**Systems for** *ex-vivo* **Isolation and Culturing of Neural Stem Cells**

Simona Casarosa, Jacopo Zasso and Luciano Conti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55137

#### **1. Introduction**

During neural development, a relatively small and formerly considered homogeneous population of Neural Stem cells (NSCs) gives rise to the extraordinary complexity proper of the Central Nervous System (CNS). These represent populations of self-renewing multipotent cells able to differentiate into a variety of neuronal and glial cell types in a time- and regionspecific manner throughout developmental stages and that account for a weak regenerative potential in the adult brain [1].

In the adult mammalian CNS, the presence of NSCs has been extensively investigated in two regions, the SVZ and the SGZ of the hippocampus, two specialized niches that control NSCs divisions in order to physiologically regulate their proliferative (symmetrical divisions) *vs* differentiative fate (asymmetrical divisions) [2].

In the early '90s it was shown that NSCs could be extracted from the developing and adult mammalian brain and expanded/manipulated/differentiated *in vitro* (Fig. 1)*.*

This has represented a key step in the field, since the obtainment of *in vitro* NSC sys‐ tems has been very useful in the last years in order to progress toward disclosure of the complex interplay of different extrinsic (signaling pathways) and intrinsic (transcription factors and epigenetics) signals that govern identity and functional properties of brain tissue-specific stem/progenitors [3]. Furthermore, it will also be a key step towards their exploitation for a better dissection of the molecular processes occurring in neurodegenera‐ tion [4]. Finally, NSC systems might represent major tools for the potential development of new cell-based and pharmacological treatments of neurodegenerative disorders and for assaying their toxicological effects [5].

© 2013 Casarosa 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.

ed by secretion of an array of BMP inhibitors, chordin, noggin and follistatin produced by an embryonic structure called "organizer" [6, 7].The organizer alsoproduces inhibitors oftheWnt signaling pathway, such as Dickkopf, frzb and cerberus [8]. Neural induction has shown a remarkable evolutionary conservation and a "default" model has been proposed, which states thatectodermalcellshaveanintrinsicpredispositiontodifferentiateintoneuroectoderm,unless inhibited by BMP signaling [9]. While in certain conditions this seems to be the case, in other assays positive inducers are needed, such as FGFs [10]. Finally, more recent studies show that inhibition of Activin/Nodal pathways also seems to be important for neural induction [8].

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

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5

Progresses in cell culture technologies combined with a better understanding of these devel‐ opmental progressions have allowed now to recapitulate these processes *in vitro* through neuralization of mouse and human pluripotent cells, i.e. Embryonic Stem cells derived from blastocyst stage (ESC; [11]) and reprogrammed cells (iPSC; [12, 13]), leading to the generation of populations of EARLY NEUROEPITHELIAL CELLS (Fig. 2). These cells give rise to all of the neural cells in the mature CNS thus denoting their extensive multipotential aptitude in terms of different cellular subtypes they can produce. Sox1 is the earliest identified marker of neural precursors in the mouse embryo and is present in dividing neural precursors from the NEURAL PLATE and NEURAL TUBE stages [3]. Studies on pluripotent cells support the "default" model for mammalian neural induction. *In vitro* studies have in fact shown that during neuronal differentiation, ESCs and iPSCs undertake gradual lineage restrictions analogous to those observed through *in vivo* fetal development, and a variety of distinctive progenitors can be generated. Accordingly, mouse and human pluripotent cells differentiate into sox 1 positive neuroepithelial cells (note that in human the earliest neuroepithelial marker is represented by pax6 that precedes sox1 expression) when grown in serum-free conditions in the absence of patterning signals [14-16]. ESCs and iPSCs neural induction can be enhanced by the addition of BMP-, Nodal- and Wnt-inhibitors, to minimize endogenous signals pro‐ duced by ESCs/iPSCs themselves and recent studies have shown that paracrine signals (i.e.

**Figure 2.** The different NSC populations that can be obtained in vitro correspond to stage-specific neural progenitors

FGF4) are also needed for neurulation [17, 18].

present at defined in vivo developmental stages.

**Figure 1. Process of NSC self-renewal and differentiation.** NSCs are tri-potent cells. These cells during the differen‐ tiation process give rise to transiently dividing progenitors (transit amplifying progenitors) that subsequently undergo lineage restrictions toward neuronal, astrocytic and oligodendroglial mature cells.

Here we will review the functional properties of different *in vitro* NSC systems, providing also a direct comparison with NSCs present *in vivo*. Furthermore, we will discuss some of recent advancements in the development of *in vitro* systems that try to re-create *in vitro* some of the aspects of the physiological NSCs niches.

#### *2. In vivo* **and** *in vitro* **developmental heterogeneity of NSCs populations**

Vertebrate neural development starts with the process of neural induction, during and after gastrulation, which allows the formation of NEUROECTODERM from the dorsal-most part of the ectoderm. The molecular nature of the inductive signals that drive this process has been unveiled by studies in *Xenopus laevis*. These have shown that neural differentiation is promot‐ ed by secretion of an array of BMP inhibitors, chordin, noggin and follistatin produced by an embryonic structure called "organizer" [6, 7].The organizer alsoproduces inhibitors oftheWnt signaling pathway, such as Dickkopf, frzb and cerberus [8]. Neural induction has shown a remarkable evolutionary conservation and a "default" model has been proposed, which states thatectodermalcellshaveanintrinsicpredispositiontodifferentiateintoneuroectoderm,unless inhibited by BMP signaling [9]. While in certain conditions this seems to be the case, in other assays positive inducers are needed, such as FGFs [10]. Finally, more recent studies show that inhibition of Activin/Nodal pathways also seems to be important for neural induction [8].

Progresses in cell culture technologies combined with a better understanding of these devel‐ opmental progressions have allowed now to recapitulate these processes *in vitro* through neuralization of mouse and human pluripotent cells, i.e. Embryonic Stem cells derived from blastocyst stage (ESC; [11]) and reprogrammed cells (iPSC; [12, 13]), leading to the generation of populations of EARLY NEUROEPITHELIAL CELLS (Fig. 2). These cells give rise to all of the neural cells in the mature CNS thus denoting their extensive multipotential aptitude in terms of different cellular subtypes they can produce. Sox1 is the earliest identified marker of neural precursors in the mouse embryo and is present in dividing neural precursors from the NEURAL PLATE and NEURAL TUBE stages [3]. Studies on pluripotent cells support the "default" model for mammalian neural induction. *In vitro* studies have in fact shown that during neuronal differentiation, ESCs and iPSCs undertake gradual lineage restrictions analogous to those observed through *in vivo* fetal development, and a variety of distinctive progenitors can be generated. Accordingly, mouse and human pluripotent cells differentiate into sox 1 positive neuroepithelial cells (note that in human the earliest neuroepithelial marker is represented by pax6 that precedes sox1 expression) when grown in serum-free conditions in the absence of patterning signals [14-16]. ESCs and iPSCs neural induction can be enhanced by the addition of BMP-, Nodal- and Wnt-inhibitors, to minimize endogenous signals pro‐ duced by ESCs/iPSCs themselves and recent studies have shown that paracrine signals (i.e. FGF4) are also needed for neurulation [17, 18].

Here we will review the functional properties of different *in vitro* NSC systems, providing also a direct comparison with NSCs present *in vivo*. Furthermore, we will discuss some of recent advancements in the development of *in vitro* systems that try to re-create *in vitro* some of the

**Figure 1. Process of NSC self-renewal and differentiation.** NSCs are tri-potent cells. These cells during the differen‐ tiation process give rise to transiently dividing progenitors (transit amplifying progenitors) that subsequently undergo

*2. In vivo* **and** *in vitro* **developmental heterogeneity of NSCs populations**

Vertebrate neural development starts with the process of neural induction, during and after gastrulation, which allows the formation of NEUROECTODERM from the dorsal-most part of the ectoderm. The molecular nature of the inductive signals that drive this process has been unveiled by studies in *Xenopus laevis*. These have shown that neural differentiation is promot‐

aspects of the physiological NSCs niches.

4 Neural Stem Cells - New Perspectives

lineage restrictions toward neuronal, astrocytic and oligodendroglial mature cells.

