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Neural transplantation for the treatment of Parkinson disease

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									      Neural transplantation
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  Neural transplantation for the treatment of
  Parkinson’s disease
  Anders Björklund, Stephen B Dunnett, Patrik Brundin, A Jon Stoessl, Curt R Freed, Robert E Breeze,
  Marc Levivier, Marc Peschanski, Lorenz Studer, and Roger Barker


  Neural transplants in Parkinson’s disease:                              trials showed unequivocally that human fetal dopaminergic
  do they work?                                                           neurons can survive and function for more than 10 years in
  Anders Björklund and Stephen B Dunnett                                  the striatum of patients with PD and show no signs of being
  Cell transplantation to replace lost neurons is a new approach          affected by the ongoing disease process. These studies
  to the treatment of progressive neurodegenerative diseases.             have also provided a clear indication that grafted fetal
  Replacement of dopaminergic neurons in patients with                    dopaminergic neurons can be therapeutically effective. On the
  Parkinson’s disease (PD) has spearheaded the development of             basis of the limited, but encouraging, observations in these
  this approach and was the first transplantation therapy to be           early open-label trials,1 the National Institutes of Health
  tested in the clinic. The success of cell replacement for               (NIH) decided to support a second phase; two double-blind
  the treatment of PD is based on two hypotheses: first, the              sham-surgery controlled trials were launched in the early
  predominant symptoms of PD are dependent on                             1990s, the results of which have only recently been reported
  the dysfunction or loss of the dopaminergic neurons in the              and turned out to be more disappointing than expected.2,3
  nigrostriatal pathway; and second, dopaminergic neurons                      Meanwhile, the on-going open-label trials have continued
  grafted into the dopamine-deficient striatum can replace those          to generate more data and further questions that designed
  neurons lost as a result of the disease process and can reverse,        trials may be able to address. The most recent data from four
  at least in part, the major symptoms of the disease.                    centres (Sweden, France, USA, and Canada), which includes a
       Extensive animal experiments over the past two decades             total of 26 patients with moderately severe PD, shows a
  have provided the basic proof of principle for cell replacement.        60–70% increase in striatal uptake of fluorine-18-labelled
  Immature dopaminergic neurons or neuroblasts—taken from                 dopa (figure 1) and overall improvements in Unified
  the mesencephalon of 13–15 day old rat fetuses or 6–8 week              Parkinson’s Disease Rating Scale (UPDRS) motor scores of
  old aborted human embryos—survive and re-establish                      6–40%.1 These improvements developed gradually during the
  dopaminergic innervation when transplanted into the                     first 6–24 months after transplantation. Moreover, clinical
  denervated striatum, and restore baseline dopamine synthesis            improvements have been associated with recovery of
  and release in the reinnervated area. Striatal dopaminergic             movement-related activation of the premotor and
  innervation can be restored to 10–40% of normal, which is               supplementary motor cortex, a deficit of which is thought to
  similar to the extent of dopamine depletion seen in the early           underlie the hypokinetic symptoms of PD.4
  stages of PD. Although these results are clear-cut, these studies            A major issue, however, is that the results of these trials
  were done in rodent and non-human-primate models of the                 have been quite variable, not only between centres but, more
  disease, in which parkinsonian symptoms are induced by the              importantly, among groups of patients at the same centre
  selective destruction of the nigrostriatal dopaminergic                 (who received grafts of the same type of cell preparation and
  pathway by means of a neurotoxin (ie, the lesion is acute               underwent the same surgical technique). Some patients
  rather than progressive, “cleaner”, and more circumscribed              showed minor benefit, or no benefit at all, whereas others had
  than in PD). Moreover, the functional improvements seen                 substantial clinical improvement with reductions in UPDRS
  after intrastriatal transplantation of dopaminergic neurons, in         motor scores of up to 50–60%.1 Transplant-induced increases
  both rats and monkeys, are limited to certain basic motor
  functions whereas impairments in more complex behaviours
                                                                          AB is at the Wallenberg Neuroscience Center, Section of
  are resolved to a lesser extent, if at all. This may be because the     Neurobiology, Lund University, Sweden. SBD is at the Brain Repair
  transplanted cells are placed in the striatum—the target of             Group, School of Biosciences, Cardiff University, Wales, UK. PB is at
  dopaminergic neurons and where dopamine exerts its                      the Wallenberg Neuroscience Center, Section for Neuronal Survival,
  action—rather than into the substantia nigra, from where the            Department of Physiological Sciences, Lund University, Sweden.
                                                                          AJS is at the University of British Columbia, Pacific Parkinson’s
  normal dopaminergic neurons are lost in PD. This ectopic
                                                                          Research Centre, Vancouver, BC, Canada. CRF and REB are both at
  placement is necessary for extensive reinnervation of the               the Departments of Medicine and Neurosurgery, University of
  striatum by the transplanted neurons, but may limit the                 Colorado School of Medicine, Denver, CO, USA. ML is at the Service
  functionality of the graft.                                             de Neurochirurgie, Hôpital Erasme, Brussels, Belgium. MP is at
       Clinical trials of fetal-cell transplantation in PD have           INSERM U 421, Faculté de Médecine, Créteil, France. LS is at the
                                                                          Developmental Biology Program and the Division of Neurosurgery,
  developed in two phases. In 1987, a series of small open-label          Sloan Kettering Institute, New York, NY, USA. RB is at the Cambridge
  trials, in which only carefully selected patients were included,        Centre for Brain Repair, Cambridge, UK.
  was initiated to obtain evidence to support the viability of            Correspondence: Dr Marc Peschanski, INSERM U 421, Faculté de
  transplantation as a therapy for PD. These early open-label             Médecine, F-94010 Créteil, France. Email peschanski@im3.inserm.fr



