Download Fifty Ways to Make a Neuron:* Shifts in Stem Cell Hierarchy and

Journal of Neuropathology and Experimental Neurology
Copyright q 2002 by the American Association of Neuropathologists
Vol. 61, No. 2
February, 2002
pp. 101 110
Fifty Ways to Make a Neuron:* Shifts in Stem Cell Hierarchy and Their Implications for
Neuropathology and CNS Repair
MARIUS WERNIG, MD
AND
OLIVER BRÜSTLE, MD
Abstract. During embryogenesis, the developmental potential of individual cells is continuously restricted. While embryonic
stem (ES) cells derived from the inner cell mass of the blastocyst can give rise to all tissues and cell types, their progeny
segregates into a multitude of tissue-specific stem and progenitor cells. Following organogenesis, a pool of resident ‘‘adult’’
stem cells is maintained in many tissues. In this hierarchical concept, transition through defined intermediate stages of decreasing potentiality is regarded as prerequisite for the generation of a somatic cell type. Several recent findings have challenged this view. First, adult stem cells have been shown to adopt properties of pluripotent cells and contribute cells to a
variety of tissues. Second, a direct transition from a pluripotent ES cell to a defined somatic phenotype has been postulated
for the neural lineage. Finally, nuclear transplantation has revealed that the transcriptional machinery associated with a distinct
somatic cell fate can be reprogrammed to totipotency. The possibility to bypass developmental hierarchies in stem cell
differentiation opens new avenues for the study of nervous system development, disease, and repair.
Key Words:
Embryonic germ cells; Embryonic stem cells; Glia; Neurons; Progenitor cells; Transplantation.
INTRODUCTION
Regarded from the stem cell perspective, mammalian
development represents a highly hierarchic pedigree composed of a multitude of cell lineages (Fig. 1). In this
pedigree, ultimate totipotency is restricted to the fertilized
oocyte. In the blastocyst, potentiality segregates into 2
lower order compartments. Cells of the blastocyst wall
give rise to the trophoblast, whereas embryonic stem cells
(ES cells) of the inner cell mass generate all somatic cell
types of the embryo proper. Since ES cells lack the potential to generate extra-embryonic tissues, they are regarded as pluripotent rather than totipotent. Giving rise
to the 3 germ layers, ES cells are ancestors to primordial
germ cells and all tissue-specific fetal and adult stem
cells. Stem cells within different tiers of this hierarchical
system differ with respect to their proliferation and differentiation capacity.
During the last 5 years, ES cells have been increasingly
explored as potential donor source for transplantation. A
large variety of cell types has been derived from mouse
ES cells in vitro, including neural cells (4–11), cardiomyocytes (12–14), insulin-producing cells (15, 16), hematopoietic progenitors (17–19), and several others (20, 21).
Many of these in vitro-generated cells have already been
used for transplantation studies in animal models (13, 16,
22). The availability of human ES cells (23, 24) has initiated tremendous interest in translating these promising
findings into clinical strategies. A variety of somatic cell
types has been obtained by in vitro differentiation of human ES cells, including neurons, cardiomyocytes, and insulin-producing cells (23, 25–29). Further characterization of human ES cell-derived neurons revealed
GABAergic and glutamatergic phenotypes (23) and expression of dopamine and serotonin receptors (27).
Embryonic Germ Cells (EG Cells)
Embryonic Stem Cells (ES Cells)
Embryonic stem cells (ES cells) exhibit 2 unique properties. First, in culture, they can be multiplied to virtually
unlimited numbers (1). Second, they are pluripotent and
can differentiate into all somatic tissues and cell types.
When ES cells are injected into another blastocyst, they
contribute to all tissues of the developing embryo (2).
Combined with gene targeting, this strategy has opened
an avenue for the generation of gene-deficient mice—one
of the most efficient tools in experimental biology (3).
*With apologies to Paul Simon.
From the Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany.
Correspondence to: Oliver Brüstle, MD, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Strasse 25,
D-53105 Bonn, Germany.
Embryonic germ cells (EG cells) are cell lines derived
from primordial germ cells during embryonic and fetal
development. Their properties resemble those of ES cells.