**Figure 2.** The different NSC populations that can be obtained in vitro correspond to stage-specific neural progenitors present at defined in vivo developmental stages.

Soon after neural induction process, pluripotent cell-derived neuroepithelial cells give rise to NEURAL ROSETTE structures, in which cells elongate and align radially, in a manner that mimics neural tube formation [19]. *In vivo*, the neural tube is formed after neurulation from the newly-induced neural plate and, as it closes, it is regionalized along the antero-posterior (A/P) axis (Fig. 3A), giving rise to four main areas: forebrain, midbrain, hindbrain and spinal cord. In amniotes, dorso-ventral (D/V) patterning takes place only after A/P patterning has occurred, after neural tube closure. The variety of neuronal cells that will be generated will have specific functions according to their position along these two axes.

Several evidences suggest that primary neural induction obtained by BMP inhibition generates anterior neural tissue, while to obtain tissue with posterior characteristics other molecules, known as "transformers", are needed. Three molecules with posteriorizing activities are known: retinoic acid (RA), Fgfs and Wnts [20, 21]. These signals are produced by the sur‐ rounding axial and paraxial mesoderm and endoderm, in addition two secondary signaling centers exist within the neural tube [22]. These are the Anterior Neural Ridge (ANR), located at the border between the forebrain and the non-neural ectoderm, and the isthmic organizer, located at the mid-hindbrain boundary. The ANR secretes the organizer molecules noggin and chordin, the resulting BMP signaling inhibition activates Fgf8, which in turn induces the expression of the transcription factor FoxG1 (Bf1), necessary for forebrain development [23]. The isthmic organizer is located at the boundary between the expression domains of the transcription factors Otx2 and Gbx2, and it is formed and maintained by an intricate regulatory network among these and other (En1/2, Pax2/5/8) transcription factors. The isthmic organizer secretes Fgf8, and the feedback loop that is set up assures the maintenance of the tissue identity [22]. RA and Wnts are produced by paraxial mesoderm with a high-posterior/low-anterior gradient and they are responsible for the patterning of midbrain, hindbrain and anterior spinal cord. Among the genes differentially regulated by varying concentrations of RA are the Hox genes, necessary for hindbrain and spinal cord A/P patterning [24, 25]. D/V patterning is mediated by signaling molecules secreted by the surrounding tissues (Fig. 3B). The overlying ectoderm produces TGFβ-family molecules that promote the formation of the roof plate in the dorsal neural tube, while the underlying notochord secretes SHH, that induces the ventral neural tube to become the floor plate. The roof plate and the floor plate in turn become a source of TGFβ and SHH, respectively. This creates two opposing gradients that provide positional information along the D/V axis, regulating the expression of key transcription factors. These will then act in a combinatorial manner to regulate the differentiation of specific neuronal and glial cell types in the correct position [26].

These *in vivo* studies have ultimately revealed that different neural progenitor populations can exist in a time and space-dependent manner and that their fate is greatly influenced by the interplay between specific extrinsic and intrinsic signaling molecules. ESCs- and iPSCsderived neuroepithelial cells are able to perceive the positional information of patterning signals. These progenitors, when obtained in conditions that minimize endogenous signals, intrinsically acquire anterior identity, while they can be caudalized by the addition of FGFs, Wnts, RA [1, 19, 27, 28].

Some studies have shown that NEUROEPITHELIAL CELLS cannot be maintained *in vitro* by the exposure to commonly used mitogens, i.e. basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF). These indeed convert these cells into radial glia populations characterized by a limited potentiality in neuronal sub-types they can give rise to. Nonetheless, it has been shown that a neuroepithelial population that grows in rosette-like structures (termed "R-NSCs") can be generated *in vitro* from human and mouse pluripotent cells when exposed to SHH/FGF8 signalling coupled to a N-Cadherin/Forse-1 cell sorting-based protocols [19]. These cells can be maintained *in vitro* for some passages by exposure to SHH and Notch

organizer. RP: roof plate. FP: floor plate.

**Figure 3.** Regional patterning of the neural tube. Schematic diagrams showing antero-posterior (A) and dorso-ventral (B) patterning of the neural tube. The patterning process is driven by opposing gradients of signaling molecules that induce the expression of region-specific transcription factors in discrete areas. ANR: anterior neural ridge. IsO: Isthmic

(a)

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Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

(b)

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells http://dx.doi.org/10.5772/55137 7

Soon after neural induction process, pluripotent cell-derived neuroepithelial cells give rise to NEURAL ROSETTE structures, in which cells elongate and align radially, in a manner that mimics neural tube formation [19]. *In vivo*, the neural tube is formed after neurulation from the newly-induced neural plate and, as it closes, it is regionalized along the antero-posterior (A/P) axis (Fig. 3A), giving rise to four main areas: forebrain, midbrain, hindbrain and spinal cord. In amniotes, dorso-ventral (D/V) patterning takes place only after A/P patterning has occurred, after neural tube closure. The variety of neuronal cells that will be generated will

Several evidences suggest that primary neural induction obtained by BMP inhibition generates anterior neural tissue, while to obtain tissue with posterior characteristics other molecules, known as "transformers", are needed. Three molecules with posteriorizing activities are known: retinoic acid (RA), Fgfs and Wnts [20, 21]. These signals are produced by the sur‐ rounding axial and paraxial mesoderm and endoderm, in addition two secondary signaling centers exist within the neural tube [22]. These are the Anterior Neural Ridge (ANR), located at the border between the forebrain and the non-neural ectoderm, and the isthmic organizer, located at the mid-hindbrain boundary. The ANR secretes the organizer molecules noggin and chordin, the resulting BMP signaling inhibition activates Fgf8, which in turn induces the expression of the transcription factor FoxG1 (Bf1), necessary for forebrain development [23]. The isthmic organizer is located at the boundary between the expression domains of the transcription factors Otx2 and Gbx2, and it is formed and maintained by an intricate regulatory network among these and other (En1/2, Pax2/5/8) transcription factors. The isthmic organizer secretes Fgf8, and the feedback loop that is set up assures the maintenance of the tissue identity [22]. RA and Wnts are produced by paraxial mesoderm with a high-posterior/low-anterior gradient and they are responsible for the patterning of midbrain, hindbrain and anterior spinal cord. Among the genes differentially regulated by varying concentrations of RA are the Hox genes, necessary for hindbrain and spinal cord A/P patterning [24, 25]. D/V patterning is mediated by signaling molecules secreted by the surrounding tissues (Fig. 3B). The overlying ectoderm produces TGFβ-family molecules that promote the formation of the roof plate in the dorsal neural tube, while the underlying notochord secretes SHH, that induces the ventral neural tube to become the floor plate. The roof plate and the floor plate in turn become a source of TGFβ and SHH, respectively. This creates two opposing gradients that provide positional information along the D/V axis, regulating the expression of key transcription factors. These will then act in a combinatorial manner to regulate the differentiation of specific neuronal and

These *in vivo* studies have ultimately revealed that different neural progenitor populations can exist in a time and space-dependent manner and that their fate is greatly influenced by the interplay between specific extrinsic and intrinsic signaling molecules. ESCs- and iPSCsderived neuroepithelial cells are able to perceive the positional information of patterning signals. These progenitors, when obtained in conditions that minimize endogenous signals, intrinsically acquire anterior identity, while they can be caudalized by the addition of FGFs,

have specific functions according to their position along these two axes.

glial cell types in the correct position [26].

Wnts, RA [1, 19, 27, 28].

6 Neural Stem Cells - New Perspectives

**Figure 3.** Regional patterning of the neural tube. Schematic diagrams showing antero-posterior (A) and dorso-ventral (B) patterning of the neural tube. The patterning process is driven by opposing gradients of signaling molecules that induce the expression of region-specific transcription factors in discrete areas. ANR: anterior neural ridge. IsO: Isthmic organizer. RP: roof plate. FP: floor plate.

