Physiol Rev 94: 709 –737, 2014
Leda Dimou and Magdalena Götz
Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for
Stem Cell Research, HelmholtzZentrum, Neuherberg, Germany; and Munich Cluster for Systems Neurology
(SyNergy), Munich, Germany
Dimou L, Götz M. Glial Cells as Progenitors and Stem Cells: New Roles in the Healthy and
Diseased Brain. Physiol Rev 94: 709–737, 2014; doi:10.1152/physrev.00036.2013.—
The diverse functions of glial cells prompt the question to which extent specific subtypes
may be devoted to a specific function. We discuss this by reviewing one of the most
recently discovered roles of glial cells, their function as neural stem cells (NSCs) and
progenitor cells. First we give an overview of glial stem and progenitor cells during development;
these are the radial glial cells that act as NSCs and other glial progenitors, highlighting the
distinction between the lineage of cells in vivo and their potential when exposed to a different
environment, e.g., in vitro. We then proceed to the adult stage and discuss the glial cells that
continue to act as NSCs across vertebrates and others that are more lineage-restricted, such as
the adult NG2-glia, the most frequent progenitor type in the adult mammalian brain, that remain
within the oligodendrocyte lineage. Upon certain injury conditions, a distinct subset of quiescent
astrocytes reactivates proliferation and a larger potential, clearly demonstrating the concept of
heterogeneity with distinct subtypes of, e.g., astrocytes or NG2-glia performing rather different
roles after brain injury. These new insights not only highlight the importance of glial cells for brain
repair but also their great potential in various aspects of regeneration.
forming the insulating myelin sheaths and the most recent member of the club, the NG2-glia discovered only
recently by the antibody NG2 (152) recognizing the
chondroitin sulfate proteoglycan 4 (CSPG4). NG2-glia
are widely dispersed through the adult brain and CNS
parenchyma, and their functions are not yet known.
One of the most basic classifications of cells in the nervous system is the distinction between neurons and glia.
Glia were originally defined as nonneuronal cells with
support functions, as the word nervenkitt (the glue between the neurons/nerves) implies (128, 283). Accordingly, glial cells were implicated in supporting neurons as
well as maintaining stability and the overall architecture
of the nervous system. Such a role is still prevalent and is
reflected in the diverse support functions of glial cells that
have been allocated to the different glial subtypes that
were subsequently discovered over the last centuries.
These are the star-shaped astrocytes, largely implicated
in ion and metabolic brain homeostasis, the highly
branched surveilling microglia with key functions in
phagocytosis and defense, the myelinating glial cells, oligodendrocytes in the central nervous system (CNS), and
Schwann cells in the peripheral nervous system (PNS),
Also in regard to neural information processing, neurons
were considered as the main players because of their allegedly unique capacity to generate action potentials and communicate via chemical synapses. Conversely, glial cells were
considered to clean up thereafter, by removing excess ions
or transmitters after neuronal activity in the vicinity of synapses, a task primarily allocated to astrocytes or nonmyelinating Schwann cells enwrapping synapses in the CNS and
PNS, respectively. This view has changed by now as it has
become clear that glial cells actively contribute to synaptic
communication as direct synaptic partners. The so-called
tripartite synapse consists of either two neuronal elements
(the pre- and postsynaptic endings) or a presynaptic neuronal element and cells of the target organ in the periphery,
such as muscle cells, and a glial part (the astrocyte or terminal Schwann cell processes in direct contact with these
elements that often ensheath the entire synapse). Moreover,
also glial cells that form and receive synapses were discovered, such as the NG2-glia (29, 37, 243), the newly discovered glial cell type that is also involved in forming the
0031-9333/14 Copyright © 2014 the American Physiological Society
perineuronal nets (PNNs), dense arrangements of extracellular matrix considered to firmly stabilize synapses (74,
286). In the meantime also other glial cells have been observed as synaptic partners with microglial processes found
in close vicinity of synapses which have been suggested to
play key roles in synapse remodeling (127, 244). Indeed, the
multiple roles of several glial cell types in synaptic communication are well consistent with their particular increase in
number in brains with higher complexity, eventually outnumbering neurons by severalfold (for recent review, see
Refs. 98, 129). A close and life-sustaining cross-talk between glia and neurons also occurs at the neuronal axon,
where myelinating glia exert trophic support to neurons
(189). The axon is indeed a further site of neural computation, rather than a sheer propagating cable. In this context,
it is of particular interest that specific glial cells, the NG2glia, can also be a direct postsynaptic site for neuronal
processes, e.g., axons, as is the case in various brain
regions including the corpus callosum (243). Strikingly,
however, the very same type of glial cells also acts as
oligodendrocyte progenitor cells (OPCs) in the adult
brain proliferating and generating oligodendrocytes particularly frequently in the corpus callosum (61, 282).
This prompts a short comment on the confusing use of
the term oligodendrocyte precursor/progenitor cells. The
term progenitor refers to a proliferating cell, while precursor refers to an immature state, which is why we use
the term oligodendrocyte progenitor cell as we are referring to proliferating cells. This is an important issue in
regard to the key question of how such roles in synaptic
communication on one side and as dividing progenitor
cells on the other side can be combined by the same cells.
Alternatively, there may be subtypes of NG2-glia, one
acting as dividing progenitors and the other as synaptic
Indeed, this is a central question which has contributed to
ignore the role of glial cells as stem and progenitor cells. In
part, the difficulty arose from the view that dividing cells
retract all their processes during M-phase, in which case all
the synaptic and/or other connections formed by glial processes with neurons would be retracted and would have to
be newly established. One way out of this dilemma would
be to postulate a division of labor with some subsets of glia
performing stem and progenitor cell functions and the other
specialising on other tasks; alternatively, however, all functions may be performed at the same time if proliferating
cells maintain their processes including those involved in
synaptic contacts (79, 143). A further intriguing possibility
may be that glial cell division with process retraction could
be a means to remodel the synaptic connections of glia and
hence contribute to brain plasticity. Similar questions also
arise for astrocytes that perform stem cell functions in specific brain regions and after injury (236), besides their classical roles executed by processes involved in synaptic communication and endfeet surrounding the blood vessels me-
diating the neurovascular coupling. How can such a cell
divide or are astrocytes with stem cell functions a special
kind? In the most extreme case, glial cells with stem or
progenitor cell function may have just been mistaken for
glial cells due to the expression of “a few markers” and
there would be a deep divide between a mature glial cell and
any type of stem or progenitor cell.
These will be the questions we shall discuss in this review.
To set the stage for these central issues of glial functions
and heterogeneity, we will proceed in a sequence following normal development and first review the progenitor
and stem cell roles of glial cells in the developing brain,
the stage at which the concept that glial cells also act as
stem and progenitor cells was originally discovered (166,
185, 198). We shall review the evidence for radial glial
cells, the ubiquitous glial cell type in the developing vertebrate CNS, acting as neural stem and progenitor cells in
a rather wide-spread manner (87, 139) and then turn to
the second type of glia to appear in the developing CNS,
the OPCs. Thereafter, we shall describe the emergence of
progenitors for the other macroglial cell types (for reviews on microglia, see Ref. 127). Afterwards we will
turn to the adult stage and discuss glial cells that still act
as stem and progenitor cells, which are largely radial glial
cells or NG2-glia occurring at different frequency in different vertebrate groups. Finally, we shall turn to the
injury condition and review glial cells acting as stem and
progenitor cells in various injury paradigms. Notably,
some glial populations that are postmitotic or quiescent
in the healthy brain reactivate proliferation and acquire a
broader potential after specific types of injury. This leads
to the important discrimination of two terms that are
often used in a confusing manner: lineage and potential.
It is fundamental to discriminate between the progeny
that these glial cells generate in vivo, their normal lineage, from their potential, i.e., the progeny the very same
glial cells can generate when exposed to a different environment in vivo or in vitro.
The reactivation of stem and progenitor properties in
some apparently differentiated glial cells will bring us
back to the issue of heterogeneity, asking to which extent
these glial cells with a broader potential may be a special
subtype and which roles this tentative subtype and its
proliferation may play in the context of brain injury. This
will provide new insights into a profound and surprising
heterogeneity within the same population of glial cells
reacting to brain injury not only opening new approaches
to improve the outcome of the glial reaction and minimize scar formation after injury, but also identifying
novel avenues towards employing stem and progenitor
cells right within the injury site for neuronal replacement.
This will close the circle to the roles that glial stem and
progenitor cells play during development, namely, generating neurons.
Physiol Rev • VOL 94 • JULY 2014 •
A. Radial Glial Cells During Development
Radial glial cells are the first glia to appear, in ontogeny and
phylogeny. Radial glial cells have been described early in
the deuterostome lineage, such as in echinodermata and the
lancelet (for recent review, see chapter 5 in Ref. 129). In
ontogeny, radial glial cells appear in all regions of the brain
at the onset of neurogenesis when the first differentiated cell
types are generated. In some regions, however, such as the
rat spinal cord, radial glial cells appear only at later stages,
at the onset of gliogenesis (175). In vertebrates, radial glial
cells develop from the earlier neuroepithelial cells, the epithelial cells forming the neural plate and neural tube. Both
these cell types are epithelial in nature as they possess a clear
apico-basal polarity with an apical membrane domain that
contains, e.g., the glycoprotein Prominin1 and is delineated
by junctional complexes containing tight and adhesion
junction proteins and a basolateral membrane domain with
contact to the basement membrane (BM) at its most basal
extension. For radial glial cells, this is the BM at the brain
surface delineating the brain parenchyma from the meninges (FIGURE 1A). Given this structure, both neuroepithelial
cells as well as the later differentiating radial glia provide a
stable radial architecture throughout the developing brain.
Radial glial cells differentiate with the onset of neurogenesis
from neuroepithelial cells by acquiring glial hallmarks, such
as expression of vimentin, the glial fibrillary acidic protein
(GFAP), the two astrocyte-specific glutamate transporters
(Slc1a3, also referred to as EAAT1 or GLAST, and Slc1a2
also referred to as EAAT2 or GLT-1), glutamine synthase
(GS) as well as ultrastructural criteria of glycogen granules
and filaments (see chapter 5 in Ref. 129). As the brain
further grows in thickness during development, radial glial
cells have to further extend their radial processes reaching
out up to several millimeters in the human cerebral cortex.
Accordingly, the first role of these cells to be discovered was
their support function as stable radial structures also providing guidance for migrating neurons. Indeed, this role is
important for various migration disorders and often mutations in genes resulting in neuronal dysplasia affect radial
glia morphology and process stability (see, e.g., Ref. 39).
Given this functional importance of the long radial processes, it was difficult to perceive that these cells should
divide at the same time despite their DNA synthesis as demonstrated by [3H]thymidine incorporation (184). Only
when several labs provided evidence for radial glial cell
division and contribution to neurogenesis did this concept
catch on. This was demonstrated first by isolating radial glia
by fluorescent-activated cell sorting (FACS) and following
their progeny in vitro that included many neurons (164,
166), then by live imaging in slice cultures following radial
glial cell divisions generating bipolar cells migrating to-
wards the neuronal layers reminiscent of neurons (185,
198), and finally by in vivo tracing of radial glia progeny by
genetic fate mapping (165). Genetic fate mapping allows
tracing of the entire progeny of a given cell type by expressing a DNA recombinase (cre, flp, or others) in this cell type.
This allows turning on a reporter gene encoding a marker
protein, e.g., a fluorescent protein that is separated by a stop
cassette flanked by sites recognized by the recombinase
from a ubiquitous promoter. When expression of the recombinase is turned on, e.g., by the GFAP promoter in
radial glial cells (165), it excises the stop cassette and thus
the marker gene expression is turned on in this specific cell
type. This is then inherited to all cell types that may ever be
generated from this cell population as the excision of the
stop cassette is irreversible and hence the complete progeny
will also express this marker. Importantly, the recombinase
can be further modified to increase temporal specificity
which was essential for fate mapping the progeny of glial
cells in the adult brain. Temporal specificity is achieved by
fusing the recombinase to a ligand binding receptor such
that it can only be active when the ligand is provided, allowing temporal control over the recombination (for review, see Refs. 113, 281). Regional and further cell type
specificity can be achieved by combinatorial means, either
combining different recombinases (104) or splitting the recombinase into two halves expressed under control of two
different promoters (102, 103) such that recombination
and subsequent expression of the reporter gene is only mediated in cells with both activated promoters. For example,
this allowed the discrimination of adult neural stem cells
(NSCs) expressing both GFAP and Prominin1 from surrounding astroglia (GFAP⫹, Prominin1-) and neighboring
ependymal cells (GFAP⫺, Prominin1⫹; Ref. 16).
