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THE ANATOMICAL RECORD (NEW ANAT.) 257:137–148, 1999
FEATURE ARTICLE
Multipotent and Restricted Precursors
in the Central Nervous System
MAHENDRA S. RAO*
Acquisition of cell type-specific properties in the nervous system is likely a process of sequential restriction in
developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and
EGF-dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent
stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the
generation of intermediate lineage-restricted precursors that differ from each other and from multipotent stem cells.
Neuronal precursors termed neuronal-restricted precursors (NRPs), multiple classes of glial precursors termed
glial-restricted precursors (GRPs), oligodendrocyte-type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and
PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted
precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non-fetal source of such cells.
Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the
possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat
Rec (New Anat): 257:137–143, 1999.
KEY WORDS: stem cell; neuronal stem cell; NRP; GRP; oligodendrocytes; astrocytes; differentiation; mouse; rat
Development of the nervous system
involves differentiation, migration to
an appropriate location, projection to
an appropriate target, synaptogenesis,
and acquisition of an appropriate rostrocaudal and dorsoventral identity.
Our knowledge of the sheer number of
cells present in the nervous system
and an increasing awareness of neural
diversity has led to an appreciation of
the magnitude of the problem a developing organism faces in trying to
specify the appropriate neuronal and
glial fates of each precursor cell.
Seminal work by a number of laboratories has led to rapid advances in
our understanding of phenotypic speci-
Dr. Rao obtained his medical degree
from Grant Medical College in India and
his Ph.D. from Caltech. He is currently
an Assistant Professor at the University
of Utah Medical School. His laboratory
focus continues to be neural stem cell
biology.
*Correspondence to: Mahendra S. Rao,
M.D., Ph.D., Department of Neurobiology and Anatomy, University of Utah
Medical School, 50 North Medical Drive,
Salt Lake City, UT 84132. Fax: 801-5814233; e-mail: Mahendra.Rao@hsc.utah.
edu
fication.18,29 Of particular importance
has been our growing appreciation of
the similarities between neurogenesis
and tissuegenesis in the liver, skin, and
bone marrow.29 In each of these systems, development proceeds by a progressive restriction in developmental
potential. In the hematopoietic system, for example, differentiation into
macrophages, granulocytes, erythrocytes, lymphocytes, and platelets occurs by a sequential differentiation of
progressively more committed cells.
Primitive stem cells (PSCs) generate
lymphoid and myeloid (committed)
progenitor cells (see Fig. 1). Committed progenitor cells undergo self-renewal and give rise to a large number
of identical cells by a process termed
clonal amplification. These more restricted cells then undergo differentiation in response to environmental and
non cell autonomous cues to generate
particular subclasses of differentiated
cells (Fig. 1). The validity of this model
of differentiation in hematopoiesis has
been confirmed by in vivo transplant
experiments and identification of cell
surface markers that clearly distinguish between classes of restricted pre-
cursors and their differentiated progeny.
If differentiation in the nervous system is indeed similar to differentiation
in other tissues, one should be able to
identify pluripotent stem cells and restricted stem cells, differentiate between classes of stem cells using markers, establish a lineage relationship
between classes of stem cells, and identify growth factor and differentiation
signals. Considerable progress has
been made on each of these fronts.
This article will focus on results from
our laboratory and cite relevant related literature that demonstrates differentiation does indeed follow a pattern of sequential fate determination.
The types of cells described and their
antigenic differences are summarized
in Table 1.
MULTIPOTENT STEM CELLS ARE
PRESENT IN THE NERVOUS
SYSTEM
Using single cell clonal analysis, retroviral tracing techniques and transplant assays, several groups have unambiguously demonstrated the presence
of multipotent stem cells (reviewed in
138 THE ANATOMICAL RECORD (NEW ANAT.)
FEATURE ARTICLE
ments, to my knowledge, have not
been undertaken in the central nervous system or the peripheral nervous
system. Labeling cells, harvesting a
small population and reisolating and
transplanting cells are still major technical challenges that need to be solved
before one can demonstrate self-renewal in the CNS. Nevertheless, the
overall consensus is that EGF- and
FGF-dependent progenitor cells are
self-renewing stem cells that likely selfrenew in vivo. Demonstrating this unambiguously is likely to be simply a
matter of time.
Many factors that can promote neuron growth, differentiation, and survival have been described. Because
this article’s focus is on stem cell lineages dependent on EGF or FGF, the
reader is directed to reference 18 for a
more complete discussion of other
Figure 1. Hematopoietic stem cell differentiation. A model of how differentiated cells of the
hematopoietic system arise is shown. Note the initially pluripotent cells undergo a progressive
restriction in developmental potential. PSC, primitive stem cell; MSC, myeloid stem cell; LSC,
lymphoid stem cell. Adapted from Pathology, Rubin and Farber, eds.44
Kalyani and Rao18) in the central nervous system (CNS). At least two major
classes of stem cells are present in the
CNS (Fig. 2). One class of cell has been
termed a neurosphere and was first
described by Reynolds et al.42 Neurosphere stem cells isolated from multiple regions of the brain have been
shown to undergo extensive self-renewal and to differentiate into neurons, astrocytes, and oligodendrocytes. Data from multiple laboratories
show that epidermal growth factor
(EGF)-dependent neurosphere cells
are multipotent, self-renewing stem
cells that can generate all major cell
types in the brain. Neurosphere stem
cells are characterized by nestin immunoreactivity, growth in suspension culture, and by the requirement of high
doses of EGF (50 ng/ml) for isolation.
These cells may subsequently be supported by fibroblast growth factor
(FGF) but neurosphere stem cells cannot be isolated using any growth factor other than EGF and transforming
growth factor-alpha (TGF-alpha).52
Another class of stem cells identified
in the nervous system is FGF-dependent cells. FGF-dependent stem cells,
like neurospheres, are self-renewing
and can generate neurons, astrocytes,
and oligodendrocytes (Fig. 3). FGF-
dependent stem cells appear to grow
in both suspension and adherent cultures and have been shown to selfrenew for months16 or years.41 EGF
cannot substitute for FGF during the
process of isolation or maintenance
and both acidic and basic FGF seem
equally capable of maintaining FGFdependent stem cells.
