Prospects for Neural Stem Cell-Based Therapies for
Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts 02115
Summary: Neural stem and progenitor cells have great po-
tential for the treatment of neurological disorders. However,
many obstacles remain to translate this field to the patient’s
bedside, including rationales for using neural stem cells in
individual neurological disorders; the challenges of neural
stem cell biology; and the caveats of current strategies of
isolation and culturing neural precursors. Addressing these
challenges is critical for the translation of neural stem cell
biology to the clinic. Recent work using neural stem cells
has yielded novel biologic concepts such as the importance
of the reciprocal interaction between neural stem cells and
the neurodegenerative environment. The prospect of using
transplants of neural stem cells and progenitors to treat
neurological diseases requires a better understanding of the
molecular mechanisms of both neural stem cell behavior in
experimental models and the intrinsic repair capacity of the
injured brain. Key Words: Neural stem cells, neural progen-
itors, neurospheres, self-renewal, multipotency.
Multipotent and self-renewing precursors termed neu-
ral stem cells (NSCs) reside in specialized molecular
microenvironments in the adult mammalian brain. The
fundamental properties of stem/progenitor cells are self-
renewal, multipotency, and migration.1,2Due to these
intrinsic properties, neural stem and progenitor cells of-
fer the clearest potential for cellular therapy in the brain.
Neural stem cell-based therapy encompasses all the strat-
egies to use the biology of NSCs to ameliorate human
neurological diseases and should not be limited to trans-
plantation. For example, alteration in neural progenitor
cell properties of migration and proliferation is the basis
of some CNS human disorders.3,4
The interest in NSCs has been driven primarily by the
prospect of using them to treat acute and chronic neuro-
degenerative diseases of the CNS. For this goal to be
feasible, NSCs must demonstrate the ability to differen-
tiate into multiple lineages, migrate long distances, and
survive in the environment of the injured brain. Further-
more, NSCs must not display oncogenic transformation.
Additional properties of NSCs have been recently dis-
covered, including a beneficial paracrine effect that can
be achieved by transplanted NSCs and embryonic stem
cells.5–7NSCs have also been shown to have immu-
These additional properties
make NSCs an attractive source of material for clinical
transplantation; however, these properties may be mod-
ified by in vitro culturing, which is therefore a matter of
great concern in the field.9,10
One of the crucial questions in regenerative medicine
is whether exogenous or endogenous NSCs are an ap-
propriate source of cells for repair. In the CNS, several
studies that have examined the responses of endogenous
stem cells in the injured brain have found neuron re-
placement to be small.11Several strategies have been
proposed to expand the pool of endogenous progeni-
tors.12–18The success of these techniques remains to be
determined. However, even if endogenous stem cells
could be recruited to yield relevant neural cells, several
challenges remain, such as generating adequate numbers
of cells with proper phenotypes and integrative capacity.
The objective of this review is to examine the current
evidence and the challenges of NSCs, as well as how
new discoveries in NSC biology are helping to delineate
their limitations and potential for brain repair. It should
be emphasized that the study of the intrinsic properties of
NSCs and biological basis of host-NSCs interactions are
Address correspondence and reprint requests to: Jaime Imitola, MD,
77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: jimitola@rics.
Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics
Vol. 4, 701–714, October 2007 © The American Society for Experimental NeuroTherapeutics, Inc.
of critical importance when assessing the clinical appli-
cations of NSCs, which provide a framework for realistic
applications of NSCs to human neurological disorders.
Properties of NSCs for therapeutical use
Self-renewal of NSCs.
NSCs as therapeutic vehicles stems from their intrinsic
properties.19The term self-renewal is used to define
the capacity of a clone to generate new stem cells with
identical properties from generation to generation, a
characteristic that can be promoted by cytokines such
as epidermal growth factor (EGF), b-fibroblast growth
factor (bFGF), and leukemia inhibitor factor among
others (FIG. 1).19Progenitor cells are a step further
along than NSCs in the differentiation process; they
have committed to a particular lineage. Some neural
progenitor cells, such as oligodendrocyte progenitor
cells (OPCs), may exhibit multipotency in vitro in
certain circumstances.20–22Some investigators have
The major interest of
found stem-like cells within a “glial” population iso-
lated from the postnatal and adult cortex, as well as the
adult optic nerve; such immature “glia” give rise to
neurons as well as mature oligodendrocytes and astro-
cytes.23–25However, controversy exists about the mul-
tipotency of other cells such as ependymal cells.14
NSCs will self-renew in response to EGF and/or
bFGF. This proliferative response can be used to isolate
and expand stem-like cells from the CNS where they
form clusters of cells termed neurospheres from primary
dissociated neural cultures. It should be emphasized that
a single cluster does not equal a clone unless it has been
proven to derive from a single isolated cell in a single
well.14,26–32Self-renewal implies that a single NSC can
give rise to differentiated cells as well as to other NSCs
that can be similarly placed into an isolated well and give
rise to both differentiated and undifferentiated progeny.
The previously mentioned assessment constitutes an op-
FIG. 1. Diagram shows self–renewal and multipotency of neural stem cells (NSCs). The NSCs can be isolated from the subventricular
zone (SVZ), a strip of tissue around the ventricles in the adult CNS. These areas exhibit the following cellular organization: type B cells
(blue) are considered the bona-fide NSCs, express glial fibrillary acidic protein (GFAP), and are believed to be a remnant of the radial
glial progenitors during neural development. The type C cell is a transit-amplifying progenitor (green) that is a highly proliferative cell and
generates type A cells that are neuronal progenitors, which migrate to the olfactory bulb in the mice brain and express double cortin.
