**Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment**

Emília Madarász

[61] Steiner, B., Klempin, F., Wang, L., Kott, M., Kettenmann, H. and Kempermann, G. (2006). Type-2 cells as link between glial and neuronal lineage in adult hippocampal

[62] Stump, G., Durrer, A., Klein, A. L., Lutolf, S., Suter, U. and Taylor, V. (2002). Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell popula‐

[63] Suh, H., Consiglio, A., Ray, J., Sawai, T., D'Amour, K. A. and Gage, F. H. (2007). In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural

[64] Suhonen, J. O., Peterson, D. A., Ray, J. and Gage, F. H. (1996). Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature. 383,

[65] Tavazoie, M., Van der Veken, L., Silva-Vargas, V., Louissaint, M., Colonna, L., Zaidi, B., Garcia-Verdugo, J. M. and Doetsch, F. (2008). A specialized vascular niche for

[66] van Praag, H., Shubert, T., Zhao, C. and Gage, F. H. (2005). Exercise enhances learn‐ ing and hippocampal neurogenesis in aged mice. J Neurosci 25, 8680-8685.

[67] Venere, M., Han, Y. G., Bell, R., Song, J. S., Alvarez-Buylla, A. and Blelloch, R. (2012). Sox1 marks an activated neural stem/progenitor cell in the hippocampus. Develop‐

[68] Wilson, A., Laurenti, E., Oser, G., van der Wath, R. C., Blanco-Bose, W., Jaworski, M., Offner, S., Dunant, C. F., Eshkind, L., Bockamp, E., Lio, P., Macdonald, H. R. and Trumpp, A. (2008). Hematopoietic stem cells reversibly switch from dormancy to

[69] Zhao, C., Deng, W. and Gage, F. H. (2008). Mechanisms and functional implications

ment. 139, 3938-3949. doi: 3910.1242/dev.081133. Epub 082012 Sep 081119.

self-renewal during homeostasis and repair. Cell. 135, 1118-1129.

tions in the postnatal mouse brain. Mech Dev 114, 153-159.

stem cells in the adult hippocampus. Cell Stem Cell 1, 515-528.

adult neural stem cells. Cell Stem Cell. 3, 279-288.

of adult neurogenesis. Cell 132, 645-660.

neurogenesis. Glia. 54, 805-814.

624-627.

44 Neural Stem Cells - New Perspectives

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55678

#### **1. Introduction**

Criteria of "neural stemness" characterize a large number of terminally non-differentiated neural tissue cells. Neural stem/progenitor cells capable for asymmetric mitoses (resulting in a similar and a differently committed daughter cell which may adopt neuronal or glial phenotypes in further development) are present during the entire life-span of vertebrates and have been found in almost all regions of the brain. With the advancement of neural tissue genesis and maturation, more and more stem/progenitor-like cells adopt "quiescent" states, but can be activated by appropriate (yet not properly understood) stimuli. Besides asymmetric (stem cell specific) division, these cells can multiply by symmetric mitoses resulting in identical progenies. Self-renewal and symmetric multiplication are responsible for maintaining or expanding stem/progenitor populations at the actual stage of neural commitment. Expanded pools of cells with similar, but yet flexible developmental potential can provide the desired number and type of cells for genesis, maintenance and repair of the nervous tissue. Except the ontogenetically and phylogenetically "oldest" pioneer and/or large projection-type neurons [1], the majority of neural tissue cells are produced through successive stem/progenitor stages [2]. The extreme cellular diversity of the mature CNS implies huge diversity in the precursor populations. Accordingly, a large number of neural stem/progenitor populations exist in different stages of neural cell fate commitment and display different cellular characteristics, developmental capability and flexibility. "Quiescent" and actively proliferating stem cells, transient amplifying progenitor populations and migrating or resident progenitor/precursor cells reside at various "niches" including the "professional" neurogenic zones, migratory routes and the neural parenchyma, as well. Drifts in cell biological features and differentiation potential of stem/progenitor/precursor cells are implemented by the advancement of devel‐ opment, by the position along the body axes and by the physiological or pathophysiological

© 2013 Madarász; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

changes of the local environment including neighbouring cells, extracellular matrix, metabo‐ lite-, oxygen- and growth factor-supply and activity-driven ionic composition.

and the non-neural ectodermal ridge of the epiblast. The positions along the anterior (A) and posterior (P) body axes -

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment

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47

During neural folding, a single layer of proliferating pre-neural stem cells called *radial neuroepithel cells* compose the neural plate and rapidly enlarge the neural primordium. These cells will give rise to primary neural stem cells, the *embryonic radial glia* (Fig.2), during the formation and closing of the neural tube (7-10 pcd in mouse). While the anterior cells of the epiblast of vertebrates were shown to generate neurons, if separated from their natural environment, ("default neural fate" [9]), or by blocking mesoderm-inducing morphogen actions, the mechanisms behind the transition of radial neuroepithel cells to radial glia cells

Embryonic radial glial cells line the lumen of the neural tube in a single cell layer and compose the primary neural germinative zone (ventricular zone; VZ). The first neurons are generated in this layer by asymmetric mitoses of radial glial cells (Fig.2). Such division produces a daughter cell, which preserves radial glia characteristics and another one, a neuronal precur‐ sor, which looses proliferation capability and migrates outward from the lumen along a radial glia neighbour [10]. These early neuronal precursors will develop mainly to large, projecting type neurons [1]. Immediate cell-to cell interactions including Notch/Delta signalling [11] and growth factor – receptor signals (as neuregulin – ErbB; [12]) play inevitable roles in the determination and maintenance of asymmetric commitment of the two daughter cells.

**Figure 2.** Formation of the primary neurogenic zone from radial neuroepithelial cells and generation of the first post‐

mitotic neuronal precursors by asymmetric division of radial glial cells in the ventricular zone.

imply regional differences among neuroectodermal cells.

are not properly understood.

With improvement of methodologies, different populations of NS cells have been isolated, propagated and investigated *in vitro*. Despite of rapidly accumulating data, however, the similarities and differences among various NS populations are not properly understood either *in vivo* or *in vitro.* This chapter intends to give an insight in the intrinsic varieties of NSC populations. As examples, fairly distinct features of in vitro propagated early embryonic and adult-derived mouse neural stem/progenitor populations will be presented.

#### **2. Diversity of neural stem cells from early embryonic tissue genesis to adult-hood cell production**

#### **2.1. Neural stem cells in the neural plate and in primary geminative zones**

The earliest neural stem populations, the cylindrical neuroepithelial cells of the early embry‐ onic neural plate (Fig.1) appear in the embryonic disc soon after embedding. As it was learned from studies on chick embryos, the anterior epiblast cells acquire "pre-neural" fate [3] as it is indicated by Sox2 expression [4] under the influence of morphogen gradients established by two early organizers, the anterior visceral endoderm (AVE) [5] and the primitive streak and later by the node. The different morphogen concentrations result in different activation of positional (region-specific) genes, predestinating regions for different future fate in both chick and mammalian embryos [6,7] (Fig.1). Accordingly, despite of the apparent morphological homogeneity [8], neuroepithelial cells are not identical from the very beginning of neural tissue genesis.

**Figure 1.** Scheme of formation and regionalization of the neural plate. The neural plate forms from the anterior epi‐ blast in response to morphogens produced by the anterior visceral entoderm (AVE), the primitive streak and nodus

and the non-neural ectodermal ridge of the epiblast. The positions along the anterior (A) and posterior (P) body axes imply regional differences among neuroectodermal cells.

changes of the local environment including neighbouring cells, extracellular matrix, metabo‐

With improvement of methodologies, different populations of NS cells have been isolated, propagated and investigated *in vitro*. Despite of rapidly accumulating data, however, the similarities and differences among various NS populations are not properly understood either *in vivo* or *in vitro.* This chapter intends to give an insight in the intrinsic varieties of NSC populations. As examples, fairly distinct features of in vitro propagated early embryonic and

**2. Diversity of neural stem cells from early embryonic tissue genesis to**

The earliest neural stem populations, the cylindrical neuroepithelial cells of the early embry‐ onic neural plate (Fig.1) appear in the embryonic disc soon after embedding. As it was learned from studies on chick embryos, the anterior epiblast cells acquire "pre-neural" fate [3] as it is indicated by Sox2 expression [4] under the influence of morphogen gradients established by two early organizers, the anterior visceral endoderm (AVE) [5] and the primitive streak and later by the node. The different morphogen concentrations result in different activation of positional (region-specific) genes, predestinating regions for different future fate in both chick and mammalian embryos [6,7] (Fig.1). Accordingly, despite of the apparent morphological homogeneity [8], neuroepithelial cells are not identical from the very beginning of neural tissue

**Figure 1.** Scheme of formation and regionalization of the neural plate. The neural plate forms from the anterior epi‐ blast in response to morphogens produced by the anterior visceral entoderm (AVE), the primitive streak and nodus

lite-, oxygen- and growth factor-supply and activity-driven ionic composition.

adult-derived mouse neural stem/progenitor populations will be presented.

**2.1. Neural stem cells in the neural plate and in primary geminative zones**

**adult-hood cell production**

46 Neural Stem Cells - New Perspectives

genesis.

During neural folding, a single layer of proliferating pre-neural stem cells called *radial neuroepithel cells* compose the neural plate and rapidly enlarge the neural primordium. These cells will give rise to primary neural stem cells, the *embryonic radial glia* (Fig.2), during the formation and closing of the neural tube (7-10 pcd in mouse). While the anterior cells of the epiblast of vertebrates were shown to generate neurons, if separated from their natural environment, ("default neural fate" [9]), or by blocking mesoderm-inducing morphogen actions, the mechanisms behind the transition of radial neuroepithel cells to radial glia cells are not properly understood.

Embryonic radial glial cells line the lumen of the neural tube in a single cell layer and compose the primary neural germinative zone (ventricular zone; VZ). The first neurons are generated in this layer by asymmetric mitoses of radial glial cells (Fig.2). Such division produces a daughter cell, which preserves radial glia characteristics and another one, a neuronal precur‐ sor, which looses proliferation capability and migrates outward from the lumen along a radial glia neighbour [10]. These early neuronal precursors will develop mainly to large, projecting type neurons [1]. Immediate cell-to cell interactions including Notch/Delta signalling [11] and growth factor – receptor signals (as neuregulin – ErbB; [12]) play inevitable roles in the determination and maintenance of asymmetric commitment of the two daughter cells.

**Figure 2.** Formation of the primary neurogenic zone from radial neuroepithelial cells and generation of the first post‐ mitotic neuronal precursors by asymmetric division of radial glial cells in the ventricular zone.

The early radial glial cells express several "marker" features (Table 1), which together with cell shape and localization may identify them. In contrast to later neural stem/progenitor populations, radial neuroepithelial and early radial glial cells express Oct4 and nanog embryonic stem cell genes and the anterior epiblast-characterizing Otx2 and En1,2 "positional" genes (see ref. in Table 1). These cells span the whole thickness of the early neural tube, and can divide without changing their spanned shape [8].

**"Marker" features References**

[refs in 1]

http://dx.doi.org/10.5772/55678

49

[23, 24 ]

receptors at the apical and basal ends; [10, 8]

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment

with spanned shape [15, 8]

epiblast and sporadically in the forming neural plate) [16]

neural plate; later restricted to the forebrain regions) [17, 18]

neural plate; later restricted to isthmus and mesencephalic, cerebellar areas) [19, 20]

neural stem cells) [4,21,22]

specific cell differentiation) [25]

transcription fragment (NICD) [11]

ErbB (Neuregulin Trk receptor); [12]

GLAST (Glutamate-aspartate transporter; astrocytes also express) [26]

molecules including small nuclear ligand molecules) [27]

cell shape Elongated, bipolar shape; Adherent junctions at the lumen-face; laminin

cellular motion interkinetic nuclear migration; mitotic nuclei at the lumen face; cell division

vimentin (characterizes many non-differentiated cells)

GFAP (characterizes astrocytes; expression by radial glia cells is species-

RC (1,2) nestin-associated protein-epitope; radial glia marker in the CNS

Oct3/4, nanog (embryonic stem cell/pluripotency genes; expressed in the

Otx2 (anterior positional gene; expressed in the anterior epiblast and in the

En1,2 (engrailed positional gene; expressed in the anterior epiblast, in the

Sox2 (codes for a HMG box transcription factor; expressed generally by CNS

Pax6 (codes for a paired-box transcription factor; plays important roles in CNS patterning and eye development; expressed by multiple neural stem/

Hes1,3,5 (code for a basic helix-loop-helix transcription factor repressing tissue-

Notch1,3 (protein product: cell surface receptor with cleavable intracellular

BLBP (brain lipid binding protein; Intracellar transporter of hydrophobic

nestin (expressed by many progenitor-type cells including muscle progenitors)

intermediate filament proteins

> master-gene expression

Cell surface receptors and transporters

Intracellular transporter

dependent )

progenitor cells)

**Table 1.** Radial glia "marker" features for characterization of radial glial cells

These early neural stem cells proliferate rapidly (with cycle time of about 14-16 hours), but not continuously, and with both symmetric and asymmetric divisions. The symmetric mitoses assure the expansion and maintenance of neural stem populations at the given stage of commitment. Intermittent non-mitotic periods help the attachment and out-migration of neuronal precursors. During the formation and closure of the tube, the neural primordium is composed by a single neurogenic zone, but with heterogeneous cellular constituents [13] comprising asymmetrically and symmetrically dividing stem/progenitor cells, and migrating, differentiating neuronal precursors.

The cellular diversity of the early neural tube is further enhanced by the time-delay in development along the anterior – posterior and dorso-ventral body axes. The tube formation and closure proceed with a delay from the zone of brachial arches (the region for future hind brain) to both, rostral and caudal directions, and the production and maturation of neural cells on the ventral face always precede those on the dorsal part in each neural domain [14]. It means that even small samples of the tissue will contain diverse populations of (stem/amplifying/ differentiating) neural cells.

The cellular heterogeneity is further increased by the ongoing regional specification, which results in well-distinguishable domains with characteristic gene expression patterns along both the anterio-posterior and dorso-ventral axes of the growing CNS [28, 29]. Differential expression of positional master genes results in diverging expression patterns of "down-hill" genes including those coding for adhesion receptors and extracellular matrix proteins, and leads to the formation of morphological boundaries between the expression domains: the developing CNS is composed by segments called neuromeres [7, 30]. Along the A-P body axis, transversal segments will delineate primary brain vesicles (prosencephalon, mesencephalon, rhombencephalon) first, and smaller neuromeres later on. Inside the larger segment bounda‐ ries, smaller developmental / morphological entities will develop as the prosomeres in the telencephalon or rhombomeres in the hindbrain. In parallel with the anterior-posterior segmentation, dorso–ventral specification will identify longitudinal domains in the develop‐ ing CNS. Dorso–ventral specification is initiated by the early lateral–medial determination of the neural plate, namely, the midline expression of sonic hedgehog and the lateral expression of bone morphogenic (BMP) proteins (Fig.3). With tube formation, progenies of midline plate cells become the ventral-most tube cells composing the notoplate. Derivatives of lateral-most neural plate cells will compose the neural crest and the closing dorsal lip of the tube, the later roof plate. Notoplate cells produce Shh, while roof plate cells produce BMPs and Wnt morphogenic factors, establishing dorso-ventral morphogen gradients throughout the developing CNS (for a recent review: [31]).

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment http://dx.doi.org/10.5772/55678 49


**Table 1.** Radial glia "marker" features for characterization of radial glial cells

The early radial glial cells express several "marker" features (Table 1), which together with cell shape and localization may identify them. In contrast to later neural stem/progenitor populations, radial neuroepithelial and early radial glial cells express Oct4 and nanog embryonic stem cell genes and the anterior epiblast-characterizing Otx2 and En1,2 "positional" genes (see ref. in Table 1). These cells span the whole thickness of the early neural tube, and

These early neural stem cells proliferate rapidly (with cycle time of about 14-16 hours), but not continuously, and with both symmetric and asymmetric divisions. The symmetric mitoses assure the expansion and maintenance of neural stem populations at the given stage of commitment. Intermittent non-mitotic periods help the attachment and out-migration of neuronal precursors. During the formation and closure of the tube, the neural primordium is composed by a single neurogenic zone, but with heterogeneous cellular constituents [13] comprising asymmetrically and symmetrically dividing stem/progenitor cells, and migrating,

The cellular diversity of the early neural tube is further enhanced by the time-delay in development along the anterior – posterior and dorso-ventral body axes. The tube formation and closure proceed with a delay from the zone of brachial arches (the region for future hind brain) to both, rostral and caudal directions, and the production and maturation of neural cells on the ventral face always precede those on the dorsal part in each neural domain [14]. It means that even small samples of the tissue will contain diverse populations of (stem/amplifying/

The cellular heterogeneity is further increased by the ongoing regional specification, which results in well-distinguishable domains with characteristic gene expression patterns along both the anterio-posterior and dorso-ventral axes of the growing CNS [28, 29]. Differential expression of positional master genes results in diverging expression patterns of "down-hill" genes including those coding for adhesion receptors and extracellular matrix proteins, and leads to the formation of morphological boundaries between the expression domains: the developing CNS is composed by segments called neuromeres [7, 30]. Along the A-P body axis, transversal segments will delineate primary brain vesicles (prosencephalon, mesencephalon, rhombencephalon) first, and smaller neuromeres later on. Inside the larger segment bounda‐ ries, smaller developmental / morphological entities will develop as the prosomeres in the telencephalon or rhombomeres in the hindbrain. In parallel with the anterior-posterior segmentation, dorso–ventral specification will identify longitudinal domains in the develop‐ ing CNS. Dorso–ventral specification is initiated by the early lateral–medial determination of the neural plate, namely, the midline expression of sonic hedgehog and the lateral expression of bone morphogenic (BMP) proteins (Fig.3). With tube formation, progenies of midline plate cells become the ventral-most tube cells composing the notoplate. Derivatives of lateral-most neural plate cells will compose the neural crest and the closing dorsal lip of the tube, the later roof plate. Notoplate cells produce Shh, while roof plate cells produce BMPs and Wnt morphogenic factors, establishing dorso-ventral morphogen gradients throughout the

can divide without changing their spanned shape [8].

differentiating neuronal precursors.

48 Neural Stem Cells - New Perspectives

differentiating) neural cells.

developing CNS (for a recent review: [31]).

(short progenitors; SNPs) or both (inner progenitors; ISVZ progenitors), and accordingly, change morphology and developmental characteristics [34, 8]. The number of primary radial glial cells decreases gradually, while their derivatives dividing at a distance from the luminar surface generate a novel germinative zone, the subventricular zone (SVZ). While a few SNP cells remain in contact with the luminar surface and thought to preserve "ancient" (primary) stem cell properties, the layer lining the ventricle wall transforms to the future ependyma and neural cell production is transposed to the secondary germinative layer, the SVZ. The SVZs along the entire neuraxis generate large number of progenitors and precursors including smaller projecting-type neurons, interneurons, astrocytes, oligodendrocytes and produces also enough stem cells to maintain an appropriate neurogenic capacity (for review see [1]). Novel precursors migrate from the SVZs to final destinations along defined routes in the developing neural parenchyma. While the lineage relations among different SVZ progenitors are not fully explored [8, 35], it is clear that evolutionary new SVZ progenitors provide the cell generating capacity for the enlarged interneuron and glia populations of the avian and mammalian CNS and provide cell-pools for the enormous expansion of the cerebellar cortex and the mammalian forebrain. With the appearance of SVZs, the developing CNS comprises a large variety of coexisting neural stem/progenitor cells both, in multiple neurogenic zones and inside / around

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment

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51

While secondary (SVZ) germinative zones form all along the wall of brain ventricles and the spinal canal, the time of formation and the cell productivity of these zones are not uniform along the neuraxis. At most parts of the developing CNS, SVZs generate neuronal and glial precursors for local tissues, and the cell-generating activity decreases with the maturation of the local neural parenchyma. Such zones, still containing "resting" stem/progenitor cells, can restore cell-generating activity in response to tissue loss or specific (not yet properly under‐ stood) physiological/pathophysiological stimuli. The most productive secondary germinative zones, including the ventral forebrain SVZ [36], the external germinative layer (EGL) of the cerebellum [37] and the subgranular zone (SGZ) of the hippocampus [38], however, generate huge amounts of novel neural cells and function for a relatively long time, in case of the ventral

The SVZ at the latero-ventral wall of the forebrain vesicles forms in a relatively early period (second embryonic week in mouse) of forebrain development [36]. It derives from the primary germinative (VZ) zone of the highly regionalized subpallial ganglionic eminences (LGE, MGE and CGE,) [39]. The enhanced cell production with preserved regional characteristics results in the formation of a variety of neuronal progenitors/precursors for developing subcortical and cortical tissue formations [39]. This zone generates neurons for the striatum and globus pallidus, provides the vast majority of interneurons of the cortex [40, 41], produces small connecting neurons for the ventral amygdala complex [42, 43] and, exclusively in humans, also for the dorsal thalamus [44]. Large number of forebrain oligodendrocytes also derives from

In contrast to the early appearance of the ventral forebrain SVZ, the external germinative layer (EGL) of the cerebellum appears around birth [47]. Secondary stem/progenitor cells from the dorsal lip of the IVth ventricle migrate on the top of developing Purkinje cells, and produce

the cell migratory routes.

forebrain SVZ and the SGZ, for the entire life-time.

the ventral SVZ complex [45, 46].

**Figure 3.** Schematic presentation of the establishment of dorso–ventral specification (longitudinal segmentation) in the neural tube. The lateral-medial (L-M) regionalization turns into dorso-ventral (D-V) regionalization when the neu‐ ral plate folds and forms the neural tube. BMP: bone morphogenic proteins; SHH: sonic hedgehog protein; Zic, Gli-3, Pax6, Emx2, Dlx, Nkx are examples of genes expressed in different dorso-ventral domains.

The position along the antero-posterior and dorso–ventral axes determines the identity of future brain regions and seems to predict the phenotype of neurons through the orchestrated expression of defined region-specific, pro-neural and neuron-specific genes [28]. Boundaries between embryonic segments provide routes for the elongation of pioneer axons and delineate the paths for future fiber tracks.

For the time being, it is not clear whether the early embryonic position can (or how far can) determine the intrinsic developmental potential of individual cells. There are contradictory results on the "positional memory" of stem/progenitor cells if removed from their original position [32]. Our own data [33] indicate that neural stem-like cells isolated from early embryonic (E9) mouse forebrain do not display regional "memory" after in vitro propagation. In the course of in vitro neuron formation they express divergent region-specific genes, those not expressed in overlapping regions of the developing CNS. The data suggest that permanent presence of region-specifying factors is required for maintaining regional commitment, at least in case of early embryonic neural stem cells. This finding, however, does not compromise the fact that in vivo, neural stem/progenitor cells of the primary germinative (VZ) zone display important molecular and cell biological differences.