While clearly both EGF- and FGFdependent stem cells can self-renew in
culture and can generate multiple phenotypes of cells under appropriate signals, it should be emphasized that
self-renewal has not yet been demonstrated in vivo. Transplants of stem
cells in vivo have clearly demonstrated
that these cells can differentiate into
neurons, astrocytes, and oligodendrocytes3 but self-renewal has been harder
to demonstrate. In the hematopoietic
system, transplanted stem cells can be
harvested, and stem cells reisolated
and then retransplanted into fresh recipients, and this entire process was
repeated to show that transplanted
stem cells undergo self-renewal in vivo
and that the daughter stem cells generated could repopulate the bone marrow with all hematopoietic phenotypes. Experiments of this sort, a
technical tour de force, clearly demonstrate self-renewal in vivo. Such experi-
While clearly both EGFand FGF-dependent
stem cells can selfrenew in culture and can
generate multiple
phenotypes of cells
under appropriate
signals, self-renewal has
not yet been
demonstrated in vivo.
effector molecules and additional references. Other classes of neuronal precursor cells have only been recently
characterized and the stage-specific
role of effector molecules remains to
be fully defined. FGF and Wnt-1, for
example, seem to play a key role in
maintaining neuroepithelial cell proliferation (e.g., see reference 41). FGF’s
appear to be the only cytokines that
promote mitosis of NEP cells. EGF,
PDGF, NT-3, and a variety of other
cytokines tested do not appear to
stimulate NEP cell division (e.g., see
references 16, 17, 52). Though FGF
promotes NEP cell proliferation, an
additional, undefined factor is required to completely prevent their differentiation.16 Another group of negative regulators, the Id family of genes,
FEATURE ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 139
TABLE 1. Neuronal precursors and neurons express distinct cell-specific antigenic markers
Name
Cell Type
Antigens Present
Antigens Absent
NEP cell
FGF-dependent, multipotent stem cell
Neurosphere
EGF-dependent multipotent stem cell
All markers expressed by
differentiated cells, EGFR,
PDGFR
All markers expressed by
differentiated cells
NCSC
Neural crest stem cell
FGFRs, Brain fatty acid protein, nestin, Brn-1, Musashi
1
EGFR, FGFR1 and 2, Brain
fatty acid protein, nestin,
Brn-1, Musashi 1
Nestin, FGFRs, p75
NRP
Neuron-restricted precursor
nestin, E-NCAM, ␤-III tubulin,
MAP2
Neuron
Differentiated postmitotic
neuronal cell
GRP
Glial-restricted precursor
cell
Late appearing markers
such as Neurofilament-H,
synaptic proteins, neutransmitter synthesizing
enzymes
A2B5, nestin, FGFR 1, 2, and
3, PLP, DM-20
O2A
Oligodendrocyte type-2
astrocyte precursor cell
A2B5, nestin, FGFR 1, 2, 3,
PLP, DM-20, PDGFR-alpha
Oligodendrocyte
Differentiated postmitotic
glial cell
O4, O1, myelination antigens, PLP, DM-20
Astrocyte Precursor Cell
(APC)
Restricted precursor that
generates only one kind
of astrocyte
A2B5, nestin, S-100␤
Type 1 Astrocyte
A differentiated glial cell
Ran 2, GFAP, S-100␤, nestin,
FGFR3
Type 2 Astrocyte
A differentiated glial cell
that has not been identified in vivo
A2B5, GFAP, S-100␤, nestin,
Musashi 1
All markers expressed by
differentiated cells
Astrocyte, oligodendrocyte
and glial precursor cell
markers. No late neuronal
markers
Astrocyte, oligodendrocyte
and glial precursor cell
markers, PCNA
No early or late neuronal
markers. GalC, myelination antigens, S-100␤,
GFAP, Ran-2
No early or late neuronal
markers. GalC, myelination antigens, S-100␤,
GFAP, Ran-2
No early or late neuronal
markers. No astrocytic
markers. Nestin, PCNA
No early or late neuronal
markers. GalC, myelination antigens, S-100␤,
GFAP, Ran-2
A2B5. No early or late neuronal markers. No GalC,
or myelination antigens
Ran2, FGFR3. No early or
late neuronal markers. No
GalC, or myelination antigens
A list of the cell types discussed and their salient characteristics. The markers listed are by no means exhaustive but are the
selection of published markers that appear to distinguish between different cell types. Note that different groups have reported
the expression of E-NCAM on NEP-like cells, GRP cells and astrocytes at specific stages of development. Note likewise A2B5
expression has been reported on subsets of neurons. The reader is referred in the text to specialized reviews for a detailed
discussion on the expression profile of these markers.
Abbreviations: FGFR, fibroblast growth factor receptor; EGFR, epidermal growth factor receptor; PDGFR, platelet growth
factor receptor; Brn-1, POU domain protein; nestin, intermediate filament protein; Musashi 1, RNA binding protein; MAP2,
microtubule associated protein 2; E-NCAM, polysialated neural cell adhesion molecule; ␤-III tubulin, a neuron-specific tubulin;
PCNA, proliferation specific nuclear antigen; A2B5, antibody that recognizes a specific non protein epitope; Ran2, an
epitope specific to type 2 astrocytes; GFAP, glial fibrillary acid protein, an astrocyte specific marker; S-100␤, an epitope
expressed predominantly by astrocytes; PLP, proteolipid protein; DM-20, an alternate transcription product of the PLP gene.
is expressed in the spinal cord and
may also play an important role in
inhibiting stem cell differntiation. Positive differentiation factors have also
been described, such as the BMPs.
Neuroepithelial stem cell differentiation into neural crest stem cells
(NCSCs) is promoted by BMPs 2 and
4; since defferentiation does not require cell division, BMPs likely play an
instructive role in the differentiation
process. Determinants have yet to be
found that bias NEP cell differentiation toward neuroblasts and glioblasts. Homologs of notch and numb
may impinge on the decision by a stem
cell to generate either a daughter stem
cell or a differentiated progenitor.
