LECTURE
The use of foetal human brain tissue as brain implants:
phenotype manipulation by genetic manipulation andbiochemical induction
Henry F. Bradford
Department of Biochemistry, Imperial College of Science, Technology & Medicine. London, UK(Received for publication on June 21, 2002)

Abstract. The use of dopaminergic mesencephalic (VM) human foetal brain tissue as implants toneurosurgically treat Parkinson’s disease has been in progress since the 1980’s. A major limitation in
the use of VM tissue is the amount of tissue available from each human embryo. Usually tissue fromabout 7 embryos is required to treat each patient unilaterally. To overcome this we have developed
various strategies. One is to convert embryonic cerebral cortex in human embryos into dopaminergic
tissue which is stable, and which will secrete dopamine in vivo once implanted. The cerebral cortex isabout 500 times larger than the VM and can therefore provide a lot more tissue for transplantation.
This can be achieved by genetic manipulation of the embryonic cerebral cortex tissue, involving thelipo-transfection of multiple copies of the human tyrosine hydroxylase gene into both neurones andglial cells. In another approach we have biochemically manipulated the development of the cerebral
cortex to direct the neurotransmitter phenotype towards the dopaminergic type, and away from otherphenotypes. This tissue, too, is stable and will synthesise and secrete dopamine when transplanted. Our
third approach has been to manipulate pluripotential neural cells which are yet to develop into neurones and glial cells. These cells can be expanded in number many-fold before treatment to direct theirdevelopment into stable dopaminergic neurones in large numbers (70%), which synthesise and release
dopamine. When used as transplants in animal models of Parkinson’s disease, these various typesof artiﬁcially induced dopaminergic tissue are very effective at reducing the Parkinsonian syndrome.
(Keio J Med 51 (3): 148–153, September 2002)
Key words: Parkinson’s disease, cell graft/transplantation, phenotype manipulation, dopamine, stem
cells

Introduction
The use of dopaminergic mesencephalic (VM) human foetal brain tissue as implants to neurosurgicallytreat Parkinson’s disease has been in progress since the1980’s.
A major limitation in the use of VM tissue is theamount of tissue available from each human embryo.
Usually tissue from about 7 embryos is required to treat
each patient unilaterally. To overcome this we havedeveloped various strategies. One strategem has been
to change the neurotransmitter phenotype of embryonic cerebral cortex in human embryos into a dop-aminergic phenotype which is stable, and which provides neural tissue which will secrete dopamine in vivo
once implanted. The cerebral cortex is about 500 times
larger than the VM and can therefore provide a lot
more tissue for transplantation.
This conversion of developing human and rat cerebral cortex into largely dopaminergic tissue in a stablefashion, and away from its genetically determined phenotype, has been achieved by two methods. One has
been the genetic manipulation of the embryonic cerebral
cortex tissue, involving the lipo-transfection of multiple
copies of the human tyrosine hydroxylase gene into
both neurones and glial cells simultaneously in culture.1

Presented at the 1253rd Meeting of the Keio Medical Society in Tokyo, May 7, 2002.
Reprint requests to: Dr. Henry F. Bradford, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London,
SW7 2AY, UK
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Keio J Med 2002; 51 (3): 148–153