The NSCs can be isolated by dissociating the SVZ and plating the cells in FGF-2 and EGF containing medium. After several days, bona
fide NSCs and type C cells generate neurospheres that contain not only stem cells but also more differentiated progenitors. NSCs can
differentiate into astrocytes, neurons and oligodendrocytes. Furthermore, NSCs can be dissociated again and replated at single cell/well
density in fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) to generate another wave of self-renewal progenitor cells.
If the resulting cells can differentiate into the three lineages of the CNS (i.e., oligodendrocytes, neurons and astrocytes), then these
cultures fulfill the criteria of containing NSCs, and the self-renewing capacity can be preserved through multiple passages. However,
suboptimal culture methodology can induce aneuploidy and transformation. The bottom panel presents a nonexhaustive list of
molecules that are associated with the different intrinsic properties of NSCs.
Neurotherapeutics, Vol. 4, No. 4, 2007
erational definition of a stem cell and remains today the
only valid way of discerning a self-renewing and multi-
potent NSC (FIG. 1).
NSCs reside in specific germinative zones in the em-
bryonic and adult mammalian CNS.2These zones in-
clude the subventricular zone (SVZ), the external germi-
nal layer of the cerebellum, and the subgranular zone of
the dentate gyrus,1from where they can be isolated.33
Recent elegant studies have demonstrated the constitu-
ents of human SVZ and the potential of NSC isolated
from this neurogenic niche.34,35The adult SVZ36is com-
posed of different types of cells (FIG. 1): 1) type A cells
migrate neuronal precursors that migrate to the olfactory
bulb and express double cortin; 2) type B cells are nestin-
positive, glial fibrillary acid protein (GFAP)-positive as-
trocytes that exhibit NSC properties; and 3) type C cells
are nestin-positive transient dlx-2 positive amplifying
progenitors cells.37Current evidence suggests that in the
adult SVZ, bona fide neural stem cells express GFAP.38
During development or when injury occurs, NSCs pro-
liferate either by symmetric division, where each cell
divides into two new NSCs, or by asymmetric division,
where the NSC gives rise to one new NSC and one
progenitor cell such as a double cortin plus A cell that
can migrate out of the SVZ to areas of injury.39
Multipotency of NSCs.
of a single NSC clone to give rise to the three major types
of cells in the CNS: 1) neurons, 2) oligodendrocytes, and
3) astrocytes. Recently, claims of pluripotency have been
made. The term pluripotency refers to the capacity of
stem cells to give rise to the full range of cells and tissues
in an organism, a unique property of embryonic stem
(ES) cells. Several controversial observations during the
past several decades have suggested that other stem cells,
including NSCs, may have such potential under extraor-
dinary nonphysiological circumstances.40,41However,
other groups failed to reproduce such findings.9Alterna-
tive explanations such as fusion, rather than authentic
transdifferentiation, may explain some of the claims of
pluripotency of neural stem cells.42–44These findings
may have implications when one is considering sources
of stem cells for repair in neurodegenerative disorders.
The rationale of choosing hematopoietic stem cells and
mesenchymal stem cells as sources of neurons for neu-
rodegenerative disorders is less robust. Mesenchymal
stem cells have shown clinical efficacy, but not convinc-
ing neuronal differentiation, and their therapeutic effects
in these models appear to be a result of their paracrine
Besides the self-renewing capacity and multipotency
of NCSs, perhaps the most critical biological property of
NSCs is their extraordinary migration toward sites of
injury.27,46–49The mechanism of this migratory capacity
is beginning to be elucidated50and makes them potent
vehicles for molecular therapy. Another promising prop-
Multipotency is the ability
erty for clinical application is the ability of NSCs to
produce bioactive molecules and growth factors, which
may help to modulate the environment and promote ben-
eficial effects in the host.51,52
Ability of transplanted human and mouse NSCs to
ameliorate neurological disorders in experimental
Human neural stem cells (hNSCs) have been iso-
lated, making it possible for the preclinical testing of
clones of hNSCs. Several laboratories have isolated
multipotent neural stem cells from fetal or adult
sources.12,35,53–55hNSCs transplanted into neonatal
immunodeficient mice proliferated, migrated, and dif-
ferentiated in a site-specific manner.18In vitro priming
procedures have been used to generate a nearly-pure
population of neurons from fetal hNSCs for transplant
into adult rat CNS.56Others have shown that human
neural stem cells, transplanted into neurogenic regions
in the adult rat brain (the subventricular zone and
hippocampus), migrated specifically along the routes
normally taken by the endogenous neuronal precursors
and exhibited site-specific neuronal differentiation in
the granular and peri-glomerular layers of the olfac-
tory bulb and in the dentate granular cell layer of the
hippocampus. The cells also migrated to the striatum
and differentiated into both neuronal and glial pheno-
Many diseases have been suggested as a target for
stem cell therapy,59and neurodegenerative disorders top
the list. Despite the enthusiasm for their use, detailed
analyses of the biology and limitations of NSCs and the
pathology of target disease are both necessary to increase
the likelihood of clinical success.60For example, in isch-
emic stroke, the goal of therapy is to restore blood flow
within minutes of the event, avoiding secondary cell
death. This objective of secondary prevention is obtained
with rapid intervention by vascular neuroradiology and
medication. Therefore, the potential role of neural stem
cells in this acute phase is limited. After this stage it is
relevant to consider the potential role of stem cells to
increase repair and facilitate recovery. In animal models
of stroke and spinal injury, neural stem cell transplanta-
tion has shown efficacy. NSCs implanted into ischemic
brain engrafted, survived, and differentiated into neurons
as well as glia, in contrast to limited differentiation of
NSCs in the normal adult to a glial phenotype.61In
ischemic injury, NSCs transplanted into the ventricles
migrated to and throughout the infarct cavity and en-
grafted and differentiated into all major neural cell
types.62,63In addition, tissue-engineering constructs of
NSCs have shown robust integration and reparative phe-
notype in a model of hypoxic-ischemic injury, with re-
duction of parenchyma loss, increase in directed neurite
outgrowth, and reduction in glial scarring.64This work
NEURAL STEM CELL-BASED THERAPY703
Neurotherapeutics, Vol. 4, No. 4, 2007
suggests injured brain can be permissive for the NSC
transplants and endogenous stem cells.65Integration and
reconstruction of circuitry is an important goal for NSC
transplants during injury. In this regard, several groups
have shown that that in vitro expanded CNS precursors,
after transplantation into the brains of rats, form electri-
cally active and functionally connected neurons66and
integrate into host cortical circuitry.61,64,67
In spinal cord injury, transplanted neural progenitor
cells integrated along axons surrounding the lesion site.