#### **2.2. Diversity of stem/progenitor cells in the secondary germinative zones of the developing brain**

With the thickening of the neural wall, increasing number of proliferation-capable cells loose contact either with the lumen-face (outer progenitors; OSVZ progenitors) or the pial surface (short progenitors; SNPs) or both (inner progenitors; ISVZ progenitors), and accordingly, change morphology and developmental characteristics [34, 8]. The number of primary radial glial cells decreases gradually, while their derivatives dividing at a distance from the luminar surface generate a novel germinative zone, the subventricular zone (SVZ). While a few SNP cells remain in contact with the luminar surface and thought to preserve "ancient" (primary) stem cell properties, the layer lining the ventricle wall transforms to the future ependyma and neural cell production is transposed to the secondary germinative layer, the SVZ. The SVZs along the entire neuraxis generate large number of progenitors and precursors including smaller projecting-type neurons, interneurons, astrocytes, oligodendrocytes and produces also enough stem cells to maintain an appropriate neurogenic capacity (for review see [1]). Novel precursors migrate from the SVZs to final destinations along defined routes in the developing neural parenchyma. While the lineage relations among different SVZ progenitors are not fully explored [8, 35], it is clear that evolutionary new SVZ progenitors provide the cell generating capacity for the enlarged interneuron and glia populations of the avian and mammalian CNS and provide cell-pools for the enormous expansion of the cerebellar cortex and the mammalian forebrain. With the appearance of SVZs, the developing CNS comprises a large variety of coexisting neural stem/progenitor cells both, in multiple neurogenic zones and inside / around the cell migratory routes.

While secondary (SVZ) germinative zones form all along the wall of brain ventricles and the spinal canal, the time of formation and the cell productivity of these zones are not uniform along the neuraxis. At most parts of the developing CNS, SVZs generate neuronal and glial precursors for local tissues, and the cell-generating activity decreases with the maturation of the local neural parenchyma. Such zones, still containing "resting" stem/progenitor cells, can restore cell-generating activity in response to tissue loss or specific (not yet properly under‐ stood) physiological/pathophysiological stimuli. The most productive secondary germinative zones, including the ventral forebrain SVZ [36], the external germinative layer (EGL) of the cerebellum [37] and the subgranular zone (SGZ) of the hippocampus [38], however, generate huge amounts of novel neural cells and function for a relatively long time, in case of the ventral forebrain SVZ and the SGZ, for the entire life-time.

**Figure 3.** Schematic presentation of the establishment of dorso–ventral specification (longitudinal segmentation) in the neural tube. The lateral-medial (L-M) regionalization turns into dorso-ventral (D-V) regionalization when the neu‐ ral plate folds and forms the neural tube. BMP: bone morphogenic proteins; SHH: sonic hedgehog protein; Zic, Gli-3,

The position along the antero-posterior and dorso–ventral axes determines the identity of future brain regions and seems to predict the phenotype of neurons through the orchestrated expression of defined region-specific, pro-neural and neuron-specific genes [28]. Boundaries between embryonic segments provide routes for the elongation of pioneer axons and delineate

For the time being, it is not clear whether the early embryonic position can (or how far can) determine the intrinsic developmental potential of individual cells. There are contradictory results on the "positional memory" of stem/progenitor cells if removed from their original position [32]. Our own data [33] indicate that neural stem-like cells isolated from early embryonic (E9) mouse forebrain do not display regional "memory" after in vitro propagation. In the course of in vitro neuron formation they express divergent region-specific genes, those not expressed in overlapping regions of the developing CNS. The data suggest that permanent presence of region-specifying factors is required for maintaining regional commitment, at least in case of early embryonic neural stem cells. This finding, however, does not compromise the fact that in vivo, neural stem/progenitor cells of the primary germinative (VZ) zone display

**2.2. Diversity of stem/progenitor cells in the secondary germinative zones of the developing**

With the thickening of the neural wall, increasing number of proliferation-capable cells loose contact either with the lumen-face (outer progenitors; OSVZ progenitors) or the pial surface

Pax6, Emx2, Dlx, Nkx are examples of genes expressed in different dorso-ventral domains.

the paths for future fiber tracks.

50 Neural Stem Cells - New Perspectives

**brain**

important molecular and cell biological differences.

The SVZ at the latero-ventral wall of the forebrain vesicles forms in a relatively early period (second embryonic week in mouse) of forebrain development [36]. It derives from the primary germinative (VZ) zone of the highly regionalized subpallial ganglionic eminences (LGE, MGE and CGE,) [39]. The enhanced cell production with preserved regional characteristics results in the formation of a variety of neuronal progenitors/precursors for developing subcortical and cortical tissue formations [39]. This zone generates neurons for the striatum and globus pallidus, provides the vast majority of interneurons of the cortex [40, 41], produces small connecting neurons for the ventral amygdala complex [42, 43] and, exclusively in humans, also for the dorsal thalamus [44]. Large number of forebrain oligodendrocytes also derives from the ventral SVZ complex [45, 46].

In contrast to the early appearance of the ventral forebrain SVZ, the external germinative layer (EGL) of the cerebellum appears around birth [47]. Secondary stem/progenitor cells from the dorsal lip of the IVth ventricle migrate on the top of developing Purkinje cells, and produce basket and stellar neurons and billiards of cerebellar granule cells [37] for the cerebellar cortex. The third "professional" secondary neurogenic zone of the mammalian brain is the subgra‐ nular zone (SGZ) of the hippocampus [38], which derives from the ventricular (primary germinative) zone of the most dorsal forebrain structure, the hem [48, 49]. In addition to astrocytes and oligodendrocytes, SGZ produces a single type of projecting neurons, the granule cells of the dentate gyrus.

resident neural cells can re-enter the cell cycle [72], can renew themselves and generate

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While such cells are regarded as neural stem cells according to "neural stemness" criteria, the time of birth, the route of migration, the conditions for settlement and quiescence are far from clear. The multiplicity of CNS derived stem/progenitor cells has been indicated also by the diversity of neural stem/progenitor cells investigated in vitro [74]; the data argue for a massive presence of tissue-resident neural cells with "stemness" properties throughout the CNS. To find single scattered quiescent cells in the healthy brain tissue or to determine their lineage relations are hard tasks, even with the use of transgenic reporter mice [35]. These neural stem / progenitor cells may have different origin, may represent different stages of neural cell differentiation and may adopt different cell physiological characteristics depending on the host environment. As they serve as resident progenitor pools for tissue repair and limited remodelling, it is of primary importance to explore their physiological characteristics and environmental requirements in order to understand local neural tissue reactions to physio‐

different – more than one - types of mature neural cells in vivo [69, 71, 73].

**3. Neural stem cells in vitro: Experimental data on diverse clones**

In vivo lineage analyses can hardly determine whether divergent fate decision could be made by a single cell or diversity was resulted by selective interactions inside a group of mixed cells [75]. Sophisticated cell isolation/propagation methods helped to prove the existence of multipotent neural stem cells. In vitro studies can describe neural stem cell features appearing under artificial conditions and can predict the *potential* phenotypes which can be acquired by the investigated cells. The in vivo fate and the phenotype manifested under in vivo conditions, however, can not be forecasted from in vitro data. For the time being, our knowledge on local microenvironmental factors governing the fate of differentiating neural cells or our under‐ standing on characteristics and cell physiological requirements of differentiating cells do not

Neural stem/progenitor cells had been isolated and investigated in many laboratories using different methods and divergent source materials including developing or adult brain tissues and embryonic stem (ES) cells. Excellent recent reviews summarize the collected data includ‐ ing the expression of various gene clusters, the inducibility of different neuronal phenotypes and the growth factor requirements of various neural stem/progenitor preparations (for a recent review see [76]). Recent knowledge on differentiation-dependent changes in require‐ ments for O2 and adhesive environment has been also summarized [77]. The diverse experi‐ mental approaches, however, hinder comparative characterization of neural stem/progenitor

Based on more than 10 different neural stem cell clones isolated and characterized in our laboratory, this chapter presents a comparative summary on important differences displayed by one-cell derived neural stem cell populations isolated from different ages and/or from

populations derived from different CNS regions and developmental ages.

logical stimuli and various pathophysiological effects.

allow extrapolating in vitro data to in vivo cell fates.

different regions of the mouse brain.

#### **2.3. Neural stem/progenitor cells in the adult mammalian brain**

At most sites, the subventricular zones cease producing neurons and reduce their gliaproduction after a short postnatal period of normal development, except the external germi‐ native layer (EGL) of the cerebellum, where cerebellar granule cells are generated for at least 10 days after birth in mouse and 1-2 years in human [47], the SVZ of the ventral forebrain [50] and SGZ of the hippocampus [51], where neuro- and gliogenic capacity is maintained for the entire life-time. The adult SVZ generates neuronal precursors mainly for regularly remodelling forebrain circuits, as the higher voice centre of singing birds [52] or the olfactory bulb in rodents [53]. Incorporation of adult SVZ-derived novel neurons, however, had been reported also in the neocortex, piriform cortex, olfactory tubercle and amygdala in rodents and in primates, as well [42, 43,54, 55]. In contrast to the SVZ, available data indicate that SGZ produces precursors solely to the dentate gyrus. Both, the SVZ [46] and the SGZ [51, 56], however, contain consec‐ utive descendent populations of progenitor cells, which seem to mutually regulate each other's generation [57].

The routes of migration and integration of newly generated "adult" neuronal precursors into olfactory and hippocampal networks have been explored in many details [51, 53, 58, 59, 60], and the exploration has been highly facilitated by the use of multiple transgenic reporter mouse straits (reviewed in [35]). Novel neurons are integrated by functioning neuronal networks and tune the physiological parameters of functional circuits in both, the olfactory bulb [61] and the hippocampus [59, 60]. As it is widely accepted, continuous neurogenesis in the adult SVZ and SGZ plays organic roles in the physiological performance of the olfactory bulb and the hippocampus of mammals. In case of SVZ-derived precursors, however, further studies should explore the mechanisms behind the detachment of novel progenitors from the common migratory paths and the acquirement of different mature phenotypes.

Outside of adult neurogenic zones and migratory routes, developing novel neurons have been described in many regions of the adult brain, both in rodents (for ref. see [62]) and primates [54]. Local neuron formation was found at the striatum [63], hypothalamus [64], associate cortex [54, 65], ventral forebrain [43] and the substantia nigra of the midbrain (for review see [66]. Beside sporadic neuron formation, ongoing astroglia production and low-rate but permanent oligodendroglia replacement [67] also indicate that differentiation-capable neural stem/progenitor cells are scattered throughout the adult nervous tissue. Many of these progenitors might derive from continuously active ("professional") adult neurogenic zones [1, 45]. Quiescent cells with stem/progenitor capabilities however are present in the wall of brain ventricles and spinal canal [68] along the entire neuraxis, in large fibre bundles [69, 70] and in the functioning CNS parenchyma [71], as well. In response to stimuli, many quiescent tissueresident neural cells can re-enter the cell cycle [72], can renew themselves and generate different – more than one - types of mature neural cells in vivo [69, 71, 73].

basket and stellar neurons and billiards of cerebellar granule cells [37] for the cerebellar cortex. The third "professional" secondary neurogenic zone of the mammalian brain is the subgra‐ nular zone (SGZ) of the hippocampus [38], which derives from the ventricular (primary germinative) zone of the most dorsal forebrain structure, the hem [48, 49]. In addition to astrocytes and oligodendrocytes, SGZ produces a single type of projecting neurons, the

At most sites, the subventricular zones cease producing neurons and reduce their gliaproduction after a short postnatal period of normal development, except the external germi‐ native layer (EGL) of the cerebellum, where cerebellar granule cells are generated for at least 10 days after birth in mouse and 1-2 years in human [47], the SVZ of the ventral forebrain [50] and SGZ of the hippocampus [51], where neuro- and gliogenic capacity is maintained for the entire life-time. The adult SVZ generates neuronal precursors mainly for regularly remodelling forebrain circuits, as the higher voice centre of singing birds [52] or the olfactory bulb in rodents [53]. Incorporation of adult SVZ-derived novel neurons, however, had been reported also in the neocortex, piriform cortex, olfactory tubercle and amygdala in rodents and in primates, as well [42, 43,54, 55]. In contrast to the SVZ, available data indicate that SGZ produces precursors solely to the dentate gyrus. Both, the SVZ [46] and the SGZ [51, 56], however, contain consec‐ utive descendent populations of progenitor cells, which seem to mutually regulate each other's

The routes of migration and integration of newly generated "adult" neuronal precursors into olfactory and hippocampal networks have been explored in many details [51, 53, 58, 59, 60], and the exploration has been highly facilitated by the use of multiple transgenic reporter mouse straits (reviewed in [35]). Novel neurons are integrated by functioning neuronal networks and tune the physiological parameters of functional circuits in both, the olfactory bulb [61] and the hippocampus [59, 60]. As it is widely accepted, continuous neurogenesis in the adult SVZ and SGZ plays organic roles in the physiological performance of the olfactory bulb and the hippocampus of mammals. In case of SVZ-derived precursors, however, further studies should explore the mechanisms behind the detachment of novel progenitors from the common

Outside of adult neurogenic zones and migratory routes, developing novel neurons have been described in many regions of the adult brain, both in rodents (for ref. see [62]) and primates [54]. Local neuron formation was found at the striatum [63], hypothalamus [64], associate cortex [54, 65], ventral forebrain [43] and the substantia nigra of the midbrain (for review see [66]. Beside sporadic neuron formation, ongoing astroglia production and low-rate but permanent oligodendroglia replacement [67] also indicate that differentiation-capable neural stem/progenitor cells are scattered throughout the adult nervous tissue. Many of these progenitors might derive from continuously active ("professional") adult neurogenic zones [1, 45]. Quiescent cells with stem/progenitor capabilities however are present in the wall of brain ventricles and spinal canal [68] along the entire neuraxis, in large fibre bundles [69, 70] and in the functioning CNS parenchyma [71], as well. In response to stimuli, many quiescent tissue-

migratory paths and the acquirement of different mature phenotypes.

granule cells of the dentate gyrus.

52 Neural Stem Cells - New Perspectives

generation [57].

**2.3. Neural stem/progenitor cells in the adult mammalian brain**

While such cells are regarded as neural stem cells according to "neural stemness" criteria, the time of birth, the route of migration, the conditions for settlement and quiescence are far from clear. The multiplicity of CNS derived stem/progenitor cells has been indicated also by the diversity of neural stem/progenitor cells investigated in vitro [74]; the data argue for a massive presence of tissue-resident neural cells with "stemness" properties throughout the CNS. To find single scattered quiescent cells in the healthy brain tissue or to determine their lineage relations are hard tasks, even with the use of transgenic reporter mice [35]. These neural stem / progenitor cells may have different origin, may represent different stages of neural cell differentiation and may adopt different cell physiological characteristics depending on the host environment. As they serve as resident progenitor pools for tissue repair and limited remodelling, it is of primary importance to explore their physiological characteristics and environmental requirements in order to understand local neural tissue reactions to physio‐ logical stimuli and various pathophysiological effects.

#### **3. Neural stem cells in vitro: Experimental data on diverse clones**

In vivo lineage analyses can hardly determine whether divergent fate decision could be made by a single cell or diversity was resulted by selective interactions inside a group of mixed cells [75]. Sophisticated cell isolation/propagation methods helped to prove the existence of multipotent neural stem cells. In vitro studies can describe neural stem cell features appearing under artificial conditions and can predict the *potential* phenotypes which can be acquired by the investigated cells. The in vivo fate and the phenotype manifested under in vivo conditions, however, can not be forecasted from in vitro data. For the time being, our knowledge on local microenvironmental factors governing the fate of differentiating neural cells or our under‐ standing on characteristics and cell physiological requirements of differentiating cells do not allow extrapolating in vitro data to in vivo cell fates.

Neural stem/progenitor cells had been isolated and investigated in many laboratories using different methods and divergent source materials including developing or adult brain tissues and embryonic stem (ES) cells. Excellent recent reviews summarize the collected data includ‐ ing the expression of various gene clusters, the inducibility of different neuronal phenotypes and the growth factor requirements of various neural stem/progenitor preparations (for a recent review see [76]). Recent knowledge on differentiation-dependent changes in require‐ ments for O2 and adhesive environment has been also summarized [77]. The diverse experi‐ mental approaches, however, hinder comparative characterization of neural stem/progenitor populations derived from different CNS regions and developmental ages.

Based on more than 10 different neural stem cell clones isolated and characterized in our laboratory, this chapter presents a comparative summary on important differences displayed by one-cell derived neural stem cell populations isolated from different ages and/or from different regions of the mouse brain.

#### **3.1. Lessons from primary brain cell cultures**

From the middle of the last century, "primary" neural cell cultures gave a statistical insight into the cellular composition of many studied brain area and gained high importance in understanding the cellular characteristics of neural tissue constituents. It became clear that neuron-enriched cultures can be easily initiated from early stages of development (embryonic brain material), while tissue samples from later stages of brain maturation provide mainly glial cultures.

embryonic source tissue, the larger frequency and rapid proliferation of non-differentiated cells allow isolating "almost primary" neural stem/progenitor cells after short-term in vitro propaga‐ tion. Using the "serial splitting" method [82], we established and cloned various neural stem cell lines from E9 - E11.5 mouse forebrain vesicles from, both, p53-/- [83] and wild-type embryos.

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55

While non-differentiated cells from the early embryonic (E9-12) brain attach readily to poly-L-lysine or collagen coated surfaces in the presence of serum, cells isolated from more developed (E14-15) mouse forebrain form aggregates under such conditions indicating that the surface of neighbouring cells provide better adherence than the provided artificial surface. The majority of cells exist inside the aggregates (e.g. in completely unknown microenviron‐

Rapid attachment of freshly seeded cells from "older" forebrain suspensions was achieved, if pre-patterned integrin-ligands were provided as adherent surfaces. Brush-like peptideconjugates built on a poly-L-lysine backbone and carrying rigid integrin-ligands (cyclic RGDfC pentapeptides) at the N-termini of regularly spaced poly-D/L lanine "spacer" side-chains (AKc[RGDfC]) [84] proved to support the attachment and serum-free propagation of a number of non-differentiated cells including mouse and human ES cells (manuscript in preparation). As neurons do not attach to the peptide-coated surface and differentiated glial cells do not proliferate in serum–free conditions, non-differentiated cells of brain cell suspensions could be selectively propagated under serum-free conditions on the peptide-coated surface (Fig.5). From forebrain suspension of transgenic mouse embryos (E14.5) carrying the human GFAPpromoter-driven GFP construct [85], GFP-labelled but GFAP-negative cells grew rapidly on AK-c(RGDfC)-coated surfaces indicating that the surface-attached populations comprised

**Figure 5.** Embryonic (E 14.5) mouse*hGFAP-GFP* forebrain cells on the 3rd day on poly-L-lysine coated surface with serum (A) and on AK-c(RGDfC) coated surface without serum (B). After two passages on AK-c(RGDfC) sureum and in serum-free conditions, the cultures were composed by GFP-expressing morphologically homogeneous, surface-attached cells (C).

ments), for a 2-3 day period before large-scale migration starts from the aggregates.

neural progenitor-type cells (Fig.5) [74].

As terminally differentiated neurons and oligodendrocytes do not divide and display poor regeneration potential, primary neural cell cultures gave a hint on the presence and distribu‐ tion of non-differentiated cells in the brain tissue, which served as precursors for the majority of cultured neurons and oligodendroglia cells. The in vitro preservation of many neuronal characteristics of the source region including the size, shape, neurotransmitter phenotypes and specific vulnerability of neurons [78] indicates either a relatively stable fate commitment of in vitro surviving progenitors/precursors or continued production of factors, which help to maintain some region-specific features.

In case of astrocytes, usually identified solely by GFAP expression, it was clear that in vitro propagation results in a "juvenile", "de-differentiated" phenotype, fairly distinct from in vivo astroglial cells [79]. In purified (90-95% GFAP-positive) mouse astroglial cultures many cells express nestin, and a few of them express also SSEA-1 stem cell antigen (Fig.4). After longer (2 -3 weeks) propagation, "epithel dome"-like structures of rapidly dividing cells and GFAPnegative process-bearing O2A bipotential glial progenitor cells [69] often appear in purified astroglial cultures (Fig.4) [80,81].

**Figure 4.** Non-differentiated cells in purified cultures of newborn (P1) mouse forebrain astroglial cells. Left: a SSEA-1 and GFAP double-immureactive cell on the 7th day in vitro. Middle: an "epithel dome"-like cellular expansion on the 21st day in vitro. Right: O2A-type progenitor cells in a 15-day old hypothalamic astrocyte culture.

As long-term propagation selects for rapidly proliferating cells, the size of such populations can be enlarged and one-cell derived clones may also emerge from such cells. Proliferation-based cloning from long-term propagated cultures, however, favours the selection for tumorigen, transformed cells and also, hinders the identification of in vivo origin of selected cells.

If primary neural cultures are prepared form early embryonic (E9-12 mouse) forebrain vesicles, clusters of rapidly proliferating non-differentiated cells occur with high frequency. From early embryonic source tissue, the larger frequency and rapid proliferation of non-differentiated cells allow isolating "almost primary" neural stem/progenitor cells after short-term in vitro propaga‐ tion. Using the "serial splitting" method [82], we established and cloned various neural stem cell lines from E9 - E11.5 mouse forebrain vesicles from, both, p53-/- [83] and wild-type embryos.

**3.1. Lessons from primary brain cell cultures**

54 Neural Stem Cells - New Perspectives

maintain some region-specific features.

astroglial cultures (Fig.4) [80,81].

cultures.

From the middle of the last century, "primary" neural cell cultures gave a statistical insight into the cellular composition of many studied brain area and gained high importance in understanding the cellular characteristics of neural tissue constituents. It became clear that neuron-enriched cultures can be easily initiated from early stages of development (embryonic brain material), while tissue samples from later stages of brain maturation provide mainly glial

As terminally differentiated neurons and oligodendrocytes do not divide and display poor regeneration potential, primary neural cell cultures gave a hint on the presence and distribu‐ tion of non-differentiated cells in the brain tissue, which served as precursors for the majority of cultured neurons and oligodendroglia cells. The in vitro preservation of many neuronal characteristics of the source region including the size, shape, neurotransmitter phenotypes and specific vulnerability of neurons [78] indicates either a relatively stable fate commitment of in vitro surviving progenitors/precursors or continued production of factors, which help to

In case of astrocytes, usually identified solely by GFAP expression, it was clear that in vitro propagation results in a "juvenile", "de-differentiated" phenotype, fairly distinct from in vivo astroglial cells [79]. In purified (90-95% GFAP-positive) mouse astroglial cultures many cells express nestin, and a few of them express also SSEA-1 stem cell antigen (Fig.4). After longer (2 -3 weeks) propagation, "epithel dome"-like structures of rapidly dividing cells and GFAPnegative process-bearing O2A bipotential glial progenitor cells [69] often appear in purified

**Figure 4.** Non-differentiated cells in purified cultures of newborn (P1) mouse forebrain astroglial cells. Left: a SSEA-1 and GFAP double-immureactive cell on the 7th day in vitro. Middle: an "epithel dome"-like cellular expansion on the

As long-term propagation selects for rapidly proliferating cells, the size of such populations can be enlarged and one-cell derived clones may also emerge from such cells. Proliferation-based cloning from long-term propagated cultures, however, favours the selection for tumorigen,

If primary neural cultures are prepared form early embryonic (E9-12 mouse) forebrain vesicles, clusters of rapidly proliferating non-differentiated cells occur with high frequency. From early

21st day in vitro. Right: O2A-type progenitor cells in a 15-day old hypothalamic astrocyte culture.

transformed cells and also, hinders the identification of in vivo origin of selected cells.

