REVIEWNeural stem cells: progression of basic research andperspective for clinical application
Hideyuki Okano
Department of Physiology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on April 5, 2002)

Abstract. It has long been thought that functional regeneration of the injured central nervous system(CNS) is impossible, as Santiago Ramóny Cajal described in the early 20th century, ‘‘once the development was ended, the fonts of growth and regeneration . . . dried up irrevocably’’. In mammalianneural development, most neuronal production (neurogenesis) occurs in the embryonic stage. However, recent ﬁndings indicate that neurogenesis continues in the olfactory bulb, hippocampus, anddentate gyrus of adult mammalian animals, from the neural stem cells (NSCs). Recently developed
techniques have made it possible to isolate, culture, and grow pluripotent self-renewing NSCs fromboth embryonic and adult brains. This basic research is attracting a lot of attention because of the hopethat it will lead to regeneration and reconstruction therapy for the damaged CNS. In this review, recent
ﬁndings on the stem cell biology of the CNS and strategies for its potential therapeutic application will
be discussed. (Keio J Med 51 (3): 115–128, September 2002)
Key words: regeneration, prospective identiﬁcation, adult neurogenesis, transplantation, embryonicstem cell

Neural stem cells (NSCs) are deﬁned as undifferentiated cells that are able to self-renew as well as generate the three major cell types that constitute the central nervous system (CNS): neurons, astrocytes, andoligodendrocytes (pluripotency) (Fig. 1). Notably, thepresence of NSCs or neural stem-like cells is not a recent concept, and a model close to the present one hadalready been proposed at the end of the 19th century:‘‘there are neural precursor cells in the ventricularzone of the neural tube, which is the primordium of the
CNS at the developmental stage (embryonic stage), andthese precursor cells differentiate to neurons and glia’’.1
These neural precursors located at the ventricular zoneof the fetal neural tube were observed in more detailin the 1960’s and named ‘‘matrix cells’’.2 Building on
the great achievements of our predecessors 100 years
ago, continued research and modern technology haveresulted in important advances, including the development of both i) a selective culture method for NSCs andii) an in situ identiﬁcation method for NSCs using selective markers. Making full use of these two developments, studies with great impact have recently been
made, which report the discovery of NSCs at unex-

pected stages and places (for example, in the adultbrain) and surprising properties of NSCs. In this review,
the basic biological properties of NSCs and the relationship of adult neurogenesis with higher brain function and psychiatric diseases are discussed. Althoughrelated topics, such as the regulation of NSCs’ selfrenewal and differentiation, the regional speciﬁcity ofNSCs, and the acquisition of speciﬁc cell fates are very
important, these issues are not described here, but they
are discussed in other reviews.3,4What Is an NSC?
Biologists studying stem cells of the hematopoietic
system, small intestine, and skin described the basic
properties of stem cells (somatic stem cells, not totipotential embryonic stem cells). These cells are: (a)undifferentiated, in which no speciﬁc differentiation
marker is expressed, (b) able to proliferate, (c) able toself-maintain their population, (d) able to differentiate
and generate many descendant cells with various functions, and (e) able to repair injured tissue.5 However, itremains to be resolved whether stem cells in the ner-

Reprint requests to: Dr. Hideyuki Okano, Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan, e-mail: hidokano@sc.itc.keio.ac.jp
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Okano H: Neural stem cellFig. 1 Neural stem cell and its lineage. The pluripotency of a single NSC is often, but not always, mediated by the generation of cell-lineagerestricted intermediate progenitor cells, including neuronal progenitor cells (NP) and glial progenitor cells (GP); this has been shown both invivo6 and in vitro.113 (Reproduced from Okano H: Seikagaku 2002; 74: 17–26 (in Japanese), Copyright 6 (2002), with permission from Nihon
Seikagakukai).114

vous system strictly meet all these criteria; thus, thedeﬁnition of NSCs differs slightly among individual
researchers. In this review, I will consider the generalcharacters of NSCs and progenitor cells to be as follows. The cell lineages generated from NSCs, which
generate neurons, astrocytes, and oligodendrocytes differ among stages and regions of the CNS. In the cerebral cortex, NSCs generate intermediate progenitor
cells that then generate speciﬁc cells6: neuronal progenitor cells generate only neurons and glial progenitor
cells generate only glial cells, as shown in Fig. 1. In thiscell fate process, pluripotential NSCs are committed toprogenitor cells of a speciﬁc lineage. NSCs, neuronalprogenitor cells, and glial progenitor cells are able toproliferate and are generally located in the periventricular area. When these three types of cells are notdistinguishable, they are collectively called, ‘‘neuralprogenitor cells’’.
NSCs can be clonogenically expanded in freeﬂoating ‘‘neurosphere’’ cultures. Researchers have used
this system to deﬁne the NSCs experimentally and toquantify their multi-lineage potency and ability to selfrenew (Fig. 2).7 However, an NSC cannot be identiﬁed
prospectively as a neurosphere-initiating cell. That is,when the culture is started, we cannot tell which cell
will form a neurosphere. Only after the neurosphere isformed can we conclude that the initial cell was an
NSC, i.e., it is a retrospective identiﬁcation.