These cells differentiated only along astro- and oligoden-
droglial lineages, supporting the notion that the adult
injured spinal cord provides molecular cues for glial
differentiation, but not for neuronal differentiation.68
However, it has been shown that neural progenitor cells
from the spinal cord may harbor neuronal capabilities,
but activation of the Notch pathway during injury may
limit their neuronal regenerative potential.69To address
this limitation, NSCs have been transplanted into the
spinal cord engineered to express neurotrophin-3. The
cells migrated long distances and differentiated into neu-
rons and glia.52
NSCs may provide substrates for cell replacement, but
there is also evidence for protective bystander effects
that improve the survival of host cells after experimental
contusion in rats and other models of injury.70,71These
effects are due to the secretion of bioactive molecules by
transplanted neural stem cells. NSCs constitutively se-
crete substantial quantities of neurotrophic factors that
can support extensive growth of host axons, as well as
host cell survival.
Models of neurodegeneration.
erative disorders represent important targets for stem cell
therapy. However, a realistic consideration of the com-
plexity of these disorders is required. For instance, in
Alzheimer’s disease (AD) the neurodegeneration is mul-
tifocal and its amelioration would require the use of
multiple transplants in each patient. This approach would
be problematic and mandates that we attempt to achieve
reasonable engraftment by systemic or intravenous ad-
ministration. It could be argued that a paracrine or by-
stander effect might be beneficial in AD to preserve
remaining neurons, even if cell replacement was not
achieved. However, more research is required to demon-
strate whether the paracrine effect observed in some
studies of neural stem cells and embryonic stem cells
could be of any benefit in AD models. The therapeutic
role of neural stem cells in such devastating and multi-
focal disorders as AD may have a coadjutant rather than
a primary role. In addition, the evidence of any benefits
of neural stem cells in preclinical testing is lacking. The
current approach to experimental therapies of AD is fo-
cused on the underlying neurobiology of the disease,
including buildup of A? and the neuronal dysfunction
that occurs. Furthermore, A? transgenic animals have an
impairment of neurogenesis due to deleterious effects of
A? to neural progenitors.72These considerations suggest
that any neural stem cell therapy for AD should be ac-
companied by efforts to decrease the altered microenvi-
ronment that may jeopardize neural stem cell survival. In
spite of widespread claims of stem cell therapies for AD,
a great deal of preclinical research will be required to
establish the potential for neural stem cell therapy for
More circumscribed neurological disease may be ame-
nable to NSC therapy, especially when a single biochem-
ical defect is apparent.59In Parkinson’s disease (PD),
fetal tissue grafts have been used in rodent and primate
models,73as well as in clinical trials.74,75However, short
graft survival and limited integration of the grafts ap-
peared to reduce the usefulness of this approach. Cells
derived from the fetal midbrain can modify the course of
the disease, but they are an inadequate source of dopam-
ine-synthesizing neurons. Immortalized rodent neural
progenitor cells were transplanted into primate and ro-
dent PD models leading to increased tyrosine hydroxy-
lase in the brains of transplanted animals.76Others trans-
planted neuronal progenitor cells derived from neonatal
rat SVZ into the striatum of adult rats after unilateral
6-hydroxydopamine lesions. The NSCs survived and mi-
grated, and many differentiated into neurons.77These
results indicate that the lesioned brain contains intrinsic
cues sufficient to direct the specific expression of dopa-
minergic traits in immature multi-potential neural stem
cells. In this context, stem cells appear to be superior to
fetal tissue grafts and represent new hope for the treat-
ment of PD. Others have shown that human neural pro-
genitor cells can differentiate into a small number of
neurons that expressed tyrosine hydroxylase and were
sufficient to partially ameliorate lesion-induced behav-
ioral deficits in some animals.78More research in pri-
mates is required to address the potential of NSCs in PD
models. In this regard, NSCs appear to prevent alteration
in dopaminergic neurons induced by 1-methyl-4-phenyl-
transplanted into brains of primates after MPTP admin-
istration appear to improve disease severity, operating
through multiple mechanisms.80
One source of stem cells that has shown great promise
is the ES cells. In vitro, an enriched population of mid-
brain neural stem cells can be derived from mouse or
human ES cells. The dopamine neurons generated by
these stem cells show electrophysiological and behav-
ioral properties expected of neurons from the mid-
brain81,82; however, they do have the potential to persist
as an undifferentiated conglomerate of cells in vivo with
the risk of tumorigenic transformation.83
(MS), oligodendrocytes and axons are destroyed and
neurons are dysfunctional.84,85To address this patholog-
In multiple sclerosis
Neurotherapeutics, Vol. 4, No. 4, 2007
ical process, transplanted NSCs would need to differen-
tiate into both oligodendrocytes and neurons. The feasi-
bility of using NSCs has been demonstrated in several
animal models of demyelinating disease. Importantly,
NSCs injected intravascularly in experimental autoim-
mune encephalomyelitis (EAE), a model of MS, success-
fully ameliorated disease,51circumventing the need for
multifocal injections in the CNS. In addition, the use of
immature multi-potent progenitors may be more benefi-
cial, given that NSCs can not only replace neurons and
oligodendrocytes but can serve as modulators of the mi-
croenvironment.51It remains unclear whether the bene-
fits of NSCs in EAE are derived from their ability to
immunomodulate encephalitogenic T cells in the periph-
