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IVth International Symposium on Neural TransplantationTranscript of the Closing Discussion (Edited)

(Session 44.0)Washington, D.C.: July 16, 1992

D. Eugene Redmond Jr. and Barry J. Hoffer (Co-Chairs)and Discussants

Eugene Redmond:

I think that the sessions have been extremelyinteresting and provocative. It would be a shame toend the meeting without an opportunity to raisefurther questions, and to tie some issues togetherbetween the various sessions, such as relationshipsbetween the clinical and basic issues. There aremany interesting loose ends, perhaps related toneural transplantation in general or to specificissues raised in the various sessions, that it wouldbe useful to try to tie together. At the same time,because we have a large audience and hopefullymany of you have important things to say, we aregoing to set some ground rules for the discussionthat will give everybody an opportunity to speak.Therefore, we have chosen some specific topics asa way of beginning the discussion, and then we willopen the discussion to the floor. We are going toask the people who are speaking to limitthemselves to 2 minutes so that everyone or nearlyeveryone can get an opportunity to speak.

The first issue in which several people haveexpressed a significant interest, is a continuation ofthe discussion concerning the design of clinicaltrials, and what do we need to do next to move thefield forward. I have had a request from PatrickAebischer to start that discussion.

Patrick Aebischer:

Several important questions have arisen duringthe course of today’s interesting sessions. One ofour major problems is the interpretation of clinicalresults. There are deficiencies in controls andethical reasons prohibit the use of sham operationsthat might provide the best results. We may,however, be able to devise a system that wouldimprove the current situation and would facilitate

the comparison of results. I found it particularlydisturbing that at the end of this afternoon I had tomake up my mind on a very subjective basis, partlybecause all the groups used different techniques,different patient populations, varying ages ofdonors and different implant sites, and also utilizeddifferent surgical techniques and evaluations. Somegroups also use cryopreservation. Under thesecircumstances, it is very difficult to draw anymeaningful comparisons, especially when thenumber of patients is three or four. I thereforebelieve that there is a need to coordinateexperiments to allow better evaluations andultimately arrive at some clear conclusions.Regarding the problem of evaluation, I noted that atleast one study made an attempt to use non-operated patients as controls. Even if the numbersare small, it might be important to use this kind ofstudy in a comparison, and to increase theirnumber. Another important method was the use ofblind evaluation. If I understand correctly, one ortwo groups tried to evaluate their patients"blindly", and this is certainly a very important andfundamental issue.

Finally, two small points. We have anotherneurosurgical alternative for the treatment ofParkinson’s disease, that is, lesions. As you know,Mahlon Delong’s work /2/ now suggests that itmight be possible to lesion the subthalamic nucleusand obtain a striking improvement, at least in theMPTP monkey model. The neurosurgicalcommunity is considering trials of this procedure inhuman patients. Could clinical lesion trials of thistype be compared to transplants in a rationalfashion?

The people who presented it may be able toexplain a little more clearly this last point. In theone patient that died there seemed to be nosurviving cells. All PET scan data, however,suggest that there is survival of the transplanted

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114 D.E. REDMOND JR. AND B. J. HOFFER

cells. Has there perhaps been an improvement inthe resolution or hardware of PET scans, so that wecould be misled just by an improvement in thetechnology?

Barry Hoffer:

Would someone like to comment on any or allof Dr. Aebischer’s questions?

Curt Freed:

We are not exactly starting from a blank pieceof paper here. The results of experiments in ratsand the related experiments in the monkey havebeen quite strong. Experiments in rats that haveincluded controls suggest that dopamine celltransplants produce effects better than adrenal cells,at least without nerve growth factor. The fetal ageof rat cells suitable for transplantation is a verynarrow and well-defined stage of development.These results from animals have been at the heartof the planning process that we have used. Thegroup from Lund has proceeded in the same way,although I don’t want to speak for someone else.Our conclusions have also been supported bymonkey experiments. We are using PET scansprior to surgery to demonstrate that the caudate isin relatively good shape and the putamen has asevere fluorodopa uptake defect. Based on thestudies by Kish, Hornykiewicz and coworkers/10/,who have measured dopamine depletion inParkinson patients, it looks like the putamen is thebest target for transplantation. Targets can beindividually chosen for each patient by using PETscans; in other words if fluorodopa uptake looksnormal in the caudate, this would indicate that it isnot necessary to use that as a target fortransplantation. While the sprouting phenomenonobserved after large lesions of the caudate isinteresting, no one has provided evidence thatsprouting occurs in the putamen after stereotaxicimplants. Thus, the fluorodopa uptake data mostlikely show growth of fibers from the fetaldopamine cells. We would argue that experimentsthat are not using 6-8 week gestation humanembryos, and which are not putting the tissue in theputamen are not going to be successful.