In vitro, murine EG cells can proliferate to large numbers
and differentiate into derivatives of all 3 germ layers,
including neurons, cardiomyocytes, skeletal muscle, and
hematopoietic cells (30–34). Similar to ES cells, murine
EG cells have been reported to contribute to chimeras
upon blastocyst injection (33, 35). Recently, EG cells
have also been isolated from human tissue (36). Studies
on rodent cells suggest that genomic imprinting in primordial germ cells and cultured EG cells differs from ES
and somatic cells (37, 38). It is currently not clear whether these findings also relate to human cells and to what
extent the lack of imprinting may affect the stability of
differentiated EG cells.
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WERNIG AND BRÜSTLE
Fig. 1. Shifts in stem cell hierarchy. Traditional concepts postulate a continuous decrease in stem cell potentiality and early
segregation into tissue-specific compartments. Recent data on nuclear reprogramming, transdifferentiation of somatic stem cells,
and the direct transition from an embryonic stem cells to a neural precursor have challenged this view. Abbreviation: NSC, neural
stem cell.
Somatic Stem Cells
Compared to ES and EG cells, the proliferation potential of both fetal and adult somatic stem cells appears to
be limited. However, they are abundant in permanently
regenerating tissues such as blood, skin, and intestine and
can generate large numbers of tissue-specific cells both
in vitro and during development (21). To ensure maintenance of multipotent progeny, somatic stem cells employ 2 modes of division. Symmetrically dividing stem
cells give rise to 2 equipotent daughter cells. In contrast,
asymmetric division yields a more differentiated daughter
cell and a multipotential stem cell (Fig. 2). Stem cells
may use both forms of division interchangeably. Lacking
the pluripotent differentiation potential of ES and EG
cells, somatic stem cells traditionally held the terminal
J Neuropathol Exp Neurol, Vol 61, February, 2002
positions in the hierarchical concept of stem cell differentiation. Recent data on the transdifferentiation of adult
stem cells have challenged this view.
STEM AND PROGENITOR CELLS IN THE CENTRAL
NERVOUS SYSTEM—THE CLASSICAL VIEW
The adult CNS is composed of a multitude of highly specialized neuronal and glial cell types. One of the fundamental
questions in developmental neurobiology is whether this heterogeneity originates from a universal stem cell population or
whether there are restricted stem cells which give rise to distinct
subtypes of neural cells. Cell lineage analyses involving low
titer retroviral infection of ventricular zone cells provided evidence for the existence of stem cells giving rise to all 3 major
cell types of the CNS—neurons, astrocytes, and oligodendrocytes (39, 40). In addition, more restricted stem cells generating
EMBRYONIC STEM CELLS: IMPLICATIONS FOR NEUROPATHOLOGY
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PDGF and, upon growth factor withdrawal, give rise to 3 different glial cells: A2B51 astrocytes, A2B52 astrocytes, and
GalC1 oligodendrocytes (66). Neuronal-restricted stem cells
show strong expression of PSA-NCAM, divide in FGF2-containing media, and give rise only to neuronal cell types (64).
Due to their characteristic surface marker expression, both stem
cell types can be efficiently enriched using immunological sorting techniques (67).
CHALLENGING HIERARCHY IN THE CNS: FROM
GLIA TO NEURONS
Fig. 2. Stem cells employ 2 modes of self-renewing divisions: Symmetric divisions give rise to equipotent progeny.
Asymmetric divisions generate a multipotent stem cell and a
more differentiated daughter cell. Both forms of division might
be used interchangeably.
only neurons and oligodendrocytes (41) or neurons and astrocytes (42) have been described in these studies.
In parallel, multipotent neural stem cells have been isolated
from various regions of the embryonic and adult CNS (43–51).
These cells self-renew in the presence of growth factors such
as FGF2 or EGF and, upon growth factor withdrawal, differentiate into neurons and glia. During in vitro differentiation,
single factors can instructively promote a specific fate. For example, ciliary neurotrophic factor (CNTF) has been shown to
efficiently recruit multipotent neural stem cells into an astrocytic fate (49, 52, 53).
The maintenance of a neuropoietic stem cell population
throughout life is supported by the observation that new neurons are continuously generated in the subventricular zone and
the hippocampus of the adult brain (47, 54–57). Recently, neurogenic cells have even been detected in the adult human hippocampus, both in vitro (58, 59) and in vivo (60). Experiments
in monkeys suggest that adult neurogenesis may even extend
to neocortical regions (61).