ery or in the CNS, to modify the destructive inflamma-
tory environment in the CNS, or to replace damaged
Several groups have shown that NSCs can differenti-
ate into mature oligodendrocytes in models of dysmyeli-
nation, including (shi) mice;87myelin deficient (md)
rats;88and the shaking (Sh) pup canine myelin mu-
tant.88,89NSCs undergo abundant oligodendrocyte dif-
ferentiation and mediate clinical improvement. ES cells
have also been shown to differentiate into oligodendro-
cytes and myelinate in vivo in shi mice90and md rat.91
Transplanted OPCs repair focal demyelinated areas in
the neonatal and adult canine mutant,92and in the md
mutant rat.93These cells can be isolated from the rat SVZ
as well and produce robust myelin after transplanta-
tion.94Other cells such as olfactory ensheathing cells and
Schwann cells have also shown efficacy. Purified popu-
lations of human olfactory ensheathing cells remyelinate
persistently demyelinated CNS axons and induce axonal
regeneration95after transplantation into experimentally
induced demyelinating lesions in the rat spinal cord.96,97
Human OPCs myelinate shiverer mouse brain; adult
OPCs myelinated more rapidly than their fetal counter-
parts, generated oligodendrocytes more efficiently than
fetal OPCs, and ensheathed more host axons per donor
cell than fetal cells.55In addition, Schwann cells have
been used for transplantation into demyelinated areas;
human Schwann cells were transplanted into the X-irra-
diation/ethidium bromide-lesioned dorsal columns of
rats showing extensive remyelination, engraftment, and
improved conduction velocity by electrophysiological
analysis several weeks after transplant.98These results
indicated that several types of progenitors and
Schwann cells may be used for remyelinating therapy
and that transplanted cells receive environmental cues
that drive their migration and differentiation toward
Cerebellar degeneration and metabolic diseases.
In an animal model of cerebellar degeneration, the
newborn meander tail mice (mea), which are character-
ized by the lack of development of cerebellar granule
cells, transplanted NSCs differentiate into neurons.100
More recent work in the Purkinje cell neurodegeneration
mutant nervous (nr) mice has demonstrated that NSCs
exert a powerful chaperone effect, physically interacting
with Purkinje neurons, reestablishing altered homeosta-
sis, and protecting these cells from neurodegeneration.5
In addition, NSCs have shown great success in models of
neurological metabolic diseases, such as mucopolysac-
charidosis VII.101,102Human and mouse NSCs have been
engineered to express the human form of the enzymes
defective in Tay-Sachs disease103,104and in Krabbe dis-
ease. It was recently demonstrated that NSCs isolated
from mouse or human sources, as well as human embry-
onic stem (hES)-derived neural progenitor cells, ap-
peared to delay disease onset and prolonged survival in a
mouse model of Sandhoff disease, suggesting that all
these cell types produce bioactive molecules that modify
neurodegenerative disease.105These diseases are typi-
cally lethal or result in severe mental retardation and
neurodegeneration. The early use of NSCs to restore
missing enzymes may have a tremendous impact on the
survival and function of affected individuals.
NSCs briskly home to brain tu-
mors,27and several groups have exploited this extraor-
dinary homing capacity to attempt to modulate glioma
growth. These studies involved injecting genetically
modified NSCs using a variety of gene products, such as
tumor necrosis factor–related apoptosis-including ligand
(a proapoptotic molecule), and the results demonstrated
glioma apoptosis and growth reduction. Others used in-
flammatory cytokines such as interleukin-12 or interleu-
kin-4 that can enhance T-cell infiltration and durable
antitumor immunity.106–108Although beyond the scope
of this review, an important consideration is the potential
origin of brain tumor from NSCs. It has been shown that
neural progenitors have the potential to undergo malig-
nant transformation into gliomas upon oncogene expres-
sion. Furthermore, infusion of growth factors can modify
the SVZ and generate low-grade glioma-like growths.109
This potential for neoplastic transformation is a crucial
aspect for the therapeutic application of neural stem
cells,110although spontaneous transformation of trans-
planted NSCs (including immortalized NSCs) into glio-
mas has not been observed. On the other hand, the po-
tential for transformation of ES cells transplanted to the
CNS remains worrisome, as shown in a recent experi-
mental trial in a PD model.83
Current challenges of neural stem cell-based
It remains to be determined whether any particular
human CNS disease will benefit from NSCs transplan-
tation.59Careful planning and extensive animal testing
will be required before clinical studies can be enter-
tained. Theoretically, diseases in which clinical effi-
NEURAL STEM CELL-BASED THERAPY705
Neurotherapeutics, Vol. 4, No. 4, 2007
cacy could be determined by a single biological mech-
anism, such as Parkinson’s or Huntington’s disease,
might have increased chances of success. MS and AD
represent higher-order challenges because their pa-
thology is widespread, affecting multiple sites in the
CNS and impairing neuronal, as well as glial, func-
tions. While targeting localized lesions that cause
great disability can be contemplated, addressing mul-
tiple lesions that extend throughout the CNS remains a
great challenge, even with migratory NSCs. Repara-
tive therapy with NSCs must be applied in concert
with other equally important traditional therapies that
aim to ameliorate degeneration and promote neuropro-
tection as informed by the molecular pathology of the
particular neurological disorder. In addition, we need
to learn the extent to which an inflammatory and neu-
rodegenerative microenvironment might promote or
hinder neural stem cell function (FIG. 2).60,72,111–113
Source of human neural stem cells: neurospheres,
immortalized, or ES-cell derived?