Patrick Aebischer:

It would be irrational to claim that rats andprimates do not yield good data. The animal datado not, however, prove the validity of the humanresults. The problem arises when you have to scaleup to humans. You are then dealing with a muchlarger structure. I think that it is important to designthese studies so that they can stand on their own. Itis difficult for those of us not doing clinical trials todraw any conclusions. To put it bluntly, should younot coordinate your clinical trials, so that you areno longer looking at 20 variables but less variableswhich are better controlled?

Don Gash:

My question follows up on Dr. Aebischer’scomments. How far along are the groups doingclinical studies in terms of adopting a uniform setof standards? As I recall, in the transplant meetingthat was held in Lund several years ago, acommittee was formed to set standards forevaluating patients. These standards have beenpublished /11,12/. Are the groups doing clinicaltransplants following those standards?

Olle Lindvall:

The Core Assessment Program for IntraeerebralTransplantation, that is, the "CAPIT", has beenpublished in a book from the Lund meeting/9,11/and in Movement Disorders /12/. Several groupsare now following CAPIT. In Europe we haveorganized a Network for IntracerebralTransplantation and Restoration called"NECTAR". A major task for NECTAR is tocoordinate clinical trials with neural grafting inEurope. All groups in NECTAR will use CAPITfor evaluating their patients before and aftertransplantation.

One point that Dr. Aebischer brought up wasrelated to PET scans. We have experience overseveral years with PET scans using the samemachine in exactly the same way. We havedefinitely not seen improvement of fluorodopauptake in all patients, and we have not seenimprovement in all parts of the striatum.

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TRANSCRIPT OF THE CLOSING DISCUSSION 115

Ignacio Madrazo:

In relation to the clinical effect caused by thelesion, we have the experience of having useddifferent kinds of grafts; so we have patients thathave received fetal transplants, co-transplants, andadrenal transplants. The time course of evolutionfor every group of patients is completely different.So, we can clearly state that the clinical response isrelated to the grafts, because they have acompletely different course of clinical evolution inthe different kinds of grafts.

Scientists are calling on the clinicians to getorganized. I will also call on scientists to getorganized, because I have heard a lot of differencesin the results of different groups in different labs.That’s something that happens in science at alllevels, so I think that it can also happen in clinics.

Patrick Aebischer:

Should we compare transplant studies to lesionstudies, especially those following MahlonDelong’s studies /2/? I know that several groupshave started to lesion the globus pallidus internal(GPI), and are thinking about lesioning thesubthalamic nucleus. Could we not compare theseprocedures in clinical trials?

Edward Hitchcock:

Those interested in the lesion trials will almostcertainly use the CAPIT scheme, so there will be aregularity and uniformity of approach, in whichcase the lesions can be compared to transplantationstudies. As long as we all do the same assessments,it will be all right it does not have to be the samegroup doing it which is the whole point of theCAPIT scheme.

Olle Lindvall:

It is important to state that, even if from theneurobiologist’s point of view it is extremelyexciting that we can restore neuronal circuits in thebrain, the main clinical question regarding neuraltransplantation is whether and how much thepatients get better. If you treat patients, it is yourresponsibility to test and compare different newpossible approaches in a similar way. In my

opinion the lesion data are clearly interesting,though this strategy has not been conclusivelyshown to be clinically valuable.

Juan J. Lopez.Lozano:

To me the crucial point at this time in the fieldof neural transplantation is, after searching amongthe audience, I have the feeling that we are losingour audience among neurologists. I think it issecondary to the way that we have conductedneurotransplantation and clinical studies. Theproblem of the lack of credibility of the resultsobtained in humans lies in several importantfactors, particularly as mentioned before. I thinkthe situation of neurotransplantation in Parkinson’sdisease from the neurologic point of view is filledwith serious difficulties. People from differentcountries have been using different techniques,different donor tissues, in different types ofParkinson’s patients. All of these differences occurbecause of the lack of using common protocols orfollow-up.

So, all of us are speaking our own language butnevertheless we are all getting good results. I cannot understand why, using different kinds oftissues, in different people and using differentfollow-ups let us say using adrenal medulla orfetal mesencephalon we are getting such similarresults. There are also puzzling differences in thetime course of the clinical changes. I notice thatsome people using fetal ventral mesencephalon aregetting good results two months after surgery, andfor other groups the changes require 24 months.Using adrenal medulla, improvement sometimeslasts for two months and in other cases lasts 40months. I would like somebody to explain to mehow this can be possible.

Patrick Aebischer:

To be the Devil’s advocate, I would like to drawyour attention to the small improvements that areobserved with the various procedures and tissuetypes used. I would then be worried aboutsomething called the placebo effect.