While non-differentiated cells from the early embryonic (E9-12) brain attach readily to poly-L-lysine or collagen coated surfaces in the presence of serum, cells isolated from more developed (E14-15) mouse forebrain form aggregates under such conditions indicating that the surface of neighbouring cells provide better adherence than the provided artificial surface. The majority of cells exist inside the aggregates (e.g. in completely unknown microenviron‐ ments), for a 2-3 day period before large-scale migration starts from the aggregates.

Rapid attachment of freshly seeded cells from "older" forebrain suspensions was achieved, if pre-patterned integrin-ligands were provided as adherent surfaces. Brush-like peptideconjugates built on a poly-L-lysine backbone and carrying rigid integrin-ligands (cyclic RGDfC pentapeptides) at the N-termini of regularly spaced poly-D/L lanine "spacer" side-chains (AKc[RGDfC]) [84] proved to support the attachment and serum-free propagation of a number of non-differentiated cells including mouse and human ES cells (manuscript in preparation). As neurons do not attach to the peptide-coated surface and differentiated glial cells do not proliferate in serum–free conditions, non-differentiated cells of brain cell suspensions could be selectively propagated under serum-free conditions on the peptide-coated surface (Fig.5). From forebrain suspension of transgenic mouse embryos (E14.5) carrying the human GFAPpromoter-driven GFP construct [85], GFP-labelled but GFAP-negative cells grew rapidly on AK-c(RGDfC)-coated surfaces indicating that the surface-attached populations comprised neural progenitor-type cells (Fig.5) [74].

**Figure 5.** Embryonic (E 14.5) mouse*hGFAP-GFP* forebrain cells on the 3rd day on poly-L-lysine coated surface with serum (A) and on AK-c(RGDfC) coated surface without serum (B). After two passages on AK-c(RGDfC) sureum and in serum-free conditions, the cultures were composed by GFP-expressing morphologically homogeneous, surface-attached cells (C).

The "selective adhesion and serum free propagation" method allowed isolating and cloning cell lines from various parts of the embryonic (E 14-15) and adult (P50-75) mouse brains (Table 2).

Multiple differences were found among the characterized cell lines indicating important

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Early embryonic neuroectoderm-derived (NE-4C, NE-7C2 and WNE) cells display epithel morphology (Fig.6), and produce uniform monolayers. These cells divide continuously (16 hour cycle-time in average) on poly-L-lysine in 10% FCS containing medium, but can not be propagated in serum-free conditions on AK-c(RGDfC) coated surfaces. NE cells express nestin intermedier filamentum protein [82,86] and two-third of the cells carries the mouse stem cell antigen, SSEA-1 [88]. In non-induced stage, the cells express Oct4, carry *blbp* and *glast* mRNAs (proteins could not be demonstrated), but do not express *pax6*, *mash1* or *neurogenin*s and do

In contrast to NE cells, embryo-derived AK-c(RGDfC)-adherent cells (RGl-1, A2, C4) prolifer‐ ate on AK-c(RGDfC)-coated surfaces in serum-free, EGF (20 ng/ml) containing medium (cycletime: 18-20 hrs) (Fig.6). The cells show elongated morphology, display nestin, RC2 and Sox2 immunreactivity, but are immune-negative for GFAP. They transcribe large amount of *blbp, glast, pax6, olig2* and also *gfap* mRNAs [74]. All clones express *mash1*, while neurogenin (*ngn2*) is not transcribed in the ventral forebrain-derived A2 clone. *Oct4* or *nanog* expression was never detected. According to the above characteristics, these cells were identified as embryonic radial

Adult-derived AK-c(RGDfC) adherent cells were prepared from the ventral forebrain SVZ (clones: SVZ\_I, SVZ\_K, SVZ\_M, SVZ\_T) from the hippocampus (HC\_A), from the parie‐ tal cortex far from corpus callosum (CTX\_H) and also from the dorsal midbrain parenchy‐ ma (colliculus superior), far from the wall of the ventricle (MES\_D). As it was expected, AK-c(RGDfC) adherent cells were much less frequent in the adult brain-derived cell suspensions, than in the embryonic preparations. The selective adhesivity and EGFsupported proliferation, however, resulted in several clones from the adult mouse brain. Isolation of CTX\_H and MES\_D clones indicates that cells with stem/progenitor features are present in the non-neurogenic adult brain parenchyma at a frequency high enough to

Regardless of origin, adult-derived clones display GFAP-, nestin- and RC2 immunpositivity, and carry immunocytochemically detectable Sox2 and Pax6 proteins [74]. None of the cloned cells express *Oct4* or *nanog* pluripotency genes. According to these features, the cells are regarded as adult-derived RGl cells (aRGl). Adult RGl cells grow in two dimensional clusters, where elongated cells line up along each other (Fig.6 right panel) and divide with a cycle-time of 20-22 hours in average. By today, eRGl and aRGl clones are over 50-80 passages without pheno- or genotypic changes; they preserved 2n (40 chromosome) euploidity, morphological

*3.2.1. Morphological and cell biological characteristics of cloned stem/progenitor cell populations*

diversity of neural cells, those fulfilling the criteria of "stemness".

not produce GFAP and RC2 proteins [87].

glia-like (eRGl) cells.

sort them out easily.

features and neural inducibility.

#### **3.2. Comparative characterization of neural stem/progenitor clones**

Our first neural stem cell clones (NE-4C *[ATTC CRL 2925]* and NE-7C2) were established 15 years ago, from early embryonic (E9) anterior brain vesicles of p53-deficient mouse embryos [82,86]. The lack of p53 tumour suppressor protein did not prevent the in vitro formation of postmitotic neuronal precursors and later neurons [82], as did not hinder normal neural development of transgenic animals despite of increased tumour frequency in aging animals [83]. For control, however, similar cell lines had been established from wild-type mouse embryos (WNE cell lines) and were proved to display similar characteristics including morphology, chromosome-stability, cell cycle-time, regional gene expression and the schedule of in vitro neuron and glia formation. NE-4C, NE-7C2 and WNE cells showed similarities to P19 EC and embryoid body-derived ES cells in many aspects [33,87], and showed marked differences if compared to AK-c(RGDfC) adherent stem/progenitor cells isolated either from late embryonic or adult brain regions (Table 2). All clones shown on Table 2 can be propagated in vitro without differentiation or changing phenotype, and can generate neurons, astrocytes and oligodendrocytes in response to appropriate inducing stimuli.


**Table 2.** Neural stem/progenitor clones investigated in the presented studies

Multiple differences were found among the characterized cell lines indicating important diversity of neural cells, those fulfilling the criteria of "stemness".

#### *3.2.1. Morphological and cell biological characteristics of cloned stem/progenitor cell populations*

The "selective adhesion and serum free propagation" method allowed isolating and cloning cell lines from various parts of the embryonic (E 14-15) and adult (P50-75) mouse brains (Table 2).

Our first neural stem cell clones (NE-4C *[ATTC CRL 2925]* and NE-7C2) were established 15 years ago, from early embryonic (E9) anterior brain vesicles of p53-deficient mouse embryos [82,86]. The lack of p53 tumour suppressor protein did not prevent the in vitro formation of postmitotic neuronal precursors and later neurons [82], as did not hinder normal neural development of transgenic animals despite of increased tumour frequency in aging animals [83]. For control, however, similar cell lines had been established from wild-type mouse embryos (WNE cell lines) and were proved to display similar characteristics including morphology, chromosome-stability, cell cycle-time, regional gene expression and the schedule of in vitro neuron and glia formation. NE-4C, NE-7C2 and WNE cells showed similarities to P19 EC and embryoid body-derived ES cells in many aspects [33,87], and showed marked differences if compared to AK-c(RGDfC) adherent stem/progenitor cells isolated either from late embryonic or adult brain regions (Table 2). All clones shown on Table 2 can be propagated in vitro without differentiation or changing phenotype, and can generate neurons, astrocytes

**Cell clone tissue origin age of source tissue method of isolation**

E 14.5

P 50-75

E 9 Serial splitting;

serum-supported growth on poly-L-lysine

AK-c(RGDfC) adherence; serum-free growth on AKc(RGDfC) coated surfaces in the presence of EGF

**3.2. Comparative characterization of neural stem/progenitor clones**

and oligodendrocytes in response to appropriate inducing stimuli.

p53-/- mouse embryo

ventral forebrain SVZ

superior)

**Table 2.** Neural stem/progenitor clones investigated in the presented studies

WNE forebrain E 11.5

NE-4C

56 Neural Stem Cells - New Perspectives

NE-7C2

SVZ\_I

SVZ\_K SVZ\_T SVZ\_M

(ATTC;CRL-2925) anterior brain vesicles;

RGl-1 whole forebrain

A2 ventral forebrain C4 dorsal forebrain HC\_A hippocampus

CTX\_H parietal cortex

MES\_D dorsal mesencephalon (colliculus

Early embryonic neuroectoderm-derived (NE-4C, NE-7C2 and WNE) cells display epithel morphology (Fig.6), and produce uniform monolayers. These cells divide continuously (16 hour cycle-time in average) on poly-L-lysine in 10% FCS containing medium, but can not be propagated in serum-free conditions on AK-c(RGDfC) coated surfaces. NE cells express nestin intermedier filamentum protein [82,86] and two-third of the cells carries the mouse stem cell antigen, SSEA-1 [88]. In non-induced stage, the cells express Oct4, carry *blbp* and *glast* mRNAs (proteins could not be demonstrated), but do not express *pax6*, *mash1* or *neurogenin*s and do not produce GFAP and RC2 proteins [87].

In contrast to NE cells, embryo-derived AK-c(RGDfC)-adherent cells (RGl-1, A2, C4) prolifer‐ ate on AK-c(RGDfC)-coated surfaces in serum-free, EGF (20 ng/ml) containing medium (cycletime: 18-20 hrs) (Fig.6). The cells show elongated morphology, display nestin, RC2 and Sox2 immunreactivity, but are immune-negative for GFAP. They transcribe large amount of *blbp, glast, pax6, olig2* and also *gfap* mRNAs [74]. All clones express *mash1*, while neurogenin (*ngn2*) is not transcribed in the ventral forebrain-derived A2 clone. *Oct4* or *nanog* expression was never detected. According to the above characteristics, these cells were identified as embryonic radial glia-like (eRGl) cells.

Adult-derived AK-c(RGDfC) adherent cells were prepared from the ventral forebrain SVZ (clones: SVZ\_I, SVZ\_K, SVZ\_M, SVZ\_T) from the hippocampus (HC\_A), from the parie‐ tal cortex far from corpus callosum (CTX\_H) and also from the dorsal midbrain parenchy‐ ma (colliculus superior), far from the wall of the ventricle (MES\_D). As it was expected, AK-c(RGDfC) adherent cells were much less frequent in the adult brain-derived cell suspensions, than in the embryonic preparations. The selective adhesivity and EGFsupported proliferation, however, resulted in several clones from the adult mouse brain. Isolation of CTX\_H and MES\_D clones indicates that cells with stem/progenitor features are present in the non-neurogenic adult brain parenchyma at a frequency high enough to sort them out easily.

Regardless of origin, adult-derived clones display GFAP-, nestin- and RC2 immunpositivity, and carry immunocytochemically detectable Sox2 and Pax6 proteins [74]. None of the cloned cells express *Oct4* or *nanog* pluripotency genes. According to these features, the cells are regarded as adult-derived RGl cells (aRGl). Adult RGl cells grow in two dimensional clusters, where elongated cells line up along each other (Fig.6 right panel) and divide with a cycle-time of 20-22 hours in average. By today, eRGl and aRGl clones are over 50-80 passages without pheno- or genotypic changes; they preserved 2n (40 chromosome) euploidity, morphological features and neural inducibility.

*3.2.2. Generation of neural tissue-type cells by NE and RGl type neural stem/progenitor cells*

NE cells, even if glia genesis starts only 7-10 days after RA-priming [87].

**Figure 7.** The scheme of RA-primed neural differentiation of NE cells.

as groups of SSEA-1 immunreactive epithelioid cells (Fig.8.) [33, 82, 87].

itor cells (Table 3).

Studies on neuron and glia formation revealed further important differences between early embryonic neuroectoderm (NE) derived and embryonic or adult radial glia like stem/progen‐

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Neural differentiation of NE cells is induced by retinoic acid (all-trans retinoic acid; RA; 10-8-10-6 M) [82, 86, 89]. A short (6-hour) RA treatment initiate aggregation of cells and the rate of aggregation increases at higher RA-concentrations and/or longer treatment. Forced aggre‐ gation of cells without RA, however, does not induce neural development, and, if initial aggregation is prevented, RA-treatment alone results in severe cell decay without differentia‐ tion [89]. It seems, that RA primes the cells for intercellular inductive signalling, what takes places inside RA-primed aggregates. After RA-priming, neural differentiation proceeds along an apparently stable program (Fig.7), in the absence of RA, in both serum-containing and serum-free culture conditions. RA-priming is required also for the formation of astrocytes from

20 hours after RA-priming, RC2 radial glia marker protein appears in the aggregated NE cells, and the first IIIβ-tubulin- and MAP2-positive neuronal precursors appear on the 3rd– 4rd day of induction, inside the aggregates. Expression of the proneural bHLH transcription factor *ngn2* is detected soon after induction (24-48 hours), increases during the first 5 days and decreases thereafter, when the "neuron-specific" *math2* transcription starts and increases gradually. The proportion of neurons reaches 50-55% of total cells during the second week of induction, and the first GFAP-positive cells appear around the 9th day of induction. Nondifferentiated stem/progenitor cells persist during the entire period of neural differentiation

In contrast to NE cells, tissue-type differentiation of RGl cells is not induced by RA-treatment, regardless of the age and region of the source tissue [74, 90]. In RGl cells, neuronal differen‐

**Figure 6.** Phase-contrast view of NE-4C early embryonic neuroectodermal stem cells, the embryo-derived radial glia like RGl-1 cells and the adult cortex-derived CTX\_H cells.


**Table 3.** Similarities and differences between NE and RGl cells

Beside marked differences in shape, proliferation activity, pluripotency gene expression and GFAP and RC2 immunreactivity, RGl cells display different electrophysiological properties in comparison to early embryonic neuroectodermal (NE) cells. While both NE and RGl cells exist in multiple gap junction coupling, and consequently, display symmetric passive conductance in response to current injections [88], voltage-dependent K-currents (KDR) can be registered in RGl cells [74], but not in NE cells.

#### *3.2.2. Generation of neural tissue-type cells by NE and RGl type neural stem/progenitor cells*

Studies on neuron and glia formation revealed further important differences between early embryonic neuroectoderm (NE) derived and embryonic or adult radial glia like stem/progen‐ itor cells (Table 3).

Neural differentiation of NE cells is induced by retinoic acid (all-trans retinoic acid; RA; 10-8-10-6 M) [82, 86, 89]. A short (6-hour) RA treatment initiate aggregation of cells and the rate of aggregation increases at higher RA-concentrations and/or longer treatment. Forced aggre‐ gation of cells without RA, however, does not induce neural development, and, if initial aggregation is prevented, RA-treatment alone results in severe cell decay without differentia‐ tion [89]. It seems, that RA primes the cells for intercellular inductive signalling, what takes places inside RA-primed aggregates. After RA-priming, neural differentiation proceeds along an apparently stable program (Fig.7), in the absence of RA, in both serum-containing and serum-free culture conditions. RA-priming is required also for the formation of astrocytes from NE cells, even if glia genesis starts only 7-10 days after RA-priming [87].

**Figure 7.** The scheme of RA-primed neural differentiation of NE cells.

**Figure 6.** Phase-contrast view of NE-4C early embryonic neuroectodermal stem cells, the embryo-derived radial glia

nestin + + + RC2 - + + GFAP - - + βIII-tub - - -

Oct 4 + - - Nanog + - - Sox2 + + + Pax6 - + + Blbp + + + GLAST + + + Ngn2 - + + Mash1 - + + Math2 - - -

Beside marked differences in shape, proliferation activity, pluripotency gene expression and GFAP and RC2 immunreactivity, RGl cells display different electrophysiological properties in comparison to early embryonic neuroectodermal (NE) cells. While both NE and RGl cells exist in multiple gap junction coupling, and consequently, display symmetric passive conductance in response to current injections [88], voltage-dependent K-currents (KDR) can be registered in

**NE-4C RGl-1; A2 HC\_A; SVZ\_M**

EGF withdrawal (neurons); serum (astrocytes); PDGF+FGF+forskolin/T3+AA (oligodendrocytes)

like RGl-1 cells and the adult cortex-derived CTX\_H cells.

Induction of neural differentiation retinoic acid

RGl cells [74], but not in NE cells.

**Table 3.** Similarities and differences between NE and RGl cells

Cytochemical markers

58 Neural Stem Cells - New Perspectives

Gene expression

20 hours after RA-priming, RC2 radial glia marker protein appears in the aggregated NE cells, and the first IIIβ-tubulin- and MAP2-positive neuronal precursors appear on the 3rd– 4rd day of induction, inside the aggregates. Expression of the proneural bHLH transcription factor *ngn2* is detected soon after induction (24-48 hours), increases during the first 5 days and decreases thereafter, when the "neuron-specific" *math2* transcription starts and increases gradually. The proportion of neurons reaches 50-55% of total cells during the second week of induction, and the first GFAP-positive cells appear around the 9th day of induction. Nondifferentiated stem/progenitor cells persist during the entire period of neural differentiation as groups of SSEA-1 immunreactive epithelioid cells (Fig.8.) [33, 82, 87].

In contrast to NE cells, tissue-type differentiation of RGl cells is not induced by RA-treatment, regardless of the age and region of the source tissue [74, 90]. In RGl cells, neuronal differen‐

Regardless of origin, RGl cells give rise to neurons in a 5-day period after withdrawal of EGF, to astrocytes in 3 days after treatment with serum, and to oligodendrocytes 8 days after the

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**Figure 10.** Neurons and oligodendrocytes generated by adult hippocampus- and mesencaphalon-derived (HC\_A and MES\_D) RGl cells are hown after neuron-specific IIIβ-tubulin and oligodendroglia-indicating O4 immunostaining, re‐

RGl cells generate neurons in smaller proportion than NE cells; the ratio of neurons among total cells does not exceed 20-30%, and the yield varies among the different clones. Significant differences among the RGl clones were found also in the rate of oligodenroglia production [74]. The time-course of formation of "mature" phenotypes, however, is shorter in RGl cells than

The retinoid-dependent and independent neuron formation is a striking difference between NE and RGl clones, respectively [90]. During development, RA regulates the formation of the neural tube and interferes with the patterning of the future hindbrain and spinal cord [92]. Important morphogenic roles of retinoids were demonstrated also in the developing forebrain [93], and the presence and production of RA were described in the developing lateral gan‐ glionic eminence (LGE) [94,95]. RA-responsive cells persist in the main neural stem cell niches of the postnatal rodent brain including the SVZ and the dentate gyrus [96]. Depletion of RA in adult mice results in impaired neuronal differentiation in the dentate gyrus [97,98] and RA synthesis seems to regulate proliferation and gene transcription of at least a subset of neural stem cells in the SVZ [99,100]. As retinoid signalling seems to influence the fate of neural stem cells throughout life, the fundamental differences in RA-responsiveness between NE and RGl

*3.2.3. RA-production and RA-responsiveness of NE and RGl cells*

type neural stem/progenitor cells have been investigated in details.

onset of oligodendroglia induction (Fig.9 and 10).

spectively.

in NE cells.

**Figure 8.** Immunocytochemical characterization of differentiating NE cells cells and the expression of some develop‐ mental neuronal "master" genes in the course of in vitro induced neural development. Developmental stages are indi‐ cated by days after RA-priming.

tiation is induced by withdrawal of EGF from the medium of dense cultures. Large-scale astroglia formation is initiated by adding serum (10% FCS), and oligodendroglia production is achieved by treating the cells with FGF, PDGF and forskolin followed by treatment with thyroid hormone (T3) and ascorbic acid, according to the protocol of Glaser [91] (Fig.9.).

**Figure 9.** The scheme of in vitro neural tissue-type differentiation of RGl cells.

Regardless of origin, RGl cells give rise to neurons in a 5-day period after withdrawal of EGF, to astrocytes in 3 days after treatment with serum, and to oligodendrocytes 8 days after the onset of oligodendroglia induction (Fig.9 and 10).

**Figure 10.** Neurons and oligodendrocytes generated by adult hippocampus- and mesencaphalon-derived (HC\_A and MES\_D) RGl cells are hown after neuron-specific IIIβ-tubulin and oligodendroglia-indicating O4 immunostaining, re‐ spectively.

RGl cells generate neurons in smaller proportion than NE cells; the ratio of neurons among total cells does not exceed 20-30%, and the yield varies among the different clones. Significant differences among the RGl clones were found also in the rate of oligodenroglia production [74]. The time-course of formation of "mature" phenotypes, however, is shorter in RGl cells than in NE cells.

#### *3.2.3. RA-production and RA-responsiveness of NE and RGl cells*

tiation is induced by withdrawal of EGF from the medium of dense cultures. Large-scale astroglia formation is initiated by adding serum (10% FCS), and oligodendroglia production is achieved by treating the cells with FGF, PDGF and forskolin followed by treatment with thyroid hormone (T3) and ascorbic acid, according to the protocol of Glaser [91] (Fig.9.).

**Figure 8.** Immunocytochemical characterization of differentiating NE cells cells and the expression of some develop‐ mental neuronal "master" genes in the course of in vitro induced neural development. Developmental stages are indi‐

**Figure 9.** The scheme of in vitro neural tissue-type differentiation of RGl cells.

cated by days after RA-priming.

60 Neural Stem Cells - New Perspectives

The retinoid-dependent and independent neuron formation is a striking difference between NE and RGl clones, respectively [90]. During development, RA regulates the formation of the neural tube and interferes with the patterning of the future hindbrain and spinal cord [92]. Important morphogenic roles of retinoids were demonstrated also in the developing forebrain [93], and the presence and production of RA were described in the developing lateral gan‐ glionic eminence (LGE) [94,95]. RA-responsive cells persist in the main neural stem cell niches of the postnatal rodent brain including the SVZ and the dentate gyrus [96]. Depletion of RA in adult mice results in impaired neuronal differentiation in the dentate gyrus [97,98] and RA synthesis seems to regulate proliferation and gene transcription of at least a subset of neural stem cells in the SVZ [99,100]. As retinoid signalling seems to influence the fate of neural stem cells throughout life, the fundamental differences in RA-responsiveness between NE and RGl type neural stem/progenitor cells have been investigated in details.