It is generally accepted that multipotent neural stem cells
give rise to progenitor cells of more restricted developmental
potential. A classic example is the O-2A progenitor derived
from the neonatal optic nerve. This glial-restricted precursor
self-renews in the presence of FGF2 and PDGF and, upon
growth factor withdrawal, differentiates into oligodendrocytes
and type-II astrocytes, depending on the culture conditions (62,
63).
Recently, glial-restricted and neuronal-restricted stem cells
have also been isolated from the neural tube and the developing
spinal cord (64, 65). Similar to O2-A progenitors, these spinal
cord-derived glial-restricted stem cells express the marker antigen A2B5. They proliferate in the presence of FGF2 and
Neurons and glial cells have long been considered as
final stages of neural stem cell differentiation. The only
accepted exception to this rule is radial glial cells, a specialized, transient cell type with a long process extending
from the ventricular zone to the pial surface (68). Extensive work by Rakic and colleagues has shown that these
radially organized processes serve as a guiding scaffold
for young migrating neuroblasts (69, 70). After completion of neuronal migration, radial glial cells were thought
to reenter the mitotic cycle and transform into ependymal
cells and astrocytes (71). Whereas lineage analyses using
retroviral vectors have long suggested a relationship between radial glia and neurons (40, 72, 73), the results of
several recent studies point to a direct conversion. Götz
and colleagues have isolated radial glia from the developing cortex based on morphological and molecular criteria (74). The differentiation into individual cell fates
was then followed in clonal culture conditions. Whereas
only a small number of clones consisted of neurons and
glia, the majority (.95%) differentiated into either neurons or glia. With proceeding development, the proportion of glial-restricted clones increased, while neuronalrestricted clones became fewer. These findings suggest
that radial glial cells represent a heterogeneous population of precursors capable of glial- and neuronal-restricted differentiation. In an elegant time lapse study, Noctor
et al recently captured the direct transition of proliferating
radial glia cells into migrating neurons in vivo (75).
Striking evidence for a postnatal transition between the
glial and the neuronal lineage comes from a new study
on O-2A progenitors. In this experiment, proliferating O2A cells were transiently differentiated into type-2 astrocytes by the addition of serum. Subsequent serum withdrawal and further propagation in FGF2 caused many
cells to revert to a multipotent stem cell capable of generating neurons, astrocytes, and oligodendrocytes (76).
Studies in the adult rodent brain suggest that glial cells
within the ventricular wall can exhibit properties of neural stem cells. Johansson et al observed that isolated ciliated ependymal cells multiply in culture and give rise to
glial and neuronal phenotypes (77). They concluded that
ependymal cells of the adult brain are multipotent stem
cells. This view was challenged by others, who claim that
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multipotent stem cells associated with the ventricular system are in fact astrocytes within the subventricular zone
(78, 79). Leaving discrepancies concerning the precise
location aside, these data indicate that multipotent neural
stem cells of the adult brain can exhibit a glial phenotype.
Very recently, Rietze et al succeeded in preparing highly
enriched stem cell populations from both the ventricular
and the subventricular zone of the adult mouse brain.
Using a minimum size of 12 mm, binding of peanut agglutinin and expression of heat-stable antigen as criteria,
they were able to generate populations containing more
than 80% cells with stem cell properties (80).
It is tempting to speculate about the nature of multipotent stem cells in other non-neurogenic regions of the
CNS. Mimicking a differentiated glial phenotype and
lacking an environment promoting the generation of neurons, these cells would go unnoticed in vivo. It would be
only in vitro that these cells exhibit their multipotent nature. Such a scenario could explain why multipotent, neurogenic stem cells have been derived from almost all
brain regions, whereas neurogenesis is restricted to few
selected sites. It would also imply that, under normal conditions, neurogenesis of ‘‘glioid’’ stem cells is repressed
by environmental factors.
CELLULAR BODY SWITCHING: FROM BLOOD TO
BRAIN
Remarkably, the developmental plasticity of neural
stem cells appears to extend well beyond the nervous
system: When Bjornson et al injected neural stem cells
derived from both embryonic and adult CNS into lethally
irradiated mice, they reconstituted a variety of myeloid
and lymphoid cell types as well as early hematopoietic
cells (81). An even broader differentiation potential was
revealed when adult neural stem cells were injected into
blastocyst stage embryos. Some of the developing embryos exhibited extensive chimerism of multiple organ
systems. Donor-derived cells contributed to tissues of all
3 germ layers including gastrointestinal epithelia, cardiac
muscle, epidermis, and the central nervous system (82).