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  in putaminal 18F-dopa uptake have also
  been highly variable, which suggests that
  differences in survival and growth of the
  grafted dopaminergic neurons may be
  an important factor in the recovery of
  patients. A systematic patient-by-patient
  analysis in the larger double-blind trials
  could help to elucidate the factors
  underlying this variability.
       Another challenge is that the
  reported clinical improvements after
  transplantation may be due to
  investigator bias, placebo effects, or
  both. In this regard, the results of the two
  NIH-sponsored double-blind studies are
  reassuring in that none of the patients
  who underwent sham surgery showed
  any significant, long-term benefit of the Figure 1. F-dopa PET scan in a patient with PD after transplantation of human fetal neurons into the
                                                             18


  placebo treatment. However, these right putamen. Left: a defined area of increased F-dopa uptake is visible on the PET image (black
                                                                                                 18


  studies have raised two major concerns: arrow). Right: this area of increased uptake corresponds to the site of graft placement (red cross) in the
                                                                                               the PET image with
  only modest efficacy was observed in the putamen, as confirmed by coregistration of of Marc Levivier. stereotactic MRI used during the
                                                    neurosurgical grafting procedure. Courtesy
  transplantation groups, and a significant
  number of patients developed off-state dyskinesias after the delivery of transplants—before further double-blind trials
  transplantation.                                                             involving larger groups of patients are warranted to determine
       It is clear from the disappointing outcome of these two the true value and efficacy of neural transplantation in
  trials that we still do not fully understand how to use fetal-cell comparison with other available treatments.
  transplantation to achieve consistent, optimum results. They
  also highlight several serious issues that must be addressed. How can we make transplants of embryonic
  First, the mechanism underlying the off-state dyskinesias neural tissue more effective?
  developed by some patients needs to be investigated. Are some Patrik Brundin and A Jon Stoessl
  patients particularly sensitive5 and how can this side-effect be Two recent double-blind, sham-surgery-controlled trials,
  avoided? Second, how do we select the patients who are likely supported by funding from the NIH, of embryonic
  to derive the greatest clinical benefit? Are patients with mesencephalic transplants for the treatment of PD
  advanced disease unsuitable candidates for fetal-cell produced disappointing results. The first of these studies2
  transplantation and is age a negative factor? Third, where is the did not show any overall improvement on a subjective
  best site for graft placement? Does the transplantation surgery global rating scale. However, patients under 60 years of
  need to be tailored to each patient’s disease profile? Fourth, do age had reductions in UPDRS scores. This modest
  we need to standardise preparation of cells for transplantation? improvement was offset by the emergence of severe
  Is it feasible to store tissue for days or weeks in vitro—as done dyskinesias in 15% of the patients, which persisted despite
  in the two NIH trials—without losing functional efficacy? substantial reductions in levodopa doses. The second study
  Fifth, what about immunosuppression? Can we really exclude has yet to be published, but the preliminary findings were
  or reduce immunosupressive treatment when multiple reported by Warren Olanow at the Movement Disorders
  allogeneic donors are used and surgery is done in stages? Society meeting (November 10–14, 2002; Miami, FL, USA).3
  Finally, we need to consider the risk–benefit analysis. What is The investigators observed no significant improvement in
  the balance between clinical benefit and side-effects, and how UPDRS motor scores, activities of daily living, or percentage
  does it compare with the other treatment options available?                  of off time. More than half the patients developed
       Now that the NIH trials have been completed and the dyskinesias that persisted for 12 h after discontinuation of
  results reported, we are entering a new phase in the levodopa, but which disappeared 36 h after the last dose of
  development of cell-replacement therapy for PD. In our levodopa. However, there was some improvement after
  opinon, transplantation of fetal dopaminergic neurons is based transplantation surgery in patients with less severe disease
  on a solid rationale and open-label trials have provided ( 49 UPDRS score at baseline).
  convincing proof of principle that transplants can work—and                      Why did these trials not achieve the extent of
  work well—in some patients. However, in the light of the NIH symptomatic relief reported in previous open-label trials?
  trials, it is clear that we need carefully to reassess critical aspects Could the positive results in previous trials simply be
  of the transplantation procedure and further analyse the issues attributable to placebo effects, or were the results open to
  raised in well-designed open-label studies. Further carefully unintentional observer bias? Alternatively, could differences
  planned open-label trials are required to answer various major in transplantation methods, patient selection, or assessment
  outstanding issues—such as those relating to tissue collection protocols account for the differences between the placebo-
  and preparation, patient selection, and surgical placement and controlled and open-label studies? Finally, why were severe