Non-differentiated *NE* cells do not produce detectable retinoids [101], but differentiating daughter cells produce considerable amounts of RA [87, 90]. In contrast, RGl cells produce well-detectable amount of RA and retinoid production increases further with the advancement of neuronal differentiation. Endogenous RA production, on the other hand, does not influence the neuron formation by RGl cells: treatment with retinoic acid receptor (RAR) antagonist (AGN193109) does not result in significant changes in the number or morphology of RGlderived neurons [90]. Expression of genes coding components of the retinoid metabolism and signalling revealed significant differences between NE and RGl cells. NE and RGl cells express non-identical sets of retinaldehyde-dehydrogenases (RALDHs) and nuclear retinoid receptor subunits. Moreover, some defined retinoid transporters (as STRA6), and catabolising enzymes (as CYP26s) are not expressed by RGl cells [90]. In accordance with Haskell and LaMantia [99], we concluded that retinoid metabolism and responsiveness are distinctive characteristics of defined subtypes of neural stem/progenitor populations.

(*vglut*) expression and VGLUT-immunpositivity [102] however was found only in the hippo‐ campus-derived HC\_A clone. As HC\_A neurons express only *vglut1* [74]*,* it seems that these cells preserve some regional, neurotransmitter-related identity, even after long-term in vitro propagation. Similarly, all adult SVZ-derived clones give rise to tyrosin-hydroxylase(TH) expressing neurons [74]. This observation might be in accord with the known production of

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment

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63

The embryo-derived RGl-1 cells, on the other hand, express both, *vglut1*, *vglut2* and also TH. The finding may indicate a less advanced stage of commitment of these cells, but needs further

Available sporadic results do not allow deciding on the preservation or loss of regional identity of in vitro propagated stem/progenitor cells. As clones comprise progenies of single cells, for far-range conclusions we need further studies on statistically sufficient number of clones from

**4. Distinct neural stem/progenitor stages require distinct environmental**

One cell derived, cloned populations are useful subjects to show the types of cells which can or can not derive from a given stem cell population, but neither of such studies can predict what sort of phenotypes can be manifested in vivo. In the course of in vivo tissue genesis, differentiating cells and their environment change in an orchestrated way: the conditions either help tissue integration or kill the differentiating cells assuring the survival of the right types

In our experience, NE-4C cells give rise to neurons with large frequency if implanted into early embryonic brain vesicles, but produce non-differentiating, tumour-like expansions in the subcortical regions of newborn mice. Moreover, the same cells diminish from the adult mouse forebrain, except the forebrain SVZ, where some cells reside for longer than 3 weeks [104]. The data demonstrate that the fate of NE stem/progenitor cells is strictly governed by environ‐

In addition to initial requirements for stem/progenitor survival, the successive progenies need different environment and change basic physiological demands including O2 -supply [105; 77]. Under hypoxic conditions, NE-4C stem cells survive and proliferate, but do not generate neurons. Soon after the onset of in vitro neuronal differentiation (48 hrs after RA-priming), however, hypoxia severely impairs cell survival [105] indicating that the basic metabolic characteristics of cells change soon after neural fate decision. The in vitro results were supported by data obtained on the sporadic neuron formation by NE-4C cells implanted into

mental factors provided by the host tissue, and which are far from being explored.

the adult mouse forebrain in response to hyperbaric oxygenation [105].

TH-positive olfactory neuron-precursors [103] in the adult SVZ.

studies.

each brain region.

**conditions**

and number of cells at the right places.

#### *3.2.4. "Regional memory"*

NE-4C cells, in non-induced "stem cell stage", express only *otx2* and *en* from the investigated "region-specific", positional genes. In differentiated NE-4C cultures, however, many positional genes including *hoxb2* are actively transcribed (Table 4) [33] indicating that these early embryon‐ ic stem cells are "open" for a variety of "position-determined" development. Accordingly, glutamatergic, GABAergic and also serotonin producing neurons develop from NE-4C cells.

Non-induced RGl cells, on the other hand, express *gbx2*, *dlx2*, *emx2*, but not *hoxb2* or *nkx2*.*1* (Table 4), regardless of origin. The pattern of investigated genes indicates an "anterior to hindbrain" (lack of active *hoxb2*), and a "not caudo-ventral" (lack of *nkx2*.*1* expression) origin, but does not distinguish between forebrain regions and between forebrain and mesencephalon derivatives.


**Table 4.** Positional gene expression by non-induced stem/progenitor cells.

The neurotransmitter phenotype of neuronal progenies, on the other hand, still indicates preservation of some fine region-restricted commitment. All RGl cells give rise to GABAproducing, VGAT-positive neurons. In adult-derived clones, vesicular glutamate transporter (*vglut*) expression and VGLUT-immunpositivity [102] however was found only in the hippo‐ campus-derived HC\_A clone. As HC\_A neurons express only *vglut1* [74]*,* it seems that these cells preserve some regional, neurotransmitter-related identity, even after long-term in vitro propagation. Similarly, all adult SVZ-derived clones give rise to tyrosin-hydroxylase(TH) expressing neurons [74]. This observation might be in accord with the known production of TH-positive olfactory neuron-precursors [103] in the adult SVZ.

Non-differentiated *NE* cells do not produce detectable retinoids [101], but differentiating daughter cells produce considerable amounts of RA [87, 90]. In contrast, RGl cells produce well-detectable amount of RA and retinoid production increases further with the advancement of neuronal differentiation. Endogenous RA production, on the other hand, does not influence the neuron formation by RGl cells: treatment with retinoic acid receptor (RAR) antagonist (AGN193109) does not result in significant changes in the number or morphology of RGlderived neurons [90]. Expression of genes coding components of the retinoid metabolism and signalling revealed significant differences between NE and RGl cells. NE and RGl cells express non-identical sets of retinaldehyde-dehydrogenases (RALDHs) and nuclear retinoid receptor subunits. Moreover, some defined retinoid transporters (as STRA6), and catabolising enzymes (as CYP26s) are not expressed by RGl cells [90]. In accordance with Haskell and LaMantia [99], we concluded that retinoid metabolism and responsiveness are distinctive characteristics of

NE-4C cells, in non-induced "stem cell stage", express only *otx2* and *en* from the investigated "region-specific", positional genes. In differentiated NE-4C cultures, however, many positional genes including *hoxb2* are actively transcribed (Table 4) [33] indicating that these early embryon‐ ic stem cells are "open" for a variety of "position-determined" development. Accordingly, glutamatergic, GABAergic and also serotonin producing neurons develop from NE-4C cells. Non-induced RGl cells, on the other hand, express *gbx2*, *dlx2*, *emx2*, but not *hoxb2* or *nkx2*.*1* (Table 4), regardless of origin. The pattern of investigated genes indicates an "anterior to hindbrain" (lack of active *hoxb2*), and a "not caudo-ventral" (lack of *nkx2*.*1* expression) origin, but does not distinguish between forebrain regions and between forebrain and mesencephalon derivatives.

defined subtypes of neural stem/progenitor populations.

**NE RGl**

**Table 4.** Positional gene expression by non-induced stem/progenitor cells.

**NE-4C Embryonic (E14.5) Adult (P 50-75)**

Gene expression Emx2 - + + + + + + + + + + Nkx2.1 - - - - - - - - - - - Gbx2 - ni ni + + + + + + + + Dlx2 - + + + + + + + + ni ni Hoxb2 - ni ni - - - - - - ni ni Ngn2 - - + + + + + + - + + Mash1 - + + + + + + + + + +

The neurotransmitter phenotype of neuronal progenies, on the other hand, still indicates preservation of some fine region-restricted commitment. All RGl cells give rise to GABAproducing, VGAT-positive neurons. In adult-derived clones, vesicular glutamate transporter

**A2 C4 RGl-1 HC\_A CTX\_H MES\_D SVZ\_I SVZ\_K SVZ\_T SVZ\_M**

*3.2.4. "Regional memory"*

62 Neural Stem Cells - New Perspectives

**Clone gene**

(ni: non investigated)

The embryo-derived RGl-1 cells, on the other hand, express both, *vglut1*, *vglut2* and also TH. The finding may indicate a less advanced stage of commitment of these cells, but needs further studies.

Available sporadic results do not allow deciding on the preservation or loss of regional identity of in vitro propagated stem/progenitor cells. As clones comprise progenies of single cells, for far-range conclusions we need further studies on statistically sufficient number of clones from each brain region.

## **4. Distinct neural stem/progenitor stages require distinct environmental conditions**

One cell derived, cloned populations are useful subjects to show the types of cells which can or can not derive from a given stem cell population, but neither of such studies can predict what sort of phenotypes can be manifested in vivo. In the course of in vivo tissue genesis, differentiating cells and their environment change in an orchestrated way: the conditions either help tissue integration or kill the differentiating cells assuring the survival of the right types and number of cells at the right places.

In our experience, NE-4C cells give rise to neurons with large frequency if implanted into early embryonic brain vesicles, but produce non-differentiating, tumour-like expansions in the subcortical regions of newborn mice. Moreover, the same cells diminish from the adult mouse forebrain, except the forebrain SVZ, where some cells reside for longer than 3 weeks [104]. The data demonstrate that the fate of NE stem/progenitor cells is strictly governed by environ‐ mental factors provided by the host tissue, and which are far from being explored.

In addition to initial requirements for stem/progenitor survival, the successive progenies need different environment and change basic physiological demands including O2 -supply [105; 77]. Under hypoxic conditions, NE-4C stem cells survive and proliferate, but do not generate neurons. Soon after the onset of in vitro neuronal differentiation (48 hrs after RA-priming), however, hypoxia severely impairs cell survival [105] indicating that the basic metabolic characteristics of cells change soon after neural fate decision. The in vitro results were supported by data obtained on the sporadic neuron formation by NE-4C cells implanted into the adult mouse forebrain in response to hyperbaric oxygenation [105].

#### **5. Conclusions**

The presented results on some selected NS populations demonstrate that neural stem/ progenitor cells derived from different brain regions and different ages may display significant diversity in many aspects including cell physiological and developmental features. The selected clones represent derivatives of a single stem cell and had been propagated under fairly artificial in vitro conditions; accordingly, none of them can represent "*the" stem cells in general* of the region of origin. From the data obtained on 3 different NE and 10 RGl cell clones, however, we could conclude that early embryonic neuroectoderm-derived stem cells display distinct *in vitro* features from those isolated by selective adhesion from later CNS tissues. In case of RGl cells, the adhesive preference-based selection and EGF-supported growth, imply a strong selection. Cells corresponding to such selection could be isolated from fairly distinct brain regions including areas not listed among "professional" neurogenic zones. *Correspond‐ ingly, RGl cells may not represent a defined lineage of stem/progenitor population, rather a common stage of neural progenitor-succession, which may appear in many lineages.* The integrin-based adhesion, EGF-supported proliferation, voltage-dependent K+ -flux, RA-production and RAinsensitivity may be characteristics of stem/progenitor cells in a defined stage of tissue integration.

**Author details**

Emília Madarász

**References**

and Developmental Neurobiology, Hungary

Nat Rev Neurosci., 10(10): 724–735

mental Biology 301: 489–503

in mammals. Cell 96,195 -209.

opment;130:5091-5101

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human brain: a review.. Brain Res., 62: 1-35.

Anat Embryol Cell Biol. 2007;190:1-65.

Institute of Experimental Medicin of Hungarian Academy of Sciences, Laboratory of Cellular

Diversity of Neural Stem/Progenitor Populations: Varieties by Age, Regional Origin and Environment

http://dx.doi.org/10.5772/55678

65

[1] Rakic P. Evolution of the neocortex: Perspective from developmental biology 2009.

[2] Kriegstein A, Alvarez-Buylla A. 2009. The glial nature of embryonic and adult neural

[3] Albazerchi A., Stern C.D. 2007. A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak. Develop‐

[4] Chapman SC, Schubert FR, Schoenwolf GC, Lumsden A. 2003. Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm. Devel‐

[5] Beddington, R. S. and Robertson, E. J. 1999. Axis development and early asymmetry

[6] Schoenwolf, G.C., 2001. Cutting, pasting and painting: experimental embryology and

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While many NS populations have been characterized in vitro [76], were implanted into different brain regions of healthy and disease-model animals and have been considered also as potential tools for clinical cell therapy (for critical review: [106, 107]), the basic physiology of such cells, their needs for survival in successive developmental stages as well as the harmonization of such needs with conditions provided by the host tissue are rarely taken into account. As a further task, physiological demands of various NS populations and descending progenies should be explored and the roles of basic physiological factors in tuning differen‐ tiation should be understood.

#### **Acknowledgements**

I thank my colleagues, especially to Zsuzsanna Környei and Károly Markó at the Cellular and Developmental Neurobiology Unit of IEM-HAS, and Katalin Schlett at the Dept. of Physiology and Neurobiology, Eötvös L. Univ., for common work, thinking and discussions. I wish to thank the Nikon Microscopy Center at IEM, Nikon Austria GmbH and Auro-Science Con‐ sulting Ltd for kindly providing microscopy support. I apologise to Authors whose excellent publications were not referred here because of space limitations. The presented studies were supported by National Science Foundation (OTKA) (K 68939, K106191), National Innovation Office (Bio\_Surf) and Eu Fp6 (BioDot) grants.

#### **Author details**

**5. Conclusions**

64 Neural Stem Cells - New Perspectives

integration.

tiation should be understood.

**Acknowledgements**

Office (Bio\_Surf) and Eu Fp6 (BioDot) grants.

The presented results on some selected NS populations demonstrate that neural stem/ progenitor cells derived from different brain regions and different ages may display significant diversity in many aspects including cell physiological and developmental features. The selected clones represent derivatives of a single stem cell and had been propagated under fairly artificial in vitro conditions; accordingly, none of them can represent "*the" stem cells in general* of the region of origin. From the data obtained on 3 different NE and 10 RGl cell clones, however, we could conclude that early embryonic neuroectoderm-derived stem cells display distinct *in vitro* features from those isolated by selective adhesion from later CNS tissues. In case of RGl cells, the adhesive preference-based selection and EGF-supported growth, imply a strong selection. Cells corresponding to such selection could be isolated from fairly distinct brain regions including areas not listed among "professional" neurogenic zones. *Correspond‐ ingly, RGl cells may not represent a defined lineage of stem/progenitor population, rather a common stage of neural progenitor-succession, which may appear in many lineages.* The integrin-based

insensitivity may be characteristics of stem/progenitor cells in a defined stage of tissue

While many NS populations have been characterized in vitro [76], were implanted into different brain regions of healthy and disease-model animals and have been considered also as potential tools for clinical cell therapy (for critical review: [106, 107]), the basic physiology of such cells, their needs for survival in successive developmental stages as well as the harmonization of such needs with conditions provided by the host tissue are rarely taken into account. As a further task, physiological demands of various NS populations and descending progenies should be explored and the roles of basic physiological factors in tuning differen‐

I thank my colleagues, especially to Zsuzsanna Környei and Károly Markó at the Cellular and Developmental Neurobiology Unit of IEM-HAS, and Katalin Schlett at the Dept. of Physiology and Neurobiology, Eötvös L. Univ., for common work, thinking and discussions. I wish to thank the Nikon Microscopy Center at IEM, Nikon Austria GmbH and Auro-Science Con‐ sulting Ltd for kindly providing microscopy support. I apologise to Authors whose excellent publications were not referred here because of space limitations. The presented studies were supported by National Science Foundation (OTKA) (K 68939, K106191), National Innovation


adhesion, EGF-supported proliferation, voltage-dependent K+

Emília Madarász

Institute of Experimental Medicin of Hungarian Academy of Sciences, Laboratory of Cellular and Developmental Neurobiology, Hungary

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**Chapter 4**

glial progenitors of the optic nerve, and

**Reactive Muller Glia as Potential Retinal Progenitors**

Regenerative medicine has become a driving force in the treatment of disease and injury over the last decade [1]. This is due to the accumulation of knowledge in several key areas; 1) the mechanisms of disease processes, 2) creation of stem cells/induced pluripotent stem cells that might be used for therapeutic purposes, and 3) factors that are necessary for the proper differentiation of specific cell types. In any tissue, it might be possible to regenerate lost cells from exogenous stem cells, endogenous stem or progenitor cells, or endogenous cells that can dedifferentiate, proliferate and re-differentiate. Several endogenous populations of cells localized to the eye have been shown to be capable of replacing some or all retinal cell types in various species; 1) an endogenous population of progenitor cells in the periphery of the eye referred to as the ciliary marginal zone (CMZ), 2) the retinal pigmented epithelium, 3) non-

finally 5) Müller glia of the retina [2]. This chapter will focus specifically on the responsiveness of Müller glia to disease or injury to the retina with a special emphasis on signals that have been shown to lead to the injury response and changes to the extracellular matrix that play a

Müller Glia, named after their discoverer Heinrich Müller, were first described in 1851 [3]. Müller Glia are a unique blend of radial glia, astrocytes, and oligodendrocytes that span the width of the mature retina from the outer limiting memberane in the outer nuclear layer to the inner limiting membrane at the edge of the retina and vitreous humor [4]. Müller cells are one of three possible macroglial cells that can be found in the retina. Astrocytes also migrate into

and reproduction in any medium, provided the original work is properly cited.

© 2013 Belecky-Adams et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Teri L. Belecky-Adams, Ellen C. Chernoff,

http://dx.doi.org/10.5772/55150

**1. Introduction**

Additional information is available at the end of the chapter

pigmented cells adjacent to peripheral retina, 4) NG2+

role in dedifferentiation and proliferation.

**2. Müller Glial cell basics**

Jonathan M. Wilson and Subramanian Dharmarajan


## **Reactive Muller Glia as Potential Retinal Progenitors**

Teri L. Belecky-Adams, Ellen C. Chernoff, Jonathan M. Wilson and Subramanian Dharmarajan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55150

#### **1. Introduction**

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vitamin A deprived rats. PLoS One 3:e3487.

Regenerative medicine has become a driving force in the treatment of disease and injury over the last decade [1]. This is due to the accumulation of knowledge in several key areas; 1) the mechanisms of disease processes, 2) creation of stem cells/induced pluripotent stem cells that might be used for therapeutic purposes, and 3) factors that are necessary for the proper differentiation of specific cell types. In any tissue, it might be possible to regenerate lost cells from exogenous stem cells, endogenous stem or progenitor cells, or endogenous cells that can dedifferentiate, proliferate and re-differentiate. Several endogenous populations of cells localized to the eye have been shown to be capable of replacing some or all retinal cell types in various species; 1) an endogenous population of progenitor cells in the periphery of the eye referred to as the ciliary marginal zone (CMZ), 2) the retinal pigmented epithelium, 3) nonpigmented cells adjacent to peripheral retina, 4) NG2+ glial progenitors of the optic nerve, and finally 5) Müller glia of the retina [2]. This chapter will focus specifically on the responsiveness of Müller glia to disease or injury to the retina with a special emphasis on signals that have been shown to lead to the injury response and changes to the extracellular matrix that play a role in dedifferentiation and proliferation.

#### **2. Müller Glial cell basics**

Müller Glia, named after their discoverer Heinrich Müller, were first described in 1851 [3]. Müller Glia are a unique blend of radial glia, astrocytes, and oligodendrocytes that span the width of the mature retina from the outer limiting memberane in the outer nuclear layer to the inner limiting membrane at the edge of the retina and vitreous humor [4]. Müller cells are one of three possible macroglial cells that can be found in the retina. Astrocytes also migrate into

© 2013 Belecky-Adams et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the retina from the optic nerve and some species also contain oligodendrocytes in the nerve fiber layer [5]. However, Müller glia are the only glial cells that are derived from retinal progenitors. Müller cells play a wide variety of roles in both the developing and mature retina. In order to consider the full effect of gliosis in the diseased or injured retina, we must first understand their function in the normal retina.

#### **2.1. Retinal histogenesis**

Lineage analysis of retinal progenitors using various techniques have shown that many retinal progenitors have the capacity to produce all retinal cell types [6-10]. Retinal cells undergo a stereotypical pattern of differentiation in which some cells leave the cell cycle (are born) very early in retinal histogenesis, such as cone photoreceptors, ganglion cells, and horizontal cells, while other cells are generated at later timepoints [6, 7, 9-12]. Müller glia are born in the group of cells that are generated late in the ontogenic period.

Vertebrate retinal cells are arranged in a specific fashion in both layers and in columns [6-8, 13-17]. Figure 1 shows the arrangement of mature retinal cells in the outer, inner and ganglion cell layers. However, some of the cells are also arranged in a columnar fashion. The later-born cells, which include the rods, bipolar, and subpopulation of the amacrine cells, all migrate along the radially arranged Müller glial cells. These cells remain in close contact with the Müller glia even as differentiation continues and are thought to comprise a metabolic and/or processing circuit within the retina [17]. The early-born cells are not a part of this columnar unit. Rather than relying on the Müller glia to migrate to the correct layer of the retina, these cells undergo nuclear translocation in the relatively thinner early retina [18, 19].

Müller glia also share properties that allow them to organize the laminar structure of the retina. Cultured Müller glia or Müller glial conditioned-medium are capable of organizing the retinal neurospheres into a layered pattern which closely resembles that seen in the mature retina [20, 21]. While these experiments suggest that there may be a secreted factor which may mediate the organizational properties of Müller glia, recent experiments done in zebrafish suggest that the apico-basal polarity that is inherent in the development of Müller glia is also a critical part of its organizational capacity [22]. A disrupted apical Müller glial cell process in zebrafish mutated in the P50 subunit of dynactin leads to a disruption in the normal laminar develop‐ ment of the retina [22]. In mice, disruption of the outer limiting membrane that is comprised of the apical Müller glial endfeet disrupts the placement of photoreceptors such that misplaced photoreceptor nuclei are found adjacent to the retinal pigmented epithelium, in a region where photoreceptor outer segments would normally be located [23].