Prospective Identiﬁcation and Isolation of NSCs
Although there are highly selective marker molecules for NSCs, such as Musashi1 (an RNA-binding
protein),8–13 nestin (an intermediate ﬁlament)14,15
and members of Sox-family (transcription factors),16
these markers, unlike those for hematopoietic stem
cells, are not cell surface antigens and therefore could
not be used to identify viable NSCs prospectively
using ﬂuorescence-activated cell sorting (FACS), until
recently.3,17 To enable the prospective identiﬁcationof viable NSCs, we ﬁrst prepared a transgenic mouse(nestin-EGFP mouse) in which a ﬂuorescent molecule, enhanced green ﬂuorescent protein (EGFP), isexpressed under the control of the 2nd intronicenhancer of the nestin gene, which acts highly selectively for NSCs. We then isolated populations of EGFPexpressing cells from the brains of these mice according
to the expression level (i.e., ﬂuorescence intensity) using FACS.18–20 The isolated cells were then investigated for the properties of NSCs, using mitotic activity(BrdU uptake), the efﬁciency of neurosphere formation, and self-renewal and pluripotency in low-density
culture as indices. The GFP ﬂuorescence intensity wascorrelated with these NSC-like biologic activities (Fig.
3).18 Because this method concentrates NSCs using
FACS, without involving in vitro growth-factor-induced
expansion (e.g., neurosphere culture), it can be used forKeio J Med 2002; 51 (3): 115–128

117

Fig. 2 Neurosphere formation assay. The major breakthrough for research on stem cell biology of the CNS was the development of the clonogenic expansion of NSCs in ﬂoating culture, called ‘‘neurosphere culture,’’ within a serum-free deﬁned medium containing EGF and/or
FGF2.7 A neurosphere derived from a single cell is capable of generating the major 3 lineages of the CNS, i.e., neurons, astrocytes, and oligodendrocytes, indicating the multipotency of the neurosphere-initiating cell, upon the differentiation assay. If the neurosphere is dissociatedinto single cells, each cell starts to form a secondary neurosphere again with high frequency. (Reproduced from Okano H: Seikagaku 2002; 74:
17–26 (in Japanese), Copyright 6 (2002), with permission from Nihon Seikagakukai).114

Fig. 3 Nestin-EGFP transgenic mice and isolation of NSCs. A) For the prospective identiﬁcation and isolation of NSCs, a nestin-EGFP transgene was constructed by placing the EGFP cDNA under the control of the nestin 2nd intronic enhancer and minimum promoter of the hsp68
gene.18 EGFP expression at embryonic day 10 of the nestin-EGFP mouse is shown below; it can be detected all through the neural tube withoutbias along the anteroposterior or dorsoventral axis. (right) Time lapse video recording of the neurosphere culture starting from a single nestinEGFP cell. (Reproduced from Kawaguchi A, et al: Mol Cell Neurosci 2001; 17: 259–273, Copyright 6 (2001), with permission from Academic
Press),18 B) A single nestin-EGFP positive can give rise to neurons, astrocytes and oligodendrocytes upon clonal monolayer culture, indicating
its multilineage potency.

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Okano H: Neural stem cell

Localization of NSCs at Various Developmental Stages
Several studies have been performed to determine
where and when NSCs exist within an organism. In thissection, I describe the localization of NSCs in neurogenesis.
Before neurulation

Fig. 4 Nestin-EGFP and CD24 expression in SP/MP cells. CD24negative, nestin-EGFP positive, and SP cells are likely to be associated with NSC-activities, respectively.18,25 Here, these threeparameters are compared with one another, by characterizing the expression of CD24 (y-axis of the plots on the right) and nestin-EGFP(x-axis of the plots on the right) of the SP cells from mouse brain. The
majority of mouse brain SP cells are nestin-EGFP positive and CD24
negative, although non-overlapping populations are also present.
Thus, combinations of these 3 parameters might enable the further
enriched isolation of NSCs compared with previous reports.18,25
Alternatively, NSCs could exist in various populations in a heterogeneous fashion.27 The original data was provided by Dr. Yumi Matsuzaki (Department of Physiology, School of Medicine, Keio University).

analyzing various biological properties of NSCs, such as
their time of appearance and regional speciﬁcity, whichhave not been well characterized. Furthermore, because it allows the identiﬁcation of viable NSCs, thismethod may be very useful for analyzing intrinsic (cellautonomous) and external (non cell-autonomous) factors involved in the proliferation and differentiation of
NSCs. It is also possible to introduce the same EGFPtransgene by lipofection or recombinant adenoviral
vector into human brain-derived cells and to isolate
neural progenitor cells using FACS,21–23 and thereby
supply donor human cells for therapeutic transplantation.
Within the past few years, there has been remarkable progress in determining surface antigen-based
immunoselection strategies for sorting live NSCs.24,25
Furthermore, stem-like cells can be puriﬁed from various organs as side population (SP) cells, based on theirproperty to exclude Hoechst 33342.26 To establish amore deﬁned strategy for the prospective identiﬁcationof NSCs, we have compared separation methods in detail cytometrically, characterizing the cell size (forward
scatter; FSC)/granularity (side scatter; SSC) proﬁle, andexamining the nestin-EGFP expression, SP phenotype,and cell-surface antigens in developing mouse brains
and found that the character of NSCs is not uniform
and changes during development (Fig. 4).27