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                                                                                                                                       become increasingly denervated later in the disease process and
     Rights were not granted to include this                                                                                           may therefore have a more important role in advanced cases;
                                                                                                                                       within the putamen, the pattern of denervation also changes as
       image in electronic media. Please                                                                                               the disease progresses. Perhaps these facts need to be taken into
           refer to the printed journal.                                                                                               account when targets for implantation are selected.
                                                                                                                                            The transplanted tissue is itself crucial to the function of
                                                                                                                                       the transplant: previous studies have used different numbers
                                                                                                                                       of donor embryos (2–9 per patient) of various ages
                                                                                                                                       (5·5–9·0 weeks); the storage or culture of tissue before surgery
                                                                                                                                       has varied from a few hours to 4 weeks; and different methods
                                                                                                                                       of tissue preparation have been used to produce different
                                                                                                                                       types of transplants (tissue strands, chunks, or cell
                                                                                                                                       suspensions).2,6,7 The most recent NIH-funded trial reported
                                                                                                                                       by Olanow and colleagues3 suggests that the use of tissue
                                                                                                                                       chunks from four embryos may be better than transplantation
                                                                                                                                       of tissue from only one embryo, but the differences between
                                                                                                                                       these two types of transplants were not significant. Age of
                                                                                                                                       donor tissue and preparation method are known to influence
                                                                                                                                       survival of transplanted dopaminergic neurons. These factors
                                                                                  Catherine Pouedras/Eurelios/Science Photo Library.




                                                                                                                                       may also affect other cells in the graft that could influence
                                                                                                                                       functional outcome. Dopaminergic neurons transplanted as a
                                                                                                                                       cell suspension produce a more homogenous reinnervation
                                                                                                                                       than transplants of tissue chunks. A homogenous
                                                                                                                                       reinnervation may reduce the risk of dyskinesias and increase
                                                                                                                                       the chance of clinically beneficial effects. Storage or culture of
                                                                                                                                       donor tissue before transplantation increases the probability
                                                                                                                                       of dyskinesias.5 The handling of donor tissue may also affect
                                                                                                                                       survival or maturation of non-dopaminergic neurons in the
                                                                                                                                       graft that either directly influence the host striatum or change
  Figure 2. Patient with PD undergoing stereotactic surgery to transplant fetal
  neurons into the striatum.
                                                                                                                                       the activity of the dopaminergic neurons in the graft.
                                                                                                                                       Consequently, storage and culture of donor tissue should be
                                                                                                                                       avoided. Many of these technical issues may require years of
  off-medication dyskinesias not observed in the studies done                                                                          further careful experimentation to develop an optimum
  before 2001? In this article, we discuss factors that might                                                                          protocol for clinical transplantation.
  have contributed to the disappointing findings in the NIH-                                                                                The immune response of the host may have an
  funded studies, and suggest ways to improve the outcome of                                                                           important effect on graft function. Although the brain is an
  future cell-transplantation trials in patients with PD.                                                                              immunologically privileged site, intracerebral transplants
      Patient selection is an essential factor. Freed and co-                                                                          undergo immune rejection in certain circumstances. In the
  workers2 suggested that younger patients might benefit more                                                                          first double-blind study, patients were not given any
  from cell transplantation. Subsequent meeting reports indicate                                                                       immunosuppressive treatment2 and in the second study only
  that levodopa responsiveness at baseline (before surgery) is an                                                                      a low dose of ciclosporin was given for 6 months. In the
  important predictor of positive response to transplantation.                                                                         latter study, evidence of functional grafts was found at
  The unpublished study by Olanow and colleagues3 suggests                                                                             6 month follow-up, but then gradually disappeared, which
  that patients with less severe disease show some clinical benefit.                                                                   could be consistent with an immune-mediated rejection of
  In an open-label trial done in Lund, Sweden, the severity of                                                                         the graft. However, PET scans revealed continued 18F-dopa
  off-medication dyskinesias after transplantation was inversely                                                                       uptake at graft sites 24 months after surgery. Earlier open-
  correlated with striatal 18F-dopa uptake before surgery.5 Thus,                                                                      label trials used a more extensive immunosuppression
  more severe pathology at baseline may reduce clinical benefit                                                                        regimen with three drugs,5,7 which was maintained in some
  and predispose patients to the development of dyskinesias.                                                                           patients for several years. Future clinical trials should
      Graft location may also be crucial. Different approaches                                                                         reassess immunosuppression protocols and investigate
  have been used to target transplants to the putamen (figure 2).                                                                      immune and inflammatory responses around the grafts by
  Sagittal stereotactic injections along the longitudinal axis of the                                                                  use of imaging techniques.
  putamen2 result in a pattern of reinnervation different to that                                                                           What of the dyskinesias? One might speculate that the
  obtained with multiple, dorsoventral injections into the                                                                             grafts produce an excess of dopamine that causes the
  postcommissural putamen6 or into a greater volume of this                                                                            involuntary movements. Two patients in the Tampa–Mount
  structure.5,7 Could imaging studies provide clues as to which                                                                        Sinai open-label pilot study6 showed a greater and longer
  patients might respond better to transplantation? Every patient                                                                      increase in striatal dopamine concentrations in response to
  with the disease has a unique profile of dopaminergic                                                                                levodopa, as assessed by PET scans with raclopride, than is
  denervation in the forebrain. Regions outside the putamen                                                                            typical for patients with PD.8 In five patients with dyskinesias


  THE LANCET Neurology Vol 2 July 2003              http://neurology.thelancet.com                                                                                                                   439