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Don Gash:

There is also the possibility of commonmechanisms underlying recovery, if people areseeing very similar effects. Now, from thissymposium, I am wondering if we could have somecomments on the differences in the degree ofrecovery seen using adrenal medulla versussubstantia nigra grafts. The other day, there wereseveral groups who described results of co-grafttrials. If Ray Watts is here, perhaps he could talkabout using the co-graft approach and howpromising those results have been. While a placeboeffect is one possibility, those are other alternativeswhich are best studied in non-human primatemodels. Trophic factors stimulating residual hostdopaminergic pathways, for example, mightprovide for a common mechanism underlyingfunctional recovery in both adrenal medulla andfetal tissue graft recipients.

Roy Bakay:

I think that there is a feeling among many of usnot directly involved with clinical fetal graftinginvestigations that we are unsure of the degree ofimprovement in these studies. If you look at thereports from everyone who presented today, fromthe first patient to the last patient, there has been aremarkable evolution. None of the investigators isstill using the same techniques as on the firstpatient. The investigators are changing things,moving things around, and in each case they aredoing something different. We must recognize thisas an evolving process, and there is not a"technique" that can be applied on a large scale.One way to look at this question is to ask howmany of us out here would ask one of theseinvestigators to operate on our mother if she had asevere case of Parkinson’s disease. am not sure ifthere are a lot of people in the audience who areready to make that decision before more studies areperformed.

Ultimately a multicenter controlled study willbe needed, but before that can be considered wehave to develop a surgical technique that we can allagree is truly effective. The only way we can get tothat point is to be able to compare the results ofdifferent groups in different countries using

different techniques through use of the CAPITprotocol /11,12,20/. Only by making the datacomplete, accurate, reliable and comparable can wecompare results directly and not have to go througha series of indirect analyses. There is a bigdifference between a pharmacologic study and asurgical study. Surgical studies always go throughthis kind of evolution to refine a technique beforeimplementing a randomized study or a controlledstudy. Even so, you can never randomize in thesame way as a drug study. There are a lot ofreasons for this, which I won’t get into, but thereasons should be obvious.

Curt Freed:

Roy, at the same time I like the idea that wehave the CAPIT set of standards, the fact thatParkinson’s disease is variable means that therealso has to be enough data on each individualpatient to say that those measurements meansomething in that particular patient. If you see apatient at isolated intervals, you get very littleinformation, so you would have to say we need 50CAPIT studies per patient, or something like that.

Roy Bakay:

The CAPIT standards deal with these issues ofhow many evaluations you need, how closetogether, and that sort of thing. Those factors mustbe considered in designing any study. The CAPITrecomendations are for the minimum requirementsand the greater the number of pre-operativemeasures the better the assessment of the patient’sseverity and variability of symptoms.

Tom Freeman:

think the critcisms that there should be moreunification in methods is a little severe at this point.For a number of reasons, there are about 15different variables that could influence the successof transplantation trials. It is healthy to try toapproach this as each individual investigator seesfit, based on their own experience and their ownlaboratory data. It is premature to say that any oneparticular method is optimal, since no one has hadan overwhelming success at curing all or even most



symptoms of Parkinson’s disease in the clinic, or inanimals for that matter.

Another issue that I think is being neglected isthat there seem to be at least two differentmechanisms that may be involved clinically, bothof which may correct the symptoms of Parkinson’sdisease. In some cases, the graft may not havesurviving dopaminergic neurons but may induce atrophic response in the host brain. It is also possiblethat clinical improvement may be obtained byhaving optimal survival of neurons that reinnervatethe brain. I think that investigators should be clearas to which of these two strategies they are tryingto optimize. Transplantation of fetal nigral tissueusing techniques that do not produce survivinggrafts in the laboratory, under the premise that itwill create a neuronal hook-up of new circuits, isnot particularly fair to the patients. If induction oftrophic effects is the goal, there are several othertypes of tissues which are far safer, easier and moreeffective to use and may produce the same endresult. Thus, we should attempt to assessbeforehand what we are trying to achieve with ourtransplants.

Barry Hoffer:

Let’s take one more comment on this topic and Iguess Dr. Sladek, that means you are going to getthe last word.

John Sladek:

I am glad Dr. Freeman raised that point becauseI think that we may be unduly harsh on ourselves,even though the objective of a scientist is to beself-critical. In fact, that is what research is allabout, a great variation in design, approaches andtechniques. Often usefully, and sometimes not sousefully, there are also differences inintrepretations but that is also what the purpose ofthis meeting is about. In the United States, our owngovernment, in its infinite wisdom, chose not tofund this kind of research and I think that, had theychosen to do so, we might be sitting here after a 5year coordinated clinical trial with morestandardized approaches and more standardizedinterpretations. At the present level of knowledge,however, the lack of a coordinated clinical trial is

not necessarily bad and I think the kind of self-analyses and critical analyses we are having willclearly lead the field to a very positive endpoint.