#### **2.2. Synapse formation**

The role of astrocytes in synaptogenesis in the CNS has been established by many investigators [24-26]. Müller glial cells have been considered by many to be astrocyte-related cells (See Table 1), hence Müller glia may play some role in synapse formation and/or maintenance in the retina. This idea has been tested in zebrafish retina with somewhat contradictory results [27, 28]. While it appears that the Müller glial cell processes do not invade the outer plexiform later until after synapses have already formed and deletion of Müller glia during early retinal development does not affect cone synaptogenesis, a separate study examining the role of harmonin (USH1C) in zebrafish which is found in the retinal Müller glia, have disrupted ribbon synapses [27, 28]. Until this conflict can be resolved and the role of these cells have been

**Figure 1.** Organization of the mature retina: The retinal cells, which consist of neurons and glia, are organized into the outer nuclear layer (ONL), inner nuclear layer (INL) and the ganglion cell layer (GCL). The ONL consists of the rod and cone photoreceptor cells. The INL is made up of the horizontal cells, amacrine cells as well as the bipolar cells. The Müller glial cell bodies are also present in this layer. However, the processes of the Müller glial cells extend outward into the adjacent layers, extending throughout the thickness of the retina. The GCL is primarily consists of the gan‐

Reactive Muller Glia as Potential Retinal Progenitors

http://dx.doi.org/10.5772/55150

75

investigated in other species, the role of Müller glia remains open.

glion cells which send out their axons out of the eye through the optic disc.

the retina from the optic nerve and some species also contain oligodendrocytes in the nerve fiber layer [5]. However, Müller glia are the only glial cells that are derived from retinal progenitors. Müller cells play a wide variety of roles in both the developing and mature retina. In order to consider the full effect of gliosis in the diseased or injured retina, we must first

Lineage analysis of retinal progenitors using various techniques have shown that many retinal progenitors have the capacity to produce all retinal cell types [6-10]. Retinal cells undergo a stereotypical pattern of differentiation in which some cells leave the cell cycle (are born) very early in retinal histogenesis, such as cone photoreceptors, ganglion cells, and horizontal cells, while other cells are generated at later timepoints [6, 7, 9-12]. Müller glia are born in the group

Vertebrate retinal cells are arranged in a specific fashion in both layers and in columns [6-8, 13-17]. Figure 1 shows the arrangement of mature retinal cells in the outer, inner and ganglion cell layers. However, some of the cells are also arranged in a columnar fashion. The later-born cells, which include the rods, bipolar, and subpopulation of the amacrine cells, all migrate along the radially arranged Müller glial cells. These cells remain in close contact with the Müller glia even as differentiation continues and are thought to comprise a metabolic and/or processing circuit within the retina [17]. The early-born cells are not a part of this columnar unit. Rather than relying on the Müller glia to migrate to the correct layer of the retina, these

Müller glia also share properties that allow them to organize the laminar structure of the retina. Cultured Müller glia or Müller glial conditioned-medium are capable of organizing the retinal neurospheres into a layered pattern which closely resembles that seen in the mature retina [20, 21]. While these experiments suggest that there may be a secreted factor which may mediate the organizational properties of Müller glia, recent experiments done in zebrafish suggest that the apico-basal polarity that is inherent in the development of Müller glia is also a critical part of its organizational capacity [22]. A disrupted apical Müller glial cell process in zebrafish mutated in the P50 subunit of dynactin leads to a disruption in the normal laminar develop‐ ment of the retina [22]. In mice, disruption of the outer limiting membrane that is comprised of the apical Müller glial endfeet disrupts the placement of photoreceptors such that misplaced photoreceptor nuclei are found adjacent to the retinal pigmented epithelium, in a region where

The role of astrocytes in synaptogenesis in the CNS has been established by many investigators [24-26]. Müller glial cells have been considered by many to be astrocyte-related cells (See Table 1), hence Müller glia may play some role in synapse formation and/or maintenance in the retina. This idea has been tested in zebrafish retina with somewhat contradictory results [27, 28]. While it appears that the Müller glial cell processes do not invade the outer plexiform later

cells undergo nuclear translocation in the relatively thinner early retina [18, 19].

photoreceptor outer segments would normally be located [23].

understand their function in the normal retina.

of cells that are generated late in the ontogenic period.

**2.1. Retinal histogenesis**

74 Neural Stem Cells - New Perspectives

**2.2. Synapse formation**

**Figure 1.** Organization of the mature retina: The retinal cells, which consist of neurons and glia, are organized into the outer nuclear layer (ONL), inner nuclear layer (INL) and the ganglion cell layer (GCL). The ONL consists of the rod and cone photoreceptor cells. The INL is made up of the horizontal cells, amacrine cells as well as the bipolar cells. The Müller glial cell bodies are also present in this layer. However, the processes of the Müller glial cells extend outward into the adjacent layers, extending throughout the thickness of the retina. The GCL is primarily consists of the gan‐ glion cells which send out their axons out of the eye through the optic disc.

until after synapses have already formed and deletion of Müller glia during early retinal development does not affect cone synaptogenesis, a separate study examining the role of harmonin (USH1C) in zebrafish which is found in the retinal Müller glia, have disrupted ribbon synapses [27, 28]. Until this conflict can be resolved and the role of these cells have been investigated in other species, the role of Müller glia remains open.


**Astrocytes Müller glia**

The blood-brain barrier refers to the separation between the circulating blood and extracellular fluid found within the central nervous system. In the brain, this barrier is formed through the interactions between astrocytes and endothelial cells that form the vasculature [29]. In the eye, the blood-retinal barrier is maintained at two junctures; 1) an "outer barrier" in the form of the retinal pigmented epithelium (RPE), and 2) the "inner barrier" that is comprised of the endothelial cells of the retinal vasculature [30]. The endothelial cells of the retinal vasculature form tight junctions that are selectively permeable to hydrophobic molecules such as O2, CO2, and hormones, while restricting the entrance of bacteria and large or hydrophilic molecules (See Fig 2). Endothelial cells and pericytes that adhere to the outside of the endothelial cells are both encompassed by a basal lamina as well as the astrocytic endfeet. There is evidence that inner barrier is induced and maintained by both Müller glial and retinal astrocytic endfeet that ensheath retinal blood vessels [31]. The processes of retinal astrocytes, however, are limited to the never fiber and ganglion cell layer and can only interact with superficial

Müller glia (as well as retinal astrocytes and retinal pigment epithelium) express factors that are critical to the formation of the deep plexus vasculature in the retina [33]. Angiogenesis is the result of a balance between the pro-angiogenic factor vascular endothelial growth factor (VEGF) and anti-angiogenic factor pigment-epithelium derived factor (PEDF) [33, 34]. The ratio of these factors carefully controls the growth of the deep plexus retinal vasculature. Not surprisingly, misregulation of these factors can lead to pathological neovascularization, a topic which will be covered later in this chaper. Many other interactions between Müller glia/ astrocytes and the vasculature have been proposed and/or documented. For instance, Paulson and Newman simulated a process whereby the activity of neurons indirectly regulated blood vessel dilation [35]. In a process referred to as siphoning, the Müller glia are proposed to take

proximity to the vasculature [35]. Thus the astrocyte can effectively redistribute the K+ from the neuron, which may be some distance away from the nearest blood vessel, to a region immediately adjacent to the arteriole in a manner that is faster than would otherwise take place

Following retinal injury –

• Following targeted ablation of photoreceptor and ganglion cells, regeneration of the respective cell types was observed from the Müller glia [111,

proliferate [111, 114]

247]

• Müller glial cells re-enter cell cycle and can

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77

at the endfeet that are in close

• Cells dedifferentiate and have the potential to

• Begin to express proteins associated to neural stem cells or radial glia (NG2, BLBP, nestin, DSD1,

re-enter cell cycle [111]

CD15) [99]

**Table 1.** Comparison of Astrocyte and Müller glial Characteristics

vasculature near the inner surface of the retina [32]

released by active neurons and then release the K+

up K+

**2.3. Blood retinal barrier development and maintenance**

Stem cell potential Following injury –


**Table 1.** Comparison of Astrocyte and Müller glial Characteristics

**Astrocytes Müller glia**

Morphology • Have a stellate or star like morphology [226] • Have a radial morphology [227]

• Found exclusively in the inner nuclear layer of the retina with the process spanning the entire width of the retina

• Originate from the neural retinal progenitor cells [227, 228]

• Serve as a scaffolding for retinal

to the photoreceptor cells [240] • Help in recycling photopigments [241] • Aid in the formation of the blood retinal

• Help direct light through the retinal layers

• Similar to astrocytes serve as a source of nourishment and energy reserve in the form of glucose and lactate respectively

• Help in maintenance of homeostasis and removal of toxic metabolites in a manner

• Neurotransmitter receptors (AMPA, GluR4, NMDA, GABA-A etc.), transporters and modulators (GLAST, GS, GAT etc.) help in neutransmitter recycling and also aid in glia-neuron communication [115]

• Similar to astrocytes following retinal damage, cells hypertrophy, change morphology and upregulate various

• Based on the ability or the lack of cells to proliferate, Müller cell gliosis is referred to as non conservative or conservative gliosis,

• Glial scar is not a prominent feature of gliosis of the Müller glia [114, 115]

similar to astrocytes [115]

markers [246]

respectively [115]

organizations [227]

barrier [242]

[227]

[227, 228]

Location • Throughout the nervous system, including the retina and optic nerve [226]

Origin • Originate from the glial restricted neural stem

Functions • Scaffolding for migration of developing neurons [230]

[229]

76 Neural Stem Cells - New Perspectives

[234]

[235, 236]

transmission[232, 239]

the "glial scar" [245]

[243]

Changes during reactive gliosis

cells or the bipotent O2A progenitor cell type

• Aid in the formation of synapses [231-233] • Aid in the formation of the blood brain barrier

• Serve as a source of nourishment and energy reserve for the neurons by providing glucose and storing excess glucose in the form of glycogen

• Possess various channels and transporter (Na +/K+ channels, aquaporins etc.) which aid in the maintenance of homeostasis, pH levels and removal of toxic metabolites [237, 238] • Possess transporters for neurotransmitters (such as GABA, glycine, glutamate)which aid in clearance and release of these molecules into the synaptic space which can affect synaptic

• Changes in gliosis based on extent of injury which ranges from mild to moderate to severe

• Cells hypertrophy (particularly by increasing the expression of GFAP), change in morphology

• Increase proliferation and in severe cases form

and upregulate various markers [244]

#### **2.3. Blood retinal barrier development and maintenance**

The blood-brain barrier refers to the separation between the circulating blood and extracellular fluid found within the central nervous system. In the brain, this barrier is formed through the interactions between astrocytes and endothelial cells that form the vasculature [29]. In the eye, the blood-retinal barrier is maintained at two junctures; 1) an "outer barrier" in the form of the retinal pigmented epithelium (RPE), and 2) the "inner barrier" that is comprised of the endothelial cells of the retinal vasculature [30]. The endothelial cells of the retinal vasculature form tight junctions that are selectively permeable to hydrophobic molecules such as O2, CO2, and hormones, while restricting the entrance of bacteria and large or hydrophilic molecules (See Fig 2). Endothelial cells and pericytes that adhere to the outside of the endothelial cells are both encompassed by a basal lamina as well as the astrocytic endfeet. There is evidence that inner barrier is induced and maintained by both Müller glial and retinal astrocytic endfeet that ensheath retinal blood vessels [31]. The processes of retinal astrocytes, however, are limited to the never fiber and ganglion cell layer and can only interact with superficial vasculature near the inner surface of the retina [32]

Müller glia (as well as retinal astrocytes and retinal pigment epithelium) express factors that are critical to the formation of the deep plexus vasculature in the retina [33]. Angiogenesis is the result of a balance between the pro-angiogenic factor vascular endothelial growth factor (VEGF) and anti-angiogenic factor pigment-epithelium derived factor (PEDF) [33, 34]. The ratio of these factors carefully controls the growth of the deep plexus retinal vasculature. Not surprisingly, misregulation of these factors can lead to pathological neovascularization, a topic which will be covered later in this chaper. Many other interactions between Müller glia/ astrocytes and the vasculature have been proposed and/or documented. For instance, Paulson and Newman simulated a process whereby the activity of neurons indirectly regulated blood vessel dilation [35]. In a process referred to as siphoning, the Müller glia are proposed to take up K+ released by active neurons and then release the K+ at the endfeet that are in close proximity to the vasculature [35]. Thus the astrocyte can effectively redistribute the K+ from the neuron, which may be some distance away from the nearest blood vessel, to a region immediately adjacent to the arteriole in a manner that is faster than would otherwise take place if the K+ was undergoing simple diffusion. Further, this could also concentrate K+ released over a wider area to the smaller area of the endfeet.

[40]. Glial-derived neurotrophic factor (GDNF) and neurturin are also released by Müller glia and appear to enhance barrier function as measured by transendothelial resistance [41].

Communication between Müller glia and endothelial cells is not a one-way street. There also appear to be inductive signals released from the endothelial cells that effect Müller glial differen‐ tiation/function. It is well established that leukemia inhibitory factor (LIF) is secreted from endothelial cells and that it helps to induce astrocyte differentiation in optic nerve astrocytes [42, 43]. LIF and ciliary neurotrophic factor (CNTF) share a part of their receptor complex and intracellular signaling pathway; therefore it is not surprising to find that CNTF has also been shown to have effects on astrocyte development [44, 45]. Both CNTF and LIF are present in the developing retina and CNTF does increase the production of Müller glia [46]. However, an increase in the expression of LIF from the lens during retinogenesis inhibited the development of retinalvasculatureandincreasedtheexpressionofVEGFinretinalastrocytesandMüllerglia[47].

The brain is a high energy consuming organ, using approximately 25% of the glucose present in the human body [48]. There is very tight coupling between the demand and supply in the central nervous system (CNS), and most of this expenditure is due to neuronal activity [48, 49]. Howev‐ er, neurons do not store much glycogen and therefore are reliant upon external sources to fuel their oxidative metabolism. In the retina, this need is met by both the Müller glia and retinal astrocytes. Glucose enters Müller glia via glucose transporter-1 (GLUT-1) and is phosporylated by hexokinase to produce glucose-6-phosphate (Fig 2). From here, part of the glucose-6-phos‐ phate is stored with the Müller glial cell body as glycogen and the rest is metabolized to various carbohydrate intermediates [50-52]. Neurons can use a variety of substrates to fuel their oxida‐ tive metabolism, including lactate, pyruvate, alanine, glutamine, and glutamate [53, 54]. Müller glia metabolize glucose and glycogen deposits predominantly to pyruvate and lactate which is released to the extracellular milieu by the monocarboxylate transporter MCT2 [55, 56]. Neurons canthentakeuppyruvateanduseitdirectlyintheKrebscycletocompensateduringtimesoflow glucose [50, 57]. Lactate generated by Müller glia is converted by lactate dehydorgenase and

> + , K+

via an ammonia transporter (AMT) [59, 60]. In addition to transporting glutamate

Müller glial cells and are either disposed of or recycled [4]. Glutamate is an excitatory neuro‐ toxin, even at low extracellular concentrations, and is tightly regulated by Müller glia in the retina [58]. Müller cells take up glutamate via the glutamate/aspartate transporter, GLAST,

further stimulates glycolysis [4, 62]. Both the NH4+ and glutamate are use to create L-glutamine by glutamine synthetase [60, 63-65]. The glutamine produced by Müller glia is then transported back to neuronal cells to aid in the synthesis of neurotransmitters glutamate and GABA [54].

Müller glia, in part by increasing the expression levels of glutamine synthetase [54, 66, 67].

, and CO2, all of which are taken up by the

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79

ions and one H+

have a combined action of increasing glycolysis by the

/K+

and counter-

ATPase which

Hence it is unclear whether LIF plays a role in Müller glial cell differentiation.

**2.4. Metabolic coupling with neurons**

pyruvate kinase to pyruvate to power the Krebs cycle [55].

into Müller glial cells, the GLAST protein co-transports 3Na+

transports one K+ [61]. The influx of Na+ into the Müller cell activates the Na+

+

Active neurons, in turn, release glutamate, NH4

The presence of glutamate and NH4

and NH4

+

**Figure 2.** Aquaporin-4 (AQ-4), bipolar cell/ horizontal cell (BC/HC), excitatory amino acid transporter (EAAT), excitato‐ ry amino acid carrier (EAAC), glial cell derived neurotrophic factor (GDNF), glutamate transporter (GLT), glycine trans‐ porter(GlyT), glutamine synthetase (GS), interleukins (IL), L-type amino acid transporter (LAT), matrix metalloproteinases (MMP), photoreceptor cell (PC), pigment epithelium derived factor (PEDF), Na+ coupled neutral amino acid transporter (SNAT), glutamate aspartate transporter (GLAST), vascular endothelial growth factor (VEGF).

Müller glia are also known for releasing many growth factors, and many of these factors effect the endothelial cells. Transforming growth factor β1 (TGFβ1) is released by Müller glia and can increase the expression of tissue plasminogen activator inhibitor-1, which could potentially have the protective effect of reducing hemorhaging in the brain [36-38]. TGF-β1 also has been shown to have a morphological effect on cultured endothelial cells, inducing them to form capillary-like structures [39]. Mice with a loss of the integrin αVβ8 that is necesary for TGF-β activation within the retina also have abnormal superficial as well as deep plexus formation [40]. Glial-derived neurotrophic factor (GDNF) and neurturin are also released by Müller glia and appear to enhance barrier function as measured by transendothelial resistance [41].

Communication between Müller glia and endothelial cells is not a one-way street. There also appear to be inductive signals released from the endothelial cells that effect Müller glial differen‐ tiation/function. It is well established that leukemia inhibitory factor (LIF) is secreted from endothelial cells and that it helps to induce astrocyte differentiation in optic nerve astrocytes [42, 43]. LIF and ciliary neurotrophic factor (CNTF) share a part of their receptor complex and intracellular signaling pathway; therefore it is not surprising to find that CNTF has also been shown to have effects on astrocyte development [44, 45]. Both CNTF and LIF are present in the developing retina and CNTF does increase the production of Müller glia [46]. However, an increase in the expression of LIF from the lens during retinogenesis inhibited the development of retinalvasculatureandincreasedtheexpressionofVEGFinretinalastrocytesandMüllerglia[47]. Hence it is unclear whether LIF plays a role in Müller glial cell differentiation.

#### **2.4. Metabolic coupling with neurons**

if the K+

78 Neural Stem Cells - New Perspectives

a wider area to the smaller area of the endfeet.

was undergoing simple diffusion. Further, this could also concentrate K+

**Figure 2.** Aquaporin-4 (AQ-4), bipolar cell/ horizontal cell (BC/HC), excitatory amino acid transporter (EAAT), excitato‐ ry amino acid carrier (EAAC), glial cell derived neurotrophic factor (GDNF), glutamate transporter (GLT), glycine trans‐ porter(GlyT), glutamine synthetase (GS), interleukins (IL), L-type amino acid transporter (LAT), matrix metalloproteinases (MMP), photoreceptor cell (PC), pigment epithelium derived factor (PEDF), Na+ coupled neutral amino acid transporter (SNAT), glutamate aspartate transporter (GLAST), vascular endothelial growth factor (VEGF).

Müller glia are also known for releasing many growth factors, and many of these factors effect the endothelial cells. Transforming growth factor β1 (TGFβ1) is released by Müller glia and can increase the expression of tissue plasminogen activator inhibitor-1, which could potentially have the protective effect of reducing hemorhaging in the brain [36-38]. TGF-β1 also has been shown to have a morphological effect on cultured endothelial cells, inducing them to form capillary-like structures [39]. Mice with a loss of the integrin αVβ8 that is necesary for TGF-β activation within the retina also have abnormal superficial as well as deep plexus formation

released over

The brain is a high energy consuming organ, using approximately 25% of the glucose present in the human body [48]. There is very tight coupling between the demand and supply in the central nervous system (CNS), and most of this expenditure is due to neuronal activity [48, 49]. Howev‐ er, neurons do not store much glycogen and therefore are reliant upon external sources to fuel their oxidative metabolism. In the retina, this need is met by both the Müller glia and retinal astrocytes. Glucose enters Müller glia via glucose transporter-1 (GLUT-1) and is phosporylated by hexokinase to produce glucose-6-phosphate (Fig 2). From here, part of the glucose-6-phos‐ phate is stored with the Müller glial cell body as glycogen and the rest is metabolized to various carbohydrate intermediates [50-52]. Neurons can use a variety of substrates to fuel their oxida‐ tive metabolism, including lactate, pyruvate, alanine, glutamine, and glutamate [53, 54]. Müller glia metabolize glucose and glycogen deposits predominantly to pyruvate and lactate which is released to the extracellular milieu by the monocarboxylate transporter MCT2 [55, 56]. Neurons canthentakeuppyruvateanduseitdirectlyintheKrebscycletocompensateduringtimesoflow glucose [50, 57]. Lactate generated by Müller glia is converted by lactate dehydorgenase and pyruvate kinase to pyruvate to power the Krebs cycle [55].

Active neurons, in turn, release glutamate, NH4 + , K+ , and CO2, all of which are taken up by the Müller glial cells and are either disposed of or recycled [4]. Glutamate is an excitatory neuro‐ toxin, even at low extracellular concentrations, and is tightly regulated by Müller glia in the retina [58]. Müller cells take up glutamate via the glutamate/aspartate transporter, GLAST, and NH4 + via an ammonia transporter (AMT) [59, 60]. In addition to transporting glutamate into Müller glial cells, the GLAST protein co-transports 3Na+ ions and one H+ and countertransports one K+ [61]. The influx of Na+ into the Müller cell activates the Na+ /K+ ATPase which further stimulates glycolysis [4, 62]. Both the NH4+ and glutamate are use to create L-glutamine by glutamine synthetase [60, 63-65]. The glutamine produced by Müller glia is then transported back to neuronal cells to aid in the synthesis of neurotransmitters glutamate and GABA [54]. The presence of glutamate and NH4 + have a combined action of increasing glycolysis by the Müller glia, in part by increasing the expression levels of glutamine synthetase [54, 66, 67]. Müller glia also act as a sink for excess extracellular K+ in the retina, which is taken up by the inwardly rectifying K+ (Kir) channels and the Na+ /K+ ATPase of the Müller cells [62]. This elevation of K+ concentration increases the glycogenolysis in cultured Müller glia, tightly coupling the breakdown of glycogen to neuronal activity [17]. The K+ is then disposed of by passing K+ into the subretinal space, the vitreous body, or the blood. [68, 69]. Finally, carbonic anhydrase converts CO2 to bicarbonate which is then released by way of the H+ /HCO3 exchanger into the vitreous or blood (Fig 2) [70-73].

glutamate, only their uptake mechanism and potential processing within Müller glia will be discussed here. GABA is used by horizontal and amacrine cells within the retina and termina‐

transporters (GATs)foundinpresynapticneurons,Müllerglia,andretinalastrocytes [76,84,85]. AfteruptakeintoMüllerglia,GABAcanbeconvertedtoglutamineviaglutaminesynthetaseand, as specified above, is returned to neurons to act as substrates for neurotransmitters [86]. Müller glia also express glutamate decarboxylase which catalyzes the decarboxylation of glutamate to

Dopamine performs a large number of functions in the developing and mature retina that are well out of the scope of this chapter. A full discussion of this topic can be found elsewhere [87]. Both the transporter and enzymes necessary for converting tyrosine to dopamine are expressed in Müller glia [88]. Likewise, ATP also performs a large number of functions in the developing and mature retina [89-91]. Müller glia express a subset of the P2X and P2Y ATP receptors and they also have the ability to convert ATP to adenosine and release both ATP and adenosine

Müller glia also carry glutamate, GABA, purinergic, glycine, dopaminergic, noradrenergic and cholinergic receptors [76]. In some instances these receptors have been shown to coordinate release of neurotransmitters by neurons with enzymatic activity and or gene regulation in the Müller glial cells. An excellent example of this coordination is the regulation of glutamate receptors on GLAST activity and expression of GLAST. When glutamate receptors are activated on Müller glial membranes it leads to an increase in intracellular Ca2+ and protein kinase C (PKC). The activation of metabotropic glutamate receptors in Müller cells leads to an increase in Ca2+ and protein kinase C, and phosphorylation of GLAST by PKC leads to an increase in transport of glutamate [82, 93]. The increased transport of glutamate through GLAST appears to regulate activation of mammalian target of rapamycin (mTOR), which activates DNA binding of the transcription factor activator protein-1 (AP-1) and an increase

Müller glia perform a variety of other functions beyond those already mentioned. For instance,

for the transport of water that accumulates in the tissue as the end product of ATP synthesis

, is released into the bloodstream. Müller cells are also involved in phagocytosis of debris in

Studies using reactive astrocytes have shown the potential to dedifferentiate into cells having neural progenitor or stem cell like properties (Table 1) [98, 99]. Following stimulation, these cells show activation of signaling pathways such as EGF, FGF, SHH and Wnts, previously

[95]. The movement of water is specifically coupled to the movement of Na+

the retina and in the release of antioxidant glutathione [96, 97]

**3. Properties that are similar to stem cells/astrocytes**

released by retinal neurons, the Müller glia are also responsible

and K+

and, like

/Cl-

Reactive Muller Glia as Potential Retinal Progenitors


81

http://dx.doi.org/10.5772/55150

tionofGABAactivityisbroughtaboutthroughtheuptakeofGABAbyNa+

into the intracellular space [91, 92].

in GLAST mRNA [94].

in addition to siphoning K+

**2.6. Other**

K+

GABA. It is unclear, however, whether GABA can be released by Müller glia [76].