However, no single molecule that ‘‘instructs’’ stem cells to become neuroblasts or glioblasts has yet been found.
Factors regulating postmitiotic neuron survival have been identified. NT-3,
LIF, and CNTF each may have tropic
140 THE ANATOMICAL RECORD (NEW ANAT.)
Figure 2. Possible lineage relationships between EGF- and FGF-dependent stem cells. The
possible lineage relationship between EGF-dependent neurosphere stem cells and FGFdependent stem cells is shown. FGF-dependent stem cells arise earlier in development and
may generate EGF-dependent neurosphere cells. Alternatively both classes of stem cells may
arise from a common precursor whose properties remain to be determined. ?? indicates that
no specific growth factor or cytokine has been identified as regulating this stage of differentiation. See Table 1 for definitions of terms. The reader is referred to Kalyani and Rao17 for a more
detailed discussion on possible candidate molecules.
effects (see 18 and references therein).
In addition, motoneurons—the earliest developing neurons—have a unique
set of factors in addition to FGF that
affect their survival. The key takehome message is that the effect of a
particular cytokine(s) is stage specific,
and when used in combination, they
may have distinct effects. For example,
sonic hedgehog (Shh) ventralizes neural tube cells, promoting both motoneuron and oligodendrocyte differentiation, but may act on other cell types
to promote mitosis.
WHEN AND WHERE ARE
EGF-DEPENDENT NEUROSPHERE
STEM CELLS AND
FGF-DEPENDENT STEM CELLS
PRESENT?
If stem cells play a role in normal
development, these cells should be present prior to the onset of neurogenesis
and gliogenesis. In the spinal cord of
rats, neurogenesis begins at or around
embryonic day (E) 12.5–13.0 and proceeds rostrally (headwards) and caudally (tailwards), with most neurogenesis being completed by E17. Select
regions of the brain show neurogenesis at later stages. Cerebellar granule
cell are born perinatally and neurogenesis is completed during the first two
weeks of postnatal life.13 Olfactory bulb
and hippocampal neurogenesis appear to continue throughout life with
a peak in the first postnatal week. The
presence of precursor cells has been
correlated with these developmental
stages and the ability of stem cells to
contribute to ongoing neurogenesis
has been determined. FGF-dependent
stem cells have been shown to be
present prior to the onset of neurogenesis. In rat spinal cord as well as in the
cortex, multipotent stem cells are present as early as embryonic day E10.516
(our unpublished results). We and others51 have shown that FGF-dependent
FEATURE ARTICLE
stem cells are present at equivalent
stages in embryonic mouse neural
tubes. Indeed, we have noted16 that at
early stages the neural tube is a homogenous population of FGF-dependent,
pluripotent stem cells and that these
FGF-dependent stem cells do not respond to EGF and do not express
detectable levels of EGF receptor.19
Both groups report that no EGFdependent neurosphere stem cells
could be detected at this stage, making
it likely that only FGF-dependent stem
cells contribute to neurogenesis at this
early developmental stage.
The absence of EGF-dependent stem
cells at these early stages of development is also supported by other, independent experiments. Weiss et al.52
have noted that EGF-dependent neurosphere cells cannot be isolated earlier
than E14.5, a time period following
the onset of neurogenesis. At earlier
developmental stages, dividing cells
[as identified by bromodeoxyuridine
(BRdU) incorporation] are present in
the ventricular zone1,11,34 and neurons
are being born. Early-born neurons
are therefore likely to arise from non
EGF-dependent stem cells. These observation are consistent with results
seen in transgenic animals, where
EGF-R expression was abolished.49 In
these animals, the ventricular zone
develops normally and no proliferative or stem cell abnormalities were
reported. Further, early neurogenesis
is unaltered though neuronal heterotopias were observed, suggesting that
while stem cell proliferation may not
be affected, EGF may be important in
regulating neuronal migration.49 Similar conclusions can be drawn from the
EGF receptor overexpression studies,
where increasing the EGF response
biases differentiation towards the astrocytic fate. Overall, the data suggest
that stem cells requiring EGF for proliferation are not present at early developmental stages.
Isolation of stem cells from E14.5
onwards suggests that both EGF- and
FGF-dependent stem cells are present
at appropriate locations and may contribute to neurogenesis at later stages
of development. Transplanting both
stem cell types has provided evidence
that stem cells can contribute to ongoing neurogenesis. The question of
whether endogenous, neurosphere
stem cells or FGF-dependent stem cells
FEATURE ARTICLE
contribute to ongoing neurogenesis is
still under investigation. An interesting set of experiments were performed
to address this question. Endogenous
stem cell proliferation was enhanced
by infusion of either FGF or EGF and
differentiation of newly born cells was
assessed by BRdU pulse labeling and
staining for the appearance of neuron
specific markers. The results suggest
that FGF-dependent stem cells may
contribute significantly more to neurogenesis. Both EGF and FGF infusion
causes expansion of the ventricular
zone and an increase in the number of
BRdU-incorporating (dividing) cells.7,20
EGF stimulates cell division to a larger
extent but the percentage of neuronal
cells that differentiate is quite small7
or undetectable.20 Cells stimulated to
divide by EGF generate predominantly
glial cells while FGF-treated cells generated both neurons and glia.20 Other
laboratories have like wise shown that
THE ANATOMICAL RECORD (NEW ANAT.) 141
cultured EGF-dependent stem cells
generate predominantly astrocytes
while FGF-dependent stem cells generate both neurons and astrocytes. Taken
together, these results suggest that
EGF-dependent stem cells are present
at late developmental stages and do
not contribute to early neurogenesis.
At later developmental ages both
classes of stem cells can contribute to
neurogenesis though FGF-dependent
stem cells appear to generate more
neurons than EGF-dependent stem
cells.