Some studies have shown that NEUROEPITHELIAL CELLS cannot be maintained *in vitro* by the exposure to commonly used mitogens, i.e. basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF). These indeed convert these cells into radial glia populations characterized by a limited potentiality in neuronal sub-types they can give rise to. Nonetheless, it has been shown that a neuroepithelial population that grows in rosette-like structures (termed "R-NSCs") can be generated *in vitro* from human and mouse pluripotent cells when exposed to SHH/FGF8 signalling coupled to a N-Cadherin/Forse-1 cell sorting-based protocols [19]. These cells can be maintained *in vitro* for some passages by exposure to SHH and Notch agonists while showing a rostral BF1+ neuroepithelial identity evocative of the signalling that *in vivo* are required for the induction of the anterior neuroepithelium. R-NSCs are characterized by a comprehensive differentiation potential toward CNS and PNS fates, supporting the idea that the R-NSCs represent neural precursors of the neural plate stage.

BPs are less comprehensive. Transitory induction of neurogenic Tbr2-positive BPs has been reported during the differentiation of ESCs to glutamatergic cortical neurons [27]. It has also been shown that BPs can be isolated from a subgroup of RG populations characterized by a high immunoreactivity for prominin that can make neurons only indirectly through the

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At the end of neurogenesis (in mice approximately at birth), neurogenic RG cells are exhausted and the remaining RG convert into astrocytes. The presence of stem cells has been reported in two regions of the adult mammalian brain, the SVZ and the SGZ of the hippocampus. Fatemapping studies have shown that these adult NSC populations are represented by the type B astrocytes that directly derive from subpopulations of fetal RG cells. Therefore, RG and type B astrocytes appear to form a continuous lineage with stem cell potential [2]. These *in vivo* studies find a parallel indirect proof from the fact that *in vitro* adult-derived NSCs reacquire

The study of different types of stem cells has greatly beneficed from *in vitro* approaches that allow the reduction the intrinsic complexity of tissues. In order to allow stable maintenance *in vitro*, cells have to be immortalized, a procedure that blocks the progres‐ sion of developmental programmes by pushing the cells to remain in enduring prolifera‐ tion. Immortalization can be achieved by means of various methods, most usually by viral transduction of immortalizing oncogenes such as c-myc or SV40 Large T Antigen. Several immortalized murine and human NSC lines have been reported and, interestingly, it has been shown that they maintain many equivalences to non-immortalized lines, exhibiting neglectable signs of transformation both *in vivo* or *in vitro* [42-45]. Nevertheless, the physiological relevance of these lines might be weakened by the expression of potential‐

In the developing CNS, exponential cell division occurs only for brief developmental windows and NSCs represent transient populations. In the brain, NSC division is rigorous‐ ly regulated by many factors of the "NICHE". The niche represents the particular cellu‐ lar microenvironment that provides the appropriate milieu to support self-renewal and that controls the balance between symmetrical proliferative (producing two stem cells) and asymmetric cell divisions (generating one stem cell and one committed progenitor). Accordingly, for a stem cell to give rise to a clonal cell line, the physiological hindrances to continuous cell division have to be bypassed. However, until few years ago, it has been extremely difficult to stably propagate homogenous cultures of NSCs without oncogene-

In the last two decades, oncogene-free procedures based on the use of soluble factors for selection and expansion of NSCs have been developed, permitting long-term mainte‐ nance of NSCs. The first report was from Reynolds and Weiss that in 1992 showed that the fetal and adult rodent brains contain cells competent for continuing *ex vivo* prolifera‐

generation of BPs [41].

ly transforming oncogenes.

mediated immortalization procedures.

fetal characteristics, such as radial glia markers.

*3. In vitro* **systems for NSCs isolation and expansion**

Another population of hESC-derived Sox1 positive self-renewing neuroepithelial cells named "lt-hESNSCs", has been described [29]. These cells can be grown as a nearly homogeneous population exhibiting clonogenicity and stable neurogenic potential. Remarkably, they can be maintained for many *in vitro* passages in the presence of FGF-2 and EGF and they preserve some properties of the R-NSCs, such as rosette-like growth, the expression of Bf1 and sensi‐ tivity to instructive signals that stimulate their conversion into distinct neuronal subpopula‐ tions. Molecular analyses have shown that lt-hESNSCs partly maintain rosette properties, possibly embodying an intermediate developmental stage between rosette-organized neuro‐ epithelial cells and radial glia (see below).

As development proceeds, neuroepithelial cells lose sox1 expression and convert themselves into another transitory stem cell type, the so-called "RADIAL GLIA" (RG). This rapidly constitutes the main progenitor cell population in late development and early postnatal life while disappearing at later postnatal and adult stages [30, 31]. Large numbers of RG cells are found in primary cell cultures from dissociated E10.5-18.5 CNS tissue. Different populations of RG, characterized by lineage heterogeneity, with both regional and temporal varieties, give rise to sequential waves of neurogenesis, gliogenesis and oligodendrogenesis that build up the CNS. The *in vivo* developmental heterogeneity of RG has been also revealed by *in vitro* primary cultures studies that have shown a temporal constraint from neurogenesis to gliogenesis from RG isolated at initial or later developmental periods, respectively [32, 33].

The transition of neuroepithelial cells to RG cells is well recapitulated *in vitro* during neural differentiation of pluripotent cells. RG populations can be efficiently generated from ESCs/ iPSCs using differentiation protocols that differ in major aspects between them. Bibel and collaborators generated transient (not expandable) populations of homogeneous RG cells that mature into glutamatergic neurons, as occurring during cortical development [34]. A different population of ESCs/iPSCs-derived RG cells can be obtained by exposing neuroepithelial cells to EGF and FGF-2. These rapidly lose Sox1 expression and acquire RG markers as BLBP and RC2 giving rise to RG-like cells which can be long term expanded in monolayer and at homogeneousness [35]. This conversion is dependent on Notch activity and on the exposure to EGF and FGF-2 [19, 35]. These self-renewing RG cells (called "NS cells") retain the marker signature of RG and the full capacity for tri-lineage neural differentiation, although their neuronal differentiation is limited to the GABAergic lineage [36-38]. These results indicate that pluripotent cells can be differentiated into distinct subtypes of RG – a non self-renewing type with aptitude to generate glutamatergic neurons, and a subtype that self-renews and exhibits a GABAergic differentiation. Such radial glial subtypes can also be found in the developing CNS *in vivo* although RG expansion *in vivo* is restricted to a defined time window.

Along with RG, a further immature population of cells with neuronal-restricted potential is represented by the BASAL PROGENITORs (BPs) that are located in the subventricular zone (SVZ) and can be generated both by neuroepithelial cells and RG [39, 40]. *In vitro* studies on

BPs are less comprehensive. Transitory induction of neurogenic Tbr2-positive BPs has been reported during the differentiation of ESCs to glutamatergic cortical neurons [27]. It has also been shown that BPs can be isolated from a subgroup of RG populations characterized by a high immunoreactivity for prominin that can make neurons only indirectly through the generation of BPs [41].

At the end of neurogenesis (in mice approximately at birth), neurogenic RG cells are exhausted and the remaining RG convert into astrocytes. The presence of stem cells has been reported in two regions of the adult mammalian brain, the SVZ and the SGZ of the hippocampus. Fatemapping studies have shown that these adult NSC populations are represented by the type B astrocytes that directly derive from subpopulations of fetal RG cells. Therefore, RG and type B astrocytes appear to form a continuous lineage with stem cell potential [2]. These *in vivo* studies find a parallel indirect proof from the fact that *in vitro* adult-derived NSCs reacquire fetal characteristics, such as radial glia markers.