I would add only one point of minordisagreement regarding reversing all the signs andsymptoms in any of the patients. In addition to thefine studies that were presented today, I think thestudy by Dr. Widner and colleagues in MPTPpatients /21/ is probably the one that allows theclearest interpretation; after all, those are patientswith a non-progressive form of parkinsonism.These MPTP patients more closely parallel thecondition seen in the animal models that we haveused, particularly the non-human primates. Therewe have seen several years of an exceptional levelof improvement that is undoubtedly due to thesurvival of dopamine nerve cells from the graftedfetal neuroblasts. I hope we are not too hard onourselves.

Barry Hoffer:

That is a good note on which to close the firstpart of the discussion. Let us now move on to asecond topic that a number of people have talkedabout informally, and which may be appropriate forthis round table. That is, we have focused onParkinson’s disease in particular and a few otherdiseases during the several days of this meeting,but what about other diseases? What kinds ofdiseases have we not discussed so far, or discussedonly briefly, where transplantation of nerve cellsmight be of importance. Does anyone have anythoughts about that?

Ignacio Madrazo:

I don’t have thoughts, I have cases. We havedone already two cases of Huntington’s disease.The first one is a patient now followed for morethan one year after surgery and who has obtained amoderate benefit from surgery. She is stillimproving after one year. The second case was justdone very recently. So, that is a new approach, anda different disease application of transplantation.

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Oile Lindvall: William Freed:

There are a number of neurological disorderswhere there could be a potential usefulness of theneurografting techniques. From the clinical point ofview, it is of course a dilemma for the doctorbecause, in many of these disorders, we have notreatment at all, and the symptoms are very severe.Nevertheless, in most of these disorders, there is noscientific basis for clinical trials even in desperatecases. In my opinion, the only disorder besidesParkinson’s disease where presently one couldmotivate clinical trials is Huntington’s disease.Huntington’s disease is a fatal disorder, withprogressive dementia. The patients die within 10 to15 years, and there is no treatment. The lack ofadverse effects in the Parkinsonian patientsimplanted with fetal dopamine cells, the evidencefor growth, survival and function of the grafts inthe human brain, as well as the importance of evena minor symptomatic improvement in Huntington’sdisease by neurografts would help to justify thefirst clinical studies with implantation of fetalstriatal neurons into the striatum in Huntington’sdisease. However, in contrast to Dr. Madrazo, Idon’t think that we are at the stage yet when suchstudies should be initiated. There are a number oftechnical and scientific problems that we mustsolve before we can start even the first clinicaltrials in Huntington’s disease. The animalexperimental data are very promising: there is goodevidence that striatal grafts can survive in theibotenic acid-lesioned striatum, that they are atleast partly anatomically and functionallyintegrated into the host neuronal circuitries, andthat these grafts can improve cognitive and motordeficits. However, we know very little about howmuch tissue we should implant, what is the optimaldonor age, and other technical aspects of criticalimportance for a clinical application.

There is also a major drawback at present in thatwe don’t know how we shall assess cognitive andmotor function in the Huntington’s patients beforeand after transplantation. Furthermore, it is criticalthat we have methods to assess graft survival inthese patients. When these problems have beensolved, I think that we can justify clinical trialswith neural grafting in a few patients withHuntington’s disease.

Apparently, Jackie Sagen is not here, but shemight want to say something about her data. Theclinical trials in patients with chronic pain that shehas described/17/are extremely interesting and Ithink that continued work along these lines iscertainly justified.

Aside from actual clinical trials per se, anotherissue that might be worthwhile to discuss is whatother diseases potentially could provideapplications for transplantation in the future. Oneof the diseases for which a very good case could bemade, it seems to me, is epilepsy. In the case ofepilepsy, I am not suggesting in any sense thatclinical trials should be done now. However, itseems to me that this is a good possibility for thefuture. The abnormality can be localizedanatomically and it originates from small areas ofthe brain. One of the major obstacles in epilepsymay be that the animal models are not adequate orare not sufficiently similar to human epilepsy.Would anyone care to comment on that perhaps?

Olle Lindvall:

We have been working with transplantation inexperimental epilepsy for 7 years and have goodevidence that neural grafts can suppress epilepticphenomena in the brain /1,13/. In our modelsystem, we have used kindled seizures whichresemble temporal lobe epilepsy in humans. Weimplant noradrenergic neurons and can suppressthe development of seizures in the kindling model/1,13/, which may suggest clinical applications.However, it has not yet been shown that grafts canhave also an anticonvulsant effect on establishedseizures.

