Medicine Meets Millennium
World Congress on Medicine and Health
Hannover, Germany
21 July - 31 August 2000

This text is an updated version of a chapter to be published in:
Neuraltransplantation in neurodegenerative disease: Current status and new
directions. Novartis Foundation Symposium 231 (J Gray and SB Dunnett, eds),
John Whiley & Sons, in press

Cell Replacement Strategies for Neurodegenerative Disorders

Anders Björklund
Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund
University, Sölvegatan 17, S-223 62 Lund, Sweden
Telephone: +46-46 222 05 41
Fax:  +46-46 222 05 59
Email: [log in to unmask]

Abstract

Cell transplantation has over the last two decades emerged as a promising
approach for restoration of function in neurodegenerative diseases,
inparticular Parkinson´s and Huntington´s disease. Clinical trials have so
far focused on the use of implants of embryonic mesencephalic tissue
containing alredy fate-committed dopaminergic neuroblasts with the capacity
to develop into fully mature dopamine neurons in their new location in the
host brain However, the recent demonstration that immature neural progenitor
cells with multipotent properties can be isolated from both the developing
and adult central nervous system (CNS) and that these cells can be
maintained and propagated in culture, has provided a new interesting tool
for restorative cell replacement and gene transfer therapies (see Gage 1998,
Snyder 1998, Svendsen and Smith 1999). Embryonic stem cells, obtained from
the early stages of embryonic development, and neural stem cells, obtained
from the developing brain, may provide renewable sources of cells for
therapeutic purposes, and could eventually offer a powerful alternative to
primary fetal CNS tissue in clinical transplantation protocols. The purpose
of this review is to discuss the prospects of the emerging progenitor cell
technology for cell replacement and restorative therapies in
neurodegenerative diseases, and consider some of the critical issues that
must be solved in order to make progenitor cells useful in studies of brain
repair.

Introduction

Neural transplantation as a tool for replacement of lost neurons and
reconstruction of damaged circuitry in the mammalian CNS is still in its
infancy. Transplantation of neuronal tissue is a classic approach in
neuroembryology, and this technique has been extensively used as an
experimental tool for the study of neuroregeneration and repair in
submammalian vertebrates. Pioneering work in amphibians and fish, which was
carried out above all by Matthey, Stone and Sperry, was the first to
demonstrate that grafted neurons have a capacity to substitute both
structurally and functionally for lost axonal connections, and that afferent
and efferent connections can be established with a high degree of
specificity between grafted neurons and denervated targets in the host brain
(see, e.g., Björklund and Stenevi 1984, for review).
The first attempts to apply neural grafting in animal models of
neurodegenerative disease were made in the late 1970s (Björklund and Stenevi
1979, Perlow et al 1979). Subsequent studies in rodents have shown that the
ability of grafted neurons to become functionally integrated into the host
brain depends on not only on the developmental potential of the implanted
cells, but also on the plasticity of the host environment, i.e., the
capacity of the brain tissue to accept new cellular elements to become
integrated into the developing or established neuronal circuitry. Both these
factors are greatly dependent on the developmental stage of donor and host:
The brain becomes less plastic as it matures, and neurons removed from their
normal context survive and grow well after intracerebral transplantation
only if they are immature, i.e. at the stage of their development when they
have become terminally differentiated but before they have formed extensive
axonal connections (see Dunnett and Björklund 1994, for review). In the
adult brain the capacity of transplanted fetal neurons to grow, integrate
and establish functional efferent and afferent connections is in many cases
substantially increased when the host circuitry is damaged, suggesting that
some of the plastic properties that are present during development, such as
mechanisms that regulate and guide axonal growth and synaptogenesis, can be
reactivated by lesions or neurodegenerative changes. These lesion-induced
cellular and molecular changes, which have yet to be clarified, form the
basis for the remarkable ability of the lesioned adult brain to incorporate
new functional elements, and thus to a degree rebuild itself. Cell
transplantation as an approach to replace lost or damaged brain cells, is
thus in its most effective form a technique for reconstruction of damaged
neuronal circuitry.