The question of why two types of
multipotent stem cells coexist in the
CNS remains unresolved. It should be
noted, however, that even in the hematopoietic system, primitive and defined multipotent stem cells have been
identified. In this system, the role of
multiple classes of stem cells also remains to be defined. Another issue
that remains unresolved is the question of localizing stem cells at different stages of development. At early
stages, it is clear that stem cells are
present in the ventricular zone and
persist in the subventricular zone15
(and below). At later stages, stem-cell
isolation experiments from whole cortex, temporal lobe and other brain
Figure 3. Multipotent FGF-dependent neural
stem cells can differentiate into CNS and PNS
derivatives. NEP cells were harvested and
grown in clonal culture (A) or in mass (B,C) for
7–15 days. NEP clones were processed for
immunocytochemistry (A) or immunopanned
to isolate A2B5-immunoreactive glial precursor (B) or P75-immunoreactive NCSC (C) cells.
The immunopanned cells were grown in
clonal culture and processed for immunocytochemistry after seven additional days in
culture. A:Neuroepithelial stem cell (NEP)derived clone that has been triple labeled for
GFAP (blue; an astrocyte marker), ␤-III tubulin
(green; a neuronal marker), and GalC (red;
an oligodendrocyte marker). Note that a
single precursor type can generate neurons,
astrocytes and oligodendrocytes. B:DAPI
(blue; nuclear stain) and GalC (red; an oligodendrocyte marker) staining of a glial-restricted precursor (GRP) clone. Note an NEPderived GRP cell can differentiate into
oligodendrocytes. C:NEP-derived neural crest
stem cell (NCSC) clone that has differentiated into ␤-III Tubulin-immunoreactive neurons (Green) and smooth muscle actin-immunoreactive (Red) smooth muscle cells. These
results indicate that NEPs can generate both
central (CNS) and peripheral nervous system
(PNS) derivatives and that differentiation proceeds via the generation of more restricted
precursors.
The question of why two
types of multipotent stem
cells coexist in the CNS
remains unresolved.
regions suggest that dividing cells are
present outside the ventricular zone as
well. For example, in a recent manuscript, Kessler and his colleagues26
showed that polysialated NCAM⫹
(PSA-NCAM) multipotent precursors
were present in the adult cortex. Since
PSA-NCAM expression is absent from
the subventricular zone, this would
suggest that these cells are present
outside the ventricular zone. BRdU
incorporation experiments have also
localized dividing cells in the cortex,
providing independent supportive evidence.
More recently, Johansson and colleagues15 have suggested that stem
cells are indeed present in the adult
brain and that they are localized to the
ependymal layer lining the ventricular
system. In a series of elegant experiments, the authors showed that ependymal cells are a slowly dividing stem
cell population that generate a more
rapidly dividing ‘‘transit cell’’ that is
localized to the subventricular zone.
The rapidly dividing cell can subsequently generate neurons and astrocytes. It is unclear from their experiments whether the EGF-dependent
neurosphere or the FGF-dependent
stem cell represented the transit cell
population or the ependymal stem cell
population. These results are consistent with earlier work30 and are exciting, since identifying and localizing
stem cells to a specific location in vivo
provides an unprecedented opportunity to examine the response of stem
cells to in vivo manipulations.
ARE EGF-DEPENDENT
NEUROSPHERE STEM CELLS AND
FGF-DEPENDENT STEM CELLS
LINEALLY RELATED MULTIPOTENT
CELLS?
The possible lineage relationships between EGF- and FGF-dependent stem
cells are summarized in Figure 2. Important characteristics of FGF-depen-
142 THE ANATOMICAL RECORD (NEW ANAT.)
dent neuroepithelial (NEP) stem cells
appear to be their ability to grow in
adherent cultures in vitro and their
absolute requirement of FGF for survival. Neither EGF nor any other cytokine tested can substitute for
FGF,15,19,52 whether cells are grown in
adherent or in suspension (neurosphere-like) cultures. Examination of
epidermal growth factor receptor
(EGF-R) expression in NEP cells provides an explanation for their failure
to respond to EGF. EGF-R expression
is not seen on FGF-dependent NEP
cells by either PCR or by immunocytochemistry, both in vitro or in vivo.16,19
In addition to the difference in growth
factor dependence, another important
difference appears to be the frequency
of neuron generation. NEP stem-cells,
as well as other FGF-dependent stem
cells, appear to generate neurons at
high frequency, whereas EGF-dependent neurospheres have a very low
frequency of neuron generation and
appear to lose the ability to generate
neurons after multiple passages. Based
on these differences, we have argued
that EGF-dependent neurospheres and
FGF-dependent stem cells represent
two distinct classes of stem cells.
Apart from their phenotypic and
other differences, perhaps the most
compelling supportive arguments are
the reports of Santa-Ollala and Covarrubias45 and Tropepe et al.51 Both
groups showed that both FGF- and of
EGF-dependent stem cells co-exist in
similar brain regions at later stages of
development. Using population and
statistical analyses, both groups argued that these represent two distinct
populations of cells. The coexistence
of these two populations suggests that
the FGF-dependent stem cell does not
necessarily transform into an EGFdependent stem cell; rather, both populations coexist.
In a technically difficult set of experiments, Tropepe and colleagues51 constructed chimeras from fibroblast
growth factor receptor 1-null (FGFR1⫺)
and normal animals and showed that
EGF-dependent stem cells must go
through a FGF-dependent stage. These
data argue that FGF-dependent cells
are the precursors of the EGF-dependent neurosphere stem cells. These
results are compelling but do not ad
dress the stage at which the two lineages diverge. Some groups have suggested that FGF-dependent neurospheres will generate EGF-dependent
neurospheres arguing that FGF-dependent cells are indeed the precursor of
the EGF-dependent stem cell. Why
these two populations co-exist and
what regulates the transition from one
multipotent stem cell to another, however, remains unknown.