These exciting observations suggest that neural stem cells
are not irreversibly locked into a neural fate but can contribute to a large variety of non-neural tissues.
Would the reverse be true? In 1997, Eglitis and Mezey
detected donor-derived astrocytes in the brains of bone
marrow-transplanted mice (83). This remarkable observation was extended by 2 recent studies. Following bone
marrow transplantation into neonatal mice incapable of
developing cells of the myeloid and lymphoid lineages,
donor-derived cells expressing the neuronal marker NeuN
were found in various brain regions (84). Similar observations were made when genetically labeled bone marrow cells were implanted into lethally irradiated normal
adult mice (85). In this experiment, some of the donor
cells were even found to activate the transcription factor
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cAMP response element-binding protein (CREB). It
would be interesting to know to what extent bone marrow-derived cells integrate in the neuronal circuitry of
the host. Another key question to be addressed is whether
the expression of selected neuronal antigens truly reflects
transdifferentiation rather than an aberrant expression
pattern mimicking some regional traits in response to local cues. It also remains possible that different tissues
share a common population of immature pluripotent stem
cells highly susceptible to local recruitment mechanisms.
Yet, the data currently available challenge our concepts
of stem cell commitment and raise many new questions.
Are hematopoietic cells providing a continuous supply of
stem cells, neurons, and glia for the CNS? How would
such a pathway respond to neurological disease? Could
an imbalance in this system itself lead to neuronal loss?
And, most importantly, what are the signals regulating
this transdifferentiation process and can they be remodeled in vitro?
NEURAL DIFFERENTATION—A DEFAULT PATHWAY
IN EARLY EMBRYOGENESIS?
During normal development, the epiblast gives rise to
endo-, meso- and ectoderm. Neural specification of the
ectoderm is thought to be initiated by mesoderm-derived
signals (86, 87). This model, which would require generation of at least 2 germ layers for neural induction, has
been challenged by a recent report of Tropepe et al (88).
In their study, they provide evidence for a direct conversion of ES cells into primitive neural stem cells. When
plated at low density in defined media containing leukemia inhibitory factor (LIF), 0.2% the ES cells generated multipotential neural spheres. The sphere-forming
ability was found to be critically dependent on endogenous FGF signaling. Single cells derived from these
spheres could be subcloned to secondary spheres, which,
upon serum addition, gave rise to neurons, astrocytes, and
oligodendrocytes. It is currently not clear to what extent
these cells resemble native neural precursors. In contrast
to primary neuroepithelial cells isolated from the developing CNS, they also express the endodermal marker
GATA4 and efficiently generate chimeras upon blastocyst
injection (88). These observations could point to a more
‘‘primitive’’ stage of neural differentiation as compared
to neural tube-derived cells.
A direct conversion of ES cells into neural precursors
challenges the classic concept of active neural induction
(87). The phenomenon rather suggests that neural differentiation is a default pathway in early embryogenesis
(Fig. 1). The generation of neuroectoderm in the absence
of mesoderm is also supported by earlier studies on the
mesoderm inducer activin. While a truncated activin receptor inhibits mesoderm induction in Xenopus embryos,
the mutants still show neural differentiation (89). Taken
EMBRYONIC STEM CELLS: IMPLICATIONS FOR NEUROPATHOLOGY
together, these findings support the notion that epiplast
differentiation is biased towards a neural fate.
REVERSION OF THE IRREVERSIBLE
The transdifferentiation of one adult cell type into another could involve both a direct transition or a retrodifferentiation into a pluripotent phenotype with subsequent
differentiation into a different cell type. While transdifferentiation on the whole cell level is still poorly understood, nuclear reprogramming studies have revealed a remarkable potential for retrodifferentiation. Already in the
1960s, studies in amphibians had revealed that adult nuclei implanted into an enucleated oocyte are reprogrammed to totipotency (90). In 1996, Campbell et al
successfully cloned sheep from a fetal cell line by transplanting cell nuclei into enucleated oocytes (91). The
generation of Dolly showed that nuclear reprogramming
can even be applied to adult mammalian cells (92).
Meanwhile, nuclear transfer techniques have been successfully used to clone a variety of mammalian species
including mice, cattle, goats, and pigs (93–96). Nuclear
reprogramming has far-reaching implications for both basic science and medicine. More than any other finding in
the biomedical field, it challenges the traditional concept
of a continuous and irreversible restriction of the transcriptional machinery during development.