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In another approach we have biochemically manipulated the development of human and rat cerebral cortex to direct the neurotransmitter phenotype towards
the dopaminergic type, and away from other phenotypes.2–6 This tissue, too, is stable and will synthesise
and secrete dopamine after transplantation into rat
models of Parkinson’s disease. Our third approach has
been to manipulate pluripotential neural cells (oncecalled ‘‘neural stem cells’’) which are yet to develop
into neurones and glial cells. These cells can be expanded in number many-fold before treatment to direct
their development into stable terminally fully differentiated dopaminergic neurones in large numbers(70%), which will synthesise and release dopamine.7
When used as transplants in animal models of Parkinson’s disease, these various types of artiﬁcially induced
dopaminergic tissue are very effective at reducing theParkinsonian syndrome.
The Use of Fresh Human Primary Ventral
Mesencephalon
It was during the 1980’s, that fresh human embryonicventral mesencephalon, isolated by dissection, was ﬁrst
transplanted into the striatum of patients with Parkinson’s disease. Some 1,000 patients have subsequently
received this surgical treatment, mainly in Sweden,
USA and the UK. The various techniques employed inthis surgery, and the overall outcome for the patients,
have been reviewed on various occasions in the last
decade.8–10
The use of rodent, and other, animal models of Parkinson’s disease to assess the effectiveness and mechanism of action of such dopaminergic tissue implants has
been the rule. In the early 1990’s, we published11 thecuring effect of implanting freshly isolated human and
rat embryonic ventral mesencephalon tissue into theneostriatum of rat models of Parkinson’s disease. The
procedure was 100% effective. However, freshly isolated, but untreated, embryonic human cerebral cortex
was completely ineffective, though it formed stable andvery viable grafts, surviving at least 9 months (Fig. 1).
Of course, these animal models of Parkinson’s disease
are too simple to give accurate predictions of theoutcome of such dopaminergic tissue implants into
patients. Moreover, the patients, allowed by Hospital
Ethics committees to receive this essentially experimental treatment, are advanced cases with many many
years progression into knock-on complications of Parkinson’s disease. These include disease progression andthe nervous system’s accommodation to long-term drug
treatment. Interlinked neurotransmitter systems (e.g.,cholinergic, serotonergic, aminoacidergic, peptidergic)
develop changes in their responses to the declining
dopaminergic input, and thereby alter their balance andFig. 1 The effect of grafts of cultured human foetal tissue in preventing metamphetamine-induced rotational, circular, movement inrats whose nigrostiatal pathways had been unilaterally removed byunilateral 6-hydroxydopamine injection into the brain. Rotation wasalmost entirely towards the lesioned side of the rat where the graft
tissue was injected, and was counted by an automatic computer controlled system.11 (Reproduced from Walters AM, et al: Neurochem
Res 1992; 17: 893–900, used with permission)equilibrium within the complex cross-talk of the basal
ganglia and other brain structures.
All this weighs against a high level of positive outcome in the patients selected to receive the tissue
implants. Also, these considerations highlight the inadequacies of the animal models as predictors of theextent of positive clinical outcome in these patients.
The animal models are tested within a relatively shortperiod (6 weeks) after unilateral degeneration of their
dopaminergic nigrostriatal pathways, and before thedevelopment of any linked permanent changes in otherneurotransmitter systems. Moreover, rat basal ganglia
are at a far less complex stage of evolution comparedwith the equivalent human system, as well as being200 smaller.
Tissue Source, Condition and Supply
In practice, the ventral mesencephalon from some 7
to 8 human embryos is required to unilaterally treat
each patient. It is clearly difﬁcult to coordinate theavailability of such relatively large numbers of humanembryos of the correct age (7–9 weeks), dissect out theVM, test for disease agents such as AIDS virus, andhave fresh tissue ready for implantation. So the dissected tissue has often been stored frozen. Unfortunately, 60% of the cells often die on thawing. So theviability of the implanted cells is severely compromised.

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Bradford HF: Inducing dopaminergic phenotype in foetal brain tissue

This large number of human embryos is required because they supply the required number of dopaminergic
neurones for the human neostriatum. Thus, there areabout half a million dopaminergic neurones in the human substantia nigra, and therefore this is the number
which surgeons have tried to implant into the putamen
on each side, though only half of these project to theputamen. Actually, only about 5% of these neurones
survive transplantation and produce the required
relieving action on the disease. Fresh VM tissue dissected out and stored in culture has only occasionallybeen used.8–10
Another problem is that since 15 different humanembryo donors are required to treat the putamen oneach side of the patient’s brain, this will present an
immunorejection challenge.
In conclusion, these and many other challenges arebarriers to the future development of the use of multiple batches of fresh human embryonic VM tissue fromdifferent donors to neurosurgically treat one human
Parkinson’s disease patient.

Table 1 The effects of BDNF, Dopamine and Forskolin, alone andin combination, on the numbers of tyrosine hydroxylase-positive cells
in cultures of human foetal cerebral cortex

The Use of Human Embryonic Dopaminergic Tissue
Produced from Cerebral Cortex
In 1994 we published2,3 a method which allows thebiochemical induction of large numbers of neurones(7%) expressing the full dopaminergic phenotype inembryonic rat cerebral cortex which, under the normal

Fig. 2 Effects of combined actions of neurotrophins (BDNF, NT-3,
NT-4) and dopamine on tyrosine hydroxylase (THþ) induction in
foetal rat cortical neurones. The histobars show the percentage ofTHþ neurones in cultures treated as shown 2 Indicated values represent mean þ SEM for triplicate cultures. In two separate platings.
NTs ¼ BDNF þ NT-3 þ NT-4. Statistical signiﬁcance is denoted by *ie P < 0.05, compared with untreated foetal cortex (CX), or withventral mesencephalon (VM).2 (Reproduced from Zhou J, et al: Eur J
Neurosci 1996; 8: 2828–2339, used with permission)