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  from the first NIH-funded study, 18F-dopa uptake was                 formed our standard transplantation protocol. We have now
  increased in small focal areas in the grafted striata.9 However,     done this operation in 60 patients. To reduce the number of
  in the Swedish study, there was no association between               needle passes into the brain, we developed a method in which
  dyskinesia and increased 18F-dopa uptake in the whole                the needle trajectories pass through the forehead above the
  putamen.5 This suggests that an uneven, patchy innervation           frontal sinus and project through the long axis of the putamen
  rather than a generalised overproduction of dopamine could           (35–40 mm) bilaterally. Four needle passes are made in total,
  be the source of the dyskinesia problem. However, graft-             one dorsal and one ventral on each side of the brain (separated
  induced dyskinesias may also depend on other mechanisms—             by a distance of about 7 mm). We also kept donor tissue in
  eg, an inflammatory response around the transplants due to a         culture for up to 1 month before use.
  low-grade, chronic immune response. Three patients from the               In our study, mesencephalic dopaminergic neurons from
  Swedish study, who received immunosuppression for a                  four aborted human embryos were transplanted and strands
  restricted time, had maximum off-phase dyskinesias 2–4 years         of tissue from a single embryo were grafted along each needle
  after surgery, whereas clinical benefit reached a peak after         track. 40 patients were recruited, 20 were under 60 years of age
  1 year.5 The concept of late-onset inflammation as a                 and 20 were over 60 years of age. The aim of the study was to
  contributing factor in graft-induced dyskinesias can be tested       compare the sham-surgery control group with the
  in animal models of PD and ought to be addressed before              transplantation group, in both younger and older patients. No
  large-scale clinical trials are reinitiated.                         immunosuppression was given. At the end of the trial, patients
       To understand which variables are important for clinical        in the sham-surgery group were given the option of receiving
  efficacy of neural transplants in patients with PD, further trials   a transplant; 14 of the 20 patients in the control group took
  in patients are required. However, these ought to be limited to      this option so, in total, 34 patients received transplants.
  small series of patients with well-defined scientific goals, and          18
                                                                              F-dopa PET scans 1 year after transplant surgery revealed
  should be based on improvements in graft technology that can         survival of grafts in 85% of patients.2 Survival was the same in
  only be developed by meticulous experiments in animals.              the younger and older subjects. The younger group showed
                                                                       improvement in UPDRS motor off scores (34%) and in
  Transplantation of embryonic dopaminergic                            Schwab and England off scores (31%) compared with no
  neurons in PD: the Colorado experience                               change in the sham-surgery control group or in the older
  Curt R Freed and Robert E Breeze                                     patients who received transplants. There were no changes in
  Since transplantation of embryonic dopaminergic cells was            best on scores, which indicates that transplants produce
  first reported in human beings in the early 1990s,10,11 several      symptomatic improvements similar to, but not better than,
  open-label clinical trials have confirmed the benefits of            the best effects of levodopa. Interestingly, the longer the tissue
  transplantation. Outcomes of these trials have been similar,         was kept in culture before transplantation, up to a maximum
  despite the use of various experimental techniques and               of 4 weeks, the better the clinical outcome.
  transplantation protocols. The validity of these studies has              Subsequent analysis has shown that preoperative
  been uncertain because of small patient numbers, variable            response to levodopa, not patient age, determines the
  inclusion criteria, and the absence of control groups.               magnitude of clinical benefit after transplantation. As a
       After the election of Bill Clinton as President of the USA in   group, the older patients had more variable responses to
  1992, the NIH agreed to fund a controlled clinical trial. In         levodopa before transplant surgery. Older patients who had
  collaboration with Stanley Fahn (Columbia University) and            good responses to levodopa (>60% improvement in motor
  David Eidelberg (North Shore University Hospital), we                scores) showed a similar response to transplantation to that
  designed a double-blind, placebo-controlled clinical trial to        of the younger patients. Reduced efficacy of levodopa in some
  assess the efficacy of neural transplantation in patients with       older patients may indicate a more extensive disease process
  PD. This was the first double-blind study in neurosurgery.           in non-dopaminergic systems that the transplants were
  Although there was opposition at the time,12 we felt that valid      unable to affect.
  conclusions could only be made from a large study in which                Patients in the sham-surgery group who subsequently
  patients undergoing transplant surgery were compared with a          opted to receive transplants were compared with patients
  control group who would undergo sham surgery.                        given transplants in the initial, blinded protocol. Clinical
       Before we did our first transplant into a human being in        improvement over the first year was identical in these two
  1988, studies in rodent and non-human primates had shown             groups, which confirms that there was no measurable placebo
  that only dopaminergic neurons at an early stage of their            effect of the sham surgery.
  development (7 to 8 weeks after conception in human beings)               Graft development and maturation in individual
  could survive and re-innervate the host brain. Experiments in        patients is uncertain, mainly because most transplanted
  animals also showed that immunosuppression was probably              dopaminergic cells die from apoptotic cell death in the first
  unnecessary for these allografts (grafting of tissue between         few days after transplantation. Pretreatment of embryonic
  individuals of the same species). This finding was critically        tissue with neurotrophic factors before transplantation can
  important because the long-term costs and complications of           reduce this cell loss and may make the effects of
  immunosuppression are difficult to justify for a symptomatic         transplantation more uniform. The biggest limitation to
  treatment. Because the putamen is most affected by dopamine          extensive testing and application of neural transplantation is
  depletion in idiopathic PD, and the structure has many links to      the limited availability of human embryonic dopaminergic
  and from the cortex, bilateral implants into the putamen             neurons. Laboratory production of large quantities of


