types will need to be systematically tested in vivo. Second,follow-up studies of patients treated with fetal tissue graftsrevealed that GIDs could be caused, in part, by the presence ofserotonergic cells or at least a high ratio of SER to DA neurons[68, 99, 100]. This suggests that purification to remove this celltype and to further enrich for cell types that produce A9 DA neu-rons is desirable. Consequently, the effects of sorting and purifi-cation on cell transplantation efficacy are an important futurearea of study.

Scale-Up

A crucial consideration for the clinical application of these cell-based therapies is the scale-up of laboratory protocols tomanufacturing processes that are reproducible and predictableand that produce large numbers of cells that are clinicallyeffective, cost effective, and compliant with current goodmanufacturing practices. Furthermore, the development ofstrategies for large-scale three-dimensional (3D)manufacturingthatmore closelymimic the native developmental environmentare under way, and advances have been made in the develop-ment of small-scale 3D approaches using engineered, chemi-cally defined biomaterials [101, 102]. Importantly, thesesynthetic environments offer superior control over cell cultureconditions while producing desired cell types with significantlyimproved efficiency, consistency, and reproducibility [102]. Itis likely that these bioengineered platforms, together withimprovements in small-molecule and other non-integration-based methods for cell differentiation, will lay the foundation

for future large-scale cell bioprocessing. This latter step willlikely involve partners in industry because most academic labo-ratories lack the resources for such large-scale manufacturing.Furthermore, an effort should be made to establish collabora-tions between biology and bioengineering laboratories becauseit will be necessary to rigorously characterize and test the cellsproduced using these methods in preclinical models of PD.Nonetheless, the progress initiated and achieved by academiclaboratories has been impressive and will be essential to con-tinue this work.

Animal Models to Evaluate Cell Therapy for PD

Preclinical studies in disease models of PD are required to bet-ter define the risk-benefit ratio associated with investigationalcell therapy products. In addition, use of disease and injury mod-els provides the opportunity for possible identification of activity-risk biomarkers that may be applicable for monitoring in clinicaltrials [103]. Animal models of PD are good models for motor def-icits and include neurotoxic models using compounds (6-OHDA,MPTP, paraquat, or rotenone) that damage the DA systemand genetic models that express mutations linked to PD inhumans [104]. Neurotoxin-based models produced by 6-OHDAor MPTP administration are the most widely used toxic models,whereas paraquat and rotenone are more recent additions tothe stable of toxic agents used to model PD but can be difficultto use. Unilateral injection of 6-OHDA has been shown tocause nigral DA neuron loss, depletion of dopamine, and behav-ioral deficits that can be quantified easily (e.g., abnormal rota-tional behavior in response to amphetamines). Easy behavioralevaluation makes the rodent 6-OHDA lesion the most commonlyused animal model. In the 1980s, the 6-OHDA model was usedto establish proof-of-principle for human ventral mesencephalictissue transplantation [105]. However, the 6-OHDA lesion doesnot recapitulate the clinical features of PD and does not rec-reate pathologies such as Lewy-like inclusions but is a sensitivemodel to assay DA nigrostriatal integrity. Furthermore, the com-paratively small size of rodent brains makes addressing theissue of scalability challenging in rodents alone. Nonetheless,these model systems are very valuable as a first step towardestablishing preclinical proof of concept for cell-replacementtherapies.

A second lesionmodel involves the administration of the neu-rotoxin MPTP [106], a compound that is selectively taken up bycatecholaminergic neurons, including DA neurons, and causesthem to die. In primates, MPTP exposure leads to the hallmarkbehavioral characteristics of PD, including tremors, motor dys-function, SNc DA neuron damage, and responsiveness of symp-toms to L-DOPA treatment [106, 107].

Important early studies by Redmond and colleagues [108,109] were the first to demonstrate the efficacy of ventral mesen-cephalic grafts in primate MPTP models of PD. Similar primatemodels have since been used to show that hESC-derived DA pre-cursors, generated by the floor-plate method, can also efficientlyengraft and produce long-lasting benefit [93]. MPTP administra-tion in nonhuman primates is an important model system for val-idating therapeutic efficacy and scalability of cell-replacementtherapies for PD.