MULTIPLE CLASSES OF GLIAL
PRECURSORS EXIST
Oligodendrocyte-type 2 astrocyte (O2A)
cells, which represent one of the best
defined glial precursors of the CNS,
were initially isolated from the postnatal rat optic nerve, and subsequently
from the postnatal cortex and spinal
cord. O2A cells have a default pathway
of differentiation into oligodendrocytes and this differentiation can be
modulated by growth factors.6 In culture, O2A cells can also differentiate
into type 2 astrocytes. Type 2 astrocytes differ from the more common
type 1 astrocyte in their expression of
A2B5 immunoreactivity and the absence of Ran 2 immunoreactivity40
(see Table 1). O2A cells will not differentiate into neurons under any culture
condition and upon transplantation8
will differentiate into myelinating oligodendrocytes. O2A cells thus represent glial-restricted precursor cells that
can generate a subset of the glial population present in the adult brain.
ASTROCYTE-RESTRICTED
PRECURSORS
Another class of precursor cells restricted to glial differentiation are astrocyte restricted precursors (APCs).
Seidman et al.46 have described astrocyte restricted precursor cells isolated
from the E16 mouse cerebellum that
do not express glial fibrillary acid protein (GFAP), an astrocyte marker, and
are EGF dependent. Upon differentiation, the cells begin to express high
levels of GFAP but do not differentiate
into oligodendrocytes. APCs are not
A2B5 immunoreactive and the astrocytes that differentiate appear to be
type 1 astrocytes. Mi and Barres28
FEATURE ARTICLE
have independently provided unambiguous evidence for an astrocyte precursor cell or APC. The authors show
that this cell expressed A2B5 immunoreactivity and expresses some, but not
all, astrocytic markers. This cell can
differentiate into astrocytes under appropriate culture conditions. The authors argue that since these APC cells
do not default to an oligodendrocyte
pathway of differentiation and but
rather differentiate into type 1 and not
type 2 astrocytes, they are clearly distinct from O2A cells. The authors did
not, however, show that this cell could
not make oligodendrocytes under conditions in which the O2A precursor
cells do, raising the possibility that
this might be a distinct type of oligodendrocyte-astrocyte precursor cell,
rather than solely an astrocyte-restricted precursor cell.
Examining oligodendrocyte differentiation from spinal cord, Richardson
and colleagues38 noted that while oligodendrocyte differentiation occurred in
the ventral cord and required plateletderived growth factor receptor (PDGFR)alpha expression, astrocytes can be
generated from dorsal spinal cord from
cells that do not express the PDGFRalpha. Their results suggested the existence of an astrocyte precursor that is
present in the dorsal spinal cord. The
authors also noted that at E13.5 A2B5
immunoreactive/PDGFR-alpha⫺ cells
were present in the dorsal spinal cord,
raising the possibility that the spinal
cord astrocyte precursor may be A2B5
immunoreactive, as compared to APCs
from the brain.
GRP CELLS ARE
GLIAL-RESTRICTED PRECURSOR
CELLS THAT CAN DIFFERENTIATE
INTO OLIGODENDROCYTES AND
TYPE 1 ASTROCYTES
While glial restricted precursors
(GRPs) clearly exist, almost all data
suggest that these are late-glial precursors. The questions that remained unanswered were: did an embryonic glialrestricted precursor exists prior to the
first generation of oligodendrocytes
and astrocytes and did this precursor
arise from a multipotent stem cell. In a
series of in vitro experiments, we39,40
identified and characterized a glial-
FEATURE ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 143
matter of time. Indeed, preliminary
results from our laboratory suggest
that GRP cells generate astrocyte restricted precursor cells that differentiate solely into type 1 astrocytes.
NEURON-RESTRICTED
PRECURSORS ARE PRESENT IN
MULTIPLE REGIONS OF THE BRAIN
Figure 4. Hypothetical relationship between glial restricted precursor cells. A model of how
NEP cells, GRPs, O2A cells, and astrocyte precursor cells are related is shown. NEP cells may
generate GRPs that subsequently differentiate into an even more restricted glial precursors the
O2A cell and APC cells. These cells can be distinguished from each other on the basis of their
antigenic properties. Note: Direct lineage relationships between GRPs, O2As, and APCs still
need to be established. See Table 1 for definitions of terms.
restricted precursor (GRP) that is present in the developing spinal cord. GRPs
can be identified as early as E12 by
their A2B5 and nestin immunoreactivity. Dividing glial precursors with similar properties can be identified as late
as postnatal day 2. GRP cells lack
PDGFR-alpha immunoreactivity (at
least initially) and synthesize detectable levels of PLP/DM-2050 (see Table
1). GRPs arise ventrally from a restricted region of the proliferating neuroepithelium, although the potential
to generate GRPs appears much more
widespread.5,12,16 GRP cells differ from
a previously characterized, later-appearing O2A precursor.40 An important difference is the absence of
PDGF-R alpha expression on GRP
cells. Perhaps the most significant difference, however, is the ability of GRP
cells to generate two kinds of astrocytes, while O2A cells generate only
one kind of astrocyte. This observa-
tion is important as it provides a possible lineage relationship between oligodendrocytes and type 1 astrocytes
(Fig. 4).
GRPs can generate type-1 astrocytes, type-2 astrocytes, and oligodendrocytes, while O2A cells and APCs
generate subsets of these populations.
Further, all three classes of glial precursors are present in the developing spinal cord at different stages of development. It is therefore tempting to
suggest a lineage relationship between
these precursor cell types. A hypothetical relationship is schematized in Figure 3. While such a relationship is
intellectually appealing, no direct evidence exists suggesting that this is
indeed the case. However, clear enunciation of the similarities and differences between the cell types and the
identification of cell surface markers
suggests that determining whether a
lineage relationship exists is simply a
In addition to neural crest stem cells
(NCSCs) and GRPs, precursors limited in their differentiation potential
to neurons have also been reported to
exist in the developing brain. MayerProschel et al.27 were able to isolate a
neuronal restricted precursor from the
developing spinal cord. The authors
showed that this neuronal restricted
precursor (NRP) expresses E-NCAM
(high polysialic-acid NCAM) and is
morphologically distinct from neuroepithelial (NEP) cells,16 NCSCs,32 and
spinal GRPs.40 The authors showed
that E-NCAM-immunoreactive neuronal precursors can be maintained as
undifferentiated precursors for over 3
months and can differentiate into multiple neuronal phenotypes but cannot
differentiate into oligodendrocytes or
astrocytes. The authors used clonal
analysis to show that individual neuroblasts could generate excitatory, inhibitory, and cholinergic neurons that synthesized multiple neurotransmitter
receptors and that they establish synapses in culture. These results were
complemented by in vivo retroviral
labeling experiments which confirm
that neuron-restricted clones are present in the chick spinal cord and that
these clones may contain multiple
kinds of neurons.