#### **2.5. Regulation of neurotransmission**

In the retina, glutamate is the primary excitatory neurotransmitter [74]. Müller glia have transporters for a wide variety of transmitters, including glutamate, GABA, Glycine, D-serine, dopamine, and ATP [75, 76]. The Müller glia take up neurotransmitters and other neuroactive substances and convert them to substances that can be supplied to retinal neurons as neuro‐ transmitters or neurotransmitter precursors (Fig 2). The modulation of neuronal excitability through regulation of neurotransmitter availability is thought to serve three functions; 1) termination of neuronal signaling, 2) prevention of neurotransmitter spread to adjacent synapses, and 3) prevention of neurotoxicity resulting from prolonged presence of a trans‐ mitter at a synapse [4, 75]. In this section, we will briefly cover transport of the major retinal neurotransmitters into Müller glia, processing of the transmitter by the Müller glia and transport of products back to retinal neurons.

Müller glia express several glutamate transporters, depending upon the species, including the previously mentioned GLAST protein (also known as excitatory amino acid transporter 1 or EAAT1). In humans for instance, the dominant transporter is EAAT1, but EAAT2 and 3 can also be found [77]. Glutamate is the most widely used neurotransmitter used by retinal neurons, including photoreceptors, bipolar and ganglion cells. Both the photoreceptors and the bipolar cells have graded potentials, hence the amount of neurotransmitter released is directly correlated to the amount of stimulus. In addition, photoreceptors are wired a little differently than other neurons that transduce sensory information; they release glutamate in the dark and less glutamate upon transduction of light signals. Hence, removal of glutamate from the synaptic region is critical for normal tranmission of light signals to take place. Knockdown and knockout studies in the retina have indicated that a loss of GLAST leads to a loss of the electroretinogram b-wave, primarily because it aids in signal processing between photoreceptors and bipolar cells, rather than any neurotoxicity associated with high levels of glutamate [78, 79]. Consistent with the idea that Müller glia are critical for clearing away glutamate released at synapses are studies in which clearance of D-aspartate was tracked first to Müller glia followed by a redistribution into other neuronal cell types of the retina [80]. Glutamate can be converted to glutamine by glutamine synthetase, and is then transported back to neurons as a precursor to glutamate [63, 81]. Loss of glutamine synthetase activity leads to a loss of glutamate content in retinal neurons which leads to functional blindness within 2 minutes [82, 83].

There are several other neurotransmitters used in the retina, such as GABA, glycine, and dopamine. Since the interactions of these neurotransmitters are not as heavily studied as

glutamate, only their uptake mechanism and potential processing within Müller glia will be discussed here. GABA is used by horizontal and amacrine cells within the retina and termina‐ tionofGABAactivityisbroughtaboutthroughtheuptakeofGABAbyNa+ /Cl- -dependentGABA transporters (GATs)foundinpresynapticneurons,Müllerglia,andretinalastrocytes [76,84,85]. AfteruptakeintoMüllerglia,GABAcanbeconvertedtoglutamineviaglutaminesynthetaseand, as specified above, is returned to neurons to act as substrates for neurotransmitters [86]. Müller glia also express glutamate decarboxylase which catalyzes the decarboxylation of glutamate to GABA. It is unclear, however, whether GABA can be released by Müller glia [76].

Dopamine performs a large number of functions in the developing and mature retina that are well out of the scope of this chapter. A full discussion of this topic can be found elsewhere [87]. Both the transporter and enzymes necessary for converting tyrosine to dopamine are expressed in Müller glia [88]. Likewise, ATP also performs a large number of functions in the developing and mature retina [89-91]. Müller glia express a subset of the P2X and P2Y ATP receptors and they also have the ability to convert ATP to adenosine and release both ATP and adenosine into the intracellular space [91, 92].

Müller glia also carry glutamate, GABA, purinergic, glycine, dopaminergic, noradrenergic and cholinergic receptors [76]. In some instances these receptors have been shown to coordinate release of neurotransmitters by neurons with enzymatic activity and or gene regulation in the Müller glial cells. An excellent example of this coordination is the regulation of glutamate receptors on GLAST activity and expression of GLAST. When glutamate receptors are activated on Müller glial membranes it leads to an increase in intracellular Ca2+ and protein kinase C (PKC). The activation of metabotropic glutamate receptors in Müller cells leads to an increase in Ca2+ and protein kinase C, and phosphorylation of GLAST by PKC leads to an increase in transport of glutamate [82, 93]. The increased transport of glutamate through GLAST appears to regulate activation of mammalian target of rapamycin (mTOR), which activates DNA binding of the transcription factor activator protein-1 (AP-1) and an increase in GLAST mRNA [94].

#### **2.6. Other**

Müller glia also act as a sink for excess extracellular K+

exchanger into the vitreous or blood (Fig 2) [70-73].

**2.5. Regulation of neurotransmission**

transport of products back to retinal neurons.

(Kir) channels and the Na+

/K+

coupling the breakdown of glycogen to neuronal activity [17]. The K+ is then disposed of by passing K+ into the subretinal space, the vitreous body, or the blood. [68, 69]. Finally, carbonic

In the retina, glutamate is the primary excitatory neurotransmitter [74]. Müller glia have transporters for a wide variety of transmitters, including glutamate, GABA, Glycine, D-serine, dopamine, and ATP [75, 76]. The Müller glia take up neurotransmitters and other neuroactive substances and convert them to substances that can be supplied to retinal neurons as neuro‐ transmitters or neurotransmitter precursors (Fig 2). The modulation of neuronal excitability through regulation of neurotransmitter availability is thought to serve three functions; 1) termination of neuronal signaling, 2) prevention of neurotransmitter spread to adjacent synapses, and 3) prevention of neurotoxicity resulting from prolonged presence of a trans‐ mitter at a synapse [4, 75]. In this section, we will briefly cover transport of the major retinal neurotransmitters into Müller glia, processing of the transmitter by the Müller glia and

Müller glia express several glutamate transporters, depending upon the species, including the previously mentioned GLAST protein (also known as excitatory amino acid transporter 1 or EAAT1). In humans for instance, the dominant transporter is EAAT1, but EAAT2 and 3 can also be found [77]. Glutamate is the most widely used neurotransmitter used by retinal neurons, including photoreceptors, bipolar and ganglion cells. Both the photoreceptors and the bipolar cells have graded potentials, hence the amount of neurotransmitter released is directly correlated to the amount of stimulus. In addition, photoreceptors are wired a little differently than other neurons that transduce sensory information; they release glutamate in the dark and less glutamate upon transduction of light signals. Hence, removal of glutamate from the synaptic region is critical for normal tranmission of light signals to take place. Knockdown and knockout studies in the retina have indicated that a loss of GLAST leads to a loss of the electroretinogram b-wave, primarily because it aids in signal processing between photoreceptors and bipolar cells, rather than any neurotoxicity associated with high levels of glutamate [78, 79]. Consistent with the idea that Müller glia are critical for clearing away glutamate released at synapses are studies in which clearance of D-aspartate was tracked first to Müller glia followed by a redistribution into other neuronal cell types of the retina [80]. Glutamate can be converted to glutamine by glutamine synthetase, and is then transported back to neurons as a precursor to glutamate [63, 81]. Loss of glutamine synthetase activity leads to a loss of glutamate content in retinal neurons which leads to functional blindness within 2

There are several other neurotransmitters used in the retina, such as GABA, glycine, and dopamine. Since the interactions of these neurotransmitters are not as heavily studied as

anhydrase converts CO2 to bicarbonate which is then released by way of the H+

concentration increases the glycogenolysis in cultured Müller glia, tightly

inwardly rectifying K+

80 Neural Stem Cells - New Perspectives

elevation of K+

minutes [82, 83].

in the retina, which is taken up by the

ATPase of the Müller cells [62]. This

/HCO3 -

> Müller glia perform a variety of other functions beyond those already mentioned. For instance, in addition to siphoning K+ released by retinal neurons, the Müller glia are also responsible for the transport of water that accumulates in the tissue as the end product of ATP synthesis [95]. The movement of water is specifically coupled to the movement of Na+ and K+ and, like K+ , is released into the bloodstream. Müller cells are also involved in phagocytosis of debris in the retina and in the release of antioxidant glutathione [96, 97]

#### **3. Properties that are similar to stem cells/astrocytes**

Studies using reactive astrocytes have shown the potential to dedifferentiate into cells having neural progenitor or stem cell like properties (Table 1) [98, 99]. Following stimulation, these cells show activation of signaling pathways such as EGF, FGF, SHH and Wnts, previously shown to be associated with the neural stem cells [98, 100-102]. Similarly, activated Müller glial cells following retinal injury have also shown the capacity to dedifferentiate into retinal progenitor cells [103]. Studies in lower vertebrates such as fish, amphibians and birds have shown the presence of a stem cell niche in the ciliary marginal zone (CMZ) of the retina [104-107]. Mammals, however, do not have a CMZ [108]. In mammals, a small group of cells in the non-pigmented portion adjacent to the retina can proliferate up to postnatal day 21, but these cells are low in abundance and are not thought to generate many cells [103, 109]. It may be more feasible to generate many retinal progenitor cells from activated Müller glia. Expres‐ sion profiling of proliferating Müller cells suggests a stem cell like role for these cells [110, 111]. Culture of the Müller cells in an enriched medium generated "multipotent neuro‐ spheres", elucidating the stem cell role of Müller cells *in vitro*. Further transplantation of enriched Müller glial cells into injured retina generated cells with neuron like characteristics [112]. Müller cells have been shown to dedifferentiate, proliferate and give rise to amacrine cells, bipolar cells, retinal ganglion neurons as well as the photoreceptor cells. [110, 111, 113]. One important factor aiding the transformation of the Müller glial cells is the membrane depolarization due to a reduction of potassium ion conductance, primarily due to downregu‐ lation of the Kir channels in the Müller cell [114]. The dowregulation of the Kir channels leads to a decrease in the p27kip1 cyclin kinase inhibitor, which is then succeeded by re-entry into cell cycle. The downregulation of the Kir channels pushes these cells towards the proliferative stage [115].

**•** Can the reactive gliosis be used to "supply" multipotent stem cells to the retina to replace

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83

**•** Can the multipotent stem cells that arise from Müller glial cells be directed in their differ‐ entiation in vivo and can the number of progenitor cells differentiating into cell types other

There appears to be a continuum in the states of reactive gliosis, from mild to severe. In the mild to moderate forms of gliosis, the cells may hypertrophy and show some changes to their functionality, but, if the trigger is removed, the cells may revert back to their former condition without altering the tissue [116]. In the more severe forms of reactive gliosis, cells hypertrophy, lose functionality, form glial scars that are inhibitory to axonal regeneration and neuronal surivival, and may also proliferate [116, 117]. The severe state is marked by the persistance of these characteristics. Within the mammalian retina, both the Müller glia and retinal astrocytes display reactivity to injury and disease. In this section we will talk about triggers of Müller glia, evidence that BMP7 may also be a trigger, and the changes in retinal homeostasis that

Müller glial reactivity can be found in every identified disease and injury that plagues the eye, including diabetic retinopathy, glaucoma, age-related macular degeneration, retinitis pig‐ mentosa, and many many others [118-122]. In considering reactive gliosis, there appears to multiple levels of complexity. For instance, there are a wide range of factors which have been shown to trigger reactive gliosis in Müller glia (Figure 3 and Table 2). Some of these triggers can have concentration-dependent effects upon astrocytes [116]. Further, different triggers can lead to specific molecular and functional changes in the Müller glia that may correspond to the various aspects of reactive gliosis [123]. Not only are there multiple triggers, but there is

Studies in the injured spinal cord have indicated a role for another family of growth factors; the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-β superfam‐ ily of growth factors. The receptors include two basic types, Type I and Type II, both of which are serine-threonine kinases. Receptors from each type must form heterodimers in order for signaling to occur, although the Type I receptors are downstream of the Type II. There are two non-canonical signaling pathways, BMP-MAPK and FRAP-STAT, that have more recently been identified in addition to the canonical SMAD-related pathway [45, 124-129]. Three type I receptors have been associated with the BMPs, activin-like kinase 2 (ALK2), ALK 3 and ALK6. Accumulated evidence has shown that in regards to the Type I receptor, BMP 6 and 7 activate the ALK2 receptor preferentially, whereas BMPs 2 and 4

heterogeneity in the response of Müller glia to the same factor [118].

**5.2. Bone morphogenetic proteins in Müller cell gliosis**

dead or dying neurons?

than Müller glia be increased substantially?

result from reactive gliosis in the retina.

**5. Triggers of reactive gliosis**

**5.1. Known triggers**

#### **4. Response of Müller Glia to injury or disease states**

When there is injury or disease within the CNS, astrocytes respond by entering a state referred to as reactive gliosis. Reactive gliosis is an ill-defined set of molecular changes that alters the homeostatic role of the cells and their interactions with neurons, vasculature, and the immune system. Reactive gliosis is thought to be the result of signals received from the injured or diseased tissue that begins a molecular cascade within the glial cells resulting in a change of state [103]. There are a mulitude of questions that have arisen as a result of our limited understanding of gliois, and investigators are currently working to answer these questions. Among them:


There appears to be a continuum in the states of reactive gliosis, from mild to severe. In the mild to moderate forms of gliosis, the cells may hypertrophy and show some changes to their functionality, but, if the trigger is removed, the cells may revert back to their former condition without altering the tissue [116]. In the more severe forms of reactive gliosis, cells hypertrophy, lose functionality, form glial scars that are inhibitory to axonal regeneration and neuronal surivival, and may also proliferate [116, 117]. The severe state is marked by the persistance of these characteristics. Within the mammalian retina, both the Müller glia and retinal astrocytes display reactivity to injury and disease. In this section we will talk about triggers of Müller glia, evidence that BMP7 may also be a trigger, and the changes in retinal homeostasis that result from reactive gliosis in the retina.

#### **5. Triggers of reactive gliosis**

#### **5.1. Known triggers**

shown to be associated with the neural stem cells [98, 100-102]. Similarly, activated Müller glial cells following retinal injury have also shown the capacity to dedifferentiate into retinal progenitor cells [103]. Studies in lower vertebrates such as fish, amphibians and birds have shown the presence of a stem cell niche in the ciliary marginal zone (CMZ) of the retina [104-107]. Mammals, however, do not have a CMZ [108]. In mammals, a small group of cells in the non-pigmented portion adjacent to the retina can proliferate up to postnatal day 21, but these cells are low in abundance and are not thought to generate many cells [103, 109]. It may be more feasible to generate many retinal progenitor cells from activated Müller glia. Expres‐ sion profiling of proliferating Müller cells suggests a stem cell like role for these cells [110, 111]. Culture of the Müller cells in an enriched medium generated "multipotent neuro‐ spheres", elucidating the stem cell role of Müller cells *in vitro*. Further transplantation of enriched Müller glial cells into injured retina generated cells with neuron like characteristics [112]. Müller cells have been shown to dedifferentiate, proliferate and give rise to amacrine cells, bipolar cells, retinal ganglion neurons as well as the photoreceptor cells. [110, 111, 113]. One important factor aiding the transformation of the Müller glial cells is the membrane depolarization due to a reduction of potassium ion conductance, primarily due to downregu‐ lation of the Kir channels in the Müller cell [114]. The dowregulation of the Kir channels leads to a decrease in the p27kip1 cyclin kinase inhibitor, which is then succeeded by re-entry into cell cycle. The downregulation of the Kir channels pushes these cells towards the proliferative

**4. Response of Müller Glia to injury or disease states**

**•** Is reactive gliosis one condition, or a host of related conditions?

**•** What are the molecular triggers of gliosis?

mediate multiple changes?

of gliosis related?

When there is injury or disease within the CNS, astrocytes respond by entering a state referred to as reactive gliosis. Reactive gliosis is an ill-defined set of molecular changes that alters the homeostatic role of the cells and their interactions with neurons, vasculature, and the immune system. Reactive gliosis is thought to be the result of signals received from the injured or diseased tissue that begins a molecular cascade within the glial cells resulting in a change of state [103]. There are a mulitude of questions that have arisen as a result of our limited understanding of gliois, and investigators are currently working to answer these questions.

**•** Do all the triggers that appear to be involved in gliosis converge on one or two pathways that mediate the changes in Müller glial state, or, are their multiple pathways that can

**•** Do different signals mitigate mild, moderate or severe reactive gliosis? How are these forms

**•** Can severe reactive gliosis be attenuated, even when triggers are chronically present?

stage [115].

82 Neural Stem Cells - New Perspectives

Among them:

Müller glial reactivity can be found in every identified disease and injury that plagues the eye, including diabetic retinopathy, glaucoma, age-related macular degeneration, retinitis pig‐ mentosa, and many many others [118-122]. In considering reactive gliosis, there appears to multiple levels of complexity. For instance, there are a wide range of factors which have been shown to trigger reactive gliosis in Müller glia (Figure 3 and Table 2). Some of these triggers can have concentration-dependent effects upon astrocytes [116]. Further, different triggers can lead to specific molecular and functional changes in the Müller glia that may correspond to the various aspects of reactive gliosis [123]. Not only are there multiple triggers, but there is heterogeneity in the response of Müller glia to the same factor [118].

#### **5.2. Bone morphogenetic proteins in Müller cell gliosis**

Studies in the injured spinal cord have indicated a role for another family of growth factors; the bone morphogenetic proteins (BMPs). The BMPs are members of the TGF-β superfam‐ ily of growth factors. The receptors include two basic types, Type I and Type II, both of which are serine-threonine kinases. Receptors from each type must form heterodimers in order for signaling to occur, although the Type I receptors are downstream of the Type II. There are two non-canonical signaling pathways, BMP-MAPK and FRAP-STAT, that have more recently been identified in addition to the canonical SMAD-related pathway [45, 124-129]. Three type I receptors have been associated with the BMPs, activin-like kinase 2 (ALK2), ALK 3 and ALK6. Accumulated evidence has shown that in regards to the Type I receptor, BMP 6 and 7 activate the ALK2 receptor preferentially, whereas BMPs 2 and 4


**Table 2.** Triggers of Müller Glia Cell Activation

activate either ALK3 or ALK6 [130]. In addition to the canonical SMAD pathway, ALK3 and 6 also activate the BMP-MAPK and FRAP-STAT pathways [45, 124, 129]. The BMPs have been shown to act as a gliosis trigger in penetrating spinal cord injuries, and a differential role for ALK3 and 6 receptors has been ascribed to various aspects of gliosis, including hypertrophy, inflammation, and tissue loss [131, 132]. While BMPs have been studied in retinal injury, primarily as a survival factor for retinal neurons, it has not been studied as a potential trigger for reactive gliosis in Müller glia [133].

**Figure 3.** Schematic representation of various signaling mechanisms which trigger and regulate reactive gliosis in Müller glia. Growth factors such as TGF-β, BMP, EGF and CNTF; interleukins; as well as reactive oxygen species and free radicals are known factors to trigger gliosis in Müller glial cells. Activator protein-1 (AP1), adenosine triphosphate (ATP), bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), calcineurin (CN), cAMP response ele‐ ment binding protein (CREB), epidermal growth factor (EGF), endothelin 1 (ET1), extracellular-signal-regulated kinase (ERK), fibroblast growth factor (FGF), interleukin (IL), inhibitor of differentiation (ID), janus kinase (JAK), mitogen acti‐ vated protein kinase (MAPK), mammalian target of rapamycin (mTOR), nuclear factor of activated T-cells (NFAT), nu‐ clear factor kappa-light-chain-enhancer of activated B cells (NF-κB), nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), uridine triphosphate (UTP), uridine diphos‐

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phate (UDP).

My lab has investigated the role of BMP7 as a potential trigger for reactive gliosis in Müller glia and retinal astrocytes. We and others have documented changes in BMP expression and signal‐ ing following injury or disease in the retina and optic nerve [134]. We have determined expres‐ sionlevelsofBMPsandBMPintracellularsignalingpathwaymembersinadiabeticmousemodel, the Akita mouse model (InsAKITA). These mice contain a naturally occurring missense mutation in

**Figure 3.** Schematic representation of various signaling mechanisms which trigger and regulate reactive gliosis in Müller glia. Growth factors such as TGF-β, BMP, EGF and CNTF; interleukins; as well as reactive oxygen species and free radicals are known factors to trigger gliosis in Müller glial cells. Activator protein-1 (AP1), adenosine triphosphate (ATP), bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), calcineurin (CN), cAMP response ele‐ ment binding protein (CREB), epidermal growth factor (EGF), endothelin 1 (ET1), extracellular-signal-regulated kinase (ERK), fibroblast growth factor (FGF), interleukin (IL), inhibitor of differentiation (ID), janus kinase (JAK), mitogen acti‐ vated protein kinase (MAPK), mammalian target of rapamycin (mTOR), nuclear factor of activated T-cells (NFAT), nu‐ clear factor kappa-light-chain-enhancer of activated B cells (NF-κB), nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), uridine triphosphate (UTP), uridine diphos‐ phate (UDP).

activate either ALK3 or ALK6 [130]. In addition to the canonical SMAD pathway, ALK3 and 6 also activate the BMP-MAPK and FRAP-STAT pathways [45, 124, 129]. The BMPs have been shown to act as a gliosis trigger in penetrating spinal cord injuries, and a differential role for ALK3 and 6 receptors has been ascribed to various aspects of gliosis, including hypertrophy, inflammation, and tissue loss [131, 132]. While BMPs have been studied in retinal injury, primarily as a survival factor for retinal neurons, it has not been

My lab has investigated the role of BMP7 as a potential trigger for reactive gliosis in Müller glia and retinal astrocytes. We and others have documented changes in BMP expression and signal‐ ing following injury or disease in the retina and optic nerve [134]. We have determined expres‐ sionlevelsofBMPsandBMPintracellularsignalingpathwaymembersinadiabeticmousemodel, the Akita mouse model (InsAKITA). These mice contain a naturally occurring missense mutation in

studied as a potential trigger for reactive gliosis in Müller glia [133].