These techniques revealed that radial glial cells generate
neurons and later glial cells (such as astrocytes, ependymal
cells, and oligodendrocyte progenitors) in many brain regions and species (139, 218). In some regions, such as the
spinal cord and possibly the retina, radial glia differentiate
so late that they only contribute to gliogenesis (175). In
regions where they contribute to neurogenesis, this regional
specification is crucial to generate the neuronal diversity of
the brain. In the ventral midbrain, for example, radial glial
cells in the floor plate, the region defined by expression of
the morphogen sonic hedgehog (SHH) and the transcription factor Foxa2, generate the ventral midbrain dopaminergic neurons (32, 130). Radial glial cells also differ profoundly in their regional specification and the neuronal subtypes they generate in the telencephalon. In the dorsal
telencephalon, the anlage of the cerebral cortex, they generate largely glutamatergic pyramidal projection neurons
governed, e.g., by the transcription factor Pax6 (for review,
see Ref. 87). In the ventral telencephalon, radial glial cells
differentiate gradually and have only acquired full radial
glia identity by mid-neurogenesis (165, 215). They express
the transcription factors Gsx2 and Ascl1 and contribute
Physiol Rev • VOL 94 • JULY 2014 •
Basement membrane
Cerebral cortex
Basal ganglia
Migrating neurons
Basal progenitors
Radial glia
Apical surface
Par complex
FIGURE 1. Glial cells as stem and progenitor cells in the developing brain. A: schematic drawing of the developing mouse
brain. Distinct subtypes of radial glial cells
(green) can be identified based on their molecular, cellular, and lineage characteristics. For example, radial glia in the ventral
telencephalon express Gsx2 (red nuclei)
while radial glia in the dorsal telencephalon
express Pax6 (brown nuclei). When neurogenesis starts, radial glia cells in the dorsal
telencephalon divide asymmetrically, giving
rise to one daughter cell of the original fate
and one of a new fate (either one neuron
blue or one basal progenitor, green). Radial
glia possess an apico-basal polarity with an
apical membrane that is delineated by junctional complexes containing tight and adhesion junction proteins like cadherins, ␣- and
␤-catenin, and proteins of the Par complex
while the basolateral membrane contacts
the basement membrane at its most basal
extension. B: model illustrating that oligodendrocyte progenitor cells are generated
during development in three sequential
waves from different parts of the telencephalon (left). While Nkx2.1-derived progenitors (most likely comprising the plp/dm20transcript positive OPCs) get rapidly eliminated during postnatal life, Gsx2- and
Emx1-derived progenitors remain and populate the adult brain (right).
First wave of OPCs generated from Nkx2.1 precursors
Second wave of OPCs generated from Gsx2 precursors
All OPCs are
Olig2+, NG2+, and PDGFRα+
Third wave of OPCs generated from Emx1 precursors
then to the generation of GABAergic projection neurons
and interneurons (38, 70, 285). As the latter is a particularly
large population of inhibitory neurons spreading throughout the telencephalon, radial glial cells generate this larger
neuronal output by a series of intermediate progenitors that
bears striking resemblance to amplifying lineages with a
large output as observed in other species in development or
the rodent olfactory bulb (OB) neurogenesis at postnatal
and adult stages (215). Indeed, adult NSCs resemble radial
glial cells not only in this region, the ventral murine telencephalon, but also in many other regions and species (see
In most regions of the mammalian brain, however, radial
glial cells disappear by generating specialized glial cells that
remain at the ventricle, the ependymal cells. While these
remain anchored at the ventricle and maintain the junctional coupling and some degree of epithelial polarity, astrocytes and OPCs spread through the entire brain parenchyma where they homogeneously cover the neuropil with
their fine processes. This is achieved by radial glial cells that
do not directly generate postmitotic astrocytes or oligodendrocytes but rather proliferative intermediate progenitors.
These progenitors then multiply and spread throughout the
entire ventrodorsal extent (FIGURE 1B; Refs. 112, 125, 275,
Physiol Rev • VOL 94 • JULY 2014 •
289), while they sometimes respect rostro-caudal domains,
such as the rhombomeres, segmental patterns in the developing hindbrain (180). Thus especially OPCs, which often
originate in ventral regions of the brain and migrate, cover
long-distances by both migration and continued proliferation, therefore populating large regions.
Surprisingly, this is rather different for astrocyte progenitors that also delaminate from the ventricle or the pial surface and continue to proliferate in the neonatal brain parenchyma, but still remain within regional constraints as they
largely migrate radially possibly along the remaining radial
glial cells (76, 77, 105, 280). These data therefore suggest
that the spread of astrocytes at early postnatal stages results
from local proliferation rather than long-distance migration. Importantly, this implies a regional heterogeneity of
astrocytes that is of crucial functional relevance. For example, only astrocytes from the correct region are able to support dendrite development of regionally matched neurons
in vitro (58, 239), and formation of specific synapses onto
motoneurons was significantly decreased when astrocytes
were reduced only by a third in this domain (280). So,
neighboring astrocytes do not invade and compensate for
the partial loss of astrocytes within a specific CNS region,
highlighting the important concept of regional diversity of
astrocytes, similar and/or in relation to the regional diversity of neuronal subtypes.
192, 193) as well as the key transcription factors Olig1/2
and Sox10 (177, 249, 272). OPCs were first discovered in
dissociated cell cultures derived from optic nerve (224,
225), then from the spinal cord and the forebrain (223,
294). These bipolar cells were positive for several cell surface markers, such as the antibody A2B5, the proteoglycan
NG2, or the receptor PDGFR␣, and were shown to proliferate and then undergo a number of structural and biochemical changes as they mature (212) into myelin-forming
cells enwrapping and myelinating axons in cocultures with
neurons. During this differentiation process, cells lose the
above-mentioned OPC specific proteins and begin to express surface antigens that bind the monoclonal antibody
O4 (7, 260), galactocerebrosides (226), and early myelin
proteins like the 2’,3’-cyclic nucleotide phosphodiesterase
(CNP; Ref. 212) and myelin basic protein (MBP; Ref. 267).
This provided an extremely useful in vitro model to unravel
factors that either inhibit or promote proliferation and differentiation of these cells, which could be verified in vivo as
described below.
B. Oligodendrocyte Progenitor Cells During
For some time, the spatiotemporal origin of these cells remained more controversial. The origin of oligodendrocytes
was detected by some labs at multiple foci or throughout
the embryonic ventricular zone (262, 265, 289), while others observed very restricted, specialized niches at the ventricular zone of the spinal cord and the forebrain (125, 203,
221, 264, 289; for review, see Ref. 240). Both hypotheses
were supported by the expression of key factors and their
receptors promoting OPC specification and proliferation
such as SHH and PDGFR␣ that are initially localized ventrally and become more widespread at later stages (221,
263, 297) while the bone morphogenic proteins (BMPs)
that inhibit OPC development are emerging from more dorsal sources (178). Finally, genetic fate mapping revealed
that both views are correct, but temporally spaced. Indeed,
at very early stages (E9/E10), plp/dm20-transcript positive
OPCs emerge from hotspots in the ventral mouse forebrain
(OPCs described in Ref. 262) (FIGURE 1B). Interestingly,
these cells are not sensitive to PDGF and seem to mature
later. Moreover, genetic fate mapping experiments using
plp-Cre and plp-CreERT2 mice revealed that cells expressing plp-Cre at early stages contribute to neuronal and glial
lineages, while cells expressing plp-Cre at later gestational
stages (E13 and after) only generate oligodendrocytes (56).
OPCs are the progenitors of the myelinating cell type of the
CNS, the oligodendrocytes, and appear in the developing
brain almost as early as the radial glial cells discussed
above. With only a few days delay after the onset of neurogenesis, OPCs were detected in ventral regions of the developing CNS from where they spread throughout all brain
regions by migration and proliferation (FIGURE 1B). Their
hallmarks are the expression of the platelet-derived growth
factor receptor ␣ (PDGFR␣; Refs. 197, 220, 221), the proteoglycan NG2 (152, 153, 194, 195; for review, see Refs.
In a second wave, cells responsive to PDGF and hence expressing PDGFR␣ emerge (around E14) from ventral regions in the spinal cord and the forebrain and their progeny
comprises virtually exclusively more OPCs and mature oligodendrocytes (221, 289). Notably, the transcription factor
Olig2 that is key for oligodendrocyte development (161,
162, 268) is also expressed at earlier stages by motoneuron
progenitors (186, 305), but is at later stages rather specific
for glial progenitors and yet later for OPCs. In the developing spinal cord, Olig2 is expressed in the ventral region, and
This is strikingly different for the other major set of glial
progenitors, the OPCs, that readily compensate the loss of
OPCs close by or in different domains as described below
(124). Intriguingly, these differences between OPCs and
astrocyte progenitors at early postnatal stages anticipate
their respective behavior in the adult brain after injury, with
astrocytes remaining very stationary with virtually no migration towards the injury site (8, 280), as opposed to glial
progenitors of the OPC lineage that readily migrate and
accumulate around the injury site (see below and FIGURE 4).
Thus the behavior of these distinct glial progenitors differs
profoundly already at early postnatal stages, with astrocytes showing a strongly regionalized diversity, while OPCs
appear to be more promiscuous in this regard.
Physiol Rev • VOL 94 • JULY 2014 •
inducible genetic fate mapping revealed that distinct sets of
Olig2-expressing cells generate motoneurons at earlier
stages, while a separate set of later appearing Olig2-expressing radial glial cells generates OPCs (188, 296). Notably, the same is the case for progenitors expressing the transcription factor Ascl1 (former named Mash1) with a distinct neuronal lineage first and a later glial lineage (13).
Indeed, Ascl1 also labels OPCs originating from dorsal regions, and their dorsal origin has been further demonstrated
by region-specific genetic fate mapping revealing cells
spreading into ventral regions as, e.g., in the spinal cord
(306). Such experiments using region-specific Cre lines even
revealed a transient nature of the early ventrally derived
OPCs at least in the telencephalon (FIGURE 1B). While these
cells populate the entire telencephalon at neonatal stages,
later OPCs arise at more dorsal positions (labeled by Emx1Cre; Ref. 124) at postnatal stages. These cells gradually
replace the earlier OPCs such that most mature oligodendrocytes in the dorsal telencephalon, the cerebral cortex,
are derived from the dorsal telencephalic regions (FIGURE
1B). These data imply also some degree of regional diversity
of mature oligodendrocytes as well as adult OPCs that continue to proliferate life-long at least in the rodent and human brains (80, 254). Indeed, some region-specific differences in the migratory and neurogenic potential have been
observed (2), but other than this the functional relevance of
this temporal replacement and/or regional origin and diversity has yet to be discovered, as no defects could be observed
when one population replaces the other. This has been
achieved by expressing diphtheria toxin in the dorsally derived OPCs to kill them selectively (124). Intriguingly, the
remaining OPCs that were largely ventrally derived could
readily replenish the depleted pool of dorsally derived OPCs
and give rise to mature oligodendrocytes that could fully
substitute the missing cell population (124). These data obtained in the developing brain already highlight the amazing
compensatory and proliferative capacity that all of these
OPC populations have and also maintain into adulthood.
Even in the adult brain, OPCs can any time reactivate proliferation and replace lost NG2-glia or oligodendrocytes
even in large numbers as discussed below.