NRP-like cells have also been identified from other cortical regions. Several laboratories have shown that neuronal precursors exist in the hippocampus, subventricular zone and
developing cortex.25,41 Other laboratories have used retroviral labeling techniques to show that restricted neuronal precursors are present in the
developing spinal cord. Levison21 has
shown by retroviral injections that neuron-only precursors are present in the
subventricular zone. Luskin and her
colleagues25 have combined retroviral
labeling and immunocytochemistry to
show that migrating olfactory neuroblasts are E-NCAM-immunoreactive,
dividing neuroblast cells. The external
144 THE ANATOMICAL RECORD (NEW ANAT.)
Figure 5. Relationships between NEP stem cells and intermediate precursors. The relationship
between NEP cells and three intermediate precursors is shown. Neural crest stem cells (NCSCs),
glial restricted precursors (GRPs), and neuronal restricted precursors (NRPs) have all been
shown to differentiate from NEP cells and to be restricted in their developmental potential.
NCSCs, GRPs, and NRPs are self-renewing multipotential stem cells that can be distinguished
from each other and from NEP cells by their differentiation potential, cell surface markers, gene
expression, and cytokine response. Note that other possible restricted precursors could exist
and additional stages in the process of differentiation may be required. See Table 1 for
definitions of terms.
granule cell layer of the cerebellum
may also contain neuron-restricted
precursor cells.13 Thus, NRPs that generate more differentiated progeny are
likely a general feature of CNS differentiation.
Whether these neuronal precursor
cells identified from different brain
regions are related to each other, however, remains to be determined. It
would be tempting to assume that
these cells represent a common neuronal precursor, harvested from multiple
sources. Indeed, in vitro assays suggest that spinal cord neuroblasts, for
example, can generate neuronal phenotypes that are normally not present
in the spinal cord.17 Other data, however, indicate that while neuroblasts
may be morphologically similar, a bias
in the differentiation potential may
already have occurred. Progenitors
from the hippocampus, but not from
the cerebellum or midbrain, produced
hippocampal pyramidal neurons. A recent report24 showed that under appropriate conditions as many as 50% of
the neurofilament immunoreactive
neurons that differentiated from midbrain precursors appeared dopaminergic. This frequency was much higher
than that obtained from any other
neuron precursor cell population.
Transplant experiments that directly
assess the differentiation potential of
regionally distinct populations of ENCAM cells in side by side compari-
sons may reveal similarities and differences.
DIRECT LINEAGE RELATIONSHIP
BETWEEN THE MULTIPOTENT STEM
CELL AND RESTRICTED
PRECURSORS
Retroviral lineage tracing has suggested that at early developmental
stages, multipotent stem cells are present while, at later stages, colonies are
phenotypically more restricted.21 Using cultures of acutely dissociated cells
from different embryonic ages we have
shown16 that most of the rat neuroepithelium at E10.5 comprises multipotent stem cells, while a short time
later,27 more restricted precursor cells
are present. These data suggest that
differentiated cells must be derived
from an initially pluripotent stem cell
population.
How this process of differentiation
occurs is only now being clarified. We
have been able to demonstrate a direct
lineage relationship between FGFdependent stem cells and spinal cord
neuron and glial-restricted precursor
cells. We showed that NEP stem cells
can be induced to differentiate into
E-NCAM⫹ and A2B5⫹ cells by replating and reducing FGF concentration.27,39,40 In this condition, approximately 50% of the cells will differentiate
into A2B5-immunoreactive cells and a
FEATURE ARTICLE
smaller percentage will differentiate
into E-NCAM-immunoreactive cells.
These two distinct populations of cells
are morphologically and antigenically
similar to NRPs and GRPs directly
isolated from more mature neural
tubes and, like E13.5 derived A2B5⫹ or
E-NCAM⫹ cells, are glial-restricted or
neuron-restricted precursors. Equally
importantly, we were able to show by
complement-mediated lysis experiments that NEP cell differentiation
into postmitotic neurons and oligodendrocytes likely requires an obligate
transition through a restricted-precursor cell stage. We were thus able to
show a direct lineage relationship between multipotent NEP stem cells and
more restricted neuronal- and
glial-precursor cells present in vivo at
E13.5.
This finding demonstration of a transition from an NEP cell to an NRP cell
was the first evidence that restricted
neuronal precursors are an intermediate stage between pluripotent stem
cells and fully differentiated postmitotic neurons (summarized in Fig. 5).
Similar lineage relationships between
other classes of potential restricted
precursors (neuron-astrocyte or neurooligodendrocyte) have not been described and it is not clear whether
EGF-dependent neurosphere stem
cells generate neurons, astrocytes, and
oligodendrocytes via a similar mechanism of progressive cell fate restriction.
FGF-DEPENDENT STEM CELLS
REPRESENT A COMMON
CNS-PNS PRECURSOR
The neural crest contains a multipotent cell termed a neural crest stem
cell (NCSC), which can generate craniofacial mesoderm, melanocytes, and
the neurons and glia of the PNS.2
Several laboratories have analyzed the
properties of NCSCs and have shown
that these cells undergo self-renewal,
can be maintained in cultures, and
that individual cells can generate neurons, Schwann cells, and other PNS
derivatives. NCSCs do not appear to
generate CNS derivatives and transplanting NCSCs into the CNS results
in Schwann cell differentiation (unpublished results, Bronner-Fraser, personal communication). Thus NCSCs
likely represent a restricted stem cell.2
FEATURE ARTICLE
A variety of evidence from chick embryo experiments at early developmental stages further suggest that NCSCs
and differentiated CNS cells share a
common progenitor. Perhaps the most
direct evidence is from neural fold
ablations, which demonstrate that cells
of the remaining neural tube have the
regulative capacity to compensate for
the ablated neural crest cells (reviewed in Kalyani and Rao18). These
results suggest that precursor cells normally destined to form the CNS possess the ability to regulate their prospective fates to form PNS derivatives.