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  dopaminergic neurons from a stem-cell source could solve              a viable treatment for PD, and should we be able to offer this
  this problem.                                                         form of treatment to all patients with advanced PD?
      The importance of this double-blind design cannot be                   For us, the answer is no. Whatever the outcome of small-
  overstated. Other double-blind trials done after our initial          scale trials, and whatever improvements in techniques are
  NIH-funded study, which have investigated transplants of              made, transplantation of human fetal neural tissue can never
  human embryonic tissue or bovine embryonic tissue, did not            be a large-scale treatment for all patients with advanced PD.
  find clinical improvement despite promising results in small          The main reason for this rather pessimistic view is that the
  open-label trials. For cell-replacement therapies to progress,        procurement of human fetal tissue requires a chain of
  more double-blind comparisons must be done.                           expertise that cannot be “industrialised”. The use of large
      From our experience, most patients whose motor scores             amounts of non-expanded neural cells from several human
  improve in response to transplant surgery develop dyskinesias         fetuses requires an enormous investment in time,
  that resolve after reduction of levodopa or dopamine agonist          organisation, and skill for each patient with PD. There is little
  treatment.2,10 Others have subsequently made similar                  hope that these logistical problems could be overcome. There
  observations. In about 10% of cases, deep brain stimulation of        are not enough data to suggest that we may eventually be able
  the subthalamic nucleus or the internal segment of the globus         to amplify neural cells from human fetuses in “cell banks”,
  pallidus may be necessary to control persistent dyskinesias.          from which neurosurgeons could simply draw the number of
      The ideal outcome of transplantation would be for the             dopaminergic cells they require. Conversely, in this forum,
  patient to regain normal neurological function after the              Lorenz Studer and Roger Barker discuss the potential of
  discontinuation of all their medication. Our experience               human embryonic stem cells and xenogeneic fetal neural tissue
  suggests that such an outcome is only possible in patients who        as potential sources for transplantation. These alternative cell
  appeared free of parkinsonian symptoms and signs while                types may eventually be able to meet the need for a substitutive
  receiving levodopa before transplant surgery. In patients with        cell therapy for all patients with advanced PD.
  advanced disease, the reduction of symptom severity in the off             Transplantation of human fetal neural tissue is a genuine
  state, with maximum benefit about 50% of that with levodopa           ethical concern because it is not available on request and,
  treatment, is a realistic outcome; levodopa and other drugs           therefore, cannot meet its social goal. By contrast, new
  would still be required to fine tune the on state.                    neurosurgical approaches have been introduced in the past
      An intriguing question at this time is whether transplants        decade that are based on ready-to-use and commercially
  can prevent the progression of PD. At 5 year follow-up, we            available equipment—ie, deep brain stimulation of the
  have observed no progression of symptoms in our patients. It          subthalamic nucleus and, to a lesser extent, lesion or
  is possible that cell transplantation early in the disease course     stimulation of the globus pallidus.15 These approaches are
  might prevent complications caused by drug therapy                    now standard procedures in patients with advanced PD.
  (eg, drug-induced dyskinesias) although this hypothesis will          Nevertheless, their expansion to treat the entire population of
  require clinical testing.                                             patients with advanced PD is severely restricted by their
                                                                        high cost and dependence on very skilled stereotactic
  Human fetal neural transplants in patients with                       neurosurgeons. Therefore, the search for alternative
  PD: ethical considerations                                            neurosurgical approaches remains an issue.
  Marc Levivier and Marc Peschanski                                          The discussion about whether, and how, clinical trials of
  There will come a time when patients diagnosed with PD will           human fetal neural transplants should be continued must be
  be treated with therapies that target and halt the                    considered within this framework. First, whether the
  neurodegenerative processes of the disease. At present,               technique should be investigated in further experimental
  patients can be treated effectively, for a while at least, with       clinical trials is dependent on the benefit to risk ratio. At
  therapies that pharmacologically replace the missing                  present, this ratio seems sufficiently positive to continue with
  dopamine. Unfortunately, as the disease progresses, these             such trials, as discussed by Curt Freed. Whether scientists
  medications lose their efficacy and patients develop severe           should invest time and money in doing such clinical trials is
  side-effects. As a consequence, patients with advanced PD are         dependent on the scientific questions that they wish to address
  candidates for alternative therapies, such as neural                  and the worth of the conclusions they may draw. We believe
  transplantation. One of the main ethical constraints of human         that there are major questions to be answered, and that the use
  fetal neural transplants is that they can not be regarded as a        of other cell types for transplantation therapies for PD will
  first-line treatment for repairing damaged neuronal circuitry         benefit from the advances made with human fetal neural
  and rather constitute a “second chance” for patients who no           transplants. For example, one major issue that has been raised
  longer derive benefit from pharmacological therapy.                   by neural transplantation is cell survival, as discussed by Patrik
       On this basis, the success of transplants of human fetal         Brundin and Jon Stoessl. It is likely that this problem is
  tissue is a major ethical issue. We are not referring to any          independent of cell type and there may be ways to overcome
  therapeutic benefit in patients receiving this treatment:             it, which can be investigated in clinical trials of human fetal
  therapeutic benefit, and how to improve it, is discussed by           neural transplants.7 Other major issues that could be
  other authors in this forum. Some individuals who have                addressed in such trials include assessment of the dopa-
  received transplants of human fetal dopaminergic neurons              minergic system reconstruction in vivo, the immune response
  have shown clinical benefit—that is beyond any doubt.13,14 But        to cells transplanted into the brain, and the combination of
  can transplantation of human fetal neural tissue ever become          transplantation therapies with neuroprotection. Small-scale