When and where NSCs appear during ontogeny isimportant to understand the developing nervous system. In vertebrates, selective NSC markers, Musashi18,9,11,28 and nestin-EGFP,18 are expressed over theentire neural plate before the neural tube closure, inboth humans and mice. Fibroblast growth factor-2(FGF-2)-responsive NSCs ﬁrst appear in vivo at embryonic day (E) 8.5. Later, between E11 and E13, a
separate and additive population of epidermal growth
factor (EGF)-responsive NSCs arises from the earlier
born FGF-2-responsive NSCs by asymmetric divisions.29–32 However, it is not clear whether the ﬁrst
neural cells that appear during ontogeny upon ‘‘neuralinduction’’ emerge from the initial FGF-2-responsive
NSCs (reviewed by van der Kooy and Weiss33).
After neurulation
It has been long believed that NSCs are located in
the ventricular zone of the neural tube after neurulation.2 This is very consistent with the results of immunohistochemical analysis using Musashi1 (Fig. 5). In the
fetal neural tube, NSCs divide both symmetrically andasymmetrically.34 In the former, one cell divides into
two identical daughter cells. This division occurs at thestage when the number of cells constituting the neuraltube rapidly expands (one neuroepithelial cell produces
two neuroepithelial cells: proliferative symmetric division), before the generation of neurons begins. In
asymmetric division, one cell divides into two different
daughter cells. This division is necessary for generating
the both differentiating cells and a stem cell by itself,
and is a general characteristic of stem cells. In the
mammalian CNS, NSCs (or neuronal progenitor cells
that are able to generate only neurons) divide asymmetrically in the ventricular zone of the neural tube,
and neural stem (or neuronal progenitor) cells themselves and neurons are generated simultaneously. Afterneuronal generation begins, neural stem (or neuronalprogenitor) cells continue to divide asymmetrically andgenerate different types of neurons, depending on what
micro-environmental and cell-autonomous factors arepresent.35,36 The proliferative symmetric division ofneuroepithelial cells is frequently observed at the relatively early stage of neural tube genesis in the forebrain
region in mammalian development. In this type of cell

Keio J Med 2002; 51 (3): 115–128

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Fig. 5 Musashi1-expression in the periventricular region of neonate
mouse brain. Musashi1-positive cells are present in the periventricularregions lining the lateral ventricle at postnatal day0 mouse forebrain.
All through the development, Musashi1-expression can be observedin the periventricular zone.

division, the cleavage plane is vertical to the ventricularwall, whereas in asymmetrical division the cleavage
plane is horizontal to the ventricular wall; however, theunderlying control mechanism is still unknown.
In the embryonic tube, a portion of the NSCs (which
can be called ‘‘fetal NSCs’’), have a characteristic radialmorphology and are generally known as radial glia
(reviewed by Barres).37–40 Radial glia have long beenthought to be involved in guiding or providing a scaffold for the migration of newly generated neurons.41
Fetal NSCs are likely to play crucial roles in the histogenesis of the brain, in addition to the production of
new neurons and glia (reviewed by Okano4).
Adult CNS
The generation of neurons does not just occur at theembryonic stage. It continues in adult rodents in two
regions: the hippocampal dentate gyrus and olfactory
bulb42,43 (reviewed by Temple and Alvarez-Buylla,Gage, Alvarez-Buylla and Garcia-Verdugo44–46). The
localization of NSCs at the neurogenic sites in adultbrain has been shown by the selection culture method
for NSCs, the neurosphere method, and in situ identiﬁcation of NSCs using selective markers (Musashi1, nestin). It has been shown that interneurons in the olfactory bulb are generated from NSCs that are localized
to the periventricular area facing the lateral ventricle
and that neuronal cells move all the way to the olfactory bulb through a pathway called the rostral migratory stream42,47,48 (reviewed by Temple and Alvarez-

Fig. 6 Nestin-EGFP expression in the adult spinal cord. EGFP expression is seen in the ependymal cells surrounding the central canal,
consistent with the previous reports indicating NSC activities in thesecells.48,52 These EGFP-positive cells are able to produce neurospheres in vitro.