Also, we have only been able to show theantiepileptogenic effect if we first damage theintrinsic noradrenaline system. That is a problem,because there are several forms of human epilepsy,and it is not known if any of them is linked to adeficiency in the intrinsic noradrenaline system.Thus, it is unclear whether our transplantation dataobtained so far have any clinical relevance. We arenow looking at other types of cells as well, e.g.GABA producing ones. I would strongly argue



against any clinical trials with grafting in epilepsyat the present stage.

Roy Bakay:

You have to look at the severity of the disease andthe severity of the proposed treatment. Moredesperate diseases require more desperate attemptsat treatment. For a glioblastoma patient who isgoing to die within a year, there is a tremendousamount that can be done experimentally becausethe risk of the procedure can be balanced by therisks of dying from the disease. For someone with adisease in which they are going to live 20 or 30years, the risks of any investigative procedure mustbe low and the experimental data need to be verycompelling before you enter into surgical trials.

I think there are really only two other diseaseswhere grafting techniques might be applied tohumans in the near future; one is the cancer painthat Jackie Sagen’s group presented/17/, and theother would be in Huntington’s disease /cf.6,9,14,19/. In both cases, the patients die in arelatively short period of time and we don’t haveadequate therapy. We must avoid the mistakes thatwere made early on in Parkinson’s disease, wherewe grafted adrenal tissue into numerous patientsand only later found that it generally didn’t surviveand only worked for a short period of time. Fordiseases that run a benign course we need to do agreat deal of animal investigation, exactly as Dr.Lindvall was describing earlier. I think that there isabsolutely no reason to start clinical trials for theother diseases discussed today such as epilepsy. Itis important to remember that the animal modelsare not the disease. We need to understand thepathophysiology of these diseases and themechanism of graft function before applying thistechnology to "benign diseases".

Richard Bunge:

If we are to develop a catalog of potentialtransplantation applications in neuronal lossdiseases, I think it should be mentioned that, at thismeeting, there have been a number of ratherremarkable papers about the success of replacinglost somatic motor neurons in the spinal cord. Oneapproach has involved introducing fetal tissue into

regions of the spinal cord depleted of its normalmotor neuron population. In work from Francefrom Drs. Horvat and Peschanski /8,15/, theseimplanted motor neurons have been successfullyguided to nearby muscle by nerve bridges. Thiswork provides evidence that transplanted motorneurons in spinal cord in experimental animals,especially rats, can be guided to successfullyinnervate skeletal muscle. Another more extremeapproach to this kind of injury, and to motorneuron disease in particular, is to actually placemotor neurons in the peripheral nerve near adenervated muscle. The purpose in this case is toprovide absent neuromuscular innervation with thehope of driving these isolated transplanted motorneurons by functional electrical stimulation toattain functionally useful contraction /5/. Insummary, I think there have been early butpromising developments in the possibility ofreplacement of the somatic motor neuron.

Helen Hodges:

I think another case where it might be possibleto develop transplants for human therapeutic use isafter heart attack. There are animal models thatmimic very exactly the interruption of blood flowto the brain and also result in focal hippocampaldamage. This sort of damage has been identified inheart attack victims. In fact, about 20 percent ofpeople who survive heart attacks are found tosuffer from very severe memory deficits. I thinkwhat some clinicians could be doing at the momentis building up very accurate pictures of the kind ofmemory deficit and the type of cell loss that occursfollowing heart attack, so that animal models thatmimic these same patterns of damage could bedeveloped. We could thus look at the effects oftransplanting particular cell types. For instance, wehave found the CA1 cell type to be an importantfactor in improving memory performance ofanimals with CA1 cell loss following ischemia. Ithink that ischemia-induced injury is an area whereclinicans could do a lot of preparatory work whileanimal models are being developed.

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Barry Hoffer:

Let us now move on to a final topic for generaldiscussion that some people brought up informallyover the last few days. We started to see glimmersof this around the time of the Third IntenationalMeeting in Cambridge and a bit earlier, and it hasreally grown very impressively to maturity here.Perhaps not full maturity as yet, but adolescencecertainly. This is the application of geneticengineering and immortalization techniques to cellsthat are to be transplanted. Perhaps we could nowhave some comments about that.