Evaluation of the efficacy of a candidate in preclinical studieswas identified as a critical issue in the development of cell ther-apies. The correlation of DA cell loss and striatal innervation with




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performance in a variety of tests provides a useful tool for theevaluation of in vivo efficacy and performance of stem cell-derived DA neuron preparations. The most commonly used testsare the drug-induced (apomorphine, amphetamine) rotationtests and the spontaneous motor tests (cylinder and stepping).Those tests are usually performed in the severe unilaterally le-sioned ratmodel of 6-OHDA. In order to showpreclinical efficacy,workshop participants agreed that the candidate cell therapymust show complete (100%) reversal of drug-induced rotation6months after transplantation (assuming the cells have been leftin situ long enough to fully mature). Moreover, the candidateshouldpreferably be tested in twoadditionalmotor tests showingrobust recovery of function. Criteria for animal inclusion and an-imal numbers should be very clearly delineated in the experimen-tal protocols.

The lack of rigorous investigations into dose finding and earlyidentification of possible side effects is another potential contrib-utor to clinical failures in translational research. In the case of celltherapies for PD, the evaluation of potency can be considereda surrogate for efficacy measurements and will play a key rolein defining the quality of the cellular therapy product. Presently,the field does not have appropriate predictive potency assaysavailable, and participants agreed that identifying assays to mea-sure correlations between in vivo cell survival and predictivefunctional outcome should remain a developmental focus for lab-oratoriesworking in PD. Ideally, potency assays should be in placefor early clinical development, and validated assays will be re-quired for pivotal clinical trials. Long-term efficacy measures willneed tobecarefully chosen for clinical trials, and the fieldneeds toassess whether preclinical imaging or other assays can predictlong-term functional outcomes.

New Tools for Transplanting Cells Into the Human Brain

Another point of discussion during themeetingwas the evolutionof new tools for transplantation. Analysis of fetal transplant stud-ies suggested that the surgical technique has a great impacton the survival and viability of cell grafts and the expression ofGIDs [64, 110]. Improved surgical techniques first pioneered byMendez and colleagues, in which grafted material is distributedevenly across the striatum, appear to reduce the incidence ofGIDs while preserving therapeutic efficacy [111–114]. Some ofthe meeting members presented their new developments inthis area. Dr. Daniel Lim from the University of California SanFrancisco described a new surgical tool, known as a “radiallybranched deployment device,” that evenly distributes multiple,small cell grafts over a large target region within a single trans-cortical penetration. There was significant enthusiasm for thistool at the workshop because this surgical approach reducesthe likelihood of trauma to other brain regions that can arise withmultiple brain penetrations and separate injections. When for-mulated with materials compatible with functional real-time im-aging, such as interventional MRI, radially branched deploymentdevices may offer an unparalleled ability to monitor the accuracyof targeting during the surgical procedure itself, therebyminimiz-ing the risk of off-target injection and damage to other brainregions [115, 116]. Importantly, by facilitating the even distribu-tion of cell grafts, this delivery platform is likely to reduce the riskof “hot spots” of graft innervation that are thought to influencethe development of GIDs. However, the currently available deliv-ery devices used in transplant trials to date are still probably

adequate because the theory of GIDs induced by hot spots comesfrom a study using a transfrontal delivery approach with noodlesof tissue. The development of GIDs may have had as much to dowith the overall approach as with the device used to deliverthe cells.

Research suggests that the extracellular environment criti-cally affects cell survival and differentiation. Dr. David Schafferfrom the University of California Berkeley discussed researchto develop synthetic bioactive materials that emulate the ex-trinsic environment. These materials could potentially increasethe number and differentiation status of cells generated invitro and could also be transplantedwith cells as away of affect-ing their survival and integration into the brain. These extracel-lular matrix materials have not been tested in animal modelsyet. This represents another important area in which collabora-tion between bioengineers and PD researchers may provevaluable.