In summary, one of the key differences between radial glial
cells and OPCs is the persistence of the latter (at least the
last wave of OPCs) throughout the mammalian brain while
the former are largely transient and only restricted in few
niches as adult neural stem cells where they continue to
generate neurons (139). This raises not only the question to
which extent very slow cycling, alias “quiescent” or “dormant” NSCs may still persist also in the adult mammalian
CNS, possibly hidden by their altered morphology, such as
the ependymal cells still lining the ventricle (10, 179) or the
astrocytes within the grey or white matter parenchyma (35,
236, 255) but also whether the OPCs may have a broader
potential and may serve as source of other cell types when
C. Glial Progenitors With Multilineage
The above concepts thus imply that only radial glial cells
give rise to multiple cell types in vivo, while OPCs and
astrocyte progenitors seemingly serve to multiply a single
lineage, not withstanding further diversity among oligodendrocytes or astrocytes. Indeed, this is the case also within
the neuronal lineage with radial glial cells sometimes generating further amplifying neuronal progenitors as mentioned above (87, 215). However, are radial glial cells really
the only cell type with neural stem cell properties and multilineage potential, or can the OPCs, at least the early population, also give rise to more cell types than “just” oligodendrocytes?
OPCs have indeed been described to generate also astrocytes first in vitro (18, 225) and then in vivo. Genetic fate
mapping of NG2-expressing cells (using NG2-Cre transgenic mice) revealed that more than 40% of the protoplasmic astrocytes in the grey matter (GM) of the ventral forebrain were generated from NG2 expressing cells (307, 308),
while this was not the case for astrocytes in the white matter
(WM). Interestingly, a similar lineage was also observed in
the spinal cord (309). Thus the lineage of NG2-expressing
cells in the developing neural tube is not restricted to oligodendrocyte cells, and the term OPCs is not appropriate. In
contrast, it is more appropriate to refer to these cells as
NG2-glia, in light of their contribution also to other glial
lineages and of their additional functions that will be described later. Importantly, other cell types in many organs
also bear the proteoglycan NG2, but these are pericytes
(204), cells residing in the perivascular space, which are
generally not considered as glial cells as they are of mesodermal origin in most organs and regions of the neural tube.
Here we use the term NG2-glia for CNS-derived glial cells
expressing the proteoglycan NG2.
While the above genetic fate mapping experiments showed
the contribution of NG2-glia to different glial cells, they
could not resolve to which extent a single progenitor cell is
bipotent generating both oligodendrocytes and astrocytes,
or whether NG2⫹ glial progenitors comprise several pools
of cells, some generating astrocytes and others generating
oligodendrocytes. To separate such different sets of progenitor cells, various combinations of cell surface markers have
been established, allowing to preferentially enrich, e.g., progenitor cells giving rise to both astrocytes and oligodendrocytes (90, 228). These cells have been called “glioblasts,” in
analogy to the term neuroblasts, but are also often referred
to as glia-restricted progenitors (GRPs) as their fate restriction to the glial lineage has been demonstrated in vitro as
well as in vivo by transplantation experiments (99). As these
cells share their bipolar morphology and many markers
with NG2-glia, they may well represent the bipotent subset
of these. Moreover, even tripotency, i.e., the potential to
generate neurons, astrocytes, and oligodendrocytes, has
Physiol Rev • VOL 94 • JULY 2014 •
been demonstrated under certain in vitro conditions when
isolating cells enriching for NG2-glia from the developing
early postnatal brain (2, 20, 137). Interestingly, these glial
progenitors can be readily converted by application of BMP
to an astrocyte-like cell in vitro which is then able to reveal
the stem cell hallmarks of self-renewal and multipotency
when grown in appropriate growth factor conditions
(FIGURE 2; Ref. 137; see however Ref. 2 for the isolation of
OPCs that naturally generate different lineages). Importantly, such multipotent cells can also be isolated from early
postnatal rodent or even human brains by culturing cells
first under conditions for astrocyte progenitors and then
probing for stem cell hallmarks (149, 284). Taken together,
at early postnatal stages, bi- and multi-potent progenitor
cells as well as progenitors restricted to a single lineage exist
in various regions of the rodent brain. Also note that this
potency is often only revealed when cells are placed in vitro
or in a different environment, while they only generate a
single cell type in vivo, with the distinction between in vivo
lineage and in vitro potential applying here. However, by 3
wk after birth in rats and mice, the plastic cell types with a
larger potency appear to be much reduced or virtually lost
(149), prompting the question as to whether remnants of
multipotent cells may still exist in the fully adult mammalian brain and could possibly be activated by the appropriate stimuli.
A. Radial Glia in the Adult Brain: Adult
Neural Stem Cells and Neurogenesis
1. Nonmammalian vertebrates
FIGURE 2. In vitro read-out for potential: the neurosphere assay.
Adult neural stem cells get isolated, dissociated, and grown in a
serum-free medium with growth factors. Some cells form free-floating cell clusters (neurospheres) that can be passaged several times
(self-renewal). When exposed to differentiation conditions, cells
from one neurosphere can differentiate into neurons, astrocytes,
and oligodendrocytes thereby showing multipotency.
Generally, endogenous neurogenesis persists in brain regions, where radial glial cells persist. This is the case in the
mammalian brain, where radial glial cells disappear in most
regions, but persist in a few niches where also neurogenesis
persists (FIGURE 3A). In most nonmammalian vertebrates,
such as in amphibian, bony fish, rays and sharks, as well as
reptiles, however, radial glial cells remain abundantly present in a widespread manner in the adult CNS (FIGURE 3B;
Ref. 50). Most glial cells lining the ventricle possess long
radial processes, have epithelial characteristics (92), and
share many proteins with radial glial cells in the developing
brain, such as Prominin1, brain lipid binding protein
(BLBP), nestin, vimentin, GFAP, S100␤, GS, and the transcription factors Hes1/5 and Sox2 (44, 75, 114, 140, 145,
170, 210). Notably, none of these proteins is specific for
radial glia as Prominin1, vimentin, S100␤, and Sox2 are
also shared with ependymal cells and GFAP, S100␤, GS,
and Sox2 with parenchymal astrocytes in the murine brain
(16, 236). The persistence of radial glial cells at the ventricular surface implies that no specialized cuboid ependymal
cells are present (see also chapter 5 in Ref. 129). Likewise,
also astrocytes are lacking in brain regions with persisting
radial glia, such that radial glial cells encompass the function of both these mature glial cells types by lining the
ventricle as ependymal cells do and enwrapping synapses
and expressing K- and aquaporin channels for ion and water homeostasis as astrocytes do (92; see also chapter 5 in
Ref. 129).
In addition, radial glial cells also continue to enact their
function as stem and progenitor cells from development,
namely, to divide and generate neurons (for review, see
Refs. 42, 89). Indeed, neurogenesis continues in a widespread manner in the species of bony fish, reptiles, and
Physiol Rev • VOL 94 • JULY 2014 •
ral cor
Dentate gyrus
ventricle lining
Niche astrocytes
Migrating neuroblasts
FIGURE 3. Glial cells as stem and progenitor cells in the healthy adult brain. A:
endogenous neurogenesis still persists in
the adult mammalian brain in few niches
like the subependymal zone in the lateral
wall of the lateral ventricle, the subgranular
zone in the dentate gyrus, and the hypothalamus. Radial glial cells at the subependymal zone of the lateral ventricle divide and
generate fast proliferating transit-amplifying progenitors (TAPs) and neuroblasts
(NBs) that proliferate while they migrate
through the rostral migratory stream
(RMS) to their final destination, the olfactory bulb (OB), where they can differentiate
to different neuronal types. B: radial glia
cells in the adult zebrafish are more widespread than in mammals and are located
along the ventricle. The zebrafish telencephalon is everted, with the ventricle lying
between and above the two telencephalic
hemispheres. Proliferating cells (green,
based on data summary in Ref. 89) are
located in distinct regions along the entire
anterior-posterior axis.
Radial Glia
amphibians which have been examined so far (89). For
example, in the zebrafish telencephalon, a subset of radial
glial cells (⬃10%) divides typically in an asymmetric manner (238) giving rise to a radial glial cell and an intermediate
progenitor. The intermediate progenitor loses its glial characteristics, but does not yet express neuronal traits. It con-
tinues to proliferate and then generates differentiating neurons (1, 43, 238). As the zebrafish and its brain continue to
grow, it is of particular relevance that stem and progenitor
cells persist and do not deplete. This is achieved by the
asymmetric mode of division described above, and a reserve
pool of radial glial cells with only some dividing while oth-
Physiol Rev • VOL 94 • JULY 2014 •
ers remain quiescent. However, virtually all the quiescent
radial glia can be activated to enter proliferation by inhibition of the Notch signaling pathway (43; see also Alunni et
al. (4a), NOTE ADDED IN PROOF) or after injury (see below),
demonstrating that all of them have proliferative neural
stem cell potential. This is also the case in regions with low
or no proliferation and neurogenesis in adulthood, such as
the zebrafish spinal cord (229, 231) or the salamander midbrain (26).
Moreover, some CNS regions, such as the dorsal midbrain
and the retina, possess non-radial glia stem cells densely
packed in a zone at the margin of the respective tissue,
called the ciliary marginal zone in the retina. The stem cells
in this region rather resemble the earlier neuroepithelial
cells as they fail to express glial hallmarks (89). Summing
up, many vertebrates are equipped with widespread adult
NSCs that resemble the NSCs in the developing brain, either
the neuroepithelial or the radial glial cells. This has important consequences for the reaction after brain injury, not
only in regard to the repertoire of glial cells reacting to
injury which is obviously different from mammalian brains
(e.g., the zebrafish CNS largely lacks parenchymal astrocytes; see, e.g., Ref. 14) but also in regard to the capacity to
replace degenerated neurons that is largely lacking in the
mammalian CNS.
2. Mammalian (and avian) vertebrates
Interestingly, also in mammalian and avian brains radial
glial cells persist into adulthood, but in a more spatially
restricted manner mostly within the forebrain (for review,
see Refs. 89, 190). For example, radial glial cells, rather
than cuboidal multiciliated ependymal cells, line the ventricle in the hypothalamus (FIGURE 3A). These are often referred to as tanycytes, a name typically referring to radial
glial cells lining the ventricle in adult brains. Tanycytes in
the hypothalamus differ depending on their dorsoventral
location with ␤-tanycytes located most ventrally, expressing FGF10 (fibroblast growth factor) and lacking GFAP and
GLAST expression in mice, while ␣-tanycytes express
GFAP and GLAST, but not FGF10 and are located more
dorsally (93, 237). Most importantly, these different populations also exhibit functional differences recently revealed
by genetic fate mapping showing that FGF10⫹ ␤-tanycytes
generate neurons within the first two postnatal months and
upon high-fat diet (93), while GLAST⫹ ␣-tanycytes generate few neurons and are rather gliogenic in normal adult
mice (237). Most importantly, however, only the latter have
the NSC hallmarks of long-term self-renewal and multipotency in vitro (237). Thus the hypothalamus certainly contains cells with NSC properties, while neurogenesis at resting state in the adult brain appears to be rather low (FIGURE
3A). Importantly, however, it can be activated by metabolic
stimuli (93, 213), prompting the important question about
the functional consequences of this induced adult neurogenesis.
In contrast to the hypothalamus, in the most active zone of
adult neurogenesis in mice, at the ventricle, the progeny of
the actively dividing radial glial cells are so many that they
form a distinct layer, the subependymal zone (SEZ) located
below the ependyma lining the ventricle. This progeny comprises transit-amplifying progenitors (TAPs) dividing rather
fast and neuroblasts (NBs) that also continue for a while to
proliferate and amplify while migrating through the rostral
migratory stream (RMS) to their final destination, the olfactory bulb (FIGURE 3A). The radial glial cells act as slow
dividing NSCs that are integrated with a process into the
differentiated ependymal layer and their cell somata located
below in the SEZ (183). This is why this zone is best referred
to as SEZ to discriminate it from the subventricular zone
(SVZ) in the developing brain when and where no ependymal cells are yet present.