Recent work from two different laboratories has now shown that FGFdependent, pluripotent CNS stem cells
likely represent such a common CNSPNS precursor. Mujtaba et al.31 have
shown that NEP cells can generate
p75/nestin (low affinity neurotrophin
receptor/nestin)-immunoreactive cells
that are morphologically and antigenically similar to previously characterized NCSCs.47 NEP-derived, p75-immunoreactive cells differentiate into
peripheral neurons, smooth muscle,
and Schwann cells in both mass and
clonal culture. More importantly, the
authors31 showed by clonal analysis
that individual NEP cells can generate
both CNS and PNS derivatives providing evidence for the first time of a
direct lineage relationship between
these two distinct cell types.
More recently, McMay and colleagues have shown that cortical stem
cells that were isolated at a stage well
after neural crest migration has taken
place still retain the ability to differentiate into PNS derivatives.14 These results demonstrate that FGF-dependent stem cells are less restricted in
their developmental potential than was
previously believed; they have the capacity to differentiate into cells of
both the CNS and PNS, and that PNS
differentiation involves a transition
from an NEP stem to another more
limited, p75-immunoreactive, neural
crest stem cell. No data on the ability
of the EGF-dependent neurosphere cell
to generate crest or PNS derivatives
are available. Our initial attempts to
generate smooth muscle and Schwann
cells from neurospheres were unsuccessful (unpublished results). If these
results are independently confirmed,
then this may represent an additional
distinction between EGF- and FGF-
THE ANATOMICAL RECORD (NEW ANAT.) 145
dependent cells and may provide further evidence that FGF-dependent cells
represent a distinct, more pluripotent
population of stem cells.
HUMAN NEURAL DEVELOPMENT
MIRRORS THAT IN MOUSE AND
RAT
CNS differentiation in humans is similar to that described for the rat and
mouse. Analysis of human fetal development shows that initially the neural
tube is a homogenous population of
dividing, nestin-immunoreactive cells.
Proliferation is restricted to the ventricular zone and neurogenesis occurs
first, followed by differentiation of oligodendrocytes and astrocytes. Neurons and glial cells express antigens
similar to those identified in mouse
and rat and differentiate in similar
spatiotemporal locations. More recently, direct evidence has been provided for the existence of multiple
classes of stem cells from human tissue.
Culture of human fetal tissue has
shown that multipotent stem cells exist in the human CNS. These cells are
nestin immunoreactive, self-renewing
cells that can generate neurons, astrocytes, and oligodendrocytes. Pluripotent stem cells with the characteristics
of neurospheres as well as FGF-dependent stem cells have been described4
(Melissa Carpenter, Stem Cell Inc.,
Ronghao Li., Signal Pharmaceuticals,
personal communication). While some
differences exist, the overall growth
properties and cytokine responses of
human and rat stem cells appear similar. Xenotransplant experiments in
which human stem cells were transplanted into rats showed that these
cells can integrate and differentiate
into multiple types of cells in vivo.3,10
In addition to multipotent stem cells,
E-NCAM-immunoreactive neuroblasts
have been identified. We have shown
that human fetal spinal cord cultures
contain dividing neuron-restricted precursor cells. More recently, Li and
colleagues22 have reported the generation of an immortalized, human neuron-restricted precursor cell line that
is limited in its differentiation potential to multiple kinds of neurons. This
cell line, termed HSP-1 (human spinal
precursor cell) cannot differentiate
into astrocytes or oligodendrocytes un-
der conditions where other cells readily
differentiate into astrocytes. Morphologically and phenotypically the HSP-1
cell line appears similar to the rat and
mouse NRP cell, suggesting that they
have immortalized a human NRP cell.
Glial precursors have also been identified in human tissue. Rivkin et al.43
identified A2B5⫹ cells that have a typical bipolar morphology characteristic
of GRPs and O2A cells. The authors
showed that in longer-term cultures,
these cells could generate oligodendrocytes and that cells with similar characteristics exist in the human fetal
brain. Similarly we have shown (unpublished results) that A2B5⫹ cells
present in fetal spinal cord cultures
express glial but not neuronal markers. Likewise Murray et al.33 have
shown that oligodendrocyte precursor
cells expressing the markers DM-20
and O4 were present in the human
CNS (see Table 1). While additional
experiments are clearly required to
characterize glial precursors more fully
and to determine whether this cell is a
O2A precursor or a GRP cell, the available data do lead to the conclusion
that glial-restricted precursors exist in
the developing human brain and that
these cells likely express A2B5 and
DM-20. Thus, the overall evidence suggests that human neural development
involves multipotent stem cells generating more differentiated progeny via
the generation of an intermediate,
more-restricted, precursor cell. While
direct lineage relationships between
multipotent and lineage restricted human stem cells have not been established, it is likely that these exist and are
similar to the lineage relationships established in rodent stem cell differentiation.
ES CELLS MAY SERVE AS A
SOURCE OF TISSUE-SPECIFIC
CELLS
Embryonic stem (ES) cells represent
the earliest totipotent cells and are
present at least until the late blastocyst stage. ES cells in vivo likely generate ectodermal, endodermal, and mesodermal precursor cells which
generate progressively more tissue specific derivatives33,48 as fetal development proceeds (Fig. 6). ES cells, in
principle, can generate every cell type
in the embryo. Indeed, in chimeric mouse
experiments, ES cells can generate an
146 THE ANATOMICAL RECORD (NEW ANAT.)
FEATURE ARTICLE
ment.32 These results (summarized in
Fig. 6) suggest that ES cells can be used
as a source of early pluripotent and late,
more-restricted precursor cells.