#### *3. In vitro* **systems for NSCs isolation and expansion**

agonists while showing a rostral BF1+ neuroepithelial identity evocative of the signalling that *in vivo* are required for the induction of the anterior neuroepithelium. R-NSCs are characterized by a comprehensive differentiation potential toward CNS and PNS fates, supporting the idea

Another population of hESC-derived Sox1 positive self-renewing neuroepithelial cells named "lt-hESNSCs", has been described [29]. These cells can be grown as a nearly homogeneous population exhibiting clonogenicity and stable neurogenic potential. Remarkably, they can be maintained for many *in vitro* passages in the presence of FGF-2 and EGF and they preserve some properties of the R-NSCs, such as rosette-like growth, the expression of Bf1 and sensi‐ tivity to instructive signals that stimulate their conversion into distinct neuronal subpopula‐ tions. Molecular analyses have shown that lt-hESNSCs partly maintain rosette properties, possibly embodying an intermediate developmental stage between rosette-organized neuro‐

As development proceeds, neuroepithelial cells lose sox1 expression and convert themselves into another transitory stem cell type, the so-called "RADIAL GLIA" (RG). This rapidly constitutes the main progenitor cell population in late development and early postnatal life while disappearing at later postnatal and adult stages [30, 31]. Large numbers of RG cells are found in primary cell cultures from dissociated E10.5-18.5 CNS tissue. Different populations of RG, characterized by lineage heterogeneity, with both regional and temporal varieties, give rise to sequential waves of neurogenesis, gliogenesis and oligodendrogenesis that build up the CNS. The *in vivo* developmental heterogeneity of RG has been also revealed by *in vitro* primary cultures studies that have shown a temporal constraint from neurogenesis to gliogenesis from

The transition of neuroepithelial cells to RG cells is well recapitulated *in vitro* during neural differentiation of pluripotent cells. RG populations can be efficiently generated from ESCs/ iPSCs using differentiation protocols that differ in major aspects between them. Bibel and collaborators generated transient (not expandable) populations of homogeneous RG cells that mature into glutamatergic neurons, as occurring during cortical development [34]. A different population of ESCs/iPSCs-derived RG cells can be obtained by exposing neuroepithelial cells to EGF and FGF-2. These rapidly lose Sox1 expression and acquire RG markers as BLBP and RC2 giving rise to RG-like cells which can be long term expanded in monolayer and at homogeneousness [35]. This conversion is dependent on Notch activity and on the exposure to EGF and FGF-2 [19, 35]. These self-renewing RG cells (called "NS cells") retain the marker signature of RG and the full capacity for tri-lineage neural differentiation, although their neuronal differentiation is limited to the GABAergic lineage [36-38]. These results indicate that pluripotent cells can be differentiated into distinct subtypes of RG – a non self-renewing type with aptitude to generate glutamatergic neurons, and a subtype that self-renews and exhibits a GABAergic differentiation. Such radial glial subtypes can also be found in the developing

that the R-NSCs represent neural precursors of the neural plate stage.

RG isolated at initial or later developmental periods, respectively [32, 33].

CNS *in vivo* although RG expansion *in vivo* is restricted to a defined time window.

Along with RG, a further immature population of cells with neuronal-restricted potential is represented by the BASAL PROGENITORs (BPs) that are located in the subventricular zone (SVZ) and can be generated both by neuroepithelial cells and RG [39, 40]. *In vitro* studies on

epithelial cells and radial glia (see below).

8 Neural Stem Cells - New Perspectives

The study of different types of stem cells has greatly beneficed from *in vitro* approaches that allow the reduction the intrinsic complexity of tissues. In order to allow stable maintenance *in vitro*, cells have to be immortalized, a procedure that blocks the progres‐ sion of developmental programmes by pushing the cells to remain in enduring prolifera‐ tion. Immortalization can be achieved by means of various methods, most usually by viral transduction of immortalizing oncogenes such as c-myc or SV40 Large T Antigen. Several immortalized murine and human NSC lines have been reported and, interestingly, it has been shown that they maintain many equivalences to non-immortalized lines, exhibiting neglectable signs of transformation both *in vivo* or *in vitro* [42-45]. Nevertheless, the physiological relevance of these lines might be weakened by the expression of potential‐ ly transforming oncogenes.

In the developing CNS, exponential cell division occurs only for brief developmental windows and NSCs represent transient populations. In the brain, NSC division is rigorous‐ ly regulated by many factors of the "NICHE". The niche represents the particular cellu‐ lar microenvironment that provides the appropriate milieu to support self-renewal and that controls the balance between symmetrical proliferative (producing two stem cells) and asymmetric cell divisions (generating one stem cell and one committed progenitor). Accordingly, for a stem cell to give rise to a clonal cell line, the physiological hindrances to continuous cell division have to be bypassed. However, until few years ago, it has been extremely difficult to stably propagate homogenous cultures of NSCs without oncogenemediated immortalization procedures.

In the last two decades, oncogene-free procedures based on the use of soluble factors for selection and expansion of NSCs have been developed, permitting long-term mainte‐ nance of NSCs. The first report was from Reynolds and Weiss that in 1992 showed that the fetal and adult rodent brains contain cells competent for continuing *ex vivo* prolifera‐

tion upon exposure to EGF and FGF-2 and that upon mitogen withdrawal exhibit trineural lineage differentiation [46, 47]. According to this procedure, freshly dissociated SVZ cells plated at low density (roughly 103 -104 cells/cm2 ) in the absence of cell adhesion substrates and in presence of EGF and/or FGF-2 have the tendency to loosely adhere to the plastic plate. Within few days, most of the cells die except a minor fraction of them that become smooth-edged and begin to proliferate while staying attached to the plate. Later, the progeny of these proliferating cells stick to each other forming sphere-shaped clones that detach from the plate thus floating in suspension giving rise to the so-called NEUROSPHERES. This assay, named "Neurosphere Assay" has thus been widely consid‐ ered as a valuable method for isolating, enriching and maintaining embryonic and adult NSC populations *in vitro* [48]. Indeed, whereas NSCs in culture are characterized by the ability to considerably divide and self-renew thus giving rise to long-term expanding NSC lines, transit amplifying progenitors exhibit partial proliferative competence without selfrenewal potential, and are eliminated during extensive sub-culturing. Notably, only a fraction of cells composing the neurosphere (commonly 1-10% for optimal cultures, although this value greatly differs depending on the age and on the brain area consid‐ ered) are true stem cells, the remainder being differentiating progenitors at different stages, and even terminally differentiated neurons and glia [49]. Neurospheres can be subcultured by mechanical or enzymatic dissociation and by re-plating under the identical *in vitro* settings. As for the primary neurosphere culture, at every sub-culturing passage, differentiating/differentiated cells are supposed to die while the NSCs divide, generating secondary spheres that can then be further sub-cultured [50]. This procedure can be serially reiterated and, since each NSC gives rise to many NSCs by the time a neurosphere is generated, it ends in the expansion of the NSC population in culture.

Once established, neurosphere cultures can be expanded to obtain large amounts of cells that can then be cryopreserved. This permits the creation a pool of cells that can be later thawed and expanded for future experimentations. Nonetheless, several studies have shown that after few passages, the neurospheres greatly decrease their efficiency in neurogenic differentiation [51] and in the neuronal subtypes they can give rise to, mostly restricting their potential to the GABAergic lineage [52] (Fig. 4).

The accurate identification of the identity of the sphere-forming cell represents a key question. As committed progenitors are capable of only restricted proliferative capability and can generate only up to tertiary neurospheres, actually the designation of a cell as *bona fide* NSC should be retrospectively refereed only to a founder cell that self-renews extensively and can be propagated in long-term cultures. To this regard, it has been suggested that at least five sub-culturing passages are required to exclude the contribution of committed progenitors to the maintenance of the cell population. More rigorously, the assay should be performed with single dissociated cells (i.e. to plate a single cell per well) in order to avoid cell clustering and also fusion between neurospheres [53, 54].