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  clinical trials that address any one of these questions would
  undoubtedly be very useful in the development of future
  substitutive treatments based on other cell types.
      There was much hope for transplantation of human fetal
  neural cells when the results of the first clinical trials were
  reported 12 years ago. These studies were done by groups of
  researchers with strong backgrounds in basic research and
  focused on a small number of carefully studied patients. It is
  now evident that clinical application of neural transplantation
  to the entire population of patients with advanced PD is not
  feasible. There is little to be gained from the repetition of
  phase III-style studies, such as those done in the USA during
  the past decade.2 Incidentally, such “group analyses” may even
  be detrimental to the scientific goal as they mask interesting
  results of the treatment in individual patients because they
  preclude careful and thorough analysis. In contrast, it is our
  firm belief that there is an ethical need for small-scale,
  scientifically focused clinical trials of human fetal neural
  transplants. Such trials would pave the way for the
  development of other substitutive cell-based therapies that        Figure 3. Neurospheres generated from rat cortex at embryonic day 14.
  may eventually prove to be an effective treatment for all          These neurospheres are expanded in culture with mitogens such as basic
  patients with advanced PD.                                         fibrobast growth factor and epidermal growth factor. These spheres can
                                                                     be used for in vitro studies or in grafting experiments in animal models.
                                                                     Hoescht staining (blue) is used to visualise nuclei, beta III tubulin (red) is an
  Are stem cells a valid alternative to human fetal
                                                                     early marker expressed by immature neurons, and the presence of glial
  tissue?                                                            fibrillary acid protein (green) denotes glia. Courtesy of Laurent Roybon,
  Lorenz Studer                                                      Jia-Yi Li, and Patrik Brundin (Wallenberg Neuroscience Center, Section for
  There has been a lot of excitement about the potential use of      Neuronal Survival, Lund University, Sweden).
  stem cells in regenerative medicine. Cell transplantation for
  the treatment of PD has been proposed as one of the major          and induced to differentiate into functional dopaminergic
  applications for stem-cell therapy. Experience with                neurons (figure 4).18 Unfortunately, these midbrain stem cells
  transplantation of human fetal tissue has provided convincing      lose their ability to differentiate into dopaminergic neurons
  evidence that midbrain dopaminergic neurons can survive            after long-term culture, although they can still generate other
  long-term in patients with PD and can produce functionally         neuronal subtypes such as GABAergic and glutamatergic
  relevant changes in dopaminergic function.16 However,              neurons.19 The persistence of neural stem cells in certain
  disappointing clinical outcomes and the occurrence of              regions of the adult brain suggests an alternative approach—
  significant graft-mediated side-effects in two placebo-            the direct recruitment and conversion of endogenous neural
  controlled trials are raising the bar for the development of a     stem cells into dopaminergic neurons. There is, however, no
  clinically successful transplantation therapy in PD.2,3            evidence that adult endogenous midbrain stem cells can
      The first step in the development of a stem-cell based         differentiate into functional dopaminergic neurons in vivo.20
  therapy for PD is to find a renewable source of midbrain               Stem cells from non-neural tissues are a readily accessible
  dopaminergic neurons. Such a source would alleviate many           source for autologous grafting. Differentiation of stem cells
  of the ethical and practical concerns associated with the use      from various tissue sources, such as bone marrow or skin,
  of human fetal tissue, as discussed by Marc Peschanski and         into cells that express neural markers has been reported.21,22
  Marc Levivier. It would also provide a time window for             The most encouraging is a report of derivation of cells that
  cell characterisation, purification, and banking before            express tyrosine hydroxylase from bone-marrow derived
  transplantation. There are at least four types of stem cells       multipotent adult precursor cells.21 However, the
  that have been studied for this purpose: neural stem cells,        dopaminergic function of transdifferentiated cells from these
  non-neural adult or fetal stem cells (eg, bone marrow or skin      sources has not been characterised.
  cells), embryonic stem cells, and nuclear transfer or                  By contrast, mouse embryonic stem cells readily
  parthenogenetic stem cells.                                        differentiate into neurons that have stable genetic and
      Neural stem cells can be isolated from either the              functional characteristics of midbrain dopaminergic cells.23–25
  developing or the adult brain (figure 3). Methods for their        Transplantation of these cells results in a remarkable
  propagation and differentiation into neurons, astrocytes, and      functional benefit in a rat model of PD.25 The efficient
  oligodendrocytes have been well established for more than          derivation of dopaminergic neurons from human embryonic
  10 years.17 However, despite considerable efforts, the efficient   stem cells has not yet been reported, but there is little doubt
  derivation of functional midbrain dopaminergic neurons             that such protocols will be available within the next few years.
  from neural stem cells has not been achieved. Stem cells               An ideal source of cells for transplantation would provide
  isolated from the midbrain just before the time of                 autologous functional neurons in an unlimited supply. The
  dopaminergic neuron “birth” can be propagated in culture,          successful derivation of dopaminergic neurons from mice