Neuronal replacement in Parkinson´s disease

Parkinson's disease has come to serve as the primary test bed for the neural
transplantation technique, for several reasons. One important reason is that
Parkinson's disease affects primarily a circumscribed set of neurons in the
brain (the mesencephalic dopamine neurons) whose main target, striatum, is
anatomically well-defined and relatively accessible surgically. Moreover,
and most importantly, there are well characterized animal models, both in
rodents and primates, that mimic the cardinal features of the disease.
Results obtained in these animal models have repeatedly proved to have good
predictive value with respect to the human disease.
 Neuronal replacement may be most likely to work for those types of systems
which have non-specific modulatory functions in the brain. The nigrostriatal
dopamine neurons are good examples of this type. These neurons, which
normally are located in the midbrain substantia nigra, provide a dense,
diffuse innervation of one of the principal motor control centers of the
forebrain, the striatum. Dopamine released from the nigrostriatal terminals
acts in a tonic, level-setting manner to regulate motor behavior. Lesions of
the nigrostriatal dopamine neurons induce a profound akinetic state which is
at least in part due to an increased threshold for activation of the
striatal output system and initiation of movement.
Neural transplantation in Parkinson's disease is based on the idea that
dopamine producing cells implanted into the denervated striatum might be
able to substitute for those mesencephalic dopamine neurons that have been
lost as a consequence of the disease process. The grafted neurons are
proposed to function either by a "pharmacological" type of action, whereby
the released dopamine is able to diffuse over sufficient distances to
activate the denervated striatal receptors, and/or through functional
reinnervation of the denervated target neurons by the outgrowing axons of
the implanted neuroblasts, which allows released dopamine to exert its
action at defined synaptic sites (see Björklund 1992, Herman and Abrous
1994, for review).
 Intrastriatal transplants of fetal nigral neurons can reverse or ameliorate
impairments in both drug-induced and spontaneous motor behaviors induced by
damage to the nigrostriatal system. In rodent and primate models of
Parkinson’s disease grafted nigral neurons can re-establish a functional
dopamine innervation of the previously denervated striatal target neurons
and restore dopaminergic neurotransmission in the area reached by the
outgrowing axons. In vivo data show that the grafted neurons are
spontaneously active and release dopamine in an impulse-dependent manner, at
both synaptic and non-synaptic sites, although largely independent of any
regulatory afferent inputs.

Reconstruction of basal ganglia circuitry by striatal transplants

The striatal GABAergic neurons provide an inhibitory control of two major
striatal output structures, globus pallidus and pars reticulata of the
substantia nigra. Loss of this striatal inhibitory output system, as occurs
in animals with striatal lesions or in patients with Huntington’s disease,
induces a hyperkinectic syndrome in combination with cognitive deficits.
Since the striatal output neurons are functionally integrated in an
intricately organized cortico-striato-thalamic circuitry, however, proper
functioning of the striatal output neurons will depend not only on the
establishment of appropriate efferent connections but also on their access
to regulatory afferent inputs.
 On the neurochemical level, striatal neuron transplants can restore GABA
synthesis and release both within the lesioned striatum and within the
adjacent, denervated globus pallidus. More importantly, however, the
implanted striatal primordium develop a striatum-like structure at the site
of implantation, and part of the cells develop into fully differentiated
striatal projection neurons. These cells, which are of the medium-size
densely spring type, grow to establish normal synaptic contracts with the
output neurons in the globus pallidus, as well as the appropriate afferent
synaptic inputs from both thalamus, cortex and substantia nigra. Indeed, in
vivo electrophysiological and microdialysis studies in rats show that the
activity of the grafted striatal neurons is under the control of both
cortical glutamatergic and nigral dopaminergic afferents (see Björklund et
al 1994, Dunnett1995, for review). There is thus compelling evidence that
fetal striatal neurons, implanted into the lesioned striatum, can
reconstruct at least some critical elements of the damaged striatal
circuitry in animal models of Huntington´s disease.

Proof of concept in clinical trials

Since 1987 about 250 patients with advanced PD have received transplants of
mesencephalic dopamine neurons, obtained from 6–9 week old cadaver embryos
at several centra in Europe and America (Olanow et al. 1996, Lindvall 1997).
There is now convincing data to show that embryonic human nigral neurons,
taken at a stage of development when they have started to express their
dopaminergic phenotype, can survive, integrate and function over a long time
in the human brain (i.e. in a tissue environment with an ongoing disease
process). Positron emission tomography (PET) scans have shown significant
increases in 18F-fluorodopa uptake (i.e. dopamine synthesis and retention)
in the areas reinnervated by the grafted cells. This increase has been
maintained for at least 6-10 years in several of the longest-studied
patients. Consistent with the imaging data, good survival of grafted
dopamine neurons and extensive reinnervation of the surrounding host
striatum have been demonstrated by immunohistochemistry in two patients that
have come to autopsy at 18 months after surgery (Kordower et al. 1999).
Long-lasting symptomatic improvement has been observed in about 2/3 of the
grafted patients, and in the most successful cases L-dopa treatment has been
possible to withdraw (see Lindvall 1997).
 In Huntington´s disease, clinical trials using transplants of striatal
primordial, obtained from the ganglionic eminences from the developing
forebrain from either human or porcine embryonic donors, have been initiated
in four different centers. Although initial reports have described signs of
improvements in parameters of motor function and cognition, it is still too
early to tell if any of these changes indeed are due to the function of the
transplanted cells. The best indications that embryonic striatal transplants
indeed may work also in the larger and more complex primate brain come from
two recent studies in monkeys, which show that striatal transplants can
induce significant recovery in both motoric and cognitive function in
animals with toxin-induced destruction of the striatum, i.e. in the best
models of HD that we have available today (Palfi et al. 1998, Kendall et al.
1998).