Buylla45). Furthermore, recent results indicate that,
contrary to the current view, multipotential (neuronalastroglial-oligodendroglial) precursors with stem cellfeatures can be isolated not only from the SVZ but
also from the entire rostral extension, including the
distal portion within the olfactory bulb.49 These ﬁndings demonstrate that stem cells are not conﬁned to theforebrain periventricular region and indicate that stem
cells endowed with different functional characteristics
occur at different levels of the SVZ-rostral extension(RE) pathway. Given the persistence of neurogenesis
into adulthood, several attempts have been made to
identify adult NSCs. Dr. van der Kooy and his colleagues reported the possible presence of stem cells in
the SVZ facing the lateral ventricle in the adult brain,using various methods including the neurosphere
method.50 Several years ago, we showed in a collaborative study with Dr. Steven Goldman, that NSCs/
neuronal progenitor cells are present in adult human

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Okano H: Neural stem cellFig. 7 Delayed NSC-transplantation in injured spinal cord. A) Scheme for therapeutic transplantation of cultured NSCs into the injured spinal
cord. B) The advantage of the delayed NSCs-transplantation. The adult spinal cord immediately after injury is likely to present non-neurogenic environment.48 However, delayed transplantation of NSCs 9 days after injury was shown to result in generation of neurons, oligodendrocytes and astrocytes from donor cells. The illustrations in this ﬁgure were provided by Dr. Yuto Ogawa (Department of Orthopedic Surgery,
School of Medicine, Keio University).

brain tissues at the region facing the lateral ventricle.51
These cells in the SVZ/ependymal cell layer facing thelateral ventricle, including Musashi1-positive cells, divided then differentiated into neurons in culture in thecontinuous presence of FGF-2 and BDNF. This ﬁndingshows the presence of NSCs or neuronal progenitor

cells in the adult brain that have the ability to generate
new neurons upon their exposure to a speciﬁc environment. Recently, more detailed studies to determine theorigin of adult NSCs have been performed by twogroups, although they remain controversial. Johansson
et al. labeled cells using viruses carrying the lacZ gene

Keio J Med 2002; 51 (3): 115–128

and the liposoluble biological dye, DiI, and concluded
that ependymal cells in the ventricle are NSCs.48 On
the other hand, Dr. Alvarez-Buylla and his colleagues
recently used glial ﬁbrillary acidic protein (GFAP), an
astrocyte marker, and reported that GFAP-positive
cells located in the SVZ are NSCs, at least in the periphery of the lateral ventricle.47 Using transgenic miceexpressing a retrovirus receptor under the control of a
GFAP promoter, they demonstrated that the GFAPpositive cells in the SVZ gave rise to new neurons and,thus, concluded that SVZ astrocytes are NSCs.47
Whether ependymal cells or SVZ astrocytes are NSCs,
it is of interest that a cell population previously considered to be glial cells includes NSCs and is related toneurogenesis in vivo. Anti-Musashi1 antibodies recognize not only ependymal cells and SVZ neural progenitor cells but also some astrocytes in adults (Fig. 5).11
The subventricular layer/ependymal cell layer facing
the lateral ventricle, where NSCs are localized, corresponds to the neurogenic site in the adult brain. However, it is of more interest that NSCs are present evenin non-neurogenic regions (reviewed by Temple andAlvarez-Buylla44). For example, NSCs were shown toexist in the ependymal layers surrounding the central
canal of the adult spinal cord, where neurogenesis
does not occur.48,52 However, the NSCs in this site
expressed nestin-EGFP (Fig. 6), formed neurospheres,and generated new neurons in culture. Thus, neurogenesis from these NSCs is likely to be inhibited in vivo,
particularly by local environmental factors. Even after
spinal cord injury, neurogenesis is inhibited, although
the NSCs are capable of producing astrocytes,48,52
which form glial scarring thereafter. Given these experimental ﬁndings, it is unlikely that the simple introduction of NSCs into an injured region would improve
the pathology, because the endogenous NSCs generate
only glial cells and not neurons. Thus, in addition to thetransplantation of NSCs, preparation of the microenvironment to induce neurogenesis would be essential. We
recently demonstrated that delaying the transplantationof NSCs to 9 days after the spinal cord injury, when inhibitory cues that prevent neurogenesis are likely to be
down-regulated,53 is important to achieve neurogenesis
from the transplanted NSCs and functional recovery(Fig. 7).54
Neurogenesis in Adults and Its SigniﬁcanceThe long-held belief that neurogenesis does not occur in the adult brain of higher animals has been disproved. The concept that neurogenesis can occur in theadult mouse brain was actually proposed by Altman
and colleagues. 40 years ago.55 In the 1980’s, Dr. Fernando Nottebohm and his colleagues proposed that inadult male canaries, neurogenesis occurs in the breed-121