Radial glial cells in this region have a shorter radial process
compared with those in the developing brain reaching all
the way to the BM underlying the meninges. The radial
processes of adult NSCs in the SEZ typically contact the BM
surrounding neighboring blood vessels and obtain key factors from this vascular niche, promoting their self-renewal
(134, 135). As typical radial glia they also have epithelial
hallmarks with junctional coupling to their neighboring radial glia or ependymal cells and a delineated apical membrane domain containing Prominin1 (16). They also express many of the radial glia proteins highlighted above
(GFAP, GLAST, GLT-1, Sox2, Hes5, Prominin1, BLBP,
etc.), and genetic fate mapping using the split-Cre technology clearly demonstrated that the cells coexpressing GFAP
and Prominin1 act as NSCs self-renewing and contributing
to neurogenesis over several months (16). Interestingly,
there is some heterogeneity with long-term and short-term
self-renewing NSCs that can be detected by genetic fate
mapping targeting cells expressing some of the above radial
glial proteins (83). Thus it appears that adult NSCs typically
line the ventricle and possess or maintain neuroepithelial
properties. Indeed, key signals derive from the cerebrospinal fluid (CSF) in the ventricle both during development
(115, 116) and in adulthood (302), and the junctional complexes characteristic for epithelial cells act as crucial cellular
signaling centers (115). However, also cells lacking direct
ventricular contact and epithelial polarity, such as the radial
astrocytes in the dentate gyrus (DG) act as adult NSCs and
source of life-long neurogenesis in most mammalian species
including humans (133, 261). These are astroglial-like cells
with a radial morphology located in the subgranular zone
(SGZ) of the DG (for review, see Refs. 139, 236), at some
distance from the ventricle and no apicobasal polarity
which is characteristic for epithelial cells. For example, Prominin1 that is sorted to the apical membrane domain in
neuroepithelial and radial glial cells is spread along the
entire membrane domain of DG NSCs including their long
radial process (17). Interestingly, however, the combination
of GFAP and Prominin1 expression targets specifically
Physiol Rev • VOL 94 • JULY 2014 •
NSCs also in this region, highlighting still some molecular
similarity to radial glial cells during development (217) and
in the adult SEZ (16). Therefore, also glial cells resembling
more parenchymal astrocytes with a radial morphology, the
so-called radial astrocytes, can act as adult NSCs self-renewing for some time and giving rise to intermediate progenitors that then generate differentiating neuroblasts
(181). Importantly, the DG NSCs reside in a vascular niche,
the other frequent niche of NSCs (85). With regard to radial
astrocytes, it is worth mentioning the Müller glia and the
Bergmann glia, two prominent astroglia-like cell types with
a characteristic radial morphology in the retina and the
cerebellum, respectively. While they do not act as actively
neurogenic NSCs in the mammalian species examined so
far, especially the Müller glia is a very plastic cell type which
reactivates a broader potential after injury (120). This
prompts the question to which extent other radial or nonradial astrocytes may possess or acquire NSC hallmarks in
the adult mammalian brain. In the normal healthy brain,
parenchymal astrocytes do not proliferate and do not show
any NSC potential upon dissociation and culture in the
neural stem cell assay, the neurosphere conditions (FIGURE
2; see above and Ref. 255). Interestingly, however, there is
a widespread proliferating glia population throughout most
of the adult brain parenchyma in rodents and humans, the
B. NG2-Glia in the Adult Brain Parenchyma:
OPCs or Stem Cells Outside the
Neurogenic Niches?
Around 5–10% of the cells in the adult mouse brain parenchyma express NG2 (53, 158, 193, 219), PDGFR␣ (64,
234), and the transcription factors Olig2 (36, 61, 161) and
Sox10 (249, 291), highly reminiscent of the profile of these
glial progenitors during development. Indeed, these cells
proliferate, as shown by the incorporation of bromodeoxyuridine (BrdU), but also by immunolabeling for Ki67 in
actively proliferating cells (222, 254) and phosphorylated
histone H3 in M-phase (Sirko, Dimou, and Götz, unpublished data). Interestingly, NG2-glia divide very slowly in
the adult mouse forebrain with a cell cycle of several weeks
(47, 222, 254), and previously proliferating cells reenter the
cell cycle and reacquire Ki67 several weeks after previous
BrdU incorporation (254). This is notably different from
the fast division rate of these cells in the postnatal brain (see,
e.g., Ref. 222). Upon injury however, NG2-glia in the adult
brain reenter the cell cycle very fast (see below), suggesting
that their cell cycle length and activation are subject to
regulation by signaling. Importantly, virtually all proliferating cells outside the neurogenic niches in the adult mouse
brain as described above are NG2/PDGFR␣/Olig2⫹ (61,
79, 254), prompting the question to which extent their
progeny in the adult brain may resemble the progeny in the
developing brain.
Both BrdU-birthdating experiments (254) as well as inducible genetic fate mapping revealed that these proliferating
cells expressing Olig2 (61), PDGFR␣ (119, 234), and NG2
(193) generate mature oligodendrocytes expressing CC1,
GST-␲, and myelin proteins in the adult brain. Using NG2-,
PDGFR␣-, and Olig2-CreER mouse lines allows inducing
genetic recombination selectively in the adult brain by application of tamoxifen at this time and following the fate of
the genetically labeled cells over weeks and months. Interestingly, there are profound region-specific differences with
NG2-glia proliferating very little in some regions, such as
the spinal cord (119), or generating more or less mature
oligodendrocytes depending on their location. This difference is particularly pronounced between the cerebral cortex
GM and WM with the majority of NG2-glia in the WM
generating mature, myelinating oligodendrocytes, while
NG2-glia in the GM generate significantly fewer mature
oligodendrocytes (61, 118). Interestingly, recent homo- and
heterotopic transplantation experiments revealed intrinsic
differences between WM and GM NG2-glia in their propensity to generate mature oligodendrocytes (282). WMderived cells generate mature oligodendrocytes very efficiently also in the less supportive GM environment, while
GM-derived cells generate significantly fewer oligodendrocytes in the very same in vivo compartment. However, when
GM-derived NG2-glial cells are exposed to a WM environment, they increase the generation of mature oligodendrocytes, even though many of these fail to acquire a full myelinating phenotype (282). Taken together, these experiments highlight for the first time major functional
differences between NG2-glia from different, even though
neighboring regions, with profound functional implications.
One important question emerging from these data is the
role of NG2-glia that does not generate oligodendrocytes,
but rather self-renews and generates more NG2-glia. This
raises the hypothesis that NG2-glia in the adult brain has
other functions beyond being progenitors for oligodendrocytes. Importantly, they differ from other glial cells by receiving synapses from neurons with clear ultrastructural
specializations (29, 55, 243), a feature for which they are
also named as “synantocytes.” These synapses can be
GABAergic or glutamatergic, and NG2-glia possess the respective transmitter receptors, including GABAA or GluRB
receptors (159, 174, 243, 279). Notably, the synaptic potentials of NG2-glia can be changed in a long-lasting manner with repetitive stimulation resulting in their long-term
potentiation mediated by AMPA receptors on NG2-glia
(78). Interestingly, although these synapses elicit clear postsynaptic potentials in the NG2-glia (140, 168, 169), they do
not generate action potentials (according to all; Refs. 28,
29, 47, 78 except one study, Ref. 120, in the rat cerebellar
WM). Therefore, NG2-glia appear to have some physiological hallmarks, previously thought to be unique for neurons
in the CNS, such as direct synaptic innervation, but not all,
Physiol Rev • VOL 94 • JULY 2014 •
such as action potential formation. As these properties,
such as the direct synaptic input, are also not shared with
other glial cells, it is appropriate to consider the NG2-glia as
a distinct glial entity, the fifth glia population (besides astrocytes, oligodendrocytes, microglia, and ependymal cells)
in the mammalian CNS (37). Whether NG2-glia are also
present in other vertebrates, however, still remains to be
elucidated (see below and Refs. 14, 171) like the question
when this type of glia evolved during phylogeny which may
also hold some clues in regard to their prime function.
While the function of this synaptic communication is not
yet fully understood, an obvious hypothesis is that it may
regulate proliferation or differentiation of these cells. Consistent with this hypothesis, sensory deprivation of the
mouse barrel cortex during development leads to increased
NG2-glia proliferation and results in their uniform distribution in the deprived barrels (60, 169), while physical
activity of adult mice leads to an immediate cell cycle exit of
NG2-glia and their differentiation into mature oligodendrocytes in the cerebral cortex (254). Based on the above
described observations, these effects could indeed be mediated via the direct synaptic contacts that are formed between neurons and NG2-glia, even though this still remains
to be proven (for review, see Ref. 243). Interestingly, NG2glia lose their synaptic input when differentiating into oligodendrocytes (167), and myelination has long been described to be regulated in an activity-dependent manner
(12, 57, 243, 300). Therefore, increased neuronal activity
seems to lead to increased generation of myelinating oligodendrocytes via NG2-glia proliferation and differentiation
even in the adult brain.
This has obvious functional consequences as increased myelination may improve the speed of information processing
in the active brain regions (22, 67), and new generation of
myelinating oligodendrocytes in the adult brain contributes
to myelin remodeling (299). Also in nerves that are fully
myelinated, such as the optic nerve, new myelinating oligodendrocytes are generated, suggesting some degree of myelin turnover (299). Intriguingly, this leads, at least in the
optic nerve, to a change in internode length (299) with clear
implications for conduction velocity (49, 60, 295). Thus
remodeling of myelin and the ability to generate myelinating oligodendrocytes also in the adult mammalian brain
provides new mechanisms for plasticity in the adult brain.
Indeed, major plastic changes, such as the response of neurons in the visual cortex to largely one eye (ocular dominance, OD, plasticity), were previously thought to come to
a permanent end after the critical period that was thought
to terminate when myelination is completed (in mice and
rats by about 2 months of age). However, recent studies
demonstrated that OD plasticity can still be elicited, e.g., by
silencing input from one eye in fully adult rodents at much
later stages, albeit at a slower pace (106, 107, 216). The
ongoing generation of new myelinating oligodendrocytes
thus contributes to adult neuronal plasticity by myelin remodeling, possibly allowing sprouting and collateral formation during this process. Compared with the forebrain,
other areas of the CNS, such as the spinal cord, show fewer
proliferating NG2-glia and lower numbers of newly generated oligodendrocytes (109, 118), either due to fewer cells
being able to perform these features or due to longer cell
cycle length and/or differentiation duration. An interesting
prediction would therefore be that, e.g., the spinal cord may
exhibit less pronounced adult plasticity. A further fascinating concept emerging from the above considerations is that
loss of myelinating oligodendrocytes, e.g., upon injury, may
contribute to reopen a window of opportunity for rewiring,
as observed for example after spinal cord injury where detour circuits allow some recovery of function (146). Most
excitingly, the discovery of proliferating NG2-glia also in
the adult human brain (80) suggests that these events also
take place in the human brain, thereby allowing not only for
neuronal plasticity but also revealing a source of actively
dividing progenitor cells which may be useful for repair
after injury. But do these cells have the capacity to generate
more than oligodendrocytes?
C. From Lineage to Potential: Are There
Neural Stem Cells Outside the
Neurogenic Niches in the Adult
Mammalian Brain?
1. In vivo
While all studies using birthdating and fate mapping of
NG2-glia in the adult mouse brain experiments agree on
them generating oligodendrocytes in several regions of the
adult mouse brain, the evidence for contribution to other
lineages has been more controversial. Rivers et al. (234)
detected some labeled neurons appearing and increasing in
number in the piriform cortex upon eliciting genetic recombination in PDGFR␣-CreERT2 mice; however, this observation could not be reproduced by the same lab (47). Indeed, also other labs using the very same mouse line had not
observed neurons emerging from PDGFR␣⫹ cells (118),
nor were they observed in the progeny of Olig2⫹ (61) or
NG2⫹ [308; See also Huang et al. (109a), NOTE ADDED IN
PROOF] cells. Therefore, evidence from genetic fate mapping
experiments converges on NG2-glia generating further
NG2-glia or oligodendrocytes but no other cell types in the
healthy adult brain and CNS.
Likewise, BrdU labeling experiments revealed only NG2glia shortly after labeling, while longer survival times revealed the generation of mature oligodendrocytes (254).