SPIN-OFFS FOR THE NEUROPATHOLOGIST
The recent advances in stem cell biology will have
interesting implications for both experimental and diagnostic neuropathology. Similar to the hematopoietic system, tumorigenesis in the CNS is increasingly understood
in the context of stem cell biology. Whereas primitive
neuroectodermal tumors in children have long been considered to originate from multipotential precursor cells,
the maintenance of neural stem cells throughout life
could extend this hypothesis to other CNS tumors. Until
today, the question as to whether gliomas are derived
from differentiated glial cells or immature progenitors is
unresolved. The presence of perpetuating or quiescent
stem cells in different regions of the adult brain and spinal cord provides an interesting starting point to ‘‘reinvent’’ brain tumor pathogenesis. Wouldn’t a population
of proliferative precursors be prone to accumulate genomic alterations more readily than a differentiated, nondividing glial cell? Is the multifocal appearance of gliomas reflecting migratory properties of immature glial
progenitors at earlier stages of development? And, if so,
do precursor and tumor cell migration obey similar rules?
Thinking of brain tumors as precursor cell tumors
might also lead to new concepts of tumor classification.
Neuropathologists have become used to think in categories such as ‘‘astrocytoma,’’ ‘‘oligodendroglioma,’’ and
‘‘glioblastoma.’’ Yet, their every day work often represents a struggle to collect diagnostic evidence for one
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category at the expense of another; a struggle which is
largely based on the idea that brain tumors derive from
mature astrocytes or oligodendrocytes, and a struggle
which often ends undecided—in categories such as ‘‘oligoastrocytoma’’ or ‘‘glioblastoma with oligodendroglial
component.’’ The concept of a precursor cell origin of
brain tumors provides a different view. The phenotype of
a tumor is no longer attributed to a differentiated cell
type. Rather, the phenotype is determined by the developmental stage of the transformed precursor cell. A bipotential precursor would be prone to give rise to a mixed
glioma, whereas more lineage-restricted progenitors
would develop into astrocytomas or oligodendrogliomas.
It has been suggested that human glioblastoma cells, indeed, exhibit growth factor responses and differentiation
properties resembling those of bipotential O-2A progenitor cells (97, 98). Gangliogliomas may result from an
even earlier precursor still capable of neuronal and glial
differentiation. Recent data on the conversion of glial
cells into neurons may even provide an explanation for
the neuronal differentiation frequently detected in some
low-grade gliomas such as subependymal giant cell astrocytoma.
The availability of human ES cells could eventually
permit direct experimental access to human brain tumor
development. Using genetic modification and in vitro differentiation of ES cells, both oncogene activation and
suppressor gene inactivation could be simulated at defined stages of neural differentiation. Depending on the
epigenetic stability of ES cell-derived neural precursors,
such an approach may allow remodeling of the molecular
pathogenesis of human brain tumors in vitro.
The idea of using ES cells as a model system could
be extended to other neurological disorders. Candidate
mutations for neurodegenerative disorders could be introduced and the resulting precursors studied both in vitro
and following transplantation into a rodent brain (99).
The potential transdifferentiation of hematopoietic
cells into neurons and glia might lead to a new understanding of CNS involvement in hematopoietic diseases.
Can stem-like hematopoietic tumor cells give rise to aberrant neuro- and gliogenesis? Do transplanted bone marrow cells, in analogy to the rodent experiments by Mezey
and Brazelton, result in the formation of donor-derived
neurons and astrocytes in the recipients’ brain? And,
most provocatively, are some apparently neuronal or glial
disorders in fact due to transdifferentiated abnormal hematopoietic cells? Tumor and/or donor cell-specific lineage analyses in the CNS of bone marrow recipients will
provide key tools for addressing these fundamental questions.
NEW CONCEPTS FOR TREATING CNS DISORDERS
Exploiting the Endogenous Stem Cell Pool
Adult neurogenesis, the availability of embryonic stem
cells, and neural transdifferentiation of adult stem cells
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provide a new framework for the development of CNS
repair strategies. In addition, neural stem cells residing
outside neurogenic regions of the adult CNS might be
amenable to recruitment into lesioned areas. Following
chromophore-targeted laser photolysis of cortical layer
VI neurons, Macklis et al observed the generation of new
neurons within the damaged layer. Some of the newly
born neurons exhibited pyramidal morphologies and
formed long distance corticothalamic connections (100).