Some researchers consider that three-dimensional organisation and the cellular milieu of the neurosphere as the *in vitro* equivalent of the *in vivo* neurogenic compartment [55, 56]. Although this view is a pure speculation, it is broadly accepted that the issue of the complexity of the neurosphere system represents a barrier for fine biochemical and molecular studies. The prospect of refining the neurosphere culture and of developing alternative *in vitro* systems, not only to enrich but also to select and clonally expand the *bona fide* stem cell population

monolayer results in a higher neurogenic potential than neurospheres

**Figure 4. Neurospheres and monolayer NSCs can be obtained by different sources and have different neuronal differentiation efficiency.** NSCs grown in monolayer and neurospheres can be derived from ESCs or iPS cells and from the germinative areas of the fetal and adult brain. The homogenous cellular composition of the NSCs grown in

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tion upon exposure to EGF and FGF-2 and that upon mitogen withdrawal exhibit trineural lineage differentiation [46, 47]. According to this procedure, freshly dissociated SVZ

substrates and in presence of EGF and/or FGF-2 have the tendency to loosely adhere to the plastic plate. Within few days, most of the cells die except a minor fraction of them that become smooth-edged and begin to proliferate while staying attached to the plate. Later, the progeny of these proliferating cells stick to each other forming sphere-shaped clones that detach from the plate thus floating in suspension giving rise to the so-called NEUROSPHERES. This assay, named "Neurosphere Assay" has thus been widely consid‐ ered as a valuable method for isolating, enriching and maintaining embryonic and adult NSC populations *in vitro* [48]. Indeed, whereas NSCs in culture are characterized by the ability to considerably divide and self-renew thus giving rise to long-term expanding NSC lines, transit amplifying progenitors exhibit partial proliferative competence without selfrenewal potential, and are eliminated during extensive sub-culturing. Notably, only a fraction of cells composing the neurosphere (commonly 1-10% for optimal cultures, although this value greatly differs depending on the age and on the brain area consid‐ ered) are true stem cells, the remainder being differentiating progenitors at different stages, and even terminally differentiated neurons and glia [49]. Neurospheres can be subcultured by mechanical or enzymatic dissociation and by re-plating under the identical *in vitro* settings. As for the primary neurosphere culture, at every sub-culturing passage, differentiating/differentiated cells are supposed to die while the NSCs divide, generating secondary spheres that can then be further sub-cultured [50]. This procedure can be serially reiterated and, since each NSC gives rise to many NSCs by the time a neurosphere is

generated, it ends in the expansion of the NSC population in culture.

Once established, neurosphere cultures can be expanded to obtain large amounts of cells that can then be cryopreserved. This permits the creation a pool of cells that can be later thawed and expanded for future experimentations. Nonetheless, several studies have shown that after few passages, the neurospheres greatly decrease their efficiency in neurogenic differentiation [51] and in the neuronal subtypes they can give rise to, mostly restricting their potential to the

The accurate identification of the identity of the sphere-forming cell represents a key question. As committed progenitors are capable of only restricted proliferative capability and can generate only up to tertiary neurospheres, actually the designation of a cell as *bona fide* NSC should be retrospectively refereed only to a founder cell that self-renews extensively and can be propagated in long-term cultures. To this regard, it has been suggested that at least five sub-culturing passages are required to exclude the contribution of committed progenitors to the maintenance of the cell population. More rigorously, the assay should be performed with single dissociated cells (i.e. to plate a single cell per well) in order to avoid cell clustering and

Some researchers consider that three-dimensional organisation and the cellular milieu of the neurosphere as the *in vitro* equivalent of the *in vivo* neurogenic compartment [55, 56]. Although this view is a pure speculation, it is broadly accepted that the issue of the complexity of the


) in the absence of cell adhesion

cells plated at low density (roughly 103

10 Neural Stem Cells - New Perspectives

GABAergic lineage [52] (Fig. 4).

also fusion between neurospheres [53, 54].

**Figure 4. Neurospheres and monolayer NSCs can be obtained by different sources and have different neuronal differentiation efficiency.** NSCs grown in monolayer and neurospheres can be derived from ESCs or iPS cells and from the germinative areas of the fetal and adult brain. The homogenous cellular composition of the NSCs grown in monolayer results in a higher neurogenic potential than neurospheres

neurosphere system represents a barrier for fine biochemical and molecular studies. The prospect of refining the neurosphere culture and of developing alternative *in vitro* systems, not only to enrich but also to select and clonally expand the *bona fide* stem cell population without losing the original prevalent neuronal fate, has been a recurrent issue in the stem cell field.

**4. Influences of the** *in vitro* **systems on the molecular and biological**

For brain tissue, founder NSCs existing during embryogenesis do not endure in adulthood but switchtoaquiescent statefollowingcompletionofdevelopment.Therefore,itmightbeexpected that in order to achieve persistent propagation of NSCs *in vitro* it might not be merely suffi‐ cient to follow intrinsic programmed mechanisms but also modifications of the "Neural Stem Cells cellular "character" are required to adapt to the synthetic *in vitro* milieu might also be required. Indeed, the interaction of typical transient progenitor populations with the artificial *in vitro* environment (i.e. high levels of growth factor stimulation and/or different matrix or cellcell interactions) may modify their transcriptional and epigenetic status, allowing them to be

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Inthisview,whencomingtothenatureoftheNSCs,thecrucialissueisiftheydoexactlyrepresent a definite sub-population of NSC/progenitor existing *in vivo*. Currently, it is still not entirely understood if the accomplishment of the NSC status might be the effect of phenotypic altera‐ tions due to culture set and how physiologically relevant the consequent *in vitro* phenotype

Tothisregard,thepossibilitythatthemixtureofmitogensmayproduceanartificialcellcondition with a proper balance of key transcription factors able to suppress lineage commitment and allow self-maintaining divisions has to be considered. It has been shown that FGF-2 and EGF, twogrowthfactors typicallyusedforthe*invitro*maintenanceofNSCscanalterthetranscription‐ al and epigenetic phenotype. For example, expression of several genes can be directly stimulat‐ ed *in vitro* in neural progenitors by exposure to FGF-2, suggesting that these genes might exert fundamental functions in the establishment of NSCs lines [66]. Similarly, foetal neural progen‐ itors *in vitro* exposed to FGF-2, rapidly activate expression of Egfr (ErbB1) and Olig2, the latter being a bHLH transcription factor linked with the oligodendrocyte lineage and ventral CNS identity [66, 67]. Under expansion conditions with high levels of EGF and FGF-2, induction of Olig2 is required for the proliferation and self-renewal of neurosphere cells and NS cells, as demonstrated by analyses in which experimental interference with Olig2 expression severely decreases the amount andthequalityofneurospheres [68].BesidesOlig2,ithasbeenshownthat acute exposure to FGF-2 induces neural progenitors to upregulate expression of a broad set of genes (for example CD44, GLAST, Olig1, Cdh20, Adam12 and Vav3) likely playing significant roles in the phenotype of the cells [69]. Likewise, EGF has been shown to deregulate expres‐ sion of Dlx-2 in NS cells, NSC cultures and in transit-amplifying cells of the SVZ, inducing their switchintoRG-likeneurosphere-formingcells[51,61,69,70].Remarkably,stimulationofseveral ofthesegenes (forinstanceVav3andCD44)occurswithinfewhoursofFGF-2exposure,possibly indicating that mitogen-mediated action is not suggestive for a physiological developmental

NSCs *in vivo* have been shown to be tremendously heterogeneous in terms of transcriptional factors expression pattern, a feature predictable to confer a complex elaboration of positional signals [33]. To this regard, several reports have shown the occurrence *in vitro* of profound variations in the expression pattern of positional genes compared with primary precursors

might be [3]. Thus, it is preferable to refer to *in vitro* expanded NSCs as NSC-like cells.

progress but rather an acute transcriptional rearrangement [69].

**properties of NSC lines**

"turned" into NSC lines.

As an alternative to the neurosphere system, other researchers have developed monolayerbased methods [57]. In 1997, Gage and colleagues reported that progenitor cells with properties similar to NSCs from adult SVZ could be obtained from the adult hippocampus [58]. These hippocampal precursor cells propagate in monolayer and using *in vitro* procedures similar to the ones used for SVZ NSCs. Hippocampal precursors divide in response to FGF-2 and show tri-neural potential being able to differentiate into astroglia, oligodendroglia, and neurons *in vitro*. More recently, the optimization of novel and efficient strategies for the derivation and stable long-term propagation of NSCs from developing and adult neural tissue and from pluripotent cellular sources has been reported. It has been shown that transiently generated ESC-derived neural precursors, normally destined to differentiate to neuronal and glial cells, can be efficiently expanded as adherent clonal NSC lines in EGF and FGF-2 supplemented medium [19, 35]. In these growth conditions, cells undergo symmetrical division with neglectable accompanying differentiation, while shifting of the cultures to differentiative conditions prompts the cells to efficiently generate mature neurons, astrocytes and oligoden‐ drocytes, thus indicating their NSC essence. The cells obtained by this procedure have been named Neural Stem (NS) cells. Notably, these results suggest that expansion of NS cells can occur in the absence of a complex cellular niche. Accordingly, NS cell expansion in monolayer conditions restrains spontaneous differentiation and permits proliferation of homogeneous *bona fide* NSCs.