NSCs may be obtained through the following routes:
primary neurosphere cultures or primary immortalized or
ES-cell derived NSCs. Therefore, questions arise regard-
ing the best sources of stem cells and the most efficacious
methods to generate them. Pluripotent hES cells (an area
of great ethical debate) are self-renewing and pluripotent.
hES cells may provide an unlimited supply of rapidly
dividing cells that can differentiate along any lineage.
However, with hES cells we must be certain to direct
them down a given lineage and to avoid the emergence
of inappropriate non-neural cells or teratocarcinomas.
Alternative sources of NSCs and protocols for differen-
tiation are being explored. A discouraging outcome of
this line of research is the limited differentiation of NSCs
into dopaminergic neurons, a pathway that is preserved
in embryonic stem cells. Nevertheless, recent findings
suggest that human astrocytes residing in the periven-
tricular areas behave as NSCs and exhibit multi-potency.
These cells can be isolated from individuals by brain
biopsies.35Other adult human neural progenitors can be
safely isolated from cadavers,53although their ability to
generate dopaminergic neurons is unknown.
Numerous challenges confront those who would iso-
late human neural progenitors. Human somatic stem cells
are often slow to expand and may senesce, unless genet-
ically augmented. Autografts of immunologically com-
patible and less ethically problematic adult stem cells are
often proclaimed as being promising for neurodegenera-
tive diseases. Grafting a patient’s own cells may appear
to circumvent ethical and immunologic concerns; how-
ever, the practical challenges could be overwhelming
because each new patient will require the prospective
isolation, expansion, and characterization of their NSCs.
This process will be costly in time, resources, and per-
sonnel, and will be dogged by potential variability be-
tween preparations. In addition, if the etiology for dis-
ease progression resides in the host genome or is a
genetic defect (i.e., as in HD, familial amyotrophic lat-
eral sclerosis, or AD), then autologous endogenous pro-
genitors will also be genetically flawed and will not
represent a good source for repair.19,114–116If the process
is noncell-autonomous, then implanting exogenous cells
may be problematic, unless they can be resistant to the
microenvironment that caused the injury.
FIG. 2. Diagram of reciprocal interaction of neural stem cells (NSCs) with an inflamed microenvironment. The concept of injury-induced
niche. NSCs from the subventricular zone (SVZ) or exogenous stem cells can sense areas of injury in the CNS by virtue of the expression
of chemokine receptors such as CXCR4. The injured brain, especially the gliovascular interface can establish an atypical injury-induced
niche, and different types of cells may participate in these areas (i.e., perivascular astrocytes [red] and endothelial cells lining the vessel
as well as immune cells, such as T cells [blue] or microglia [green]). All these cells by virtue of the secretion of stem niche regulators may
create a permissive microenvironment during the acute phase of injury. Several stem cell regulators have been found in these areas (i.e.,
CXCL12, Noggin, and BMP-4). Furthermore, the reciprocal interaction is achieved when NSCs express growth factors that can modulate
the inflammatory response and improve the survival of altered neural cells establishing a bidirectional interaction that is the basis of the
chaperone effects of NSCs. In addition, these cells can differentiate into neurons and replace missing cells. (Modified from Ref. 50).
Neurotherapeutics, Vol. 4, No. 4, 2007
An alternative to autografts is the use of genetically
established somatic stem or progenitor cell lines. Us-
ing established immortalized NSCs, all paradigms of
neural stem cell therapy can be achieved. These cells
have been used extensively and have contributed to
our understanding of NSC behavior and therapy dur-
cently, Roy et al.16showed that retroviral overexpres-
sion of human telomerase reverse transcriptase can
immortalize neural progenitors from the human fetal
spinal cord.122The cells could be passaged without
evidence of senescence, karyotypic abnormality, or
loss of normal growth control. After transplantation
into developing rat fetal telencephalon or spinal cord,
no neoplasms formed in the small number of animals
studied and neuronal markers persisted in vivo with an
absence of glial markers.16,122The feasibility of ob-
taining immortalized hNSCs has been recently dem-
onstrated using v-myc; these hNSCs downregulate the
immortalizing gene and show neuronal differentiation.
These data suggest that gene-based methods for gen-
eration of NSC clones are not inherently dangerous.