While neurons were sometimes found very close to BrdUlabeled NG2-glia in the neocortex (254), no double-labeled
neurons were observed. While others reported the conversion of NG2-glia into GABAergic neurons (54, 269), it is
important to bear in mind that BrdU incorporation also
Physiol Rev • VOL 94 • JULY 2014 •
labels cells undergoing DNA repair (for the pitfalls of BrdU
labeling, also see Ref. 63), and DNA repair processes have
been implicated in normal regulation of transcription, i.e.,
not necessarily requiring cell damage. Likewise, genetic fate
mapping has its own technical limitations such as side effects of tamoxifen, ectopic and low level of CreER expression in other cells (e.g., neurons in this case) becoming evident only after prolonged tamoxifen, and/or slow accumulation of the reporter gene activity. Therefore, it is best to
obtain evidence by multiple approaches which is the case
for NG2-glia generating oligodendrocytes or further NG2glial cells which has been confirmed also by transplantation
experiments (282). The lack of neurogenesis in most regions
outside the dentate gyrus/hippocampus region has also been
confirmed in the human brain by yet a different technique,
namely, carbon isotope birthdating (30, 261; see also Ernst
et al. (65a) NOTE ADDED IN PROOF).
The above data then suggest that the glial progenitors outside the neurogenic niches either cannot generate other lineages (such as neurons) or that they do not do so in their
local environment but could do so if they were not inhibited
by the local signals. Thus the failure of these cells to generate neurons may be due to the potent antineurogenic environment to which these cells are exposed. Consistent with
the latter scenario, transplantation experiments showed
that neuroblasts or even neural stem cells, i.e., cells that are
well capable to generate neurons normally, largely convert
to gliogenesis when transplanted into the adult brain parenchyma (for recent review, see Ref. 190). This highlights the
potent gliogenic environment NG2-glia are exposed to and
prompts the question if they may also have a broader potential when exposed to more favorable conditions, e.g., the
environment in the neurogenic niches.
Indeed, when cells derived from regions like the spinal cord
or the substantia nigra were transplanted into the region of
their origin, they generated largely NG2-glia, but when
transplanted into the neurogenic niche of the DG they seemingly gave rise to neurons (157, 250). These data suggest
that multipotent adult progenitor cells exist in the adult
CNS in a widespread manner and that these cells can generate different cell types depending on the local niche (neurogenic versus gliogenic) and the signals emerging from this
environment. However, it is again important to bear in
mind the technical limitations of these transplantation experiments. By now we know that transplanted cells can fuse
with cells of their environment (see, e.g., Refs. 34, 51), but
this has not been assessed in these previous transplantation
experiments. Moreover, in these experiments, cells had
been labeled by BrdU, such that their death may well have
provided neighboring proliferating cells with BrdU to incorporate into their DNA when dividing. Indeed, more recent
experiments revisiting whether glial cells (even cultured as
neurospheres and exhibiting therefore some in vitro stem
cell potential, such as self-renewal and multipotency; Ref.
252) can generate neurons when exposed to the neurogenic
SEZ, failed to detect any neuronal progeny from these cells,
suggesting that they may be unresponsive to the neurogenic
cues, at least those provided by the SEZ environment. Thus
the question of to what extent the widespread progenitors
present in the adult brain parenchyma can contribute to
other lineages in vivo still remains open.
2. In vitro
Conversely, in vitro evidence for some cells exhibiting the
potential of long-term self-renewal and multipotency, i.e.,
the generation of neurons, astrocytes, and oligodendrocytes, has been achieved by several labs. As described briefly
above, the neurosphere assay is a culture system well suited
to probe for neural stem cell potential (FIGURE 2). Several
decades ago Sam Weiss (232), Perry Bartlett (233), and
colleagues placed dissociated cells from the adult rat brain
at low density free-floating in medium with epidermal
growth factor (EGF) and/or basic fibroblast growth factor
(FGF2). Under these conditions, a few cells proliferated and
formed small spheres, the “neurospheres,” especially when
cells were isolated from the striatum. These cells can selfrenew for a long time as they form new spheres when dissociated and plated in the same medium conditions for
many passages (for recent review, see Ref. 208). To probe
their differentiation potential, neurosphere cells can then be
exposed to differentiation conditions by plating them on
adherent substrates and withdrawing the mitogens. While
most neurospheres can be obtained from the regions comprising adult NSCs, in particular the adult SEZ (see, e.g.,
Ref. 255 for direct comparison of SEZ and other regions), a
few neurospheres also emerge when cells were isolated from
different areas of the brain outside the neurogenic niches
like the striatum (206), the cortex (205), optic nerve, corpus
callosum, spinal cord, etc. (157, 251, 278, 292), with higher
numbers reported for cells isolated from rat compared with
mice (compare the above and Ref. 255). Interestingly, some
of these neurosphere-forming cells have been suggested to
express NG2 and Olig2, implying that some NG2-glia may
yield a larger potential when exposed to these favorable
conditions. However, so far no clear evidence for this was
obtained, e.g., by genetic fate mapping.
Neurosphere forming multipotent stem or progenitor cells
were also identified in the human and rodent WM (82, 199,
273). These cells mainly generate oligodendrocytes when
cultivated in high density and in the presence of serum or
are implanted into the adult brain in a demyelinating paradigm (293). In contrast, when these cells isolated from the
subcortical WM are cultivated in a low density and depleted
from serum, they are able to acquire multipotency and differentiate into neurons, astrocytes, and oligodendrocytes
(199). Taken together, these data suggest that a pool of
stem or progenitor cells with larger potential exists in the
adult WM, but these cells are restricted to generate glia due
to their local environment in vivo rather than their intrinsic
Physiol Rev • VOL 94 • JULY 2014 •
fate restriction. The exciting question now is to which extent these cells may provide a useful source of cells for repair
after brain injury when the environment changes dramatically.
A. Radial Glia in Vertebrate CNS Injury
Given the widespread neurogenesis persisting into adulthood in many vertebrates, these cells may provide the best
local source for neuronal repair after brain injury. Indeed,
the zebrafish as well as some other teleost and amphibian
species are well-known models for regeneration of many
organs (132), including scarless wound healing and neuronal repair after CNS injury (for review, see Refs. 15, 69,
1. Regeneration of neurons
Upon traumatic injury inflicted into the forebrain or cerebellum by insertion of a small cannula through the skull or
the nostrils, cells undergoing neurogenesis become activated and generate additional neurons that migrate to the
site of injury (6, 14, 121, 140, 172). Genetic fate mapping of
radial glial cells by CreERT2 expressed under the control of
the regulatory elements of the Notch target gene Her4 suggests that the additional neurons derive from the radial glial
cells or their immediate progeny still expressing sufficiently
high levels of the Her4-driven Cre recombinase (140). Consistent with this, both radial glial cells and their progeny,
the TAPs, transiently increase their proliferation (14, 172),
a response mediated by inflammatory signals resulting in
upregulation of the transcription factor Gata3 (131, 144).
Thus cells lining the ventricle in the adult zebrafish brain
and already undergoing neurogenesis become activated
upon injury and increase proliferation to generate additional neurons migrating to the injury site.
Generally, this reaction appears comparable to the response
to injury in the few sites in the mammalian brain where
radial glial cells and adult neurogenesis persist into adulthood as described above. Also there, neurogenesis is increased in response to traumatic or ischemic injury close by,
and various cell types are instructed to migrate from the
neurogenic niche to the injury site (5, 23, 40, 156). However, there are major differences between this reaction in the
mouse and the zebrafish forebrain. In the zebrafish model,
many of the neurons generated in addition to the baseline
neurogenesis survive long-term and the injury site disappears by scarless wound healing (6, 14, 140, 172). Conversely, few of the neuroblasts recruited to the injury
site next to the SEZ differentiate into fully mature
(DARPP32⫹) striatal projection neurons (5), and virtually
none survives longer (274). Importantly, SEZ cells do not
normally generate striatal projection neurons in these species but rather give rise to specific olfactory bulb interneurons in a highly region-specific manner (33, 160). It is thus
conceivable that neuroblasts recruited into the striatum
may not have the appropriate neuronal subtype specification and therefore eventually succumb to cell death. Moreover, ependymal cells that do not normally proliferate and
generate neuroblasts become activated upon stroke (40)
and may not complete the full neurogenic program, thereby
generating immature or misspecified neurons during this
injury-induced neurogenesis.
However, there is also evidence for plasticity of the type of
neurons generated after injury (for review, see Ref. 236). In
the few cases where new neurons with the adequate specification have been generated (or introduced by transplantation), their survival may be compromised by the environment faced in the site of injury, e.g., the scar. Indeed, direct
evidence for this has been obtained by specific neuron ablation models in the mouse which elicit little scar formation
and allow long-term survival of endogenous or transplanted new neurons (45, 163). These data further support
a decisive role of the scarless wound healing observed in the
zebrafish lesion models to allow long-term survival of the
regenerated neurons.
Strikingly, the scar persisting for long time after mammalian brain injury continues signaling to recruit new neurons even months after the insult. The adjacent neurogenic SEZ region still supplies large numbers of neuroblasts migrating to the injury site (274), demonstrating
persistent failure of these to survive and integrate even
after the acute reaction has vanished. Thus one key difference between mouse and zebrafish brain injury is the
scar formation which persists for months in the mammalian brain, while the injury site is no longer visible after
days or weeks in the zebrafish brain depending on the size
of the injury. An intriguing possibility for signaling the
need for new neurons is the lack of functional neuron
regeneration itself. Such a signaling feedback from intact
neurons has been unraveled in the salamander midbrain
where the lack of the neurotransmitter dopamine serves
to activate the generation of new dopaminergic neurons
(25, 26). Interestingly, longer range dopaminergic signals
from the brain to the spinal cord are involved in regulating regeneration of spinal cord motoneurons (230), and
other neurotransmitters also influence neurogenesis and
gliogenesis (for recent review, see Ref. 25). Of note, in the
regenerating spinal cord, the fibers releasing dopamine
serve to further enhance the region-specific, developmental signaling pathway important for motoneuron generation and regeneration, the SHH signal. SHH is reactivated after spinal cord injury in the radial glial cells that
then resume generation of motoneurons (229, 230).
Physiol Rev • VOL 94 • JULY 2014 •
Several important conclusions emerge from the above.
First, signals from intact functional neurons provide a feedback mechanism in regard to the needs for replacement.
Second, radial glia still lining the ventricle in nonmammalian vertebrates can reactivate adequate patterning cues resulting in neurogenesis of the appropriate neuronal subtypes (26, 229, 245) also in regions no longer undergoing
neurogenesis, as is the case in the salamander midbrain and
the zebrafish spinal cord or cerebellum. This is a profound
difference to the mammalian brain, where outside the adult
neurogenic niches no neurogenesis is initiated upon injury
consistent with the absence of radial glial cells at these sites.
However, these results observed in species with full regenerative potential prompt the search for a partial response in
the mammalian species with activation of some developmental signals and partial reactivation of NSC properties
(see below).
2. Scarless wound healing
Importantly, the persistence of radial glial cells has further
consequences also for scar formation in nonmammalian
vertebrates, as astrocytes are lacking in most of the regions
exposed to injury. This is important as activated, reactive
astrocytes surround the injury site in the mammalian CNS
(see below), while such cells are absent upon injury in species with radial glial cells instead of astrocytes. Indeed, radial glia cells largely retain their cell somata at the ventricular surface also after injury, and little to no radial migration to the injury site is detected (14), except after very large
injuries (140). Thus reactive astrogliosis as present in the
mammalian brain fails to occur in these models, and this
may contribute to create a favorable environment for neuronal replacement (see also Ref. 236 for further discussion).
However, a subset of reactive astrocytes in the mammalian
spinal cord were recently observed to be derived from cells
lining the ventricle, as occurs in zebrafish spinal cord, and
these have beneficial effects on neuronal survival and
wound contraction (242) reminiscent of the role of radial
glia-derived emigrating cells in the zebrafish. Similarly, also
NG2-glia appear to be largely absent or fail to be activated
in these injury models. While cells expressing Olig2 are
present in the adult zebrafish brain and spinal cord (14,
171, 207), few of these proliferate, and many seem to be
rather differentiated oligodendrocytes. Even after injury
few of these proliferate (14) with larger injuries eliciting an
accumulation of Olig2⫹ cells around the injury site (172). It
is noteworthy, however, that the CSPG4 recognized by the
NG2 antibody and abundantly present on Olig2⫹ cells in
mammalian brains, has not yet been detected on these cells
in nonmammalian vertebrates which may have consequences for neurite regeneration in these species.