Phenotypic characterization of NS cell cultures indicates a close similarity to forebrain RG [35]. Indeed, NS cells are homogenously immunopositive for nestin, SSEA1/Lex1, Pax6, prominin, RC2, vimentin, 3CB2, Glast, and BLBP, a set of markers diagnostic for neurogen‐ ic RG. NS cells keep their neurogenic potential after extensive expansion (over 100 passages), yet retaining the capability to produce a large proportion of mature neurons (Fig. 4). These results further indicate that the acquisition of RG properties endows the cells with a "niche" that traps them in a state of symmetric cell division. Significantly, NS cells do not represent a peculiarity of ESCs and iPSCs cell differentiation [35, 59]. In fact, similar lines can also be obtained from foetal or adult CNS and established from longterm expanded neurosphere cultures [35, 60, 61]. It is therefore possible that NS cells embody the resident NSC population within neurospheres. Further characterization of different mouse NS cell lines has demonstrated a close similarity in self-renewal, neuro‐ nal differentiation potential and molecular markers, independently from their origin. NS cells are not exclusive for mouse sources but it has indeed described the possibility to generate NS cells both from human fetal neural tissue and from human ESCs [62]. Interestingly, similar cells can be developed also from brain tumors and might serve as systems for find new targets in order to develop new therapeutic approaches [63, 64]. Similarly to NS cells, also lt-hESNSCs grow in monolayer and can be long-term expand‐ ed but differently from NS cells, they maintain sox 1 expression and a wide developmen‐ tal competence [29, 65]. These aspects might be suggestive for some species-specific differences.

### **4. Influences of the** *in vitro* **systems on the molecular and biological properties of NSC lines**

without losing the original prevalent neuronal fate, has been a recurrent issue in the stem cell

As an alternative to the neurosphere system, other researchers have developed monolayerbased methods [57]. In 1997, Gage and colleagues reported that progenitor cells with properties similar to NSCs from adult SVZ could be obtained from the adult hippocampus [58]. These hippocampal precursor cells propagate in monolayer and using *in vitro* procedures similar to the ones used for SVZ NSCs. Hippocampal precursors divide in response to FGF-2 and show tri-neural potential being able to differentiate into astroglia, oligodendroglia, and neurons *in vitro*. More recently, the optimization of novel and efficient strategies for the derivation and stable long-term propagation of NSCs from developing and adult neural tissue and from pluripotent cellular sources has been reported. It has been shown that transiently generated ESC-derived neural precursors, normally destined to differentiate to neuronal and glial cells, can be efficiently expanded as adherent clonal NSC lines in EGF and FGF-2 supplemented medium [19, 35]. In these growth conditions, cells undergo symmetrical division with neglectable accompanying differentiation, while shifting of the cultures to differentiative conditions prompts the cells to efficiently generate mature neurons, astrocytes and oligoden‐ drocytes, thus indicating their NSC essence. The cells obtained by this procedure have been named Neural Stem (NS) cells. Notably, these results suggest that expansion of NS cells can occur in the absence of a complex cellular niche. Accordingly, NS cell expansion in monolayer conditions restrains spontaneous differentiation and permits proliferation of homogeneous

Phenotypic characterization of NS cell cultures indicates a close similarity to forebrain RG [35]. Indeed, NS cells are homogenously immunopositive for nestin, SSEA1/Lex1, Pax6, prominin, RC2, vimentin, 3CB2, Glast, and BLBP, a set of markers diagnostic for neurogen‐ ic RG. NS cells keep their neurogenic potential after extensive expansion (over 100 passages), yet retaining the capability to produce a large proportion of mature neurons (Fig. 4). These results further indicate that the acquisition of RG properties endows the cells with a "niche" that traps them in a state of symmetric cell division. Significantly, NS cells do not represent a peculiarity of ESCs and iPSCs cell differentiation [35, 59]. In fact, similar lines can also be obtained from foetal or adult CNS and established from longterm expanded neurosphere cultures [35, 60, 61]. It is therefore possible that NS cells embody the resident NSC population within neurospheres. Further characterization of different mouse NS cell lines has demonstrated a close similarity in self-renewal, neuro‐ nal differentiation potential and molecular markers, independently from their origin. NS cells are not exclusive for mouse sources but it has indeed described the possibility to generate NS cells both from human fetal neural tissue and from human ESCs [62]. Interestingly, similar cells can be developed also from brain tumors and might serve as systems for find new targets in order to develop new therapeutic approaches [63, 64]. Similarly to NS cells, also lt-hESNSCs grow in monolayer and can be long-term expand‐ ed but differently from NS cells, they maintain sox 1 expression and a wide developmen‐ tal competence [29, 65]. These aspects might be suggestive for some species-specific

field.

12 Neural Stem Cells - New Perspectives

*bona fide* NSCs.

differences.

For brain tissue, founder NSCs existing during embryogenesis do not endure in adulthood but switchtoaquiescent statefollowingcompletionofdevelopment.Therefore,itmightbeexpected that in order to achieve persistent propagation of NSCs *in vitro* it might not be merely suffi‐ cient to follow intrinsic programmed mechanisms but also modifications of the "Neural Stem Cells cellular "character" are required to adapt to the synthetic *in vitro* milieu might also be required. Indeed, the interaction of typical transient progenitor populations with the artificial *in vitro* environment (i.e. high levels of growth factor stimulation and/or different matrix or cellcell interactions) may modify their transcriptional and epigenetic status, allowing them to be "turned" into NSC lines.

Inthisview,whencomingtothenatureoftheNSCs,thecrucialissueisiftheydoexactlyrepresent a definite sub-population of NSC/progenitor existing *in vivo*. Currently, it is still not entirely understood if the accomplishment of the NSC status might be the effect of phenotypic altera‐ tions due to culture set and how physiologically relevant the consequent *in vitro* phenotype might be [3]. Thus, it is preferable to refer to *in vitro* expanded NSCs as NSC-like cells.

Tothisregard,thepossibilitythatthemixtureofmitogensmayproduceanartificialcellcondition with a proper balance of key transcription factors able to suppress lineage commitment and allow self-maintaining divisions has to be considered. It has been shown that FGF-2 and EGF, twogrowthfactors typicallyusedforthe*invitro*maintenanceofNSCscanalterthetranscription‐ al and epigenetic phenotype. For example, expression of several genes can be directly stimulat‐ ed *in vitro* in neural progenitors by exposure to FGF-2, suggesting that these genes might exert fundamental functions in the establishment of NSCs lines [66]. Similarly, foetal neural progen‐ itors *in vitro* exposed to FGF-2, rapidly activate expression of Egfr (ErbB1) and Olig2, the latter being a bHLH transcription factor linked with the oligodendrocyte lineage and ventral CNS identity [66, 67]. Under expansion conditions with high levels of EGF and FGF-2, induction of Olig2 is required for the proliferation and self-renewal of neurosphere cells and NS cells, as demonstrated by analyses in which experimental interference with Olig2 expression severely decreases the amount andthequalityofneurospheres [68].BesidesOlig2,ithasbeenshownthat acute exposure to FGF-2 induces neural progenitors to upregulate expression of a broad set of genes (for example CD44, GLAST, Olig1, Cdh20, Adam12 and Vav3) likely playing significant roles in the phenotype of the cells [69]. Likewise, EGF has been shown to deregulate expres‐ sion of Dlx-2 in NS cells, NSC cultures and in transit-amplifying cells of the SVZ, inducing their switchintoRG-likeneurosphere-formingcells[51,61,69,70].Remarkably,stimulationofseveral ofthesegenes (forinstanceVav3andCD44)occurswithinfewhoursofFGF-2exposure,possibly indicating that mitogen-mediated action is not suggestive for a physiological developmental progress but rather an acute transcriptional rearrangement [69].