TreatmentTHþ cells/well% THþ cells
comparedwith control

Control(BDNF 50 ng/ml)
BDNF þ dopamine
BDNF þ forskolin
BDNF þ dopamine þ forskolin

0.28 þ 0.18
16 þ 10*78.51 þ 4.90*
219.51 þ 15.85*

100
1,600
7,851
21,950

Human cortical neurones were grown in the presence of 50 ng/ml
BDNF for 5 weeks. The cultures were treated 3 times each week from
DIV 0 with 10 dopamine and 10 forskolin, either alone, or together,and were then stained for THþ cells. These numbers were expressedas percentage of the control. Data are mean þ SEM for 4 to 6 separate experiments carried out in triplicate. Statistical difference fromcontrol group is indicated as * for P < 0.001.

pattern of rat genotype expression, develops very few
dopaminergic neurones (Fig. 2). Following this, in 1998,
we published4,5 a procedure for the successful conversion of neurones (1%) in the human foetal cerebral
cortex into the dopaminergic transmitter phenotype
and away from whatever terminal transmitter phenotype was prescribed in the natural human genotype(Table 1). Although these percentage conversions are
relatively low, dopaminergic neurones develop in sufﬁcient numbers to make the cerebral cortex effective as
neostriatal implants to cure the Parkinsonian symptoms
seen in rat models of the disease. In contrast, untreatedfoetal cerebral cortex of the same age and from thesame source was ineffective in this respect. Thus theasymmetric rotation of the animals was reduced, by 40
times, to below 7 turns per minute by the converted
cerebral cortex. Moreover, this conversion to dopaminergic phenotype was stable over at least nine months.
The embryonic human cerebral cortex is about 500
times larger than the human VM. Moreover, this artiﬁcially induced dopaminergic tissue, derived from cerebral cortex by biochemical induction, is stable, in thesense that it is permanently induced. Most importantly
it has been demonstrated to synthesise and secrete
dopamine. Therefore, it should greatly ameliorate theproblem presented by the need to provide sufﬁcientvolume and genetic uniformity of human foetal embryonic dopaminergic tissue for use as implants to treat
Parkinson’s disease.
All investigation has shown that the method wehave employed induces the complete dopaminergic
phenotype. This means that the enzymes tyrosinehydroxylase, dopa-decarboxyalase, monoamine oxidase,
aldehyde dehydrogenase and catecholamine-O-methyltransferase are all induced in parallel in these neurones.
In addition, we have demonstrated that the membraneproteins expressed to provide the critical dopaminergic

Keio J Med 2002; 51 (3): 148–153

phenotype-marker, high-afﬁnity dopamine uptake, arealso expressed by these artiﬁcially induced dopamine
cells.
There are ethical objections, in many countries, tothe use, for medical treatments, of tissue from humanembryos obtained as a result of pregnancy termination.
Obviously, therefore, it is attractive to be able to provide 500 times more usable dopaminergic tissue per
embryo through this route, which involves the highjacking and redirection of the pathway of terminal differentiation of neurotransmitter phenotype.
The Use of Genetically Engineered Human Foetal
Cerebral Cortex
In a parallel study designed to genetically modify
human and rat embryonic cerebral cortex by inducing
the ability to synthesise and release dopamine, we used
lipofection as the transfection method of choice.1 The
reason for undertaking this research project was again
to make use of the relatively large size of the cerebral
cortex as a source of modiﬁable tissue to be used asbrain implants, but to adopt a completely different
method for conversion to the dopaminergic phenotype.
The successful transfection, by lipofection, of humanand rat embryonic cerebral cortex, in tissue culture, with151