ing season and replaces old and lost neurons in the highvocal center (HVC) in the neostriatum and that thisprocess is closely related to learning a new song.56–57
As described in the preceding chapter, in rodents,
neurogenesis occurs in limited regions in adults: theolfactory bulb and the hippocampal dentate gyrus.
Dr. Fred Gage and his colleagues demonstrated thatneurogenesis also occurs in adult humans.58 These
researchers took advantage of the fact that bromodeoxyuridine (BrdU), which labels cells at the proliferative stage, is administered to cancer patients forclinical diagnosis. Examination of the hippocampal tissue obtained post mortem from ﬁve patients revealed
many BrdU-labeled neurons, demonstrating that theseneurons were generated after BrdU administration andbefore death (16–781 days).58
The presence of NSCs with both pluripotency andthe ability to self-renew in adults has been shown by
neurosphere culture.7,18,50 However, the sites of neurogenesis in vivo are limited in adults. Analysis of thephysiological signiﬁcance of this ﬁnding is underway,
focusing on NSCs involved in neurogenesis in vivo. It
has been reported that experiencing of a ‘desirable environment’ increases adult neurogenesis (granule cells)
by 15% in the hippocampaldentate gyrus in mice, andalso increases the efﬁciency of space learning performance (reviewed by Kemperman and Gage59). Recent
studies have shown that newly generated neurons are
physiologically active and are able to form new synapses with older neurons.60 A fascinating thought, that‘‘an additional 15% of neurons increased learning efﬁciency’’ (reviewed by Kemperman and Gage59), may be
proposed, but how new neurons form a neuronal
network with existing neurons and whether the newneurons are really involved in learning have yet to
be determined. Interestingly, it has been shown that
inversely, a stressful experience markedly inhibits neurogenesis. It is proposed that stress-induced decreases
in neurogenesis within the hippocampal dentate gyrus
are an important causal factor in precipitating episodesof depression. Reciprocally, it is speculated that therapeutic interventions for depression that increase serotonergic neurotransmission act at least in part by augmenting dentate gyrus neurogenesis.61
The reported results on adult neurogenesis in the
neocortex have been controversial.62–64 claimed thatneurogenesis occurs in the prefrontal area of the frontal
lobe, the lower temporal association area, and the posterior parietal association area in the cerebral neocortical association area of adult macaque monkeys.
These regions are associated with higher functions suchas emotion, memory, and judgment. The detection ofneurogenesis in this area attracted attention because
it disproved previous beliefs and provided profound
implications for understanding the cellular mechanisms

122of higher functions. However, Dr. Pasko Rakic and hiscolleagues’ more recent results do not support thepresence of neurogenesis in normal adult primate cortex.64 In contrast to the results of Dr. Gould’s group,Drs. Kornack and Rakic showed that, although numerous BrdU-labeled cells were distributed throughout
the cerebral wall, including the neocortex, these wereidentiﬁed as non-neuronal cells by careful immunohistochemical observations using confocal microscopy;
evidence for newly generated neurons was limited tothe hippocampus and olfactory bulb in adult macaquemonkeys.64 Interestingly, however, this condition is
likely to change upon injury. We found that signiﬁcantneurogenesis was induced upon ischemia at least insome part of the neocortex in adult monkeys (Tonchev
AB, Yamashima T, Zhao L, Okano HJ and Okano H:
submitted).
Prospective for Cell Therapy of the Damaged CNS
Regenerative medicine using stem cell biology is
attracting a lot of attention. The adult mammalian CNS
was traditionally considered to be lacking in regeneration ability, but it is now an important target ofregenerative medicine. Strategies for regenerative
therapy using a stem cell system are roughly divided
into: i) the use of organ (or tissue) stem cells (e.g.,
NSCs), ii) the use of embryonic stem (ES) cells, and iii)
the use of dedifferentiation/transdifferentiation of differentiated cells. Therapeutic strategies should be determined based on a comprehensive consideration of
the available information. Each of these strategies isreviewed below.
Use of tissue stem cells (NSCs)
The presence of characteristic stem cells (tissue stem
cells) has been conﬁrmed in many organs or tissues
within a mature body. As discussed above, stem cells
are self-renewing undifferentiated cells with pluripotency, and the ability to contribute to tissue repair after
injury may be added to the deﬁnition. The high potential for tissue stem cells to be applied to the regenerative therapy for various organs can be predicted fromthese deﬁnitions. The ﬁrst successful organ regeneration using the tissue stem cell system was bone marrowtransplantation.65 In addition to the hematopoietic system, there is evidence for stem cells in many organs,such as the liver, intestinal tract, skeletal muscle, mammary gland, neural crest (peripheral nervous systemand smooth muscle), skin, and CNS. Indeed, NSCs are
tissue stem cells of the CNS. As described above,
several groups, including ours, have sought to establish a technique for the prospective identiﬁcation of
NSCs.17,24,25 The prospective identiﬁcation and isola-