Conversely, microglial cells readily activate their proliferation and accumulate around the injury site also in the zebrafish model (14, 140). However, their type of reaction
may well differ from those in the mammalian brain, where
both beneficial and adverse types of microglia and macrophage reactions have been identified (127, 182, 248). In line
with this, positive inflammatory signals after zebrafish
brain injury promote the proliferative response of radial
glial cells and seemingly promote neuronal repair in this
model rather than impeding regeneration as in mammals
(144). Thus the difference in the glial composition (no astrocytes, little activation of NG2-glia if at all) and the type
of their reaction (microglia activation/macrophage roles/
inflammatory response) in these models of scarless wound
healing provides first insights into the possible culprits for
scar formation in the mammalian CNS creating an adverse
environment for neuronal survival and process regeneration after injury.
B. NG2-Glia Reaction to Injury in the
Mammalian CNS
Given the above considerations, it is crucial to understand
the glial cell reaction after brain injury in the mammalian
brain to delineate beneficial from adverse roles. Indeed, the
major proliferative glial cell type in the mammalian brain,
the NG2-glia, has only recently been investigated in the
context of traumatic injury, as they were so far largely studied in the context of demyelinating conditions given their
predominant view as OPCs (41, 59, 123). However, NG2glia also show a striking reaction after traumatic injury in
the CNS with a very fast and huge increase in proliferation
within 1–3 days up to almost 100-fold their baseline proliferation rate (254). Such a traumatic CNS lesion is the stab
wound injury model that results in highly reproducible injury of a well-defined size. The size of the injury can be
chosen by the dimension of the cannula or small blade that
is penetrated into the respective CNS region. For example, a
punctate wound of 200 ␮m length or a stab wound of 1 mm
length can be generated by inserting different-sized blades
into the cerebral cortex (8). The ensuing cellular response is
limited to the injury tract and the immediate surrounding
tissue. Already within hours after injury proliferation of
microglia at the lesion edges and infiltration of the wound
site by activated macrophages occurs (84), followed by the
activation of NG2-glia undergoing hypertrophy, cell division, and migration within 24 – 48 h (254; von Streitberg
and Dimou, unpublished data; FIGURE 4). At later stages,
astrocytes react by hypertrophy and delayed proliferation
(8, 254 and below). While some of these proliferating NG2glia differentiate into mature oligodendrocytes, the majority of them remains as NG2-glia (61, 136), indicating some
degree of heterogeneity in their reaction to an injury insult
(see below and FIGURE 4). Interestingly, NG2-glia increase
in number and density at the site of injury (254), which is
particularly intriguing as their number and density is tightly
controlled by cell death and repulsive behavior in the
healthy brain, thereby maintaining a constant network of
NG2-glia under normal conditions (110).
Physiol Rev • VOL 94 • JULY 2014 •
Cortical grey matter
Cortical grey matter
FIGURE 4. Glial cell reaction after brain injury. Astrocytes and NG2-glia react after injury with hypertrophy
(swelling of the cell body and the main processes, orange), polarization (elongation of the processes, purple),
and proliferation (blue). While only a limited number of astrocytes react and in a slow manner, most NG2-glia
show a very fast reaction (nonreactive cells shown in green). Notably, only NG2-glia can migrate towards the
injury site (gray).
The original density is largely restored several weeks after
injury by a progressive decrease of the NG2-glia with increasing postlesion times (151, 254). Notably, not all injury
conditions are sufficient to overcome this strict homeostasis
(19, 110), prompting the search for the identity of the signals involved in NG2-glia homeostasis. Moreover, under
some lesion paradigms, NG2-glia also contribute to generate astrocytes (94, 272), as also observed when reactive
NG2-glia are cultured in vitro (unpublished observations).
This means that under certain conditions NG2-glia generate GFAP⫹ astrocytes in ectopic positions. It will be interesting to determine when and how, e.g., which subtype of
NG2-glia, generates this specific type of reactive astrocytes
as well as if and how these astrocytes derived from NG2glia differ from other astrocytes especially in regard to their
contribution to scar formation. Indeed, reactive astrocytes
derived from ependymal cell lining the spinal cord also perform specific and largely beneficial functions (242). In addition, the role of NG2-glia in neurite outgrowth and synapse formation has yet to be studied after injury, as these
cells are covered by the CSPG4 that has been shown to
block neurite outgrowth (for review, see Ref. 271). However, this protein also interacts with scaffolding proteins
anchoring transmitter receptors, such as GluR2/3 subunits
in the postsynaptic membrane (266), suggesting that it may
also act as an important factor for synapse formation or
function. Despite all the data published so far, much remains to be understood about the contribution of this novel
class of abundant glial cells to the traumatic injury reaction.
C. Astrocyte Reaction to Injury in the
Mammalian CNS
In contrast to the NG2-glia described above, astrocytes are
well studied after brain injury and their functional contribution to the injury reaction has been probed by various cell
ablation and gene deletion experiments (236, 259). Astrocytes serve as biosensors for almost any type of brain damage in neuropathology, based on their upregulation of intermediate filaments, such as GFAP, vimentin, nestin, and
synemin; proteins that are normally expressed at low levels
or entirely absent in adult astrocytes (for review see Refs.
209, 236, 259). Indeed, this is the most sensitive reaction of
astrocytes that occurs already in virtually all astrocytes
upon very small traumatic brain injury (8) and under all
injury conditions examined so far (255). The profound upregulation of GFAP also led to the concept that astrocytes
were the scar-forming cells as they increase in the vicinity of
the injury site, given that only few GFAP⫹ cells are present
Physiol Rev • VOL 94 • JULY 2014 •
prior to injury and many more thereafter. However, this
increase may rather reflect upregulation of GFAP than an
actual increase in astrocyte numbers as many, if not most
astrocytes do not express GFAP in many regions of the
healthy brain. Indeed, when monitored by immunohistochemistry for proteins also present in astrocytes prior to
injury (254), such as GS, S100␤, or Aldh1l1, by genetic fate
mapping (35) or live imaging (8), the total increase in astrocyte number is only 10 –20% in the adult mouse cerebral
cortex even after large stab wound injury. Importantly,
chronic live imaging of astrocytes after stab wound injury
revealed that astrocyte numbers increase solely by proliferation as reactive astrocytes did not migrate to the injury site
in vivo (FIGURE 4; Refs. 8, 187). Importantly, also the proliferative reaction of astrocytes is rather limited with only
few astrocytes (10 –20%) dividing a single time (8, 187).
This occurs in a rather delayed manner with proliferation
starting only at 3 days post injury (dpi) and peaking between 5 and 7 dpi (see also Ref. 254). Moreover, the few
dividing astrocytes reside in a special niche, as their soma is
in direct contact with the BM surrounding blood vessels,
suggestive of highly specific functions at this interface (8).
Thus a special subset of juxtavascular astrocytes divides
and increases astrocyte numbers largely in direct apposition
to the vasculature, while no astrocytes migrate towards the
site of GM injury in the cerebral cortex (for spinal cord
however, see Ref. 242). In contrast to the proliferating astrocytes showing a juxtavascular localization, proliferating
NG2-glia do not show any preference in their localization
(Dimou, unpublished observations). This is of interest as
after injury vascular endothelial growth factors (VEGF) released by endothelial cells have been linked with the proliferation and migration of NG2-glia. Indeed, it has been
shown in vitro that the behavior of postnatal OPCs can be
influenced by VEGFs, with VEGF-A to promote migration
and VEGF-C to enhance proliferation of OPCs (96, 150).
These data challenge the concept that astrocytes would considerably increase in number around the site of injury and
form the scar, even though they may of course contribute by
indirect means, e.g., via signaling mechanisms attracting
other cells. In support of this, a small reduction in astrocyte
numbers at the injury site, due to a decrease in their proliferation (8), is already sufficient to increase the number of
microglial cells in the injury site (235). Indeed, higher numbers of microglia and macrophages were also observed
upon genetic ablation of proliferating astrocytes in various
injury models using mice expressing thymidine kinase under the GFAP promoter (for review, see Ref. 259). In these
mice, proliferating astrocytes will be selectively killed when
ganciclovir is provided and is converted to an agent toxic
for proliferating cells by the enzyme selectively present in
reactive astrocytes. Intriguingly, this led to a significant increase in infiltrating macrophages and inflammatory processes, as well as a larger injury size and later blood-brain
barrier (BBB) closure (259). All of these effects are reminis-
cent of the location of the proliferating astrocytes at juxtavascular positions that are well positioned to regulate
macrophage invasion and inflammation as well as BBB closure. Interestingly, recent work highlighted different waves
and sources of invading macrophages. An earlier wave of
the classically activated macrophages (M1) performs more
adverse roles, while a later wave of invading M2 macrophages with acquired deactivation or alternative activation
exert more beneficial effects (182, 248). While the later
wave derives largely from the choroid plexus, the earlier
wave is recruited from the circulation (248), i.e., at the
interphase where the proliferating astrocytes are located
(8). Notably, other means to reduce astrocyte proliferation
after injury, such as by deleting STAT3 (100, 202, 288),
cdc42 (235), or RAS (242), also result in increased macrophage and/or microglia numbers, scar formation, and lesion
size. Interestingly, in the spinal cord, a specific subpopulation of reactive astrocytes has been observed to originate
from the cells lining the ventricle which has potent beneficial effects on injury size and compaction (242). Thus specific subsets of astrocytes perform apparently distinct functions after brain and spinal cord injury.
These considerations highlight two important new concepts. One is to which extent the subsets of astrocytes observed to exert distinct behaviors after cerebral cortex injury may differ intrinsically due to a distinct origin. Such as
the tanycyte/ependymal-derived reactive astrocytes in the
spinal cord differ from other parenchymal astrocytes (242),
also the juxtavascular astrocytes that constitute only a third
of all astrocytes but are the most proliferative subset after
stab wound injury in the cerebral cortex (8) may have a
distinct developmental origin as juxtavascular astrocytes
have been observed in distinct clones during early postnatal
development (76). This prompts the concept that, e.g., also
the further heterogeneity in the astrocyte reaction as observed by live imaging with some subsets not reacting at all,
even though located right next to the injury site (8) may
have a distinct developmental origin differing from, e.g., the
subset of astrocytes forming long polarized processes towards the injury site, the so-called palisading astrocytes
(200, 288). Alternatively or in addition, the local niche to
which these subtypes are exposed, like the close vicinity of
juxtavascular astrocytes to blood vessels, may expose them
to specific signals influencing their subtype-specific behavior [See also Martín-López et al. (169a) and NOTE ADDED IN
PROOF]. In this regard it is interesting that both astrocyte
subtypes with NSC in vitro potential identified in the cerebral cortex (255) and the spinal cord are (242) exposed to
specific niches reminiscent of the environment of NSCs in
the neurogenic zones, such as the vascular niche (255) and
the CSF (242).