NSCs *in vivo* have been shown to be tremendously heterogeneous in terms of transcriptional factors expression pattern, a feature predictable to confer a complex elaboration of positional signals [33]. To this regard, several reports have shown the occurrence *in vitro* of profound variations in the expression pattern of positional genes compared with primary precursors and progenitors *in vivo* thus leading to a mixed regional identity and limited neuronal differentiation. For example, neurospheres from the spinal cord have been shown to undergo upregulation of Olig2 and downregulation of the dorsal spinal cord transcription factors Pax3 and Pax7 [71]. Olig2 and Mash1 are also induced in E14 cortex or ganglionic eminence precursors, short- or long-term grown as neurospheres [72]. With some exceptions, a similar deregulation of the regional patterning is evident in the adherent NS cells and lt-hESNSCs cultures [29].

Even though it is still a long way to fully understand the complex physiological context of a niche, researchers are now trying to reproduce *in vitro* at least some aspects of the dynamic *in vivo* environment. A better comprehension of the mechanisms underlying the NSC niche and the development of systems aimed at the reconstruction of this milieu will fill the gap between bi-dimensional (2D) simplified *in vitro* studies and the complex but physiological conditions

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To this purpose, a synthetic NSC niche should recreate the complex interactions between NSCs and others cells, extracellular matrix, gradients of regulatory molecules and physical factors (Figure 5). In particular an ideal *in vitro* mimicked SVZ niche should contemplate the following

**4.** autonomous production of cellular and molecular factors necessary for self-renewal and

*In vitro* generation of structures grossly simulating the SVZ NSC niche have been reported from mouse ESC-derived NSCs without the administration of mitogenic factors and complex physical scaffolds. In these studies, following a neuralization process with retinoic acid and plating the NSCs at high density on an entactin-collagen-laminin coated surface, heterogene‐ ous multicellular aggregates appeared spontaneously, showing some of the characteristics postulated above, although a well-defined structural architecture was lacking [77]. In the last years, the development of new 3D culture systems that can allow to better reproduce *in vitro* structures in between standard monolayer culture and living organisms have been/are under

In this direction, standard culture methods involving petri dishes are being replaced with more accurate micro-scale devices, allowing procedures at the time and length scales of biological phenomena, enabling the control of multiple parameters, such as molecular and physical factors [78]. More attention is now focused on both the generation of morphogen-gradients, taking advantage of microfluidic systems, and three-dimensional extracellular matrix mimicscaffolds in which multiple cells can be entangled allowing spatiotemporal control of the

Microfluidic systems can reproduce a niche-like microenvironment permitting also the generation of concentration gradients of signaling molecules, often without the application of an external power source. Indeed, two different solutions can be introduced into the main channel of a microfluidic-chip by an osmotic pump. Since at this scale fluids mix only by diffusion, at the interface of the two solutions, diffusion generates a stable concentration

**2.** production of the characteristic NSC niche-signaling molecules

**3.** presence of a basal lamina and extracellular matrix

**5.** incorporation of extra-neural (i.e. endothelial cells) cells

system and satisfying all of the features of a niche [79].

**6.** spatial assembly reproducing the SVZ *in vivo* architecture.

differentiation of resident stem cells

of *in vivo* methods.

minimal requirements: **1.** presence of NSCs

investigation.

Importantly, this relaxation in the positional code might be related to a recurrent restriction in the competence to generate diverse neuronal subtypes. Indeed, NSCs have been reported to rapidly lose their original competence to generate site-specific neuronal subtypes when cultured *in vitro*, both in monolayer and in aggregation, in the presence of EGF and/or FGF-2, becoming mainly constrained to adopt a GABAergic fate [35, 52, 73, 74]. A notable exception is represented by the lt-hESNSCs [29], possibly indicating that for some reasons neuroepithelial cells derived from human pluripotent sources are more "predisposed" to long-term better preserve a broad neuronal sub-types developmental competence.

On the whole, these results might thus emphasize an artificial nature of cell culture, under‐ lining the requirement for prudence in extrapolation of *in vitro* results to normal development or physiology without corresponding *in vivo* data [3]. Alternatively, this might be due to inadequate culture conditions that are not actually competent to preserve the molecular and biological properties of genuine NSCs.

#### **5. Reconstruction of NSC niche** *in vitro*

NSC niches present distinctive features leading to diverse ways to ensure neurogenesis. In the adult SVZ, three main immature neural populations lie adjacent to a layer of ependymal cells lining the lateral ventricle wall [2]. The Type B cells, representing the NSCs, reside interposed into the ependymal layer, displaying connections with both the ventricular wall and the blood vessels-network characterizing this niche. They are relatively quiescent but capable of giving rise to transit amplifying cells (Type C cells), a more rapidly dividing population that in turn generate the third population composed by neuroblasts (Type A cells) that migrate into glial tubes to reach the olfactory bulb. Besides these populations, a vital role for the maintenance of the niche is played by ependymal cells (Type E cells), astrocytes and endothelial cells. A comparable organization has been reported also for hippocampal SGZ niche although this exhibits a more planar structure [75, 76]. For a more detailed description of the neurogenic niches refer to of this book.

It emerges that both of these neurogenic niches are arranged to allow NSCs integration and to permit a strict responsiveness to signals from the *"external world"* (blood vessels and ventricles) and the "*neighboring world*" (newly generated neuroblasts, resident astrocytes and microglia, ECM components-forming scaffolds, etc.). All of these components harmoniously interact with each other providing both positive and negative signals and feedback that regulate NSCs activity.

Even though it is still a long way to fully understand the complex physiological context of a niche, researchers are now trying to reproduce *in vitro* at least some aspects of the dynamic *in vivo* environment. A better comprehension of the mechanisms underlying the NSC niche and the development of systems aimed at the reconstruction of this milieu will fill the gap between bi-dimensional (2D) simplified *in vitro* studies and the complex but physiological conditions of *in vivo* methods.

To this purpose, a synthetic NSC niche should recreate the complex interactions between NSCs and others cells, extracellular matrix, gradients of regulatory molecules and physical factors (Figure 5). In particular an ideal *in vitro* mimicked SVZ niche should contemplate the following minimal requirements:

**1.** presence of NSCs

and progenitors *in vivo* thus leading to a mixed regional identity and limited neuronal differentiation. For example, neurospheres from the spinal cord have been shown to undergo upregulation of Olig2 and downregulation of the dorsal spinal cord transcription factors Pax3 and Pax7 [71]. Olig2 and Mash1 are also induced in E14 cortex or ganglionic eminence precursors, short- or long-term grown as neurospheres [72]. With some exceptions, a similar deregulation of the regional patterning is evident in the adherent NS cells and lt-hESNSCs

Importantly, this relaxation in the positional code might be related to a recurrent restriction in the competence to generate diverse neuronal subtypes. Indeed, NSCs have been reported to rapidly lose their original competence to generate site-specific neuronal subtypes when cultured *in vitro*, both in monolayer and in aggregation, in the presence of EGF and/or FGF-2, becoming mainly constrained to adopt a GABAergic fate [35, 52, 73, 74]. A notable exception is represented by the lt-hESNSCs [29], possibly indicating that for some reasons neuroepithelial cells derived from human pluripotent sources are more "predisposed" to long-term better

On the whole, these results might thus emphasize an artificial nature of cell culture, under‐ lining the requirement for prudence in extrapolation of *in vitro* results to normal development or physiology without corresponding *in vivo* data [3]. Alternatively, this might be due to inadequate culture conditions that are not actually competent to preserve the molecular and

NSC niches present distinctive features leading to diverse ways to ensure neurogenesis. In the adult SVZ, three main immature neural populations lie adjacent to a layer of ependymal cells lining the lateral ventricle wall [2]. The Type B cells, representing the NSCs, reside interposed into the ependymal layer, displaying connections with both the ventricular wall and the blood vessels-network characterizing this niche. They are relatively quiescent but capable of giving rise to transit amplifying cells (Type C cells), a more rapidly dividing population that in turn generate the third population composed by neuroblasts (Type A cells) that migrate into glial tubes to reach the olfactory bulb. Besides these populations, a vital role for the maintenance of the niche is played by ependymal cells (Type E cells), astrocytes and endothelial cells. A comparable organization has been reported also for hippocampal SGZ niche although this exhibits a more planar structure [75, 76]. For a more detailed description of the neurogenic

It emerges that both of these neurogenic niches are arranged to allow NSCs integration and to permit a strict responsiveness to signals from the *"external world"* (blood vessels and ventricles) and the "*neighboring world*" (newly generated neuroblasts, resident astrocytes and microglia, ECM components-forming scaffolds, etc.). All of these components harmoniously interact with each other providing both positive and negative signals and feedback that regulate NSCs

preserve a broad neuronal sub-types developmental competence.

biological properties of genuine NSCs.

niches refer to of this book.

activity.