**Growth Factors and Cytokines**

84 Neural Stem Cells - New Perspectives

248-251]

Ciliary Neurotrophic Factor/Leukemia Inhibitory Factor [86,

Epidermal growth factor/HB-EGF [84, 87, 180]

Fibroblast growth factor 2 [250, 252] Brain-derived neurotrophic factor [250]

**Transduction Pathways and Transcription Factors**

STAT3 [248, 253, 254] NF-κB [255, 256]

Toll-like receptor 2 [257]

Gp130 [249]

MEK [179, 258]

Glucose [88, 259] Amyloid Beta [260] Endothelins [261] Nitric Oxide

ATP

**Other**

**Table 2.** Triggers of Müller Glia Cell Activation

TRPV1 (Vanilloid Receptor) [85]

Epidermal growth factor receptor [87] Fibroblast growth factor receptor [179]

Oxidative Stress/Ischemia [38, 254, 255]

the insulin 2 gene that causes a switch from a cysteine to a tyrosine residue at amino acid 96, removing one of the cysteines necessary for an intramolecular disulfide bond [135]. Heterozy‐ gous mice are severely insulin deficient and become diabetic at about 6 weeks of age [135]. For these studies we used two stages; mice that are 3 weeks of age have mild to no reactive gliosis, while 6 weeks of age has moderate gliosis. Levels of BMP expression were determined by reverse transcription – quantitative polymerase chain reaction (RT-qPCR) of RNA samples from 3 and 6 week old wild type and heterozygous mice. The graphs show changes in mRNA levels in the 3 and 6 week InsAKITA mice relative to levels of mRNA in wild type samples (Fig 4A, B). Further, genes that are known downstream targets of the BMP pathway, such as inhibitor of differentia‐ tion (ID) 1, 3, and MSX2 are also increased, consistent with an increase in BMP signaling (Fig 4A, B). To verify there was an increase in canonical BMP signaling, an increase in nuclear localiza‐ tion of phospho-SMAD1 (p-SMAD1,5,8) sections through wild-type and 6 week InsAKITA retina were immunolabeled for p-SMAD1,5,8 and glutamine synthetase (Fig 4C-N). The InsAKITA retina showed a clear increase in p-SMAD1,5,8 expression in the inner nuclear layer at 6 weeks of age, some of which was coincident with cells glutamine synthetase-expressing Müller glia (Fig 4L-N).Therewasalsoclearincreaseinp-SMAD1,5,8inothercellsoftheinnernuclearlayerandcells of the ganglion cell layer.

evidence from injured spinal cord indicates axonal regeneration and functional recovery was increased in GFAP/vimentin double-knockouts in comparison to wild type controls [139]. Further, the retinas of GFAP/vimentin double knockouts were also protected from retinal degeneration following retinal detachment, and integration and neurite extension from

In addition to increased intermediate filament expression, hypertrophy is also the product of

take up K+ released by retinal neurons and release it into the bloodstream. Water in the tissue, created through the process of oxidative synthesis of ATP, is removed through the pigmented epithelium and Müller glia. The movement of water is coupled to the movement of osmolytes,

Loss of functionality is a part of the general response of the cells to undergo dedifferentiation. However, the response of the Müller cells can vary depending upon the disease or injury present. A good example of this is the regulation of the glutamate transporter in disease and following mechanical injury. Downregulation of glutamate transporters is observed in glaucoma, ischemia and diabetic retinopathy, due to a decrease in the activity of the glutamate transporter GLAST. This in turn downregulates the activity of glutamine synthetase, an enzyme involved in glutamate recycling [141]. However, following mechanical nerve injury, as seen with the optic nerve crush model, glutamine synthetase was found to localize to the ganglion cell layer, aiding in the recycling of the excess glutamate released due to neuronal

The Kir channels (potassium channels) in the Müller glial cell membrane play an important role in the gliosis response as well. Decrease in conductance of the potassium ions due to down regulation of Kir 4.1 leads to an increase in potassium ions outside the membrane. This, in turn, decreases the transport of glutamate, glucose and water across the Müller glial cell surface. Consequently, an increase in the glutamate toxicity, decrease in glutathione synthase activity and osmotic swelling were observed in the retina, which contribute to the loss of glia/neuron

There is also a reduction in the blood-retinal barrier function under hypoxic conditions. This appears to be driven by changes Müller cell expression of growth factors that regulate endothelial cell tight junctions. The balance between factors that increase endothelial cell tight junctions (PEDF, glial derived neurotrophic factor (GDNF), transforming growth factor Beta (TGFβ), thrombospondin, etc) and factors that decrease barrier function (VEGF, TNFα, FGF2, etc) is disrupted by reactive gliosis [34, 41, 147-153]. VEGF appears to be the dominant factor released from Müller glial cells in decrease of barrier function and angiogenesis that occurs in

release into the bloodstream [4]. Müller glia undergoing gliosis downregulate the K+

the blood. The end result is swelling of the Müller cell body.

conductance into the blood stream as already covered in section 1.3, Müller glia

to the vasculature, which uncouples the movement of K+

ions, and are subsequently removed from the Müller cell bodies via

channel,

87

and water into

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transplanted cells is also enhanced [140].

and K+

interactions [97, 114, 120, 143-146].

many forms of retinal injury and disease [153].

a loss of K+

including Na+

injury. [142].

Kir4.1, that delivers K+

**6.2. Loss of functionality**

To test the role of BMPs in reactive gliosis in vivo, adult murine eyes were injected intravitreally with vehicle or BMP7 and analyzed 3 or 7 days post injection. At both 3 and 7 days post vehicle injection, there were the normal low levels of GFAP expression and moderate levels of gluta‐ mine synthetase in Müller glia (Fig 5A, B, G, H). A low level of the chondroitin sulfate proteogly‐ can, neurocan, is present throughout the retina (Fig 5C, I). Three days post BMP7 injection, no increaseinGFAPwasdetected,butanincreaseinbothglutaminesynthetaseandneurocanlevels were detected (Fig 5D-F). Immunolabel of BMP7-injected eyes showed an increase of GFAP, glutamine synthetase and neurocan in comparson to vehicle-injected eyes (Fig 5J-L).

#### **6. Characteristics of reactive gliosis in Müller Glial cells**

Müller glia display many changes during reactive gliosis (Fig 6). We have grouped these changes into 6 broad categories; 1) hypertrophy, 2) loss of functionality, 3) neuroprotection, 4) inflammation, 5) proliferation, 6) remodeling.

#### **6.1. Hypertrophy**

Hypertrophy refers to the swelling of the Müller glial cell body and processes. The swelling is, in part, brought about by an increase in the expression of two type III intermediate filament genes, GFAP and vimentin. As with many changes that occur with reactive gliosis, upregula‐ tion of intermediate filaments and the ensuing hypertrophy has both good and bad charac‐ teristics associated with it. Hypertrophic glia help to form and maintain a barrier around injured tissue which helps to protect surrounding tissues from inflammatory signals [136, 137]. On one hand, there is evidence that the increased production of GFAP does not lead to diminished neuronal metabolism, eletrophysiology or visual function [138]. However, evidence from injured spinal cord indicates axonal regeneration and functional recovery was increased in GFAP/vimentin double-knockouts in comparison to wild type controls [139]. Further, the retinas of GFAP/vimentin double knockouts were also protected from retinal degeneration following retinal detachment, and integration and neurite extension from transplanted cells is also enhanced [140].

In addition to increased intermediate filament expression, hypertrophy is also the product of a loss of K+ conductance into the blood stream as already covered in section 1.3, Müller glia take up K+ released by retinal neurons and release it into the bloodstream. Water in the tissue, created through the process of oxidative synthesis of ATP, is removed through the pigmented epithelium and Müller glia. The movement of water is coupled to the movement of osmolytes, including Na+ and K+ ions, and are subsequently removed from the Müller cell bodies via release into the bloodstream [4]. Müller glia undergoing gliosis downregulate the K+ channel, Kir4.1, that delivers K+ to the vasculature, which uncouples the movement of K+ and water into the blood. The end result is swelling of the Müller cell body.

#### **6.2. Loss of functionality**

the insulin 2 gene that causes a switch from a cysteine to a tyrosine residue at amino acid 96, removing one of the cysteines necessary for an intramolecular disulfide bond [135]. Heterozy‐ gous mice are severely insulin deficient and become diabetic at about 6 weeks of age [135]. For these studies we used two stages; mice that are 3 weeks of age have mild to no reactive gliosis, while 6 weeks of age has moderate gliosis. Levels of BMP expression were determined by reverse transcription – quantitative polymerase chain reaction (RT-qPCR) of RNA samples from 3 and 6 week old wild type and heterozygous mice. The graphs show changes in mRNA levels in the 3 and 6 week InsAKITA mice relative to levels of mRNA in wild type samples (Fig 4A, B). Further, genes that are known downstream targets of the BMP pathway, such as inhibitor of differentia‐ tion (ID) 1, 3, and MSX2 are also increased, consistent with an increase in BMP signaling (Fig 4A, B). To verify there was an increase in canonical BMP signaling, an increase in nuclear localiza‐ tion of phospho-SMAD1 (p-SMAD1,5,8) sections through wild-type and 6 week InsAKITA retina were immunolabeled for p-SMAD1,5,8 and glutamine synthetase (Fig 4C-N). The InsAKITA retina showed a clear increase in p-SMAD1,5,8 expression in the inner nuclear layer at 6 weeks of age, some of which was coincident with cells glutamine synthetase-expressing Müller glia (Fig 4L-N).Therewasalsoclearincreaseinp-SMAD1,5,8inothercellsoftheinnernuclearlayerandcells

To test the role of BMPs in reactive gliosis in vivo, adult murine eyes were injected intravitreally with vehicle or BMP7 and analyzed 3 or 7 days post injection. At both 3 and 7 days post vehicle injection, there were the normal low levels of GFAP expression and moderate levels of gluta‐ mine synthetase in Müller glia (Fig 5A, B, G, H). A low level of the chondroitin sulfate proteogly‐ can, neurocan, is present throughout the retina (Fig 5C, I). Three days post BMP7 injection, no increaseinGFAPwasdetected,butanincreaseinbothglutaminesynthetaseandneurocanlevels were detected (Fig 5D-F). Immunolabel of BMP7-injected eyes showed an increase of GFAP,

Müller glia display many changes during reactive gliosis (Fig 6). We have grouped these changes into 6 broad categories; 1) hypertrophy, 2) loss of functionality, 3) neuroprotection, 4)

Hypertrophy refers to the swelling of the Müller glial cell body and processes. The swelling is, in part, brought about by an increase in the expression of two type III intermediate filament genes, GFAP and vimentin. As with many changes that occur with reactive gliosis, upregula‐ tion of intermediate filaments and the ensuing hypertrophy has both good and bad charac‐ teristics associated with it. Hypertrophic glia help to form and maintain a barrier around injured tissue which helps to protect surrounding tissues from inflammatory signals [136, 137]. On one hand, there is evidence that the increased production of GFAP does not lead to diminished neuronal metabolism, eletrophysiology or visual function [138]. However,

glutamine synthetase and neurocan in comparson to vehicle-injected eyes (Fig 5J-L).

**6. Characteristics of reactive gliosis in Müller Glial cells**

inflammation, 5) proliferation, 6) remodeling.

of the ganglion cell layer.

86 Neural Stem Cells - New Perspectives

**6.1. Hypertrophy**

Loss of functionality is a part of the general response of the cells to undergo dedifferentiation. However, the response of the Müller cells can vary depending upon the disease or injury present. A good example of this is the regulation of the glutamate transporter in disease and following mechanical injury. Downregulation of glutamate transporters is observed in glaucoma, ischemia and diabetic retinopathy, due to a decrease in the activity of the glutamate transporter GLAST. This in turn downregulates the activity of glutamine synthetase, an enzyme involved in glutamate recycling [141]. However, following mechanical nerve injury, as seen with the optic nerve crush model, glutamine synthetase was found to localize to the ganglion cell layer, aiding in the recycling of the excess glutamate released due to neuronal injury. [142].

The Kir channels (potassium channels) in the Müller glial cell membrane play an important role in the gliosis response as well. Decrease in conductance of the potassium ions due to down regulation of Kir 4.1 leads to an increase in potassium ions outside the membrane. This, in turn, decreases the transport of glutamate, glucose and water across the Müller glial cell surface. Consequently, an increase in the glutamate toxicity, decrease in glutathione synthase activity and osmotic swelling were observed in the retina, which contribute to the loss of glia/neuron interactions [97, 114, 120, 143-146].

There is also a reduction in the blood-retinal barrier function under hypoxic conditions. This appears to be driven by changes Müller cell expression of growth factors that regulate endothelial cell tight junctions. The balance between factors that increase endothelial cell tight junctions (PEDF, glial derived neurotrophic factor (GDNF), transforming growth factor Beta (TGFβ), thrombospondin, etc) and factors that decrease barrier function (VEGF, TNFα, FGF2, etc) is disrupted by reactive gliosis [34, 41, 147-153]. VEGF appears to be the dominant factor released from Müller glial cells in decrease of barrier function and angiogenesis that occurs in many forms of retinal injury and disease [153].

an increase in levels of BMP7 with a subsequent decrease in the levels of other BMP molecules, indicating a role for BMP7 in reactive gliosis in the diseased state. Immunohistochemistry was performed to determine the localization of phospho SMAD with glutamine synthetase in the retinas (**C – N**). The 3 week retinas show similar nuclear phospho SMAD levels in both the wild type and the Ins2Akita (**C, E, F and H**). In the 6 week Ins2Akita, there is a clear increase in the phospho SMAD levels in the inner nuclear layer nuclei (**L and N**) when compared to the wild type (**I and K**), possibly

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**Figure 5.** Effect of intra vitreal injections of BMP7 into normal mouse eyes – Retinal sections of eyes injected with ei‐ ther vehicle or BMP7 were analyzed 3 days (**A – F**) and 7 days (**G – L**) post injection via immunohistochemistry for the localization of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and neurocan. Retinas isolated 3 days post injections do not show an increase in GFAP (**A and D**) or neurocan (**C and F**), although GS does seem to show an increase when compared to the vehicle injected eyes (**B and E**). Retinas isolated 7 days post injection did show a clear increase in GFAP (**G and J**), GS (**H and K**) and neurocan (**I and L**) in the BMP7 injected eyes compared to the control

eyes, suggesting the BMP7 was able to trigger gliosis in these retinas.

due to the increase in BMP7 shown previously (**B**).

**Figure 4.** Analysis of retinas of the Ins2Akita diabetic mouse shows increase in BMP signaling in the diseased eye when compared to the wild type eye. **A** and **B**: qPCR results analyzing the levels of various BMP molecules shown to be regu‐ lated during reactive gliosis and some of the targets of the canonical BMP signaling pathway, using RNA obtained from whole retinas in 3 week and 6 week diseased eye, respectively, normalized to their respective wild types. At the 3 week stage (**A**), when little or no gliosis is observed (data not shown) levels of BMP 2, 4 and 6 appear to be high. At the 6 week stage (**B**) when we do seen an increase in expression of GFAP, GS and neurocan (data not shown), there is

an increase in levels of BMP7 with a subsequent decrease in the levels of other BMP molecules, indicating a role for BMP7 in reactive gliosis in the diseased state. Immunohistochemistry was performed to determine the localization of phospho SMAD with glutamine synthetase in the retinas (**C – N**). The 3 week retinas show similar nuclear phospho SMAD levels in both the wild type and the Ins2Akita (**C, E, F and H**). In the 6 week Ins2Akita, there is a clear increase in the phospho SMAD levels in the inner nuclear layer nuclei (**L and N**) when compared to the wild type (**I and K**), possibly due to the increase in BMP7 shown previously (**B**).

**Figure 5.** Effect of intra vitreal injections of BMP7 into normal mouse eyes – Retinal sections of eyes injected with ei‐ ther vehicle or BMP7 were analyzed 3 days (**A – F**) and 7 days (**G – L**) post injection via immunohistochemistry for the localization of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and neurocan. Retinas isolated 3 days post injections do not show an increase in GFAP (**A and D**) or neurocan (**C and F**), although GS does seem to show an increase when compared to the vehicle injected eyes (**B and E**). Retinas isolated 7 days post injection did show a clear increase in GFAP (**G and J**), GS (**H and K**) and neurocan (**I and L**) in the BMP7 injected eyes compared to the control eyes, suggesting the BMP7 was able to trigger gliosis in these retinas.

**Figure 4.** Analysis of retinas of the Ins2Akita diabetic mouse shows increase in BMP signaling in the diseased eye when compared to the wild type eye. **A** and **B**: qPCR results analyzing the levels of various BMP molecules shown to be regu‐ lated during reactive gliosis and some of the targets of the canonical BMP signaling pathway, using RNA obtained from whole retinas in 3 week and 6 week diseased eye, respectively, normalized to their respective wild types. At the 3 week stage (**A**), when little or no gliosis is observed (data not shown) levels of BMP 2, 4 and 6 appear to be high. At the 6 week stage (**B**) when we do seen an increase in expression of GFAP, GS and neurocan (data not shown), there is

88 Neural Stem Cells - New Perspectives

Müller cells also protect retinal neurons from oxidative stress, excitotoxicity and from dam‐ aging reactive oxygen species via conversion of glutamate to glutamine as well as synthesis and release of antioxidants such as glutathione [165, 166]. However, concomitant with an increase in the antioxidant glutathione, during hypoxia, diabetic retinopathy, hyperglycemia and ischemia there is also an increase in the expression of inducible nitric oxide synthase and cyclooxygenase-2 [167, 168]. These enzymes can lead to production of nitric oxide, prosta‐ glandins and superoxides which are detrimental to retinal neurons and may induce apoptosis in neural cells [169]. Nitric oxide also has a beneficial role as it increases blood flow by dilating blood vessels and prevents glutamate toxicity by closing *N-*methyl –D-aspartate (NMDA)

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Müller cells also play a role in the inflammatory response observed in the retina, primarily seen in the diabetic retina. Under these conditions, the activated Müller cells begin expressing pro inflammatory cytokine interleukin-6 (IL-6) and IL-1B [171, 172]. They also increase expression of TNF-α which increases the expression of the chemokine IL-8 and MCP-8, and promotes infiltration of inflammatory cells [173]. The inflammatory response is further supported by the decrease in glutamate uptake in diabetic retinas. This increases the expression of glutaredoxin, which translocates NF-κB to the nucleus and increases the expression of pro-

Dedifferentiation and proliferation of Müller glia is known to occur in many different species, including chick, fish, and even mammalians [108, 110, 174-177]. Several aspects of Müller cell proliferation are of interest here; 1) the molecular pathways that result in the release of the cells from their normally quiescent state, 2) extrinsic signals that are necessa‐ ry for the proliferative reponse, and 3) directing progenitor cells to differentiation and

Several intracellular signaling pathways have been investigated to determine those that may be important for the proliferative response in dedifferentiating Müller glia. The FGF-MAPK pathway appears to be indespensible for the proliferative activity seen during reactive gliosis [178, 179]. The heparin binding epidermal growth factor (HBEGF)-MAPK pathway is be also induced in the Müller glia found in injured areas and appears to be associated with regener‐ ation-associated genes [180]. Further, the HB-EGF pathway appears to be upstream of the WNT-β-catenin pathway, which has been very clearly associated wth re-entrance of Müller glia into the cell cycle [181]. More specifically, Müller glia that are poised to re-enter the cell cycle accumulate β-catenin in injured zebrafish retina, whereas those Müller cells that remain quiescent do not accumulate β-catenin [181]. Further, activation of the WNT/β-catenin pathway stimulates a loss of Müller glia and a concomitant increase in newly generated

receptors [170].

**6.4. Inflammation**

inflammatory proteins [141].

integration into retinal tissue.

photoreceptors [181].

**6.5. Proliferation**

**Figure 6.** Schematic representation of reactive gliosis response in Müller glia depending on the extent of the injury. Mild changes in reactive gliosis comprises of hypertrophy of the cells due to an increase in glial fibrillary acidic protein and changes to the function and morphology of the cell, with little or no proliferation which has the potential to re‐ solve once the stimulus subdues. Severe reactive gliosis occurs following tissue damage and induces Müller glial cell proliferation, overlapping of cell processes, hypertrophy, functional and morphological changes. Under severe gliosis conditions reactive Müller cells have shown the ability to dedifferentiate and give rise to some of the retinal cells types.

#### **6.3. Neuroprotection**

Reactive gliosis in Müller cells is a complex response dependent on the injury or disease. Diseases which lead to retinal degeneration such as retinal detachment, retinitis pigmentosa or physical damage to the retina elucidate such a response from the Müller cells to aid in neuoprotection and prevent apoptosis [114, 141]. A wide range of growth factors secreted by the reactive Müller cells, including bFGF, GDNF, CNTF, and VEGF [114, 141, 150, 154, 155]. Upregulation of CNTF and bFGF have been observed following mechanical injury, ischemia and NMDA mediated neuronal death [156-158]. These growth factors help to increase neuron survival and inhibit apoptosis, either directly as is the case for bFGF, or indirectly in the case of CNTF and GDNF [159, 160]. GDNF also upregulates GLAST, thereby, protecting neurons from excessive glutamate excitotoxicity [160]. VEGF is another factor which is upregulated following gliosis. Hypoxia as well as diabetes has shown to increase the VEGF secretion by Müller glial cells [161, 162]. VEGF may act directly by increasing the permeability of the endothelial cells [163]. VEGF may also be regulated by TGF-β released during hypoxia, which, along with other cytokines such as TNF-α, increase the expression of matrix metalloproteinases which can clear the basement membranes of these cells generating leaky vessels [38, 164].