Richard Robbins:

There are a number of very important issues toconsider when dealing with these kinds ofapproaches for cell transplantation. Obviously, oneof the most important is that many of the genes thatare introduced in order to help cells divide are inthe category of oncogenes, and these can actuallyinduce a condition of transformation. Suchtransformed cells may have anywhere from a smallto a large capacity to continue to divide in thecentral nervous system. The development of, forinstance, temperature-sensitive oncogenes such asthose described by Ron McKay and co-workers atthis meeting/4/is a significant advance in trying toovercome this problem. Another category of genesthat don’t normally induce a transformedphenotype, called immortalizing genes, have begunto be explored as a possible way to turn a switch onin a cell to make it divide. Such immortalized cellsmay continue to produce their own native factors orbiologically active molecules. Genes such as theadenorviral E1A gene are an example. There aresome new genes coming down the pike that mightnot have the transforming potential, and couldpotentially be useful for development of cell lines.

I think that one of the other general approachesthat might need to be considered carefully here iswhether or not we can simply transplant cells thathave been modified to overproduce one growthfactor or one neurotransmitter in an unregulatedway, as opposed to implanting cells that have aneuronal phenotype and may be able to makeappropriate synaptic connections and release either

proteins or small neurotransmitters in a regulatedway.A major issue that one should consider when

trying to create cell lines, is that there are twofundamentally different approaches. You can startwith the cell that you want and add on gene thathelps that cell divide, hopefully changing itsphenotype as little as possible. Alternatively, youcan take cells that can be genetically manipulatedquite easily, and introduce the gene, for instance,for tyrosine hydroxylase, to try to get that cell tomake dopamine. I think we are obviously going tohave to demonstrate that any cell that makes acertain factor can be transplanted not only intorodents but primates with a great deal of safetybefore we will be able to approach our institutionalreview boards or human safety committees toconvince them that we will be able to put thesecells into a human and have absolutely no chanceof forming a tumor.

Paul Carvey:

This a general observation. I see a lot of peopledoing very nifty things with gene transplantation---it is a marvelous field. Nonetheless, I am alwaysthe individual who asks the question---how fardoes the substance diffuse? I see a lot ofindividuals who are trying to develop things thatmay not be practical because the substances theyare trying to make don’t diffuse far enough fromwhat is essentially a point source. So I think that itis important that you should establish thesefundamental principles before developing aprotocol to engineer something. If you make it andimplant it into the brain, can it diffuse far enough todo any good?

Edward Baetge:

The ultimate cells for neural transplantationmay be those that can naturally give rise to specificdifferentiated phenotypes and be propagatedcontinuously in culture. There are now a number ofneuroepithelial progenitor cells which have beenimmortalized using SV40 or Myc retroviraltransgenes as recently described by Ron McKayand co-workers /4/ and Connie Cepco and co-workers /3/. Although these cells appear to



differentiate after in vivo transplantation, it remainsto be determined what true long-term in vivopotential they have and whether the immortalizingtransgenes they express are safe for prolonged invivo use. An alternative cell might have theproperties of a neural stem cell, giving rise to bothneurons and glia but also capable of continuouspropagation in vitro without the use ofimmortalizing or transforming genes. As describedearlier today, Sam Weiss and Brent Reynolds/16,18/provided evidence for such a cell that wascapable of being continuously grown in vitro anddifferentiated into a number of neuronal and glialcell types. Such cells are unmodified and mightprovide a plentiful transplantation cell sourceassuming they could mainta,.’,a their potential fordifferentiation upon transplantation or treatmentwith various mitogenic or trophic factors. Muchwork remains to determine the full potential for thein vitro/vivo generation of differentiated CNS celltypes, but for the first time we are able tocontinuously culture neuroprogenitor cells whichshould provide for rapid progress in this area.

Fred Gage:

Another way to look at the idea of gene transferfrom the central nervous system is to take it out ofthe context, if we could for the moment, of theclinical setting. There are other reasons why onewould want to introduce genes into the centralnervous system. Traditionally, the approach wehave had in these meetings has not been just toaddress diseases, but also to use transplantation as amethodology to understand something about thebrain. In that context, I think the delivery ofindividual genes, either somatically through the useof transplanted cells or directly through direct genedelivery, is one way of more specifically askingquestions about how specific proteins ortransmitters function within the central nervoussystem. To that end, we are at the early stages ofthis methodology. There are at least 10 questionsthat have been raised just by the last two speakersand I can think of 100 more that are necessary to beaddressed, not for any kind of clinical applicationbut even in terms of utilizing this as a tool. But Iwould say that from what we have seen in thismeeting in the last few days, there have been

probably 15-20 new applications of gene deliveryin the central nervous system as a method oflooking at regenerative responses and release oftransmitters.

While there are a lot of questions that remain tobe answered, I think that what we are seeing is thebirth of a field which will help us to address morespecifically the role that transmitters and trophicfactors play in the central nervous system. If wethink about it that way, rather than immediatelytrying to think about how we can apply this to aclinical setting, we might be able to resolve someof the fundamental issues first, such as what celltype to use, and ways of achieving long termregulatable gene expression, before we startdesigning things specifically for their clinicalapplications.