Of course, more extensive research is required to ver-
ify that they are not prone to form tumors or aberrant
connections and that they will differentiate appropri-
ately in a great numbers of neurodegenerative disor-
ders, a standard that has been substantially achieved
with mouse NSCs.27,61,70,77,87,101,103,117–121,123
In vitro generation of neural stem cells by neuro-
a small number of stem cells can regenerate multiple cells
types, NSCs do not regenerate progeny with the same ef-
ficiency, and it is not possible to use a small number of cells
to regenerate the entire repertoire of neurons, oligodendro-
cytes, and astrocytes. Therefore, NSCs need to be propa-
gated in vitro by using FGF-2 or EGF. One strategy is to
use neurosphere cultures; neurospheres represent a non-
clonal population of neural progenitors cells that include
progenitors in different stages and exhibit physiological
capacity to respond to FGF 2-and EGF.4The efficiency of
generating neurospheres has been used as a surrogate for
neural stem cell activity in vivo, although this approach is
controversial.124In addition, the neurosphere assay remains
under scrutiny because of the long-held assumption that
neurospheres are clonal. Although it is true that stem cells
cultured in vitro at a dilution of one cell per well can form
neurospheres that are multi-potent and self-renewing, the
clonal efficiency of these cultures is very low. Thus, most
investigators culture neurospheres under the so-called
clonal dilution of 10 to 20 cells per ?L. These neurospheres
are not clonal and a recent report demonstrated significant
fusion of neurospheres at this density.125Moreover, in vitro
expansion may modify the properties of NSCs, sometimes
after only a few passages, which can lower the threshold for
transformation and increase the potential to induce brain
tumors.126,127In this regard, aneuploidy is a recognized
phenomenon of neural stem cells cultures. In addition, an-
euploidy can be seen in ES cells dissociated with trypsin.9
Neurospheres can be transformed by culture after 10 to 15
passages,9but adult NSCs do not exhibit transformation in
vitro. Therefore, more research is needed to clarify the
appropriate methods of culturing NSCs to avoid aneuploidy
and genomic instability.128Because one or two tumorigenic
hits can render neural progenitors cells highly oncogenic
use in humans will require genetic examination to clarify
their tumorigenic potential. These studies will include
karyotyping to detect aneuploidy or comparative genomic
hybridization to detect microdeletions.
Because of the need to expand neural stem cells in
vitro, a debate is currently ongoing among researchers
as to whether in vitro manipulations change the prop-
erties of stem/progenitor cells. Some cells that are
restricted progenitors in vivo, such as dlx-2 or type C
cells, can acquire stem cell properties such as multi-
potency in vitro.37Moreover, in vitro propagation with
FGF-2 may change some of the intrinsic properties
observed in vivo.130Taking lessons from the ES cell
field, we should use the same rigorous methodology
for culturing hNSCs. More specific research is re-
quired to determine the safety of these cells in hu-
mans. Nevertheless, neurospheres isolated from indi-
viduals with genetic alterations may be useful for
research if used in the first passages, and investigators
are using neurospheres obtained from individuals with
brain abnormalities as surrogate in vitro models for
understanding CNS disease and development.3,4
New biological insights in regeneration from neural
stem cell-based transplantation: relevance for
An important area for investigation is the molecular
interactions between NSCs and the disease microenvi-
ronment. Because the paracrine and immunomodulatory
function of certain stem cells can be therapeutically valu-
able, even if integration of the cells in the CNS does not
occur, there is evidence of cross-talk between the resi-
dent CNS cells with neural stem/progenitor cells that are
activated and initiate the process of repair (FIG. 2). In
disease models with a prominent inflammatory compo-
nent, such as stroke, experimental autoimmune enceph-
alomyelitis, and experimental demyelination, the stem
cell niches appear activated and exhibit increased prolif-
eration and migration of neural progenitors.131,132Inter-
estingly, exogenously administered neural progenitors
appear to participate and modulate disease by reciprocal
interactions with the inflamed brain. Understanding the
mechanisms of resident CNS cells with NSC interaction
is important for development of new therapies to slow
the progression of neurodegenerative diseases. Until re-
NEURAL STEM CELL-BASED THERAPY 707
Neurotherapeutics, Vol. 4, No. 4, 2007
cently it was believed that the mechanisms of action of
NSCs during injury were cell autonomous and indepen-
dent of the damaged environment. However, it has been
shown that NSCs depend on an inflammatory and dam-
aged environment to function.51,64Other investigators
have demonstrated direct immunomodulatory activity of
NSCs8and mesenchymal stem cells.133In EAE, several
groups have now shown that immunomodulation with
stem cells is achievable via paracrine mechanisms.8
In addition immunogenicity of NSC is pertinent for
survival, because allogeneic neural stem cells can not
survive in the host without immunosupression.134Both
embryonic and adult neural progenitor cells may express
major histocompatibility complex class I and co-stimu-
latory molecules, but they do not express surface class II
molecules, and thus they are considered less immuno-
genic than differentiated cells. However, during inflam-
mation, molecules that render NSCs immunogenic are
upregulated.113The concern still remains that even
though NSCs are less immunogenic than other cells, their
differentiated progeny will express class II, which could
jeopardize the long-term therapeutic effects of the
cells.113More studies are needed to evaluate the potential
of chronic rejection in neural transplantation.