Such an experimental recruitment of quiescent adult stem
cells into a neuronal fate could serve as an important tool
for the identification of endogenous factors mediating lesion-induced neurogenesis. The results of several studies
indicate that exogenously applied EGF and FGF2 promote cell proliferation in the adult subventricular zone
(101, 102). In a recent report, Fallon et al reported that
infusion of transforming growth factor alpha (TGFa) into
the 6-OHDA-lesioned striatum promotes recruitment of
both neurons and glial cells from the adult subventricular
zone to the infusion site. Some of the recruited neurons
expressed tyrosin hydroxylase and dopamine transporter,
and a reduction of apomorphine-induced rotations was
noted in a subset of the operated animals (103).
Various extrinsic stimuli have been shown to modulate
adult neurogenesis. The formation of new granule neurons in the dentate gyrus, for example, is promoted by
pathological conditions such as ischemia, seizures, and
mechanical injury, but also by physiological parameters
including enriched environment, physical exercise, estrogen, and learning (104–111). In contrast, stress, increased
levels of glucocorticoids, and old age are known to decrease the rate of hippocampal neurogenesis (112, 113).
Adult neurogenesis is not merely an academic issue:
Van Praag et al have reported that physical exercise induces neurogenesis and results in improved learning behavior in the Morris water maze (114). Therapeutic exploitation of adult neurogenesis will critically depend on
the identification of factors translating complex extrinsic
stimuli into increased proliferation and survival of neuronal precursors. Considering recent evidence of neuronal
differentiation of glia (74, 76), these studies will also
have to address the question whether endogenous glial
precursors can be recruited into a neuronal fate in vivo.
Transplantation
While the exploitation of endogenous stem cell recruitment for neural repair is still in its infancy, transplantation of neural precursors has long developed into a
major field. A key problem with this approach is the
availability of suitable donor tissue. Clinically significant
numbers of donor cells could, so far, only be obtained
from fetal brain tissue. Numerous studies have shown
that multipotential neural precursors can be expanded in
vitro in the presence of growth factors (115, 116). However, more in vivo studies are required to assess the
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functional activity of long-term growth factor-expanded
neural precursor cells after transplantation. Oncogenemediated immortalization represents a second strategy for
the expansion of primary neural precursors (117). Several
studies have shown that oncogene-immortalized neural
precursors represent an efficient tool for cell-mediated delivery of trophic factors and enzyme substitution in storage disease models (118–122). Two recent reports extend
the spectrum of applications to the experimental treatment of brain tumors. Transplantation of neural precursors overexpressing interleukin 4 or cytosine deaminase,
an enzyme that converts the prodrug 5-fluorouracil to its
active form, was shown to reduce tumor size and increase
survival in rodent glioma models (123, 124). In one of
these studies, the grafted neural precursors showed significant tropism for the lesioned brain area and even
seemed to ‘‘chase down’’ infiltrating tumor cells (123).
While these results point to a potential role of oncogeneimmortalized cell lines in the treatment of CNS tumors,
the fact that these cells carry a potentially tumorigenic
gene remains a major concern.
Xenotransplantation offers an option to bypass the necessity for human donor cells. Indeed, phase I trials involving transplantation of porcine fetal neural cells into
Parkinson disease patients have provided evidence for
both donor cell survival and partial functional improvement (125). However, the potential transfer of pathogens
from animals to humans remains one of the key problems
associated with clinical xenotransplantation.
ES Cells as Universal Donor Source
In contrast to the limited proliferation potential of primary neuroepithelial cells, ES cells can be expanded to
virtually unlimited numbers. Proliferation potential and
pluripotency make this cell population an attractive donor
source for cell replacement strategies (126, 127). Upon
transplantation into the ventricles of embryonic rats, murine ES cell-derived neural precursors participate in brain
development and contribute neurons and glia to a large
variety of brain regions (9). The results of several recent
transplant studies illustrate the potential of ES cell-derived neural precursors for reconstructive neurobiology.
Glial progenitors generated from ES cells have been successfully used for myelin repair in the myelin-deficient
rat, an animal model of Pelizaeus-Merzbacher disease.
Upon transplantation into the myelin-deficient spinal
cord, the grafted cells generate myelin across several millimeters of the host dorsal columns. Intraventricular
transplantation of ES cell-derived glial precursors during
embryonic development permits simultaneous population
and myelination of several host brain compartments (22).