The second and most important new concept is the functional relevance of astrocyte’s heterogeneity in reaction to
injury. If each of these astrocyte subsets serves a specific
Physiol Rev • VOL 94 • JULY 2014 •
functional role, identifying the signals or cell types regulating this subset may allow selectively influencing specific
types of reactive astrocyte functions and not others. Given
that reactive astrocytes with NSC properties seemingly exert beneficial functions as discussed above (235, 242, 255),
the CSF composition and vascular niche signals may provide prime candidates to foster these beneficial populations
selectively. Interestingly, specific sets of pericytes have been
recently implicated in formation of the fibrotic scar (86),
prompting ideas about reciprocal signaling between pericytes and juxtavascular astrocytes such that the latter could
interfere with pericyte infiltration and fibrotic scar formation in the brain and conversely pericyte subtypes may inhibit juxtavascular astrocyte proliferation. Thus the vascular niche as an important regulator of adult NSC proliferation in the SEZ and DG (for recent review, see Ref. 85) also
holds promises for mediating key properties for subsets of
reactive astrocytes including activation of their NSC-like
Indeed, reactive astrocytes reactivate several hallmarks of
radial glial cells that have been downregulated during their
differentiation, such as the intermediate filament nestin, the
extracellular matrix component tenascin-C, or the DSD1proteoglycan, all of which are normally absent in mature
astrocytes, but present in the radial glial cells of the developing and adult brain (209, 236, 259). Moreover, some
astrocytes proceed from a quiescent or postmitotic state to
a proliferative state as described above. However, despite
these changes, most genetically traced astrocytes remain in
their lineage in vivo (10, 35). However, when the potential
of these cells is probed by the neurosphere stem cell assay
mentioned above, there is a clear and significant increase in
in vitro multipotent and self-renewing NSCs derived from
genetically fate mapped cerebral cortex GM astrocytes (35,
255, 256) or tanycytes/ependymal cells lining the spinal
cord ventricle (10, 68). While this is only a very small fraction of reactive astrocytes in the cerebral cortex and ventricular cells in the spinal cord (68), neurosphere formation
from the stab wound injured or ischemic cerebral cortex
reaches about 1/5 to 1/10 of the NSC frequency in the adult
SEZ where endogenous actively proliferating stem cells reside (255). Moreover, the appearance of these reactive astrocytes with NSC potential correlates with the degree of
proliferation in vivo, with a peak at 5 dpi in stab wound
injury and a significant decline between 3 and 6 mo of age
(255). Indeed, SHH entering the brain from plasma and
CSF after traumatic injury has recently been identified as a
key factor regulating both astrocyte proliferation and NSC
potential (255). SHH acts directly on astrocytes, as conditional deletion of the receptor smoothened only in astrocytes potently reduced both their proliferation as well as
their neurosphere formation (255). In summary, these data
highlight different sets of glial cells, juxtavascular astrocytes in the cerebral cortex (255) and ventricular tanycytes
in the spinal cord (242), with the capacity to reactivate NSC
potential after invasive injury. This is important as these
cells with NSC hallmarks are local to the injury site and
hence equipped with the adequate region-specific identity,
thereby providing exciting sources for improving the injury
outcome in various aspects.
D. Different Injuries Lead to Different Glial
Reactions in the Tissue
While we focused above on traumatic injury paradigms, it is
important to consider the profound difference of glial reaction in distinct injuries, especially as there is a most profound effect on the proliferative response of reactive glia. As
discussed above, glial cells begin to proliferate in vivo in
response to various insults such as ischemia, trauma, and
models of Alzheimer’s or Parkinson’s disease (19, 35, 66,
254, 255, 298), but there are profound differences amongst
the different types of glial cells that proliferate. While microglial cells readily proliferate in virtually all injury conditions so far examined, the proliferation of NG2-glia and
astrocytes appears to be triggered largely by lesion conditions disrupting the tissue and BBB. Most strikingly, ablation of virtually half of all neurons in the adult mouse cerebral cortex by inducible expression of p25 in neurons fails
to elicit a proliferative response from either astrocytes or
NG2-glia (255). This happens despite an otherwise profound reactive gliosis with microglia activation and proliferation, astroglia upregulating GFAP, vimentin, nestin, and
many other hallmarks of reactive astrocytes as well as becoming hypertrophic together with NG2-glia (255). However, literally no reactive astrocyte proliferates and the
NG2-glia only continue to divide at their slow baseline rate
despite the death of so many neurons and the clearance of
their debris by highly proliferative activated microglia.
Indeed, also other conditions eliciting the typical hallmarks
of astrogliosis (hypertrophy, GFAP upregulation, etc.) but
failing to activate their proliferation have been described,
such as amyloid plaque deposition in mouse models of Alzheimer’s disease (19, 118, 255). A similar reaction is observed in inflammatory conditions induced, e.g., by lipopolysacharide injection mimicking bacterial infection with astrocytes failing to activate proliferation (257). Interestingly,
in both these conditions, NG2-glia react with increased proliferation (19, 257), demonstrating that the signals regulating the proliferative response of reactive astrocytes and
NG2-glia are at least in part distinct. Conversely, chronic
inflammatory conditions, such as experimental autoimmune encephalomyelitis (EAE), activate both astrocyte and
NG2-glia proliferation. Interestingly, in EAE conditions,
the BBB also becomes permeable for cells of the innate and
adaptive immune system (27, 258). This raises the important point of cellular invasion into the brain beyond diffusible signals such as SHH that can enter in case of BBB
disruption and is sufficient to elicit pronounced reactive
astrogliosis without any signs of neurodegeneration or in-
Physiol Rev • VOL 94 • JULY 2014 •
flammation (246). Cells invading either via the CSF, the
meninges, or the blood vessels act as the source for distinct
chemokine signals that activate specific parenchymal glial
cells and exert distinct effects on neuronal survival (see
below). Taken together, the type of injury, the signals, and
the cells recruited into the brain are crucial for eliciting
and regulating the proliferative response of astrocytes and
NG2-glia. Their proliferation is distinctly regulated in specific contexts only, while proliferation of microglia is activated in a broader spectrum of injury paradigms. These
selective signals regulating, e.g., NG2-glia but not astrocyte
proliferation in some disease settings allow a novel entry
point for highly innovative therapeutic approaches. For example, if NG2-glial cell accumulation around the injury site
contributes to scar formation, it may be beneficial to selectively inhibit their proliferation. On the other hand, blockage of NG2-glia proliferation could at the same time be
detrimental as these cells generate new oligodendrocytes,
that are responsible for the remyelinating capacity of the
tissue. Taking into consideration these both faces of the
medal, the best approach would be to selectively inhibit
proliferation and promote differentiation of NG2-glia. Importantly, selective influences on distinct glial subsets no
longer apply only to the main glial subtypes, but also to
their respective heterogeneous subpopulations that may
further help to delineate beneficial from adverse effects.
A. Wound Closure and Extracellular
The amazing specificity and heterogeneity in the glial cell
behavior after brain injury described above raises the importance of distinct functional contributions made by these
specific glial subtypes with the final aim of promoting specific beneficial subpopulations and reactions, e.g., those
promoting wound healing, while inhibiting the adverse
roles, such as scar formation.
Indeed, proof of concept for distinct functional contributions of cellular subsets has been validated for the microglia
and macrophage reactions with beneficial and adverse signaling pathways and subtypes being more and more clearly
discernible. For example, microglia activation can either
favor the release of neuroprotective factors or activate
phagocytotic and cell death-promoting subtypes and signaling pathways (95, 127, 248). Likewise, two major types of
macrophages invading the CNS after injury have been identified as discussed above (248) which influence not only
neuronal survival but also remyelination and differentiation of NG2-glia into mature oligodendrocytes in demyelinating conditions (182 and below). These are important
new insights to open novel avenues of limiting the responses
promoting adverse inflammatory signals and cell death
while promoting anti-inflammatory and prosurvival signals
after injury. Likewise, distinct functions seem to emerge for
the proliferative NG2-glia and astrocytes.
1. Fast NG2-glia reaction and function
NG2-glia ensheath a wound in the mouse cerebral cortex
very fast with their processes and NG2-glia numbers increase relatively fast within 2– 4 days by fast reentry into the
cell cycle (110, 254). This leads to a considerable increase in
their number surrounding the injury site in the cerebral
cortex GM. Similarly, an accumulation of NG2-glia surrounding the fibrotic scar has been observed in the injured
spinal cord, implicating these glial cells into functions previously allocated to reactive astrocytes: wound closure, inhibition of neurite outgrowth by inhibitory chondroitin
proteoglycan presentation, and scar formation by creating a
cell dense region enriched in glial cells.
2. Astrocyte reaction & function at early stages after
Also astrocytes show a fast reaction but neither by directing
processes towards the injury site nor by proliferation, which
are both reactions occurring at later stages. Rather, the very
first reactions of astrocytes to injury consist of cellular hypertrophy and gene expression changes (301). These imply
upregulation of the phagocytic protein machinery implying
astrocytes in helping to engulf damaged cell processes or
released proteins as well as helping to reinstall the extracellular milieu, e.g., by upregulation of glutamate transporters. Importantly, the most obvious and well-examined reaction of reactive astrocytes, the intermediate filament upregulation, plays a role in this context promoting delivery of
proteins to the cell surface (154, 236). This has been demonstrated by lower glutamate transport levels in mice lacking the intermediate filament proteins GFAP and vimentin
(154). Thus one may speculate that the early reaction of
astrocytes common to all injury conditions (hypertrophy
and IF upregulation) largely encompasses functions involved in removing aberrant cell debris, ions, amino acids,
etc., and reinstalling ion and protein homeostasis, possibly
an ancient function of glial cells (see chapt. 5 in Ref. 129).
Indeed, this is also a role exerted by astrocytes in noninvasive injury conditions, such as amyloid plaque deposition
where they contribute to reduce the plaque load (214). Interestingly, this function is also severely impaired when
GFAP and vimentin are genetically deleted and cannot be
upregulated by reactive astrocytes (138). Several other fast
contributions of astrocyte activation have been suggested
after inflammatory and ischemic stimuli (301), including
inhibition of bacterial growth, peptidase inhibitors etc.
Physiol Rev • VOL 94 • JULY 2014 •
3. Reactive astrocyte functions at later stages
In regard to the focus of this review on glial progenitors, it
is important to note that genome-wide expression analysis
of reactive astrocytes found genes promoting proliferation
activated with some delay in reactive astrocytes with a peak
between 3–5 days (301). This is in full agreement with the
observations made by BrdU or Ki67 immunostaining and
live imaging that show a peak of astrocyte proliferation in
this time window (8, 35, 254, 255, 288). As mentioned
above, this is also the time window when most neurosphereforming astrocytes are present after stab wound injury.
Given the predominant location of proliferating astrocytes
at juxtavascular positions in the vascular niche, this is suggestive of a role at this interphase, either regulating vascular
remodeling, reestablishment of certain BBB properties or
invasion of blood-born cells, such as lymphocytes or macrophages as discussed above. Most importantly, however,
the discovery of subsets of astrocytes in unique locations
undergoing specific changes (e.g., cell proliferation) not
only allows to better nail down their exact function but also
to delineate separate subsets of reactive astrocytes performing distinct functions (242). Thus the approach to modulate
the outcome of brain injury by improving wound healing,
reinstalling the extracellular milieu, and promoting neuronal cell survival and process outgrowth has become more
realistic by the delineation of distinct glial subsets proliferating at different times and supposedly devoted to very
different roles. These new insights from basic research open
new therapeutic avenues by modulating specific glial subtypes and functions.
B. Contribution of Reactive Glial Progenitors
to Neurite/Axonal Regeneration
In addition to helping neurons to survive an insult, also
their processes need to survive and reconnect beyond or
around the site of damage. During development, newly generated neurons are able to grow an axon while mature neurons after injury in the mammalian CNS are incapable of
self-repair and regeneration. When an axon is damaged, the
distal segment undergoes Wallerian degeneration (48, 123)
and demyelination, while the proximal segment dies by apoptosis or undergoes chromatolysis as an attempt for repair, often by forming retraction bulbs, plastic structures
that never extend beyond the injury site. Unfortunately, and
in contrast to PNS injury, injury in the CNS is not followed
by extensive regeneration, in particular when exposed to a
fibrotic scar and the extracellular matrix factors that altogether create an unfavorable environment for axonal regeneration, counteracting the repair of axons and myelin. To
exclude a limited intrinsic ability of mature neurons to grow
an axon after injury, permissive PNS bridges (e.g., sciatic
nerve grafts or Schwann cells; Ref. 21) have been implanted
in an injured spinal cord showing the regrowth of axons
and pointing to a regeneration inhibition solely by the en-
vironment. This inhibitory environment is comprised of the
glial scar and many extracellular matrix inhibitory molecules like chondroitin sulfate proteoglycans (for review, see
Refs. 4, 247) as well as inhibitory molecules expressed by
myelin such as Nogo-A, MAG, OMgp, ephrins, etc. (24, 46,
62, 287) or NG2-glia such as CSPG4 (270, 271) or the
astrocytic and/or fibrotic scar (86, 97). Genetic deletion or
blocking of several inhibitory molecules showed an improvement in axonal regeneration, but this was never sufficient to allow the majority of injured axons to regenerate,
pointing to a rather complex inhibitory machinery elicited
by the glial cells of the CNS. Interestingly, most of these
inhibitory molecules are localized in mature, differentiated
glial cells demonstrating that one aspect of maturation of
glial cells from a progenitor state is also to downregulate
neurite outgrowth-promoting aspects. This idea could be
endorsed after transplantation of glial progenitor cells, the
GRPs, which exerted a beneficial effect on axonal regeneration after spinal cord injury (3, 101), suggestive of additional roles of progenitor cells in instructing a more plastic
environment (as discussed above).