Müller cells also protect retinal neurons from oxidative stress, excitotoxicity and from dam‐ aging reactive oxygen species via conversion of glutamate to glutamine as well as synthesis and release of antioxidants such as glutathione [165, 166]. However, concomitant with an increase in the antioxidant glutathione, during hypoxia, diabetic retinopathy, hyperglycemia and ischemia there is also an increase in the expression of inducible nitric oxide synthase and cyclooxygenase-2 [167, 168]. These enzymes can lead to production of nitric oxide, prosta‐ glandins and superoxides which are detrimental to retinal neurons and may induce apoptosis in neural cells [169]. Nitric oxide also has a beneficial role as it increases blood flow by dilating blood vessels and prevents glutamate toxicity by closing *N-*methyl –D-aspartate (NMDA) receptors [170].

#### **6.4. Inflammation**

Müller cells also play a role in the inflammatory response observed in the retina, primarily seen in the diabetic retina. Under these conditions, the activated Müller cells begin expressing pro inflammatory cytokine interleukin-6 (IL-6) and IL-1B [171, 172]. They also increase expression of TNF-α which increases the expression of the chemokine IL-8 and MCP-8, and promotes infiltration of inflammatory cells [173]. The inflammatory response is further supported by the decrease in glutamate uptake in diabetic retinas. This increases the expression of glutaredoxin, which translocates NF-κB to the nucleus and increases the expression of proinflammatory proteins [141].

#### **6.5. Proliferation**

**Figure 6.** Schematic representation of reactive gliosis response in Müller glia depending on the extent of the injury. Mild changes in reactive gliosis comprises of hypertrophy of the cells due to an increase in glial fibrillary acidic protein and changes to the function and morphology of the cell, with little or no proliferation which has the potential to re‐ solve once the stimulus subdues. Severe reactive gliosis occurs following tissue damage and induces Müller glial cell proliferation, overlapping of cell processes, hypertrophy, functional and morphological changes. Under severe gliosis conditions reactive Müller cells have shown the ability to dedifferentiate and give rise to some of the retinal cells

Reactive gliosis in Müller cells is a complex response dependent on the injury or disease. Diseases which lead to retinal degeneration such as retinal detachment, retinitis pigmentosa or physical damage to the retina elucidate such a response from the Müller cells to aid in neuoprotection and prevent apoptosis [114, 141]. A wide range of growth factors secreted by the reactive Müller cells, including bFGF, GDNF, CNTF, and VEGF [114, 141, 150, 154, 155]. Upregulation of CNTF and bFGF have been observed following mechanical injury, ischemia and NMDA mediated neuronal death [156-158]. These growth factors help to increase neuron survival and inhibit apoptosis, either directly as is the case for bFGF, or indirectly in the case of CNTF and GDNF [159, 160]. GDNF also upregulates GLAST, thereby, protecting neurons from excessive glutamate excitotoxicity [160]. VEGF is another factor which is upregulated following gliosis. Hypoxia as well as diabetes has shown to increase the VEGF secretion by Müller glial cells [161, 162]. VEGF may act directly by increasing the permeability of the endothelial cells [163]. VEGF may also be regulated by TGF-β released during hypoxia, which, along with other cytokines such as TNF-α, increase the expression of matrix metalloproteinases which can clear the basement membranes of these cells generating leaky vessels [38, 164].

types.

**6.3. Neuroprotection**

90 Neural Stem Cells - New Perspectives

Dedifferentiation and proliferation of Müller glia is known to occur in many different species, including chick, fish, and even mammalians [108, 110, 174-177]. Several aspects of Müller cell proliferation are of interest here; 1) the molecular pathways that result in the release of the cells from their normally quiescent state, 2) extrinsic signals that are necessa‐ ry for the proliferative reponse, and 3) directing progenitor cells to differentiation and integration into retinal tissue.

Several intracellular signaling pathways have been investigated to determine those that may be important for the proliferative response in dedifferentiating Müller glia. The FGF-MAPK pathway appears to be indespensible for the proliferative activity seen during reactive gliosis [178, 179]. The heparin binding epidermal growth factor (HBEGF)-MAPK pathway is be also induced in the Müller glia found in injured areas and appears to be associated with regener‐ ation-associated genes [180]. Further, the HB-EGF pathway appears to be upstream of the WNT-β-catenin pathway, which has been very clearly associated wth re-entrance of Müller glia into the cell cycle [181]. More specifically, Müller glia that are poised to re-enter the cell cycle accumulate β-catenin in injured zebrafish retina, whereas those Müller cells that remain quiescent do not accumulate β-catenin [181]. Further, activation of the WNT/β-catenin pathway stimulates a loss of Müller glia and a concomitant increase in newly generated photoreceptors [181].

In order for Müller glia to re-enter and progress in the cell cycle, the cells would also have to suppress some of the cell cycle check-points that are responsible for the quiescent state of the cells. Inhibition of the cyclin kinase inhibitor p27 has been shown to play a pivitol role in the ability of Müller glia to re-enter the cell cycle. P27 regulates the cell cycle by blocking cell cycle progression into the S-phase, and hence is necessary for maintainance of the quiescent state [182]. Knock-out mice for p27 show many of the characteristics of reactive gliosis, including an increase in GFAP expression and proliferation and migration of cells into the subretinal space [138, 182-184].

proteolytically cleaves one or more ECM molecules. The activity of MMPs is regulated by activators as well as inhibitors; the precursor molecules must be processed, either by already activated MMPs or by one of a variety of serine proteases and the MMPs can be inhibited by the tissue inhibitors of metalloproteinases (TIMPs) [219]. When activated, the MMPs degrade the existing ECM in preparation for replacement with an ECM that partially inhibits neurite outgrowth or supports abnormal neurite outgrowth [141]. In the normal adult retina heparin sulfate proeoglycans (HSPGs) are typically found on Müller glial endfeet and in retinal basal lamina, serving as a substrate for axonal outgrowth. The HSPG, via the HS chains, is also a ligand for the protein tyrosine phosphatase-sigma (PTP-σ), used in signaling in axons and growth cones in response to matrix cues. The HSPGs involved are agrin and collagen XVIII [220]. The HSPGs are lost in favor of the axonal outgrowth inhibitory molecules known as the chondroitan sulfate proteoglycans (CSPGs). The CSPGs include phosphacan, aggrecan, NG2, brevican, versican, and neurocan [221]. In addition to turning over the ECM, the degradation of the ECM also releases growth factors that are bound to the ECM, such as EGF, FGFs, BMPs,

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93

Müller glia can form new neurons in a process said to involve dedifferentiation of the Müller glia. Tenascin C (TNC), a matricellular protein, influences the dedifferentiation behavior of Müller glia in response to FGF2 in vitro, affecting the composition of the ECM. Sulfated chondroitin glycosaminoglycan chains in CSPG are the main target. Chondroitin sulfate increases in TNC-deficient mouse ECM [222]. The proteoglycan most affected by TNC is the CSPG Phosphacan/RPTPβ/ζ which bind to TNC [223]. TNC shows overlapping expression with phosphacan [224]. In a TNC knock out mouse TNC level rise. Studies using immunocy‐ tochemistry for phosphacan, Western Blots and PCR for mRNA levels show that it is the chondroitin sulfate chains that increase, not the amount of mRNA for CSPG core protein. Proliferation rates also increase in the TNC-deficient mice, but it is not clear if this affects exit

SPARC(secretedprotein,acidicandrichincysteine)*/*osteonectinisalsoamatricellular.Itinteracts with growth factors and ECM forming a link that modulates the cell cycle and other cell behav‐ ior. SPARC remains expressed at significant levels in the adult CNS, moreso than in most normal adult tissues. SPARC is widely expressed in remodeling injured tissue and in morphogenesis in development [225]. In normal newborn and adult bovine retinas SPARC is found in ganglion cell soma and in ganglion cell axons, with higher expression in the adult tissue. SPARC is thought to have a function in maintaining healthy retinas and is localized to the ganglion cell layer (GCL), nerve fiber layer (NFL) and some retinal capillaries. Müller glia showed no immunoreactivity,

The evidence to date has shown that Müller glia undergo dedifferentiation and generate retinal progenitors that may be capable of differentiating into retinal neurons. Several potential problems have arisen that impact on the ability of those progentiors to effectively be used to

insulin, and VEGFs [219].

**7. Conclusion**

from the cell cycle and differentiation [222].

but the GFAP-positive retinal astrocytes were SPARC-positive [225].

#### **6.6. Remodeling**

There appears to be three elements of the retina which can undergo remodeling as a result of gliosis; 1) vasculature, 2) the Müller glia themselves, and 3) the extracellular matrix. The neovascularization is, for the most part, due to an imbalance between the antiangiogenic factor PEDF and and the angiogenic factor VEGF [162, 185-190]. Under hypoxic conditions, tran‐ scriptional activation of VEGF occurs by translocation of the newly stabilized hypoxia inducible factor-1α (HIF-1α) and it's partner HIF-1β to the nucleus where they bind to the hypoxia responsive element (HRE) in the 5' flanking regions of the VEGF gene [191, 192]. VEGF is released into the extracellular mileu, where it penetrates the basal laminae and interacts with retinal endothelial cells. This interaction results in an increase in the release of a family of zincdependent endopeptidases called the matrix metalleoproteinases (MMPs), plasminogen activators, and other proteinases which degrade proteins, such as occludens, which necessary for the tight junction formation between endothelial cells [192-196]. The VEGF activates the MAPK pathway via phospholipase C-γ, which mediates proliferation of the endolthelial cells [197]. The MMPs also degrade the basal laminae, removing contact inhibition of the endothelial cells and permitting proliferation [38].

The Müller glia participate in remodeling themselves by extending hypertrophied processes into areas they are not typically found. For instance, processes can protrude into the subretinal space, plexiform layers, the vitreous, into occluded blood vessels, and even into the choroid [122, 198-203]. In some respects, the Müller glia are expanding into areas where degenerating neurons and/or axonal processes are found, such as the subretinal space or plexiform layers [204]. If these new processes persist, the end result is the formation of scar tissue, which can permanently block the reattachment of the retina, regeneration of outer segments or regener‐ ation of synaptic contacts in the plexiform layers [118, 122, 205-209]. The extension of processes onto the vitreal surface of the retina results in the formation of periretinal membranes that may under epithelial to mesenchymal transformation into myofibrocytes that spread and become contractile [210]. The contractility leads to folds and/or deformations in the retina, causing visual distortions at the very least, and, at worst, can cause retinal detachments [211, 212]. Glial membranes/scars are a significant issue in the treatment of visual disorders in humans, occuring in appoximately 15% of retinal detachments [213].

The last element of the retina that undergoes remodeling during reactive gliosis is the extrac‐ ellular matrix (ECM). During reactive gliosis, Müller glia upregulate expression of MMPs and the gene products are secreted and activated [196, 214-218]. Each MMP specifically targets and proteolytically cleaves one or more ECM molecules. The activity of MMPs is regulated by activators as well as inhibitors; the precursor molecules must be processed, either by already activated MMPs or by one of a variety of serine proteases and the MMPs can be inhibited by the tissue inhibitors of metalloproteinases (TIMPs) [219]. When activated, the MMPs degrade the existing ECM in preparation for replacement with an ECM that partially inhibits neurite outgrowth or supports abnormal neurite outgrowth [141]. In the normal adult retina heparin sulfate proeoglycans (HSPGs) are typically found on Müller glial endfeet and in retinal basal lamina, serving as a substrate for axonal outgrowth. The HSPG, via the HS chains, is also a ligand for the protein tyrosine phosphatase-sigma (PTP-σ), used in signaling in axons and growth cones in response to matrix cues. The HSPGs involved are agrin and collagen XVIII [220]. The HSPGs are lost in favor of the axonal outgrowth inhibitory molecules known as the chondroitan sulfate proteoglycans (CSPGs). The CSPGs include phosphacan, aggrecan, NG2, brevican, versican, and neurocan [221]. In addition to turning over the ECM, the degradation of the ECM also releases growth factors that are bound to the ECM, such as EGF, FGFs, BMPs, insulin, and VEGFs [219].

Müller glia can form new neurons in a process said to involve dedifferentiation of the Müller glia. Tenascin C (TNC), a matricellular protein, influences the dedifferentiation behavior of Müller glia in response to FGF2 in vitro, affecting the composition of the ECM. Sulfated chondroitin glycosaminoglycan chains in CSPG are the main target. Chondroitin sulfate increases in TNC-deficient mouse ECM [222]. The proteoglycan most affected by TNC is the CSPG Phosphacan/RPTPβ/ζ which bind to TNC [223]. TNC shows overlapping expression with phosphacan [224]. In a TNC knock out mouse TNC level rise. Studies using immunocy‐ tochemistry for phosphacan, Western Blots and PCR for mRNA levels show that it is the chondroitin sulfate chains that increase, not the amount of mRNA for CSPG core protein. Proliferation rates also increase in the TNC-deficient mice, but it is not clear if this affects exit from the cell cycle and differentiation [222].

SPARC(secretedprotein,acidicandrichincysteine)*/*osteonectinisalsoamatricellular.Itinteracts with growth factors and ECM forming a link that modulates the cell cycle and other cell behav‐ ior. SPARC remains expressed at significant levels in the adult CNS, moreso than in most normal adult tissues. SPARC is widely expressed in remodeling injured tissue and in morphogenesis in development [225]. In normal newborn and adult bovine retinas SPARC is found in ganglion cell soma and in ganglion cell axons, with higher expression in the adult tissue. SPARC is thought to have a function in maintaining healthy retinas and is localized to the ganglion cell layer (GCL), nerve fiber layer (NFL) and some retinal capillaries. Müller glia showed no immunoreactivity, but the GFAP-positive retinal astrocytes were SPARC-positive [225].

#### **7. Conclusion**

In order for Müller glia to re-enter and progress in the cell cycle, the cells would also have to suppress some of the cell cycle check-points that are responsible for the quiescent state of the cells. Inhibition of the cyclin kinase inhibitor p27 has been shown to play a pivitol role in the ability of Müller glia to re-enter the cell cycle. P27 regulates the cell cycle by blocking cell cycle progression into the S-phase, and hence is necessary for maintainance of the quiescent state [182]. Knock-out mice for p27 show many of the characteristics of reactive gliosis, including an increase in GFAP expression and proliferation and migration of cells into the subretinal

There appears to be three elements of the retina which can undergo remodeling as a result of gliosis; 1) vasculature, 2) the Müller glia themselves, and 3) the extracellular matrix. The neovascularization is, for the most part, due to an imbalance between the antiangiogenic factor PEDF and and the angiogenic factor VEGF [162, 185-190]. Under hypoxic conditions, tran‐ scriptional activation of VEGF occurs by translocation of the newly stabilized hypoxia inducible factor-1α (HIF-1α) and it's partner HIF-1β to the nucleus where they bind to the hypoxia responsive element (HRE) in the 5' flanking regions of the VEGF gene [191, 192]. VEGF is released into the extracellular mileu, where it penetrates the basal laminae and interacts with retinal endothelial cells. This interaction results in an increase in the release of a family of zincdependent endopeptidases called the matrix metalleoproteinases (MMPs), plasminogen activators, and other proteinases which degrade proteins, such as occludens, which necessary for the tight junction formation between endothelial cells [192-196]. The VEGF activates the MAPK pathway via phospholipase C-γ, which mediates proliferation of the endolthelial cells [197]. The MMPs also degrade the basal laminae, removing contact inhibition of the endothelial

The Müller glia participate in remodeling themselves by extending hypertrophied processes into areas they are not typically found. For instance, processes can protrude into the subretinal space, plexiform layers, the vitreous, into occluded blood vessels, and even into the choroid [122, 198-203]. In some respects, the Müller glia are expanding into areas where degenerating neurons and/or axonal processes are found, such as the subretinal space or plexiform layers [204]. If these new processes persist, the end result is the formation of scar tissue, which can permanently block the reattachment of the retina, regeneration of outer segments or regener‐ ation of synaptic contacts in the plexiform layers [118, 122, 205-209]. The extension of processes onto the vitreal surface of the retina results in the formation of periretinal membranes that may under epithelial to mesenchymal transformation into myofibrocytes that spread and become contractile [210]. The contractility leads to folds and/or deformations in the retina, causing visual distortions at the very least, and, at worst, can cause retinal detachments [211, 212]. Glial membranes/scars are a significant issue in the treatment of visual disorders in humans,

The last element of the retina that undergoes remodeling during reactive gliosis is the extrac‐ ellular matrix (ECM). During reactive gliosis, Müller glia upregulate expression of MMPs and the gene products are secreted and activated [196, 214-218]. Each MMP specifically targets and

space [138, 182-184].

92 Neural Stem Cells - New Perspectives

cells and permitting proliferation [38].

occuring in appoximately 15% of retinal detachments [213].

**6.6. Remodeling**

The evidence to date has shown that Müller glia undergo dedifferentiation and generate retinal progenitors that may be capable of differentiating into retinal neurons. Several potential problems have arisen that impact on the ability of those progentiors to effectively be used to regenerate large numbers of neurons following injury or during disease. Of the proliferating population that arise from dedifferentiated Müller glia, a very small percentage go on to become retinal neurons [4, 141]. The inability of the cells to differentiate into retinal neurons implies that either the signals and/or competence necessary for differentiation have been lost or there are signals present that direct progenitor cells away from differentiation into retinal neurons. Further, if the progenitor cells can be induced to differentiate, they will have to functionally integrate into the diseased or injured retina. This, in and of itself, will be a challenge if glial scars are present in the tissue as the glial scars will prevent ntegration by inhibiting migration, placement, and/or synapse formation. Clearly, investigators have been untangling which signaling pathways are critical for various aspects of reactive gliosis to occur. If signals that are necessary for proliferation can be separated from those necessary for glial scars to form, there is the possibility that therapeutic approaches could be engineered that will block scar formation while allowing proliferation to occur. There are many challenges ahead before the potential of Müller glia as a source for retinal regeneration can be realized.

[2] Wohl SG, Schmeer CW, Isenmann S. Neurogenic potential of stem/progenitor-like cells in the adult mammalian eye. Prog Retin Eye Res. 2012 May;31(3):213-42.

Reactive Muller Glia as Potential Retinal Progenitors

http://dx.doi.org/10.5772/55150

95

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#### **Acknowledgements**

This work was supported by grant 1R01EY019525 (TBA) and 1R15EY020816 (TBA) from the National Eye Institute and the American Health Assistance Foundation (TBA).

#### **Author details**

Teri L. Belecky-Adams1,2, Ellen C. Chernoff1,2, Jonathan M. Wilson1,2 and Subramanian Dharmarajan1,2

\*Address all correspondence to: tbadams@iupui.edu

1 Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA

2 Center for Regenerative Biology and Medicine, Indiana University- Purdue University In‐ dianapolis, IN, USA

#### **References**

[1] Daadi MM. Novel paths towards neural cellular products for neurological disorders. Regenerative medicine. 2011 Nov;6(6 Suppl):25-30. PubMed PMID: 21999259. Epub 2011/10/26. eng.

[2] Wohl SG, Schmeer CW, Isenmann S. Neurogenic potential of stem/progenitor-like cells in the adult mammalian eye. Prog Retin Eye Res. 2012 May;31(3):213-42. PubMed PMID: 22353284. Epub 2012/02/23. eng.

regenerate large numbers of neurons following injury or during disease. Of the proliferating population that arise from dedifferentiated Müller glia, a very small percentage go on to become retinal neurons [4, 141]. The inability of the cells to differentiate into retinal neurons implies that either the signals and/or competence necessary for differentiation have been lost or there are signals present that direct progenitor cells away from differentiation into retinal neurons. Further, if the progenitor cells can be induced to differentiate, they will have to functionally integrate into the diseased or injured retina. This, in and of itself, will be a challenge if glial scars are present in the tissue as the glial scars will prevent ntegration by inhibiting migration, placement, and/or synapse formation. Clearly, investigators have been untangling which signaling pathways are critical for various aspects of reactive gliosis to occur. If signals that are necessary for proliferation can be separated from those necessary for glial scars to form, there is the possibility that therapeutic approaches could be engineered that will block scar formation while allowing proliferation to occur. There are many challenges ahead

before the potential of Müller glia as a source for retinal regeneration can be realized.

National Eye Institute and the American Health Assistance Foundation (TBA).

Teri L. Belecky-Adams1,2, Ellen C. Chernoff1,2, Jonathan M. Wilson1,2 and

\*Address all correspondence to: tbadams@iupui.edu

This work was supported by grant 1R01EY019525 (TBA) and 1R15EY020816 (TBA) from the

1 Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis,

2 Center for Regenerative Biology and Medicine, Indiana University- Purdue University In‐

[1] Daadi MM. Novel paths towards neural cellular products for neurological disorders. Regenerative medicine. 2011 Nov;6(6 Suppl):25-30. PubMed PMID: 21999259. Epub

**Acknowledgements**

94 Neural Stem Cells - New Perspectives

**Author details**

IN, USA

dianapolis, IN, USA

2011/10/26. eng.

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Subramanian Dharmarajan1,2


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**Chapter 5**

**Neural Stem Cell: Tools to Unravel Pathogenetic**

Luca Colucci-D'Amato and MariaTeresa Gentile

**1.1. Neurogenesis, stem cells and cellular models of diseases**

periventricular subependymal zone and expanded in vitro [2].

properly cited.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55677

**1. Introduction**

and adult brain.

**Mechanisms and to Test Novel Drugs for CNS Diseases**

In a study published in 1992, Weiss and Reynolds at University of Calgary, demonstrated for the first time that cells isolated from the brain of adult mice have the ability to proliferate in vitro and differentiate into neurons, astrocytes and oligodendrocytes using a specific cocktails of growth factors [1]. In 1999, similar stem/progenitor cells were isolated from the human adult

Neural stem cells (NSC) are self-renewing, multipotent cells residing in the nervous system. NSC during development produce the enormous diversity of neurons, astrocytes and oligo‐ dendrocytes within the developing nervous system. However, accumulating evidence has clearly shown that a number of newborn neurons can be generated also from adult NSC, integrates into pre-existing neural circuits and is functional [3]. In the adult brain, neurogenesis is not a diffuse event and occurs in restricted regions, where classical developmental signals and morphogens such as, Bone Morphogenic Proteins (BMPs), ephrins, Noggin, Sonic hedgehog homolog (Shh), and Notch expression are maintained even after differentiation [4]. In particular, the Notch pathway is a highly conserved arbiter of cell fate decisions and is intimately involved in developmental processes [5]. Thus, besides its pivotal role in neural development, it is also involved in the control of neurogenesis, neuritic growth [6], neural stem cell maintenance [7], synaptic plasticity [8] and long term memory [9] both in the developing

In the adult brain the well-established restricted regions of neurogenesis, named niches, are the sub ventricular zone (SVZ) of the lateral ventricle wall and the dentate gyrus subgranular zone (SGZ) of the hippocampus [9]. Several reports describe that neurogenesis may also occur

and reproduction in any medium, provided the original work is properly cited.

© 2013 Colucci-D'Amato and Gentile; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