Phiilipe Horrellou:

I would like to add some comments to theprevious ones. As you know, genetic engineeringfor intracerebral transplantation is a new field.What we have achieved so far is the use of genetictools to produce and deliver neurotransmitters orgrowth factors after gene transfer to cells fortransplantation. In our efforts to compensate fordegenerative processes, for the time being, we haveat best been able to deliver the molecules thoughdiffusion from the grafted cells. There are a numberof tools and experimental conditions that are nowbeing approached in order to improve thesemethods to obtain specific and regulated releasefrom neurons, such as immortalized neuro-epithelial cells developed, for instance, by RonMcKay/4/. Beside genetic modification of cells fortransplantation, direct gene delivery into neurons isemerging with new vectors. Such vectors, forinstance those illustrated by the methods whichwere very nicely described by Joseph Glorioso inthis meeting/7/, may soon be available. We are stillat a starting point and very few applications ofthese vectors have been presented. However, wewould like to remove the viral genes which arepresent in these vectors that might induce sideeffects. I think that there is a requirement for majorimprovements in the genetic engineering systemsbefore we can go in a clinical direction. Inconclusion, gene transfer to the brain is a starting

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field and major improvments are taking place. Itrequires considerable effort to develop these toolsfor clinical applications, both in term of efficiencyand in terms of safety.

Jens Zimmer

If I may say a few words, I find it extremelyfascinating in this meeting to see that cells that arenormally in the brain can be taken out,immortalized, and then can actually be put backinto the brain again by using transplantation as atool. In the adult rat brain, within 15 days suchcells can transform into genuine CA3 pyramidalcells if you put them in the hippocampus,neocortical pyramidal cells when you put them inthe neocortex, or they can be granule cells whenyou put them in the cerebellum. With other celllines, glial cells may be formed. I am really lookingforward to the meeting in Paris, because I think thatwe are at a stage that we will soon see greatadvances and refinement in the techniques.

Going back to the clinical issue, I would saythat in many of the cases where we do not know themechanisms of a disease, we nonetheless see thatcertain nerve cell types degenerate. After cerebralischemia, the CA1 pyramidal cells in thehippocampus are, in particular, susceptible. I thinkthat in two years time in Paris, we may see celllines that when introduced into the brain, into theCA1 area, will transform into CA1 pyramidal cells.Moreover, a very interesting thing is that the adultbrain seems to have instructions, or environmentalconditions, that will make these cells differentiateand perhaps even form and this is where basicresearch comes in the right connections. So, inthat sense I am very optimistic about the prospectsfor this methodology. With refinements intechniques, and also with further clarification, wemay see large advances in cell engineering anddevelopment. Many of these we may havepredicted, but now in some cases we know for surewhat is possible.

Richard Robbins:

I would just like to extend that thought bysaying that I think that some of the newermolecular biological approaches for understanding

the commitment of precursor cells in the brain andunderstanding how to express genes after the cellshave made a terminal commitment are going to bevery dramatic. In four or eight years time, when wehave these meetings, we are not even going to betalking about transplanting fetal tissue anymore.Fetal tissue is going to become a thing of the pastvery soon and we are only going to be talkingabout transplanting engineered cells.

Curt Freed:

Dr. Robbins, this has not happened yet withkidneys, livers or hearts. I think we have to behumble when we look at the evolution of thesewonderful dopamine cells. It will not be easy tomake our own versions of normal brain cells.

Barry Hoffer:

Are there other topics or areas the audience wouldlike to explore?

Larry Kromer:

I would just like to add a comment. At the end ofthese meetings we always look for clinicalapplications. Our discussion is usually focused oncell replacement theories and the clinicalapplication of replacing degenerating neurons. Ithink that there is a whole aspect of transplantationthat has a very classical history from early in thecentury, and that is to understand how the nervoussystem is put together, the intricate interactionsbetween cells, and the role of glial cells in thisinteraction. I think that we should not lose ourappreciation for using transplants to understand thenervous system and its development. We shouldnot always have to feel it has to have clinicalapplication as a primary focus.

Barry Hoffer:

Your point is very well taken. Do we have othercomments?