Notably, the relatively low extent of regeneration cannot
explain the level of recovery in function as observed in
rodents or primates after spinal cord injury. This could be
the result of newly generated multisynaptic connections between cortical and spinal motor neurons that could restore
function. Interestingly, following spinal cord injury, new
axonal branches (sprouts) extend from corticospinal axons
above the lesion site or from the intact contralateral side
that establish contacts with interneurons forming a relay
circuit restoring the input to segments below the injury site
(9, 273). These results suggest that rearrangements in the
spared circuitry provide an indirect mechanism to achieve
endogenous repair to some extent. Importantly, glial progenitors, in particular NG2-glia, may not only help to remyelinate damaged axons, but also allow sprouting and
plastic changes by remodelling myelin as discussed above.
C. Remyelination and Axonal Survival
NG2-glia play a prime role in conditions resulting in the
destruction of myelin (demyelination) where they mediate
successful remyelination providing a good example of regeneration also in the adult mammalian CNS. The most
common demyelinating disease in human is multiple sclerosis (MS), which is believed to occur due to a chronic inflammatory response that causes focal plaques in the brain and
spinal cord (148) as the result of an autoimmune response
towards myelin proteins (108) leading to the death of oligodendrocytes. Also after spinal cord injury, one of the
pathological outcomes is the demyelination of the axons at
the lesion site due to oligodendrocyte death through necrosis or apoptosis (65, 277). Although these two demyelination paradigms are very different, the reaction of NG2-glia
being recruited to the site of injury to remyelinate denuded
Physiol Rev • VOL 94 • JULY 2014 •
axons is common (81, 122, 176). Watanabe et al. (290)
could show that endogenous NG2-glia have the ability to
proliferate and differentiate in and around a chemically
induced demyelination lesion in the spinal cord, and similar
results could be obtained after transplantation of the very
same cells into a demyelination lesion or into myelin-deficient mice (91, 304). Interestingly, also after spinal cord
injury, NG2-glia strongly proliferate and produce a high
number of new oligodendrocytes (111, 176). In general, one
can conclude that endogenous NG2-glia proliferate after
demyelination or injury to enhance remyelination. However, this new myelin formation following demyelination is
not normal, with thinner and not so compacted myelin
sheaths, abnormal g-ratio (ratio of axon circumference to
myelin circumference), and shorter internode length (211;
for review, see Ref. 73). Although remyelination is not optimal, it is still beneficial by helping to protect the axons
from further damage or even degeneration and leads to an
increased conductance velocity even if it never reaches physiological levels (147). Thus, even in this example of successful regeneration, there remains ample room for improvement.
A better understanding of the signals regulating NG2-glia
proliferation, migration, and differentiation provides an
obvious entry point to improve full myelination. In this
regard, it is important to remember the profound regional
heterogeneity in myelination capacity between WM and
GM derived NG2-glia (282). For example, if many NG2glia surrounding the demyelination foci migrated from the
GM, the cells that are not intrinsically very potent to myelinate, then myelination cannot proceed to completion and
the NG2-glia may well fail to repair the damaged myelin
sheaths in a satisfactory manner, prompting the question
which could be the factors overcoming this limitation
and/or recruit NG2-glia from the WM. Possible additional
explanations may be an inhibitory environment at the site
of demyelination that does not promote differentiation or
the lack of growth factors and signals that would lead to the
complete remyelination. An additional pitfall is that although remyelination in acute demyelinating lesions is
pretty effective, this is not the case in chronically demyelinated lesions. Again, different hypotheses have been suggested for the limited remyelination after chronic demyelination: 1) depletion of progenitors by time (173), 2) axon
damage due to the chronic demyelination (31, 227), and/or
3) age-dependent block of NG2-glia proliferation and differentiation (253). The latter idea of the age-associated decrease in remyelination efficiency is associated with an impairment of NG2-glia recruitment as well as their limited
differentiation into remyelinating oligodendrocytes (253).
These age-dependent changes could be due to the different
expression of growth factors influencing the behavior of
NG2-glia and to the changes in the inflammatory processes
associated with demyelination (72). Further studies are
needed to resolve the inability of NG2-glia to efficiently
repair myelin after demyelination that could then provide
possible therapeutic approaches.
Notably, in addition to the NG2-glia, also Schwann cells,
the myelin-forming cells of the PNS, have been suggested as
a source of remyelinating cells (11, 71, 155). A recent study
could show that after a chemically induced demyelination
injury Schwann cells do not migrate from the PNS, as speculated before, but are directly generated by PDGFR␣⫹
NG2-glia resident around the injury site (303), further supporting the important role of NG2-glia for the remyelinating properties of the CNS.
D. Neuronal Replacement From Glial Stem
and Progenitor Cells
Glial cells and their progenitors play multiple roles helping
to alleviate neurological defects after brain injury, not the
least by promoting axonal and neuronal function and
thereby fostering neuronal survival. However, once neurons have degenerated and been lost, could any of the above
glial progenitors actually be used to replace the lost neurons? And if so, can glial progenitors help to allow the
regenerated neurons connecting to their appropriate target
As discussed above, reactive glial cells by and large do not
generate new neurons endogenously in vivo in the injured
brain. This is the case for both NG2-glia as well as the
proliferating reactive astrocytes that all largely remain
within their respective lineage in vivo (35, 61, 118, 236).
However, they provide a pool of stem and progenitor cells
that are local to the injury site and could hence be instructed
towards neurogenesis. Indeed, genome-wide expression
analysis of adult NSCs and astrocytes isolated from various
forebrain regions showed that adult NSCs are more similar
to mature astrocytes and ependymal cells from neighboring
regions than to neurogenic radial glial cells from the developing brain (16). Thus cells with a predominant astroglial
gene expression profile can act as neurogenic NSCs. The
crucial differences are that the NSCs within the neurogenic
niches are primed towards neurogenesis by expressing already, albeit at low levels, key neurogenic fate determinants, such as Pax6, Dlx2, Sox11, Sox4, and others (16,
191). Accordingly, upregulation of such factors in reactive
astrocytes that resumed proliferation already should be sufficient to instruct them towards neurogenesis. This was first
shown by forced expression of the transcription factor
Pax6, which surprisingly succeeded to instruct some proliferating glial cells towards neurogenesis within the stab
wound injured cerebral cortex (36) or the ischemic striatum
(141). Moreover, factors efficient in converting even fibroblasts into neurons also proved successful in the striatum in
vivo when targeted to astrocytes in the healthy brain (196,
276). Likewise, transcription factors of the proneural basic
helix-loop-helix family Neurog2 or Ascl1 are sufficient to
Physiol Rev • VOL 94 • JULY 2014 •
instruct local glial progenitor cells (most likely NG2-glia as
they are the only population proliferating in the uninjured
CNS) towards neurogenesis in the spinal cord and cerebral
cortex (88, 201). Combination with factors promoting proliferation and dedifferentiation, such as EGF and FGF2,
further potentiates this response both in the spinal cord and
the ischemic or stab wound injured forebrain (88, 201),
supporting the concept that proliferation and neurogenic
fate determinants are key in instructing neurogenesis from
local glial stem and progenitor cells. Thus proliferating glial
cells within the injury site provide a local source of cells for
repair including the replacement of neurons.
This is particularly exciting given the regionalization of glial
cells, in particular the astrocytes as described above. Proliferating reactive astrocytes may hence be easier to instruct
towards the neuronal subtype appropriate for the respective
brain region compared with other NSCs derived from the
SEZ or the SGZ that are specialized to generate different
types of neurons, namely, olfactory bulb interneurons or
dentate gyrus granule cells. Moreover, glial cells may hold
the key to overcome also the other major problem still faced
in such attempts to replace degenerated neurons, namely,
the long-term survival of these newly regenerated neurons.
Especially after brain injury, the number of the originally
elicited neurons profoundly decreases over weeks with few
of them surviving (36, 88). But is longer survival and successful integration of new neurons into the adult brain parenchyma at all possible? The answer is yes, as shown when
an environment beneficial for survival and integration of
new neurons is provided. This is the case when neurons die
in an apoptotic manner and little reactive gliosis is elicited
(45, 163). In such conditions, both endogenously recruited
cells or neuroblasts isolated from the same region (the developing cerebral cortex) and transplanted after ablation of,
e.g., corticospinal neurons, readily differentiate into mature
neurons that send their axons for long distances throughout
the entire brain even to the spinal cord (45). Indeed, young
neurons can extend axonal processes also in the adult brain
and white matter as they are not repelled, e.g., by the
growth inhibitory components of myelin (52). These neurons survive for many months, clearly demonstrating that
young neurons can reconnect even over long distances and
integrate and survive in an adult brain, if the environment is
sufficiently supportive. However, in the context of severe
brain injury with reactive gliosis, this has not yet been
achieved. Thus a key task for the future is to understand
which glial subtypes create an adverse environment for survival of new neurons and best utilize these to turn them into
neurons by neurogenic fate determinants. Taken together,
glial stem and progenitor cells at the injury site not only
provide a local progenitor source amenable to fate conversion towards neurogenesis allowing replacement of lost
neurons, but also hold the key to provide a favorable environment for neuronal survival (see also Ref. 242) and suc-
cessful integration. And this key lies in understanding and
utilizing the diversity of glial subtypes.
Given the above exciting new avenues for repair, it is now
important to unravel which subsets of reactive glia are closest to neurogenesis and can be best turned into the appropriate neuronal subtype. Considering the similarity between adult NSCs and astrocytes or radial glia and
tanycytes, these may be the proliferating subset of reactive
astrocytes or cells lining the ventricle. Moreover, these cells
retain their regional specification. However, utilizing these
cells for neuronal replacement further supports the need to
understand their endogenous role after brain injury, e.g., in
wound or BBB repair or helping neuronal survival. Given
that they indeed exert a beneficial role, one strategy could
be to amplify these proliferative reactive glia with NSC
potential first, such that sufficient cells are still available
even after turning some into new neurons.
An alternative strategy is to turn reactive glial subtypes with
adverse and scar forming functions into new neurons,
thereby improving the environment for neuronal survival
and integration and eliciting neurogenesis locally. To which
extent NG2-glia may be this cell type remains to be experimentally tested. If the early reacting NG2-glia perform important functions in wound closure, a strategy could be to
target them with neurogenic factors with some delay first
allowing them to mediate wound closure and then turning
the additional NG2-glia that are anyhow subject to later
reduction into neurons. Importantly, the enormous capacity of NG2-glia for homeostasis will most likely lead to a
repopulation of the brain even after turning a considerable
fraction of these cells into a different cell type, in this case
neurons. Thus a sufficient number of NG2-glia will be again
available to fulfil their normal functions in the healthy
brain, such as activity-dependent communication and providing newly generated oligodendrocytes to myelinate the
axons of regenerated long-distance projection neurons.
Taken together, the new insights into glial cell diversity now
open new avenues towards best utilizing the regional, temporal, and subtype diversity of glial cells for beneficial influences and repair purposes, but much still remains to be
understood about this diversity to put it to its best use.
The following important publications appeared after the
last revision of the review: Alunni et al. (4a), Ernst et al.
(65a), Huang et al. (109a), and Martín-López et al. (169a).
Address for reprint requests and other correspondence:
M. Götz, Physiological Genomics, LMU, Pettenkoferstr.
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