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                                                                               dopaminergic neurons. Furthermore, differentiation of
                                                                               embryonic stem cells in vitro enables access to all stages of
                                                                               dopaminergic neuron development—from neural induction
                                                                               to regional specification, dopaminergic neuron “birth”, and
                                                                               maturation. Systematic transplantation of cells at each of these
                                                                               stages should reveal varied potential for phenotypic
                                                                               differentiation and host integration. Once an optimised cell
                                                                               type has been identified, we need to focus on controlling the
                                                                               interactions of these cells with the host environment.
                                                                                    The adult brain provides limited support for neuronal
                                                                               differentiation, migration, and synaptic integration, and the
                                                                               process of transplantation itself might reduce survival by the
                                                                               induction of an inflammatory response. An additional
                                                                               limiting factor in transplantation therapy for PD is the
                                                                               ectopic placement of the graft into the striatum. Despite the
                                                                               ability of such grafts to release dopamine locally, transplanted
                                                                               cells lack the appropriate afferent innervation. Fetal
                                                                               dopaminergic neurons transplanted into the neonatal
                                                                               substantia nigra have the ability to regrow axons into the
  Figure 4. Dopaminergic neurons (derived from embryonic day 12                striatum.30 However, when the same cells are grafted into the
  midbrain precursor cells) double labelled for tyrosine hydroxylase (green)   substantia nigra of a 20 day old or adult animal, they are no
  and bromodeoxyuridine (red). The presence of double-labelled cells
  confirms that these cells were generated from dividing precursor cells.
                                                                               longer able to extend axons into the striatum. The molecular
                                                                               basis of this inhibition has not been identified, but
  and non-human primates by use of nuclear transfer26 and                      chondroitin sulfate and other myelin-related factors such as
  parthenogenetic27 stem cells proves that such a source could be              Nogo may be involved. Stem-cell technology could support
  available in the future. However, progress in this area has been             strategies to modulate intracellular responses of the grafted
  delayed by ethical and political concerns, as well as by                     cells to this inhibition, or directly change the environment.
  biological challenges.28 Parthenogenetic stem cells, derived                 For example, by increasing intracellular concentrations of
  from unfertilised egg cells, are compatible with the host in                 cyclic AMP, the response of dorsal-root-ganglion cells to a
  immunological terms but might function abnormally due to                     wide range of inhibitory factors in vivo can be reversed.31,32
  homozygosity of alleles and purely maternal imprinting.                      Infusion of chondroitinase ABC into the nigrostriatal tract
       Teratoma formation is one of the defining properties of                 allows partial regrowth of axotomised nigral dopaminergic
  embryonic stem cells, prevention of which is a significant                   neurons in the adult.33 Such knowledge could be used to
  challenge in transplantation therapy. Although the incidence                 develop a stem cell-based strategy for restoring the
  of tumour formation can be reduced by the transplantation of                 nigrostriatal pathway. Concerns about the potential for graft-
  fewer undifferentiated embryonic stem cells,29 the only safe                 induced dyskinesia could be addressed by genetically
  approach is to eliminate all undifferentiated embryonic stem                 modifying cells to express an inducible negative regulator of
  cells before transplantation. Additional safety concerns will                dopamine production.
  need to be addressed before the use of human embryonic cells                      After the disappointing results of fetal tissue
  can be tested clinically. Of particular concern is that all human            transplantation in placebo-controlled trials, the stakes to make
  embryonic stem cell lines listed in the NIH registry that are                transplantation therapy a clinically viable option are high.
  eligible for federal funding have been derived in coculture with             However, recent progress in stem-cell technology provides the
  murine cells, not necessarily in accordance with the good                    necessary muscle and sophistication to take on the challenge.
  manufacturing practice standards required by the Federal
  Drug Administration.                                                         Porcine neural xenografts for the treatment of
       The therapeutic effect of dopaminergic neurons derived                  neurodegenerative diseases
  from human embryonic stem cells in various animal models                     Roger Barker
  of PD is likely to be proved experimentally over the next few                The concept of xenografting—transplantation of tissue from
  years. However, it remains unclear which parameters will help                one species into another—is not a new one. Traditionally, the
  stem cells exceed the therapeutic benefit that can be obtained               pig has been the most logical choice of tissue source. There are
  by transplantation of human fetal tissue.                                    well-established advantages to the use of pigs, such as big
       One important variable may be graft composition. Human                  litters and the potential for greater cell migration and axonal
  fetal grafts typically contain 5–10% dopaminergic neurons (the               outgrowth in the adult CNS.34 Nevertheless, there are some
  rest of the graft constitutes various other neuronal and glial cell          major disadvantages that prevent the clinical use of porcine
  types). Populations of dopaminergic neurons in grafts may                    tissue in the UK.35,36 Despite these disadvantages, in the USA,
  harbour precursors that differentiate into neurons with                      clinical trials of porcine neural xenograft for the treatment of
  distinct functional characteristics of both the substantia nigra             PD and Huntington’s disease (HD) have already begun.37–39
  and the ventral tegmental area. Stem-cell technology should                       The first main disadvantage is the risk of zoonotic
  provide the means to generate purified populations of nigral                 infection. Although pigs can be bred in pathogen-free