Okano H: Neural stem celltion of stem cells from organs by combining cell surface
antigens or speciﬁc reporter genes with cell separation
using a cell sorter is an important technique. Stem cellsof each organ will eventually become commerciallyavailable as promising therapeutics for tissue repair andorgan regeneration.
Regeneration of an organ by activation of the tissue
stem cells within it is also an important direction of
study, which may show a lot of achievement in the future, because this is considered a particularly importanttherapeutic strategy for regeneration of the CNS. Notably, recent ﬁndings have raised the possibility that thebrain has a latent capacity for self-repair in response to
injury or disease through the use of the endogenous
NSCs or neural progenitor cells.66 Dr. Jeffery Macklis
and his colleagues demonstrated that the endogenous
NSCs (or neural progenitor cells) can be induced in situ
to differentiate into cortical projection neurons inregions of the adult mammalian neocortex that do
not normally undergo any neurogenesis. Furthermore,these neurons form appropriate connections after
chromophore-targeted synchronous apoptotic degeneration of neurons is induced.66 These results indicatethat neuronal replacement therapies for neurodegenerative disease and CNS injury may be possible throughthe manipulation of endogenous NSCs in situ. Furthermore, recent results indicate that exogenous BDNF
supplied either by infusion of the recombinant protein,67 or by the intraventricular delivery of and ependymal infection by adenoviral vectors,68 induced neurogenesis from resident progenitor cells in the adultbrain, even at sites where adult neurogenesis does notoccur normally. Thus, it is likely that the adult brain
parenchyma may recruit and/or generate new neurons,
which could replace those lost as a result of injury or
disease.
In addition to the activation of endogenous tissue
stem cells, another approach for regenerative medicine
using tissue stem cells is, of course, their transplantation, as was ﬁrst performed for reconstruction of thehematopoietic system.65 There are some promisingreasons to pursue NSC-transplantation as a treatment
for CNS damage. The effects of transplanting NSCs
in an attempt to obtain functional recovery from various types of CNS damage have been reported
recently.4,19,69–74 For Parkinson’s disease (PD), clear
evidence of dopaminergic fetal graft survival andsymptomatic relief – (at least for younger patients –)has been recently provided by open label clinical
trials75 and a double-blind, sham-surgery controlled
study.76 However, 5@10 fetal brains are required totreat one PD patient, causing practical and ethicalproblems with this procedure. On these grounds, there
has been increasing interest in developing procedures
to obtain large amounts of standardized and quality-

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controlled NSCs. However, it should be noted that NSC
transplantation therapy in PD is still in its infancy. Although in-vitro-expanded human NSCs (neurospheres)derived from the fetal cortex can survive for long periods, differentiate into neurons and astrocytes, andextend long processes into the host brain, dopaminergic(DA) neurons do not arise very often in vitro or following transplantation.77,78 On the other hand, NSCtransplantation still holds much promise for some cases.
Studies have demonstrated that the fates of the transplanted NSCs or progenitor cells greatly depend on thehost microenvironment, e.g., the timing of the transplantation,54,79,80 the transplantation site (neurogenic
or non-neurogenic sites), and the cell types that are lost
within the host CNS upon injury or disease.72 We
transplanted rat fetal spinal cord-derived NSCs grownin vitro into a rat spinal cord injury model (cervicalcompression model) and successfully obtained histological and behavioral recovery.54 Further study using a
primate donor and recipient will yield useful information for the application of these ﬁndings to humans. The
range of NSC applications aimed at regeneration of the
CNS is not limited to cell therapy by transplantation.
The investigation of how endogenous NSCs are activated and induced to contribute to organ self-repair are
also important areas of research.
Use of embryonic stem cells (ES cells)
ES cells are undifferentiated stem cell lines established from the epiblasts that are present in the innercell mass (ICM) of an early embryo at the blastocyst
stage. Epiblasts differentiate into three germ layers thatwill later form the body in the developmental process
and thus, they are stem cells that are able to differentiate into the three germ layers. ES cells can be induced
to differentiate to various cells derived from the threegerm layers under various culture conditions, and human ES cell lines have been established; therefore, ES
cells have been a focus of regenerative medicine.81
To date, various cell types, including those of the nervous system, fat, smooth muscle, skeletal muscle, thehematopoietic/vascular system, and cardiac muscle
have been derived from ES cells.
Several studies have applied ES cells to the nervous system. When glial progenitor cells derived frommouse ES cells were injected into rat mutants showing
hypomyelination,82 the tissue was stained with an antibody against a myelin-speciﬁc protein marker, PLP.
Recently, Dr. Ole Isacson and his colleagues showedthat transplanting low doses of undifferentiated mouse
ES cells into the striatum of a rat PD model (6-OH DA
unilaterally-lesioned hemiparkinsonism) results in a
proliferation of ES cells into fully differentiated DA
neurons and a gradual and sustained behavioral resto-

123

Fig. 8 A new strategy for the cell therapy of Parkinson’s disease. A
large quantity of dopaminergic neurons can be prepared from invitro-expanded NSCs or ES cells, followed by effective induction into
dopaminergic neurons and their subsequent puriﬁcation using the
TH-EGFP reporter and FACS-mediated isolation. This could provide a new strategy for the cell therapy of Parkinson’s disease, overcoming the ethical and practical problems associated with the current
fetal grafts.