The concept of injury-induced niches.
and endothelial cells are considered niche cells that sup-
port neural stem cells. Astrocytes in the hippocampus
support neurogenesis,135but reactive astrocytes (gener-
ated by inflammatory stimuli) contribute to astroglio-
sis.136Although the specific mediators of this response
are unknown, inflammatory cytokines play a role in
changing the niche function of astrocytes. Microglia can
be neurogenic and enhance the neural differentiation of
NSCs.137However, lipopolysaccharide-activated micro-
glia (in some paradigms) inhibit neurogenesis.112It has
been shown that the survival of oligodendrocyte progen-
itor cells in the inflamed CNS is dependent on the acti-
vation of microglia, but that excessive microglia activa-
tion reduces the efficacy of the transplant.138The nature
of the activating signal may influence the outcome, as
microglia activated with small dosages of interferon-? or
the TH2 cytokine interleukin-4, but not lipopolysaccha-
ride, appear to increase neurogenesis and oligodendro-
genesis.139Therefore, acute inflammation changes the
intrinsic NSC properties to enhance repair, but the per-
sistence of this abnormal microenvironment may be del-
eterious to NSC survival.
It is becoming clear that the repair plasticity of the brain
requires not only NSC competence, but also the ability of
other cells to participate in the repair process. After injury,
non-neural cells located outside the typical niche start to
recapitulate a developmental process and generate an ec-
topic or atypical stem cell niche.8,50,65It has been demon-
strated that CXCL12 is one of the mediators of ectopic
niches.50,60,65These data have been reproduced and ex-
tended and have consolidated the CXCL12/CXCR4 path-
way as the first molecular pathway formally demonstrated
as a mediator of ectopic neural stem cell niches (FIG.
2).50,65,131,140–142These observations suggest that normal
brain cells far from the SVZ can create an environment for
repair independent of the known stem cell niches. If the
SVZ and dentate gyrus were the only sources of NSCs
during repair, NSCs would have to migrate very long dis-
tances to reach remote areas of injury in the cortex or spinal
cord. Interestingly, surviving astrocytes and endothelial
cells in areas of injury appear to create a local niche for
repair, whereas progenitor cells with known stem cell ca-
pacities, such as oligodendrocyte progenitor cells, are dis-
tributed in the entire brain, and especially around blood
vessels. These observations suggest the possibility that neu-
ral progenitors around vessels and far from the normal
niches can exert a facultative NSC role and the gliovascular
interface can have a facultative stem cell niche role.65
Therefore, injury may not only create neo-niches for SVZ
cells but also for exogenous NSCs. More work to prove this
hypothesis remains, but this possibility suggests that not
only the limited areas around the SVZ and DG niches but
rather the entire CNS might have the potential for repair. In
support of this concept it was shown that a small percentage
of exogenous neural cells in EAE remain undifferentiated
for a long period of time around vessels. In addition, the
injured site and infiltrating immune cells express molecules
known to have a role in normal stem cell niches.8More
research and validation of other molecular mediators of
ectopic niches is necessary (FIG. 2).
NSC biology holds tremendous potential for neurolog-
ical therapy. This approach is not limited to the use of
NSC for transplants, but includes the manipulation of
endogenous stem cells and the multiple bioactive mole-
cules that they express during reciprocal interactions
with the diseased brain. New knowledge of the molecular
biology and genetics of NSCs and their bioactive prod-
ucts, as well as the injured microenvironment, will guide
and refine our judgment of when and how to use NSCs.
In the meantime, several steps are required to move the
field toward the ultimate goal: 1) we must better stan-
dardize methods and protocols of isolation and culture of
hNSCs, 2) we must better evaluate the clinical efficacy of
NSC transplants in more adequate animal models, 3) we
must study the molecular mechanisms of the limitations
of intrinsic brain repair, and 4) we must learn to promote
the long-term survival of these cells by creating a more
permissive environment in the diseased brain and define
the molecular mechanism of the stem cell niche. All of
the aforementioned steps will help us to improve our
translation of NSC biology to the clinic.
Neurotherapeutics, Vol. 4, No. 4, 2007
GLOSSARY OF TERMS
BMI-1? B lymphoma Mo-MLV insertion region 1
Transcription factor that mediates the control of self-
renewal of NSCs.
CCR2 ? chemokine, cc motif, receptor 2
This encodes a receptor for the chemokine; monocyte
chemoattractant protein-1 (MCP1) is produced by
endothelial cells and smooth muscle cells and reg-
ulates the NSC migration.
This molecule is an integral cell membrane glycopro-
tein with a postulated role in matrix adhesion lym-
phocyte activation and lymph node homing, also
expressed by neural precursors cells.
CXCR2 ? chemokine, cxc motif, receptor 2
This is a chemokine; the receptor for growth-regu-
lated gene (GRO1, or CXCL1) expressed in oligo-
dendrocyte progenitor cells and it modulates the
CXCR4 ? chemokine, cxc motif, receptor 4
This is a seven-tramsmembrane protein involved in
migration of many cells including hematopoietic
stem cells and lymphocytes, it is expressed in
NSCs, and it mediated the migration during devel-
opment and injury.
DCX ? double cortin
Gene expressed by migratory neuroblasts, this gene is
mutated in the human X-linked lissencephaly and
double cortex syndrome.
DLX-2 ? distal-less homeobox 2
ral progenitors or type C cells, some Dlx-2 cells can
differentiate into oligodendrocytes and astrocytes.
EGF ? epidermal growth factor
Growth factor associated with proliferation or neural
EMX2 ? empty spiracles homeobox 2
This is expressed in the subventricular zone in vivo,
associated with migration and proliferation of neu-
EPO ? erythropoietin
Cytokine involved in erythropoiesis and is also a reg-
ulator of neural stem cell differentiation and pro-
FGF-2 ? fibroblast growth factor
Growth factor that mediates self-renewal and prolif-
eration of NSCs.