Neuronal progenitors

A major limitation of the fetal cell transplantation procedure is the low
survival rate of the grafted dopamine neurons (in the range of 5–20%) which
makes it difficult to obtain sufficient amounts of cells grafting in
patients. Currently, mesencephalic fragments from at least 6-8 embryos are
needed for transplantation in one PD patient. Moreover, the ethical,
practical and safety issues associated with the use of tissue from aborted
human fetuses are problematic, and severely restricts the possibility to
apply the procedure outside highly specialized centers. The emerging
possibilities to use stem cells or neuronal progenitors as a renewable
source of cells provides an attractive approach to solve these problems.
 Differentiation of immature neural progenitors into fully mature neurons
depends on an interaction between extrinsic signals, present in the cells
local environment, and intrinsic signals operating in a cell autonomous
manner within the cells themselves. With time during development neural
progenitors will gradually acquire independence from extrinsic signals and
as the cells become more restricted in their developmental potential
cell-autonomous programs of differentiation will take over (see Edlund and
Jessell 1999, for review). This is observed also in transplantation
experiments: Cells taken at a stage of development when they have become
committed to a specific neuronal fate (which appears to be around the time
of the last cell division in case of nigral dopamine neurons) will carry on
their normal development also in the fully mature brain, or when
transplanted to ectopic sites (such as the anterior eye chamber or the
kindey capsule). Uncommitted neural progenitors are able to integrate,
differentiate and develop appropriately after transplantation to the brain
in fetuses (Brustle et al. 1995, Fishell 1995, Campbell et al. 1995), but
the capacity of the host brain environment to direct the development of
uncommitted progenitors in a region-specific manner is gradually lost during
the late fetal and early postnatal development in rodents. This indicates
that critical extrinsic signals, necessary for terminal differentiation of
neuronal precursors, are lost or downregulated as the brain matures. There
are two notable exceptions to this rule: The olfactory bulb and the dentate
gyrus of the hippocampal formation where active neurogenesis continues
throughout life (Gage 1998, McKay 1998). In both these regions there is a
continuous production of new neurons (of the small, short-axoned interneuron
type) from a pool of endogenous undifferentiated progenitors located in the
subventricular zone, which supplies cells to the olfactory bulb, and in the
subgranular zone of the dentate gyrus. Indeed, undifferentiated progenitors
transplanted into either of these sites will migrate, integrate and
differentiate, along with the endogenous cells (Lois and Alvarez-Buylla
1994, Gage et al. 1995, Suhonen et al. 1996, Flax et al. 1998, Fricker et
al. 1999). In these two brain regions, at least, neurogenic signals of the
type present during development continues to be expressed also in the adult.