ration.83 However, teratoma developed in rare instances after transplantation in these cases, indicatingthat improvements to this procedure still need to be
designed and tested before it can be applied as a celltherapy for PD. Dr. Yoshiki Sasai and his colleagues at
Kyoto University reported that DA neurons are efﬁciently induced in vitro when mouse or monkey ES cells
are co-cultured with PA-6 cells (stromal cells), and thatthese neurons survive after their transplantation into
the striata of PD model mice.84,85 This method makes it
possible to derive DA neurons from ES cells, but thederived DA neurons are intermingled with other typesof cells. To apply this method to therapy, it is necessary
to purify the dopaminergic neurons and to exclude
other types of cells, including undifferentiated ES cells,
which could form a teratoma after transplantation.
We developed a reporter gene (TH-EGFP) containing
cDNA encoding green ﬂuorescence protein under thecontrol of the promoter for tyrosine hydroxylase, thekey enzyme of dopamine biosynthesis86 and designed asystem for purifying TH-positive neurons using the THEGFP reporter gene and cell sorter (Fig. 8). Neverthe-124

less, it is also important to investigate whether the
introduced ES cell-derived dopaminergic neurons arefunctional in vivo. It was recently shown that, in manyneurodegenerative diseases including PD, speciﬁc neurons are selectively lost (amyotrophic lateral atrophy[ALS], motor neurons; Huntington’s chorea, striatal
GABAnergic neurons; and cerebellar ataxia, cerebellar
neurons including Purkinje cells). This implies thattreatment of such diseases using ES technology would
require a two-step strategy: efﬁcient derivation of theseneurons from ES cells and their selective isolation.87
As human ES cells become more available, the following points should be addressed regarding their usage in medical treatment: I) The direct transplantationof ES cells induces teratoma, as observed in immunodeﬁcient animals such as SCID mice. It would be betterto induce ES cell differentiation to a more mature state,i.e., more like the cells constituting the body, thentransplant them, rather than to transplant them directly.
II) Even when cells are induced to differentiate, various
cell types are derived in the differentiation via the formation of the embryoid body, and separation of thespeciﬁc cells required for the therapy is necessary. III)The possibility of immunological rejection always exists
in transplantation therapy, except with autografts. It istheoretically possible to perform therapeutic cloning,in which human ES cell pools of each HLA type areprepared or nuclear transferred ES cells are generated(ntES cells) by denucleating an unfertilized oocyte,
injecting the somatic cell nucleus of a speciﬁc person
into the embryo, culturing the embryo until the blastocyst stage, and establishing ES cells of the speciﬁc person from the inner cell mass of the cloned embryo. The
preparation of a human embryonic clone reported by a
US venture business group, Advanced Cell Technology
Co. and was sparked widespread debate about the
ethics of this research.88 The human embryonic clone
prepared by this group divided only to the 6-cell stageand embryogenesis did not reach the blastocyst stage,
which is thought to be required for the establishment of
ES cells. However, when they induced the parthenogenesis of an unfertilized oocyte that had completed thesecond meiosis, using cytochalasin B treatment and a
calcium ionophore, the embryogenesis progressed tothe blastocyst stage. Notably, ES cells were recently
established from non-human primate parthenogenic
blastocysts (termed, ‘‘parthenogenic stem cells’’, PS
cells).89 It is still unclear whether PS cells can be used
as immune-rejection-free donor cells in regenerative
therapy, because the consequences of the homozygosityof recessive mutations in these cells are unknown.
The implications of these recently developed ES cell
technologies in therapeutic applications have only begun to be discussed. It is critical to discuss the ethicalissues associated with studies using human ES cells,

Okano H: Neural stem cellwhich are complex, and attitudes vary among countries.
Use of dedifferentiation/transdifferentiation
Tissue stem cells have been found in various organs,and these stem cells supplement differentiated cells lost
due to physiological turnover or injury. Accordingly,
until a few years ago, it was commonly thought that
endogenous tissue stem cells in each organ only generate cells that are speciﬁc to that organ. However, thisview was challenged by one study in which the differentiation and proliferation of hematopoietic cells were
derived from NSCs.90 Although there has been some
difﬁculty reproducing this particular result,91 since thisreport, other studies on the reprogramming, or ‘‘plasticity,’’ including transdifferentiation and dedifferentiation of mammalian cells have appeared in the literature.
These recent studies include: I) the transdifferentiationof bone-marrow-derived cells into neural cells,92–95 II)
the transdifferentiation between muscle- and bonemarrow-derived cells,96,97 III) the transdifferentiationof bone marrow cells98,99 or puriﬁed hematopoietic
stem cells100 into liver cells; IV) the differentiation of
puriﬁed NSCs injected into a blastocyst into various
cells other than nervous system cells,101 V) the conversion of NSCs to skeletal muscle upon co-culturing,102
VI) the dedifferentiation of oligodendrocyte progenitor
cells into NSCs under certain culture conditions,103
VII) the differentiation of skin cells (dermis) into neurons, glial cells, and fat cells,104 VIII) the differentiation of cord blood cells into neural cells,105 and themultiorgan and multilineage potential of a single bonemarrow-derived stem cell upon engraftment.106
These ﬁndings indicate that the ability of stem cells
to differentiate, which was thought to be tissue-speciﬁc,is unexpectedly ﬂexible. It has been proposed that a few
pluripotential cells like ES cells exist in each organ in amature body and that such cells differentiate into speciﬁc cells under speciﬁc conditions.107 However, somestudies reported in the last few years have been difﬁcult
to reproduce. Unfortunately, the study reporting that
NSC-derived blood was obtained from a mouse that
had received an intravenous injection of NSCs fromanother mouse has not been conﬁrmed by an additional
study. The latest thinking is that the phenomenon thatwas considered to be the plasticity of stem cells is more
likely to result from the fusion of transplanted cells andhost cells.108,109 Previous studies reporting the conversion of transplanted bone marrow cells into unexpected
cell types in vivo92,93,96,98–100,106 based the apparent
transdifferentiation on the presence of a donor-derived
marker (i.e., the Y chromosome when the donor is maleand the recipient is female or the expression of a donorspeciﬁc gene) in cells that had acquired a new phenotype within the recipient animals. However, a recent