GDNF ? glial derived neurotrophic factor
Cytokine that mediates glial differentiation of NSCs.
GFAP ? glial fibrillary acidic protein
Intermediate filament protein expressed by bona fide
neural stem cells and astrocytes.
GLI1 ? glioma-associated oncogene 1
This is implicated in the transduction of the sonic
hedgehog signal in neural stem cells.
Hes1 ? homolog of the drosophila (hairy/enhancer of
Basic helix-loop-helix (bHLH) involved in self-re-
newal of and neurogenesis of NSCs.
Hes5 ? homolog of drosophila (hairy/enhancer of
Basic helix-loop-helix (bHLH), which is associated
with neurogenesis of NSCs.
Cell adhesion molecules that are also bi-directional
signaling molecules. Many integrins are associated
with migration of neural stem cells (among them
alpha 2, 3, 6, 7 and beta1 integrin.
Ligand of the notch receptor that triggers a cascade of
proteolytic cleavage that leads to the release of the
intracellular part of the receptor from the mem-
brane, allowing it to translocate to the nucleus and
activate transcription factors important in neural
stem cell differentiation.
LIF ? leukemia inhibitor factor
Growth factor associated with self-renewal of neural
LGL1 ? lethal giant larvae-1
Putative tumor suppressor gene involved in asymmet-
ric division of NSCs; mutation of this gene gener-
ates tumor-like growth for neural stem cells in
drosophila and mice.
MMP2 ? etalloproteinase-2
This is secreted by activated endothelial cells that can
promote neural progenitor cell migration.
This molecule is a RNA-binding protein with promi-
nent expression in precursor cells in the ventricular
and subventricular zones and controls self-renewal
capacity of NSCs.
NEURAL STEM CELL-BASED THERAPY 709
Neurotherapeutics, Vol. 4, No. 4, 2007
Neural progenitor cell 1
(Niemann-Pick disease, type C1)
Mutation in this gene has been shown to decrease
self-renewal of NSCs.
Basic helix-loop-helix (bHLH) transcription factor in-
volved in determining neurogenesis.
Basic helix-loop-helix (bHLH) transcription factor
gene that plays an important role in neurogenesis
Notch proteins are single-pass transmembrane recep-
tors that regulate cell fate decisions during devel-
opment. These are involved in many stem cell
systems. In the brain these are associated with NSC
differentiation and gliogenesis.
Gene involved in neural progenitor asymmetric cell
divisions and neurogenesis. Loss of Numb causes
premature progenitor depletion and malformation
of the neocortex and hippocampus.
Olig1 ? oligodendrocyte lineage transcription fac-
Helix-loop-helix (bHLH) transcription factors ex-
pressed in oligodendrocytes and oligodendrocyte
precursors and is required for oligodendrocyte dif-
Olig2 ? oligodendrocyte lineage transcription fac-
Helix-loop-helix (bHLH) transcription factors ex-
pressed in oligodendrocytes and oligodendrocyte
precursors and is required for oligodendrocyte dif-
p21 ? also known as cyclin-dependent kinase inhib-
itor 1A or CDKN1A
Encodes a cyclin-dependent kinase inhibitor that in-
duces cell-cycle arrest.
Cyclin-dependent kinase inhibitor-2A (CDKN2A) as-
sociated with cell-cycle inhibition and senescence
and aging of neural stem cells.
p53 ? transformation-related protein 53
Tumor suppressor gene involved in a variety of
human cancers and involves in self-renewal of
PDGF ? platelet derived growth factor
Is implicated in the self-renewal and proliferation of B
cells, as well as proliferation of oligodendrocyte
PEDF ? pigment epithelium derived factor
Member of the serpin gene family (group of serine
protease inhibitors); this molecule is associated
with self-renewal of NSCs.
PSA-NCAM ? polysialylated neuronal cell adhesion
This molecule is the polysialylated form of NCAM,
which is important for migration of neuronal pro-
PTEN ? phosphatase and tensin homolog
Tumor suppressor gene associated with cell-cycle ar-
rest is mutated in a variety of human cancers and is
involved in self-renewal of NSCs.
Gene expressed neural progenitors; mutation is this
gene alters the migration, reelin is associated with
reduce migration. Furthermore, blockade of reelin
increases migration of neural progenitors.
SHH ? sonic hedgehog
Gene associated with brain morphogenesis and medi-
ates proliferation and neurogenic and oligodendro-
glial differentiation of neural progenitors.
Mammalian homologous of the drosophila gene slit; it
is a chemorepellent instead of chemoattractant of
the subventricular zone and neural progenitor cells.
SOX2 ? sex determining region y–box containing
Transcription factor associated with self-renewal of
embryonic and neural stem cells.
SOX10 ? sex determining region y–related HMG-
box gene 10
Encodes a transcription factor characterized by a
DNA-binding motif known as the high mobility
group (HMG) domain associated with oligoden-
TH ? tyrosine hormone
Steroid hormone required for terminal oligodendro-
cyte differentiation from NSCs and OPCs.
Wnt3 ? wingless-type mmtv integration site family,
Member of the canonical wnt-? catenin signal path-
ways implicated in self-renewal and neurogenesis
of neural progenitors.
Neurotherapeutics, Vol. 4, No. 4, 2007
Wnt5 ? wingless-type mmtv integration site family,
Member of the canonical wnt-? catenin signal path-
ways implicated in self-renewal and neurogenesis
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