New Tools to Evaluate Disease Progression andFunctional Recovery

Functional neuroimaging techniques such as PET have been im-portant tools for assessing the neuropathology of PD and havehelped develop understanding of the mechanisms responsiblefor the success or failure of grafting human fetal tissue in clinicaltrials [117]. During the workshop, one of the requirements thatemerged for the success of a future cell therapy trial is an opti-mized functional imaging protocol. Although functional imagingcannot currently be used as a primary endpoint in clinical trans-plantation trials, if used appropriately, it can provide researcherswith an additional valuable in vivo tool alongside clinical observa-tions. Of these neuroimaging methods, radiotracer imaging ofthe nigrostriatal DA system using 18F-DOPA PET, is among themost accepted methods for monitoring disease progression[118]. A bottleneck in the field is the search for a ligand tagginga specific DA presynaptic terminal location that would allowidentification of the survival and growth of the DA-rich graft.To date, 18F-DOPA PET remains the standard formonitoring sur-vival and growthof graftedDAcells. Studies have showna strongrelationship between striatal dopamine deficiency as measuredby 18F-DOPAPET and the severity ofmotor symptoms [117]. How-ever, it is not possible to quantitatively assess DA neuron numberusing this approach; therefore, the true relationship betweentracer uptake and tissue biology remains imperfect [119]. Still,theability of 18F-DOPAPET tomonitor ongoingdiseaseprogressionthat accurately tracks with progression ofmotor symptomsmakesthis a usable and reliable biomarker for some aspects of diseaseprogression and monitoring [119], albeit an expensive one.

RECOMMENDATIONS

As described in the Introduction, the workshop identified a seriesof critical initial steps to facilitate development of a phase I/IIclinical trial for stem cell therapy.

Define the Cell Source

Either hiPS cells or hESCs could be used in the first stem cell trial.hESCs provide amore beneficial commercialmodel because a sin-gle hESC line can be banked in large quantities to treat multiplepatients. hiPS cells can be autologous or allogeneic. Ideally, plu-ripotent stem cell banks should be matched to patients to




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minimize immune effects. Experiments to evaluate the differentcell sources are under way, and several approaches are under in-vestigation. Currently, the effort is focused on prioritizing the dif-ferent approaches andmoving forward into clinical trials with thehighest priority.

Choose a Differentiation Protocol That Can ProduceFunctional Authentic Nigral A9 DA Neurons

It will be critical to select a differentiation protocol that producesthe right type of DA neuron, both in vitro and in vivo in animalmodels. The method of differentiation is currently being opti-mized in different laboratories. Purification of DA cells fromothercells is also necessary to control the ratio of SER to DA neurons ingraft preparations and avoid dyskinesias aswell as to preclude tu-mor formation. Animal models should be used to optimize condi-tions for cell survival, integration, and functional benefits to theanimal.

Determine the Minimum Effective Dose and theMaximum Feasible Dose in Large Animal Models

Preclinical experiments in large animal models should be con-ducted on the selected cell to optimize integration with existingneural circuitry and restoration of function. Although the num-ber of cells required to replace the nigral cell loss is unclear,other cell therapy projects and fetal transplant experiments in-dicate that it is likely to be around 100,000 DA neurons, assum-ing they have the same innervation potential as fetal nigraldopamine cells.

Develop Biological Assays That Predict In VivoFunctional Activity and Biological Action

The likelihood of an effective approach for clinical applicationwillincrease if cells express the features of authentic nigral A9 DAneurons, such as the right transcriptional and neurophysiologicalprofile (i.e., pacemaker potentials), dopamine production and re-lease in response to physiological stimuli, and significant fiberoutgrowth with synapse formation. Further research is neededto define additional biomarkers and to develop potency assays

that provide correlations between cell survival and predictivefunctional outcome.

Develop a Consistent Transplantation Protocol ThatShows Low Risk of Hemorrhage and NoTumor Formation

The first trial should involve bilateral transplantation into theputamen. The physician conducting the transplant would selectthe optimal method of transplantation, which may include theuseof newtransplantationdevices, but thedevice for cell deliveryis not thought to be a rate- limiting step for the phase I/II trial.

Select Target Patients for the Phase I/II Trial

Given the results from the fetal transplants trials, it seems clearthat the target patients should ideally be younger patients whoare early in their disease course and responsive to oral dopaminereplacement therapies.

Select Outcomes and Follow-up Studies for Phase I/IITrial

Mostparticipants agreed that thepatientsmustbe followed for atleast 3 years after transplantation. Outcome parameters shouldinclude a variety of clinical measures along with MRI and PET.

ACKNOWLEDGMENT

R.C.-A. is currently affiliated with the Foundation for the NationalInstitutes of Health, Bethesda, MD.

AUTHOR CONTRIBUTIONS

R.C.-A.: manuscript writing, final approval of the manuscript;G.P.L., E.G.F., and C.P.: discussion, manuscript editing.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicated no potential conflicts of interest.

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Rosa Canet-Aviles, Geoffrey P. Lomax, Ellen G. Feigal and Catherine PriestProceedings: Cell Therapies for Parkinson's Disease From Discovery to Clinic

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