Myelin repair by grafted ES cell-derived oligodendrocytes was also observed after transplantation into shiverer
mice and foci of chemically induced demyelination (128).
EMBRYONIC STEM CELLS: IMPLICATIONS FOR NEUROPATHOLOGY
Retinoic acid-treated ES cells grafted into traumatic spinal cord lesions were even found to promote recovery of
motor function, although the mechanism of functional
restoration remains to be determined (129).
The current excitement about potential therapeutic applications of ES cells should not mask the fact that several key problems need to be solved before this technology can be applied in a clinical context. One of the major
risks associated with the use of ES cell-derived precursors is tumor formation. Not appropriately differentiated
cells and residual embryonic stem cells may form nonneural tissues or teratomas upon transplantation (130). A
second key problem is cell type specification, i.e. the predetermination of the donor cell population with respect
to the specific phenotype required for the individual application. In murine ES cells, both targeted differentiation
and lineage selection have been successfully employed to
address these problems. Concerted exposure to different
growth and regionalization factors permits the generation
of highly purified, non-tumorigenic glial precursors and
the enrichment of neuronal subtypes such as dopaminergic and serotoninergic phenotypes (11, 22, 131). Introduction of selectable markers into neural-specific genes
has been shown to enable the in vitro selection of purified
pan-neural populations (10). However, both strategies
still await successful translation to human ES cell systems.
So far, proliferation of human ES cells could only be
achieved in coculture with murine fibroblasts. A clinical
application of ES cell lines generated in this manner
would require the same precautions as xenografts, including appropriate steps to exclude transmission of murine pathogens to the transplant recipient. Very recently,
feeder-free growth of human ES cells has been reported
(132). While this system still depends on cell culture supernatant of murine cells, defined culture media or human
feeder cells could bypass the necessity of animal cells.
Careful evaluation of the stability and the epigenetic
variability of ES cells will be required to determine their
applicability in regenerative medicine. A recent study by
Humpherys and coworkers indicates that individual ES
cell lines and even subclones obtained from a single ES
cell line may exhibit significant differences in methylation patterns (133). Pronounced epigenetic variability
could complicate not only therapeutic strategies but also
other main application fields of ES cell-derived somatic
precursors such developmental, pharmacological, and
toxicological studies.
Therapeutic Cloning
While transplantation of allogeneic ES cell-derived somatic precursors still carries the risk of transplant rejection, ES cells derived from nuclear transfer embryos
could permit autologous repair strategies (134). This approach, generally referred to as ‘‘therapeutic cloning,’’
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has already been successfully applied to rodent cells.
Cloned murine ES cells were shown to give rise to a
variety of therapeutic relevant cell types including dopaminergic neurons (135, 136). Yet, it is questionable
whether nuclear transfer will, in the end, be used for clinical purposes. The frequent abnormalities observed in
cloned animals (137) indicate that reprogramming by nuclear transfer is highly variable. A recent report clearly
demonstrates a pronounced variability in gene methylation in cloned newborn mice (133). While some reprogramming errors might lead to obvious developmental
abnormalities, they might go unnoticed during proliferation and in vitro differentiation of cloned ES cells—only
to become apparent after transplantation. Thus, a detailed
understanding of the reprogramming mechanisms is essential for a clinical exploitation of this phenomenon.
Transdifferentiation of Non-Neural Adult Stem Cells
The observation that bone marrow-derived cells can
enter the CNS and acquire properties of neurons and glia
(83–85) provides attractive prospects for non-invasive
neural repair strategies. Current data on the recruitment
of hematopoietic cells to the nervous system are based
on bone marrow transplantation studies. Interesting questions to be addressed are whether endogenous bone marrow cells are recruited at similar rates and whether they
show functional integration into neuronal and glial networks. Would it be possible to promote ‘‘homing’’ of
blood-borne cells to lesioned areas, and are these cells
undergoing region-specific neuronal differentiation? And,
above all, what are the molecular mechanisms that promote the acquisition of neural expression profiles? As we
learn more about the neural differentiation of pluripotent
ES cells, we might also gain insight into signaling pathways involved in the transdifferentiation of somatic stem
cells.
ACKNOWLEDGMENTS
This work was supported by the Deutsche Forschungsgemeinschaft,
the Bundesministerium für Bildung und Forschung, the Innovationsprogramm Forschung des Landes Nordrhein-Westfalen and the Helga Ravenstein-Stiftung.
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