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      Forum                                                                                           Neural transplantation




  conditions, transmission of porcine
  endogenous retroviruses from donor to
  host cells is still a risk.40,41 It is not known
  how real this risk is, although it may be
  minimised by selective breeding of pigs
  that are unable to transmit porcine
  endogenous retroviruses to human cells.
      Indeed, a meta-analysis of all
  patients exposed to live pig tissue in
  1999 reported no adverse effects,
  despite the presence of porcine cells in
  the circulation years after exposure to
  porcine tissue.42 However, there was no
  evidence of surviving pig tissue in these
  individuals (except the few cells in the
  circulation). Transmission of porcine Figure 5. Grafts of embryonic day 27 porcine ventral mesencephalon transplanted into the
                                                   6-hydroxydopamine lesioned striatum of immunosuprressed marmosets. The graft has survived
  endogenous retroviruses, without 6 weeks post-implantation and has been visualised by use of pig specific neurofilament staining.
  adverse effect to the host, has Courtesy of Lucy Annett.
  been reported in severely combined
  immunodeficient (SCID) mice that received grafts of pig other compounds (eg, 15-DSG, FK506),53–55 although most of
  tissue.43 The xenografts survived in these mice; however, the these studies have only investigated short survival periods.
  relevance of this experimental result is debatable, because These results should be interpreted with caution because the
  xenografted patients would not be as severely drugs may only delay or slow the rejection process, rather
  immunosuppressed as SCID mice.                                            than prevent it. Nonetheless, the most successful strategies to
      Transgenic pigs that express regulators of the human date have used monoclonal antibodies targeted to
  complement cascade44 have been developed to reduce the lymphocytes or the cytokines responsible for their
  likelihood of complement-mediated hyperacute rejection of recruitment (eg, interleukin 2).56,57 Alternatively, less
  vascularised whole organ xenografts.45 However, this strategy immunogenic tissue, such as porcine neural precursors or
  may actually promote the transmission of porcine endogenous stem cells, can be used.58,59
  retroviruses by inhibition of complement mediated lysis,41                      The third disadvantage relates to the functional capacity
  although these transgenic pig lines have low expression of such and capabilities of xenografts. Data on this issue are limited
  regulators in the embryonic brain.46                                      owing to problems with graft rejection. However, in studies
      The risk of porcine-endogenous-retrovirus transmission, where grafts have survived (figure 5), basic functional deficits
  and its implications, is unresolved. This issue represents a in lesioned animals (eg, drug-induced rotation) have been
  major hurdle to the clinical adoption of this approach, reversed.60 Furthermore, several studies have now shown that
  although it is ultimately more likely to represent a political and xenografts of porcine tissue from various regions of the
  a theoretical risk than a real risk.                                      embryonic brain can extend axons, form synapses, and exert
      The second disadvantage concerns rejection of xenografts. functional benefit in the host rat brain.60,61 Studies of glial
  When grafted into the rat brain, porcine neural tissue is progenitor cells from embryonic pig brain have shown that
  rejected by a cellular and humoral process over a 5 week these cells have remyelinating qualities; this suggests that
  period.47 Humoral rejection seems to have a minor role in the embryonic pig neural cells may have a similar capacity to
  rejection process in the case of pig-to-rat xenografts because repair the CNS as their human counterparts.62
  rats do not have high numbers of circulating naturally                          Although there are issues that need to be resolved, clinical
  occurring antibodies to porcine tissue.47 However, human trials have already been initiated for xenotransplantation in
  beings do have high numbers of naturally occurring HD (although without benefit) and in PD.37–39 In a phase I
  antibodies to porcine tissue, in particular to the glycoprotein study in PD by Diacrin Inc, greater than 30% improvement in
    -galactosyl epitope, which is expressed on neural cells in the UPDRS scores was reported in 3 patients 1 year after grafting,
  embryonic pig brain.48,49 Therefore, in human beings, these although these improvements did not correlate with evidence
  antibodies can bind to porcine tissue and initiate complement of dopaminergic activity in the graft on PET scanning.37 One
  mediated lysis of the cells, similar to the response seen during patient in the trial—who died from unrelated causes
  hyperacute rejection of whole-organ xenografts.45 Various 7–8 months after receiving a graft of porcine ventral
  genetically modified pigs, designed to either express regulators mesencephalon—had a few surviving dopaminergic neurons
  of the human complement cascade or that lack -galactosyl at the graft site.63 Therefore, the basis of any beneficial effects
  epitope, have been developed.50,51 These pigs may thus be of of xenografting in this study is unknown. A phase II study by
  value for clinical xenograft programmes in the future.                    the same company, which has been reported in abstract
      Apart from these genetic approaches, the prevention of form,39 enrolled 18 patients with advanced PD who were
  porcine neural xenograft rejection by drug therapies has been randomly allocated to receive either a xenograft (and
  attempted. Monotherapy with ciclosporin A resulted in 40% ciclosporin A monotherapy) or sham surgery. Both groups of
  survival of xenografts.52 Better results have been obtained with patients showed significant improvement in their motor


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        Neural transplantation
                                                                                                                                                                  Forum

  scores, although there was no significant difference between                                     moratorium on all clinical studies of this treatment
  the grafted and the sham-operated groups. The reason for such                                    approach in the UK (and in some other countries). As a
  a large placebo effect is unclear, particularly because this has                                 result, alternative cell sources are being pursued, including
  not been reported in sham-operated groups in trials of human                                     the possible use of neural stem cells.
  fetal allografts.2,3 However, a more detailed analysis of the study
  is required before any further comments can be made.                                             Acknowledgments
       In summary, xenografts of porcine neural tissue offer                                       LS’s work cited in this article was supported by the MJ Fox Foundation
                                                                                                   and the National Institute of Neurological Disorders and Stroke (NIH).
  some hope but there are major problems with this                                                 RB’s work cited in this article was supported by the Medical Research
  approach that—at the present time—have led to a                                                  Council, the Parkinson’s Disease Society, and the Wellcome Trust.

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