Expansion of dopamine neuron precursors in vitro

Along the path to fully differentiated neurons the precursor cells pass a
critical event, i.e., they stop to divide and cannot re-enter the cell
cycle. Determination of cell fate may take place several divisions before
cell cycle exit, or – as is probably the case for the mesencephalic dopamine
neurons – at the time of or shortly after the last cell division. In the rat
ventral mesencephalon dopamine neurons are generated over a 5-day period,
from about E11 to about E15, and phenotypic markers (e.g. tyrosine
hydroxylase, TH) are expressed at the time of, or shortly after the last
cell division (see Bouvier and Mytilineou 1995). In transplants of fetal
mesencephalic tissue, grafted to the adult striatum, Sinclair et al (1999)
have shown that in grafts taken at E14, i.e. during active neurogenesis,
virtually all surviving dopamine neurons are derived from precursors that
have undergone their last cell division prior to transplantation, and hence
already been committed to a dopaminergic neuronal fate. These results are
consistent with the view that those extrinsic signals which are necessary
for the induction of mature neuronal phenotypes from uncommitted precursors
may not be present normally  in mature non-neurogenic brain regions, such as
the adult striatum.
 Bouvier and Mytilineou (1995) and Studer et al (1998) have shown that
mesencephalic dopamine neuron precursors can be expanded in a
pre-differentiated state in culture provided that they are plated at a time
close to their last cell division, but before they express a definitive
dopaminergic phenotype (i.e., the TH enzyme). These lineage-restricted
precursors do not continue to divide spontaneously in culture but can be
maintained in an undifferentiated dividing state for about a week under
stimulation with a growth factor, FGF-2. At the end of this expansion
period, Studer et al (1998) estimated that the total number of cells in the
FGF-2 treated cultures had increased 10-fold, and upon removal of the
mitogen the yield of differentiated TH-positive neurons was increased about
30-fold over the non-expanded controls. Studer and collaborators went on to
show that these in vitro expanded precursors can survive transplantation to
the dopamine-denervated striatum. However, the overall yield of surviving
dopamine neurons in the grafts was very low, above all due to the excessive
loss of cells in the differentiation and grafting steps of the procedure
(over 95%), where the expanded cells were first removed from the culture
dishes and then allowed to differentiate into dopamine neurons in
free-floating aggregate cultures prior to transplantation. This cell
survival problem should be possible to solve, however, by improvements in
the handling of the cells in the differentiation step.
The cells expanded in the Studer et al (1998) study differentiated
spontaneously to TH-positive neurons upon removal of the mitogen, which
suggests that they were committed dopamine neuron precursors. Stimulation by
FGF-2 was able to induce cell division in this precursor cell population and
delay terminal differentiation, but only for about 7-8 days (Bouvier and
Mytilineou 1995). Mesencephalic progenitors can be expanded for longer
periods of time in neurosphere cultures. The neurosphere cells express
features of multipotent progenitors, and can differentiate into both neurons
and glia, but they do not spontaneously differentiate into dopamine neurons
when placed in monolayer cultures in the absence of the mitogen (Svendsen
and Rossor 1995, Ling et al 1998). Carvey and collaborators (Ling et al
1998, Potter et al 1999) have shown that these uncommitted progenitors can
be induced to differentiate into a dopaminergic neuronal fate in the
presence of a combination of cytokines, mesencephalic membrane fragments and
striatal culture-condition medium. About 50% of all neurons, and 20–25% of
all cells, in the differentiated cultures were seen to express the TH
marker. Interestingly, the cytokine effect was seen with mesencephalic but
not striatal progenitors, suggesting that this combination of factors acted
to induce differentiation in a lineage-restricted mesencephalic precursor
cell population (Potter et al 1999). These cells expressed not only the TH
enzyme but also DOPA decarbocylase, the dopamine transporter and dopamine
itself, suggesting that they represent a mature dopaminergic neuronal
phenotype. To what extent these cells can survive and function after
transplantation is not yet clear.

Engineering cells for transplantation

In vitro expanded neural progenitors may provide a highly useful source of
cells for intracerebral transplantation in Parkinson’s disease provided that
we can reliably control or direct their differentiation towards a specific
dopaminergic neuronal fate. Ideally, these cells should not only be
dopamine-producing but they should also possess the specific features of
mature nigral dopamine neurons. This implies factors that convey both
region-specific and transmitter-specific identity on the developing
precursors. Local extrinsic signals, such as sonic hedgehog and FGF-8 (Ye et
al 1998) and cell specific transcription factors, such as Nurr1 (Zetterström
et al 1997), are likely to be critically involved in this process.
Wagner et al (1999) have recently reported that overexpression of Nurr1 in
combination with (as yet unidentified) factors from local type 1 astrocytes
is sufficient to induce a dopaminergic neuronal phenotype in
undifferentiated cells from the C17-2 immortalized neural stem cell line.
With the protocol used, over 80% of the induced cells expressed the TH
enzyme, as well as two other phenotypic markers, ADH-2 and c-ret,
characteristic for ventral mesencephalic dopamine neurons. These results
point to an important role of glia-derived factors in the process leading to
the maturation of the dopaminergic neuronal phenotype. Indeed, in primary
cultures the differentiation of mesencephalic precursors into dopamine
neurons coincides with the appearance of astrocytes in the FGF-2 treated
cultures (Bouvier and Mytilineou 1995). Moreover, it has been shown that
cortical astrocytes, stimulated with FGF-2, secrete factors that stimulate
differentiation of mesencephalic dopamine neurons (Gaul and Lubbert 1992),
and that glia-conditioned media (in combination with FGF-2) can induce TH
expression in in vitro expanded neural progenitors (Daadi and Weis 1999).
These data suggests that neurons and glia may cooperate in both regional
specification and the induction of specific neuronal identities of
developing precursors. If so, the ideal cell preparation for transplantation
in the Parkinson model will consist of a mixture of committed neuronal
precursors and regionally specified glial cells in a stage of development
where they can help to convey regional indentity to the developing
neuroblasts.