multiple copies of cDNA for human tyrosine hydroxylase was published by our research group in 2001.1
About 4% of the cultured cerebrocortical cells weretransfected, the tissue actively secreted dopamine, andwas successful in eliminating the asymmetric turning
induced by amphetamine in animal models (Fig 3).
Thus, genetic manipulation may be used to produce
a large quantity of human foetal cerebral cortex which
produces and secretes dopamine, and could therefore
be used for transplantation into the putamen of patients with Parkinson’s disease. Indeed, the grafts ofthis genetically modiﬁed neural embryonic tissue arestable. The animal models of Parkinson’s disease remain ‘‘cured’’ for at least 9 months, the longest period
tested, and the grafts are seen to remain localised, andintact. Moreover, tyrosine hydroxylase persists in theneurones at this time, indicating that expression of the
enzyme by TH-cDNA continues.1
The Use of Pluripotential Neural Progenitor Cells
Isolated from the Ventral Mesencephalon
The advantage of using pluripotential neural progenitor cells (called brain ‘‘stem cells’’ in the past) isthat a few cells isolated from human embryos (up to 8
weeks-old) can be allowed to multiply indeﬁnitely bymitosis in an expansion medium, and thereby increase
their numbers, and provide an indeﬁnite supply of cells
which are clone-like perfect neurospheres, which produce no neurites, and form clumps of many cells. A
large proportion of these expanded neurospheres cansubsequently be taken and treated with factors which
will initiate their differentiation, ﬁrst into neurones, andthen into neurones of speciﬁc neurotransmitter phenotype (Figs 4 and 5).
Using this approach we have produced large numbers of cells terminally differentiated into dopaminergic
neurones, which appear to be stable, and have beenshown to produce and release copious amounts of dopamine.7
Conclusions

Fig. 3 The effects of grafts of human foetal tissue genetically engineered, by lipofection, to increase the content of human tyrosinehydroxylase cDNA which increased the grafts’ ability to synthesise
and secrete dopamine 1. The tissue grafts were injected into the neostriatum on the side of the brain lesioned by 6-hydroxydopamine toremove the nigrostriatal pathway. The data show the number of circular rotations towards the lesioned side, counted by an automatedcomputerised system, (y-axis), on the days before and after grafting.1(Reproduced from Hurley M, et al: J Neural Transm 2000; 108 781–
792, used with permission)

The future use of human embryonic cells as implants
to treat the speciﬁc loss of dopaminergic neurones
which occurs in Parkinson’s disease currently seems assured. Freshly dissected, uncultured, unmodiﬁed, brain
tissue from 6 to 8 different human foetuses has so far
featured as the choice for these implants. These neurosurgical treatments have been judged to be successful ina sufﬁciently large proportion of patients to merit continuation.
Advances in the progress towards much greater success in these treatments will come from the changes wewill soon see in the nature of the embryonic tissue

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Fig. 4 Effects of BDNF, dopamine (DA), and forskolin (FSK) on the
induction of the dopaminergic phenotype in pluripotential neuralprecursor cells differentiated by withdrawal of bFGF. Cells weretreated for 3 weeks with different combinations of of the inducers asshown. The dopaminergic phenotype was measured as the synthesis
and secretion of dopamine. The levels of dopamine and its primarymetabolite DOPAC are shown (y-axis). Results are shown as mean
þ SEM from 5 separate harvests.7 Statistical signiﬁcance from control, untreated, cultures is denoted by * with P < 0.05. (Reproduced
from Riaz SS, et al: Dev Brain Res 2001; 136: 127–134, used withpermission)

Bradford HF: Inducing dopaminergic phenotype in foetal brain tissue

employed as cell implants into the putamen. First willbe the switch to using foetal tissue, of 8 to 9 weeks
postconception age, which has been cultured to demonstrate its capacity to produce dopamine. Secondly,
the cultured tissue will be modiﬁed either geneticallyor biochemically to induce, or increase, its capacity to
produce dopamine. This could include the use of foetal
human cerebral cortex because of its very large tissue
volume, allowing the treatment of several patients froma single embryo. Thirdly, human foetal brain tissue atmuch earlier stages of development will be used as
implants. These pluripotential neural cells can be expanded, possibly indeﬁnitely, in number, and can thenbe induced to develop into largely (e.g., 70%) dopaminergic neurones by ex vivo techniques.7 The number of patients treated from a single human embryo
could be even larger than is possible from modiﬁed
foetal cerebral cortex. Such pluripotential progenitor
cells can be immortalised and used in a variety of ways
for CNS gene transfer and repair.12
Acknowledgements: I would like to thank The Paul Hamlyn Foundation London, The Parkinson’s Disease Society London, The Edmund
Safra Foundation New York, and Hilary and Charles Elliot of Watford UK and their family and friends, and also Brenda Miles and theCapel Singers of Watford UK, for ﬁnancial support of this research
work over the decade since it began.

Fig. 5 Methods used for the expansion and differentiation of pluripotential neural progenitor cells.7 (Reproduced from Riaz SS, et al: Dev
Brain Res 2001; 136: 127–134, used with permission)

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