Keio J Med 2002; 51 (3): 115–128

report by Terada et al.108 raises the alternative possibility that donor bone marrow cells can fuse withrecipient somatic cells in vivo and that this process
may contribute to some of the apparent ‘‘donor-typed’’cells in various organs, although in this study, whichcharacterized the phenomenon in vitro, the frequencyof the spontaneous cell fusion was very low (2–11
clones per 10 6 bone marrow cells). Careful investigation of this issue using both donor- and recipientderived markers will be necessary to clarify the mechanisms involved.
Although it is difﬁcult to distinguish from reprogramming or plasticity, unexpected pluripotency has
been observed in mesenchymal stem cells (MSC) inbone marrow interstitium, and skin-derived precursors(SKP) in vitro,104 in which the phenotype is not likely
to result from cell fusion. In addition to osteoblasts,
MSCs have been shown to differentiate into hepatocytes, skeletal muscle, cardiac muscle, cartilage, fatcells, and neural cells in culture.94,95,110–112 From SKP,
neurons, astrocytes, oligodendrocytes, fat cells, andsmooth muscle cells were differentiated.104 Althoughmany issues remain to be resolved, such as whether
differentiation can cross germ layers and the difﬁculty in
distinguishing MSC’s diversity from pluripotency, it islikely that we will see the day when MSCs can berecovered by bone marrow puncture at an outpatient
clinic and applied in autografts for the treatment ofvarious organ deﬁciencies.

125

and once efﬁcacy and safety are conﬁrmed, a system bywhich groundbreaking medical trials in a small numberof patients with informed consent can take place, mustbe established.
Acknowledgements: Most of this review article was written during mystay in Harvard Square (Cambridge, USA) during March 2002, where
I presented at the 3rd conference on the prospects for neural transplantation, gene therapy, and progenitor cell biology (organized byDrs. Ole Isacson and Xandra O. Breakeﬁeld of the Departments of
Neurology and Neuroscience, Harvard Medical School/Massachusetts
General Hospital). Here I wish to thank the many excellent participants at this conference, especially Drs. Olle Lindvalle (Wallenberg
Neuroscience Center, Sweden), Curt Freed (University of Colorado
School of Medicine, USA), Clive Svendsen (University of Wisconsin,USA), Masato Nakafuku (University of Tokyo, Japan), Yoshinaga
Saeki (Massachusetts General Hospital, USA), Nobuko Uchida(Stanford University, USA), Jeffery Macklis (Children Hospital,
USA), Marie Filbin (Hunter College, USA), and Paul Reier (University of Florida, USA) for their inspiring discussions. I am also
grateful to all of the members of my laboratory and collaborators allover the world, especially Drs. Masaya Nakamura, Yoshiaki Toyama(Department of Orthopedic Surgery, School of Medicine, Keio University, Japan), Takaki Miyata (RIKEN Brain Science Institute,Japan), Yonehiro Kanemura (Tissue Engineering Research Center,
Japan), Urban Lendahl (Karolinska Medical Institute, Sweden), andSteven Goldman (Cornell University Medical College, USA) for theircreative thinking and discussions, Drs. Masaya Nakamura, Yuto
Ogawa and Yumi Matsuzaki for providing illustrations and unpublished data, Ms. Kumiko Inoue, Yoko Hanno and Akiyo Hirayama
for their assistance in preparing MS, Human Frontier Science Program (HFSP) and the Ministry of Education, Science, Sports, andCulture of Japan and Core Research for Evolutional Science andTechnology (CREST), Japan Science and Technology Corporation(JST) for their past and ongoing supports.

Perspectives for the Future
Regenerative therapies that take advantage of stem
cell technology for CNS damage including neurologicaldiseases hold tremendous hope for the future. Althoughmost studies aimed at the realization of these therapies
are still at the basic stage, researchers worldwide,
driven by the high competition in this ﬁeld, may begin
focusing more on clinical application. Obviously, it isimportant to approach this goal in a step-by-step manner, following animal experiments using progressively
modiﬁed technology with studies in small numbers ofpatients that apply well validated assessment protocols.
Many achievements in basic science have been made inWestern countries, mainly the United States, but theUnited States has not always occupied a leading position, as it did in the genome project. Nonetheless, theUnited States is investing 100 million dollars in ES cellresearch and 200 million dollars in somatic stem cellresearch and development from the federal budget
in the next ﬁnancial year (2002), which will accelerate
the research. In Japan, before medical applications of
the three strategies described above can be realized,
guidelines for stem-cell therapy, quality control systems
to conﬁrm the efﬁcacy and safety of the procedures,

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