Paul Sanberg:

I heard the comment earlier, that perhaps inabout four or five years we won’t even be



transplanting fetal cells. One area we need to thinkabout is how do you determine the "dose" of cellsto transplant? Right now we are lucky, in that weare implanting autografts of adrenal cells, orallografts of fetal cells. Perhaps 10% of themesencephalic cells that we are transplanting aredopaminergic. However, when we start usingengineered cells, we will have very large numbersof pure dopamine- secreting cells, or growth factor-secreting cells. If we are successful, and we usethese cells as a cell therapy, it is not like a drug.These cells are going to stay in there. Thus, wereally need to think about how we dose thesethings. With any drug, for example, you get acertain percentage of patients that show abnormalresponses. Some adverse responses and side effectscan be anticipated with cell transplants as well.How do you take those cells out, how can you getrid of them if the patient has an abnormal adverseresponse? We have so far been quite lucky withmost of the clinical data. With the fetal cell grafts,we have not seen many harmful effects, but I thinkas we get into these other types of cells, it’s goingto be a real critical issue and one to think about.

Barry Hoffer:

We would like to close this discussion byexpressing our graditude to the organizers, theaudience, and all of the participants for anenlightening and stimulating meeting. I think thateveryone has done a wonderful job. There was a lotof effort involved in organizing this meeting, andwe want to thank all of you very much forparticipating.

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3. Cepko C, Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartweig EA. Multipotent neural cell linescan engraft and participate in development of mousecerebellum. Rest Neurol Neurosci 1992; 4: 135.

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ADDRESSES AND AFFILIATIONS OF PARTICIPANTS:

Barry J. Hoffer (Co-Chair)Department of PharmacologyUniversity of Colorado HealthSciences Center4200 East Ninth Ave.Denver, CO 80262, USA

D. Eugene Redmond, Jr. (Co-Chair)Yale Univ. School of Medicine333 Cedar Street, P.O. Box 3333Neurobehavior LabNew Haven, CT 06510, USA

Discussants:

Patrick AebischerDepartment of NeurobiologyBrown University, Box GProvidence, R102912, USA

Edward BaetgeCytotherapeutics, Inc.4 Richmond SquareProvidence, R102906, USA

William J. FreedNeuropsychiatry BranchNIMH Neuroscience Center at St.ElizabethsWashington, D.C. 20032, USA

Thomas FreemanUniversity of South FloridaDivision of Neurosurgery#4 Columbia Dr., Suite 730Tampa, FL 33606, USA

Fred GageUniversity of California, San DiegoDepartment of Neuroscience, M-024La Jolla, CA 92093, USA

Donald GashDepartment of Neurobiology andAnatomyUniversity of Rochester MedicalCenter601 Elmwood AvenueRochester, N.Y. 14642, USA

Olle LindvallDepartment of HistologyUniversity of LundBiskopsgatan 5S-223 62 Land, Sweden

J. Lopez-LozanoLaboratory of NeurobiologyHospital Puerta de HierroINSALUDSan Martin de Porres, 428035 Madrid, Spain

Ignacio MadrazoCentro Medico Siglo XXIIMSSCamino A Santa Teresa 1055S-15 Col Heroes de PadiernaMexico, DF 10700Mexico

Richard RobbinsNeural Transplant ProgramYale University School of MedicineNew Haven, CT 06510, USA

Roy BakayEmory ClinicNeurological Surgery1327 Clifton Rd., NEAtlanta, GA 30322, USA

Richard BungeScience Director, Miami ProjectUniversity of Miami Medical School1600 NW 10th Avenue, R-48Miami, FL 33136, USA

Paul CarveyNeuropharmacology ResearchLaboratoriesDepartment of Neurological Sciences& PharmacologyRush-Presbyterian-St. Luke’s MedicalCenter1753 West Harrison Street, Suite 915Chicago, IL 60612, USA

Curt FreedDepartments of Medicine andPharmacologyUniversity of Colorado HealthSciences Center4200 East 9th AvenueP.O. Box C237Denver, CO 80262, USA

Edward HitchcockMidland Centre for Neurosurgery andNeurologyHolly LaneSmethwick, WarleyWest Midlands B67 7JX, England

Helen HodgesDepartment of PsychologyInstitute of PsychiatryDe Crespigny ParkDenmark HillLondon SE5 8AF, England

Philippe HorellouLaboratoire de NeurobiologieCNRS 1 Av. de Terrasse9119 Gif Sur YvetteFrance

Larry KromerGeorgetown Univ. School of MedicineDepartment of Anatomy3900 Reservior Rd., NWWashington, D.C. 20007, USA

Paul SanbergUniversity of South FloridaDivision of NeurosurgeryMDC-16Tampa, FL 33606, USA

John SladekThe Chicago Medical SchoolNeuroscience Institute3333 Green Bay RoadNorth Chicago, IL 60064, USA

Constantino SoteloINSERM U 106Hopital de la Salpetriere47 Blvd de L’HopitalParis 75013France

Jens ZimmerInstitute of NeurobiologyUniversity of AarhusAarhus C DK-8000Denmark

VOLUME 4, NO. 2, 1993

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