The importance of these results is
twofold. First, it may obviate the need
for fetal tissue. ES cells appear to be
spontaneously immortal and have been
passaged as undifferentiated cells for
many years. Primate and human ES
cell lines have been recently isolated35,37 and these cell lines can be
used instead of harvesting cells from
the fetus. Second, multiple ES cell
lines can be generated that can be immunologically matched to the recipient, obviating problems arising from mismatch
of ES-derived differentiated cell populations. While moral and ethical issues
with regard to the acquisition and use of
human ES cells need to be resolved, it
now appears that a source of multiple
kinds of precursor cells is assured.
THERAPEUTIC IMPLICATIONS
Figure 6. ES cells are a source of early and late neural progenitors. ES cells can generate
neurons, astrocytes, and oligodendrocytes. Recent evidence summarized in this figure suggests that differentiation involves the generation of neural specific and lineage-specific stem
cells. ?? indicate that these cell types have not been shown to derived from ES cell cultures.
See Table 1 for definitions of terms.
entire animal (including germ line cells).
ES cell lines that are spontaneously immortal were isolated from mice in the
early 1980s and have more recently been
isolated from several species.
ES cell lines have been shown to
recapitulate normal nervous system
differentiation in vitro and to generate
postmitotic neurons and glia that appear phenotypically normal and integrate and function normally after
transplantation.9,35,48 Equally important, the sequential development of
totipotent ES cells into specialized
CNS derivatives appears to involve
many of the same cytokines and transcription factors identified as being
important in normal development.
These observations raise the possibility of isolating pluripotent stem cells,
NRPs, and GRPs from ES cells maintained in culture, rather than from
developing embryos.
McKay and colleagues36 showed that
ES cell derived neural stem cells could
be isolated and maintained in culture.
These passaged, nestin-immunoreactive cells could subsequently be induced to generate multiple classes of
neurons. Li and colleagues23 used an
elegant transgenic approach to select
for ES-derived neural stem cells. Using a Sox-2/ßetageo gene system to
drive expression of betagalactosidase
and neomycin resistance in neural precursor cells, the authors selected differentiated neomycin-resistant precursor
cells and showed that the selected cells
made predominantly neurons (and occasional astrocytes). These results
raised the possibility of isolating late
neural progenitors such as NRPs and
GRPs from ES cell cultures. To directly test this possibility, we used
E-NCAM and A2B5 antibodies to immunoselect cells from differentiating,
ES-cell cultures. ES-cell derived ENCAM⫹ and A2B5⫹ cells appeared phenotypically and antigenically similar
to the neuron restricted and glial restricted cells that we had harvested at
later stages of embryonic develop-
Stem cell therapy is already well established for hematopoietic disorders, cancer treatment, and in immune disorders.
No such therapy is routinely available for
CNS disorders. However, recent advances
in identifying sources of stem cells, isolating subclasses of stem cells, maintaining
cells in culture, and the more recent
demonstration that ES cells can be used
as a source of stem cells have all provided a major impetus to the use of stem
cells for therapy in the CNS.
Several clinical trials and tests in
animal models have begun to provide
important insights into optimum cells
for transplants, methods of injection,
and criteria for assessment. What has
become clear is that no single cell type
will be universally used. The choice of
cell type, be it stem cell, neuron- or
glial-restricted precursor, will be dictated by the functional replacement
that is desired. Neuron-restricted precursors will likely be favored in diseases such as Parkinson’s, in which
specific subsets of neurons are lost,
(GRP) cells, because of their ability to
migrate over large distances in intact
brain tissue, may be the optimum cells
for delivering drugs or genes, in ganglioside disorders such as Tay-Sachs
and Sandhoff disease for example,
where replacement of a single gene
(hexosaminidase A or sialidase, respectively) is sufficient to ameliorate symptoms. ES cells and stem cells may be
FEATURE ARTICLE
used as sources of more differentiated
cells or may be used in transplants in
situations where multiple cell types
have been lost, such as in strokes or
traumatic injury to the brain. An important recent result that will likely
broaden the number of disease targets
is the demonstration that environmentspecific differentiation, neurite outgrowth, and appropriate connectivity
may be possible in the adult damaged
brain (reviewed in Kalyani and Rao18).
As our ability to further define different neural lineages is enhanced, so
will be our ability to identify and understand the different tumors derived
from these cells. Determining the relationship between NEP or NRP cells
and primitive neuroectodermal tumors such as medulloblastomas or
between GRP cells or other glial progenitor cells and different gliomas will
begin to establish a tumor classification that may in time emulate the
These exciting findings
offer the hope that stem
cell therapy will soon be
feasible for a variety of
human diseases of the
nervous system.
sophisticated tumor classification system that exists for cancers of the hematopoietic system. As the cells of origin
for various CNS tumors are identified,
markers characteristic of each tumor
will become available for diagnostics
and specific gene targets will be identified for therapeutic intervention.
SUMMARY
Stem cell therapy has long held out the
promise of totally replacing damaged
and defective tissue. This prospect,
initially farfetched, now appears much
closer to fruition because of several
new findings: 1) recent advances in the
isolation and culture of multiple classes
of stem cells, 2) the demonstration that
human nervous system development follows the same principles of progressive
fate restriction previously described in
animal models, and 3) the finding that
ES cells can generate early and late neu-
THE ANATOMICAL RECORD (NEW ANAT.) 147
ral precursors. These exciting findings
offer the hope that stem cell therapy will
soon be feasible for a variety of human
diseases of the nervous system.
ACKNOWLEDGMENTS
I thank the AAA for sponsoring the
C.J. Herrick award and for inviting me
to write this article, describing the
work that led to this award. I thank Dr.
T.N. Parks, Dr. M. Condic, Jeff Lee,
and Dr. A. Greig for their comments
on this manuscript and gratefully acknowledge the input members of our
laboratory provided through discussions and constructive criticisms. Due
to editorial constraints only a sampling of the rich literature in this field
could be cited. Thanks are due those
contributors to the field who were not
mentioned.
This work was supported by an NIH
FIRST award, an MDA award, and a
March of Dimes research grant. Some
of the work on neurogenesis was supported by a sponsored research agreement with Accorda, Inc. I thank Dr. S.
Rao for her constant support.
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