**5. Reconstruction of NSC niche** *in vitro*

cultures [29].

14 Neural Stem Cells - New Perspectives


*In vitro* generation of structures grossly simulating the SVZ NSC niche have been reported from mouse ESC-derived NSCs without the administration of mitogenic factors and complex physical scaffolds. In these studies, following a neuralization process with retinoic acid and plating the NSCs at high density on an entactin-collagen-laminin coated surface, heterogene‐ ous multicellular aggregates appeared spontaneously, showing some of the characteristics postulated above, although a well-defined structural architecture was lacking [77]. In the last years, the development of new 3D culture systems that can allow to better reproduce *in vitro* structures in between standard monolayer culture and living organisms have been/are under investigation.

In this direction, standard culture methods involving petri dishes are being replaced with more accurate micro-scale devices, allowing procedures at the time and length scales of biological phenomena, enabling the control of multiple parameters, such as molecular and physical factors [78]. More attention is now focused on both the generation of morphogen-gradients, taking advantage of microfluidic systems, and three-dimensional extracellular matrix mimicscaffolds in which multiple cells can be entangled allowing spatiotemporal control of the system and satisfying all of the features of a niche [79].

Microfluidic systems can reproduce a niche-like microenvironment permitting also the generation of concentration gradients of signaling molecules, often without the application of an external power source. Indeed, two different solutions can be introduced into the main channel of a microfluidic-chip by an osmotic pump. Since at this scale fluids mix only by diffusion, at the interface of the two solutions, diffusion generates a stable concentration

A fundamental impulse has come from the advance in the field of BIOMATERIALS. These have been greatly improved in the last few years, allowing now to finely control cell-matrix interactions, to direct cell migration and to permit the precise topographical administration of

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17

While it is quite difficult to modify only one variable with a naïve ECM component, the use of biomaterials has improved and simplified many experimental approaches. For example, when using natural matrices, decreasing the concentration of collagen leads to a decrease stiffness of the gel, nonetheless this also determines a decreasing in the concentration of adhesive ligands and an increase in diffusion, resulting in accumulation of variables to the system. This can be avoided with engineered biomaterials that enable isolation of individual variables, without varying others. Nowadays, synthetic biomaterials are greatly exploited to mimic the physical and mechanical features of the ECM. They allow to control a number of important parameters, including polymerization, degradation, and biocompatibility and to combine

Another point of control allowed by new biomaterials is the possibility to incorporate cells releasing molecules or molecules *per se* as soluble factors, such as cytokines, NFs and GFs. Indeed, these molecules are constantly synthesized, secreted, transported, and depleted in NSC niches. To this regard, Zhang and colleagues have described a 16-residues peptide capable of self-assembly into membrane upon addition of a physiological concentration of salt [88]. Now commercially available as PuraMatrix™, it has been shown to support neurite outgrowth and synapse formation [89] and more recently to regulate murine and human NSCs growth

Synthetic peptides can also be used in combination with a variety of polymers to provide materials with cell-adhesive, enzymatically degradable, and GFs-binding properties. Amino‐ acid sequences commonly include collagen-, laminin-, and fibronectin-cell-adhesive domains, these can be mixed together and with other bioactive motifs, such as proteolytically degradable sequences, to create a multifunctional peptide material with different physical properties. For instance, NSCs survival has been shown to be improved in a collagen hydrogel that incorpo‐ rates laminin-derived adhesion motifs [94]. Peptides can also be used as structural compo‐

The reconstructions of a NSC niche can be translated to multiwell-based high-throughput methods for screening compounds that can positively regulate neurogenesis and thus be developed as potential therapeutic drugs. Protein-based microarrays have been developed and applied to diverse stem-cell populations [95-97]. These devices consist of robotically spotted GFs or ECM molecules in combinations, on cell repellent substrates in order to avoid cell migration, and cell fate changes are often analyzed via immunocytochemistry assays. Platforms like these have been used to analyze human NSCs differentiation and proliferation in response to combinations of ECM components, morphogens and other signaling proteins. A joint effect of Wnt and Notch pathways to maintain human NSCs in an undifferentiated state, a dose dependent activity of Notch ligands in shifting neuronal differentiation towards glial fate and a neurogenic effect of Wnt3A have thus been reported. Consequently, it is

and differentiation following adjunction of NSCs-active molecules [90-93].

defined physical (both soluble or not) signals.

them with fully defined chemical components [81-87].

nents.





**Figure 5.** Schematic illustration of the different colture methods to reproduce *in vitro* the NSC niche.

gradient. To this regard, it has been shown that solutions of SHh, FGF8 or BMP4 are able to induce human ESC-derived NSCs neuronal differentiation, leading to the formation of a complex cellular network [80].

A fundamental impulse has come from the advance in the field of BIOMATERIALS. These have been greatly improved in the last few years, allowing now to finely control cell-matrix interactions, to direct cell migration and to permit the precise topographical administration of defined physical (both soluble or not) signals.

While it is quite difficult to modify only one variable with a naïve ECM component, the use of biomaterials has improved and simplified many experimental approaches. For example, when using natural matrices, decreasing the concentration of collagen leads to a decrease stiffness of the gel, nonetheless this also determines a decreasing in the concentration of adhesive ligands and an increase in diffusion, resulting in accumulation of variables to the system. This can be avoided with engineered biomaterials that enable isolation of individual variables, without varying others. Nowadays, synthetic biomaterials are greatly exploited to mimic the physical and mechanical features of the ECM. They allow to control a number of important parameters, including polymerization, degradation, and biocompatibility and to combine them with fully defined chemical components [81-87].

Another point of control allowed by new biomaterials is the possibility to incorporate cells releasing molecules or molecules *per se* as soluble factors, such as cytokines, NFs and GFs. Indeed, these molecules are constantly synthesized, secreted, transported, and depleted in NSC niches. To this regard, Zhang and colleagues have described a 16-residues peptide capable of self-assembly into membrane upon addition of a physiological concentration of salt [88]. Now commercially available as PuraMatrix™, it has been shown to support neurite outgrowth and synapse formation [89] and more recently to regulate murine and human NSCs growth and differentiation following adjunction of NSCs-active molecules [90-93].

Synthetic peptides can also be used in combination with a variety of polymers to provide materials with cell-adhesive, enzymatically degradable, and GFs-binding properties. Amino‐ acid sequences commonly include collagen-, laminin-, and fibronectin-cell-adhesive domains, these can be mixed together and with other bioactive motifs, such as proteolytically degradable sequences, to create a multifunctional peptide material with different physical properties. For instance, NSCs survival has been shown to be improved in a collagen hydrogel that incorpo‐ rates laminin-derived adhesion motifs [94]. Peptides can also be used as structural compo‐ nents.

The reconstructions of a NSC niche can be translated to multiwell-based high-throughput methods for screening compounds that can positively regulate neurogenesis and thus be developed as potential therapeutic drugs. Protein-based microarrays have been developed and applied to diverse stem-cell populations [95-97]. These devices consist of robotically spotted GFs or ECM molecules in combinations, on cell repellent substrates in order to avoid cell migration, and cell fate changes are often analyzed via immunocytochemistry assays. Platforms like these have been used to analyze human NSCs differentiation and proliferation in response to combinations of ECM components, morphogens and other signaling proteins. A joint effect of Wnt and Notch pathways to maintain human NSCs in an undifferentiated state, a dose dependent activity of Notch ligands in shifting neuronal differentiation towards glial fate and a neurogenic effect of Wnt3A have thus been reported. Consequently, it is

gradient. To this regard, it has been shown that solutions of SHh, FGF8 or BMP4 are able to induce human ESC-derived NSCs neuronal differentiation, leading to the formation of a

**Figure 5.** Schematic illustration of the different colture methods to reproduce *in vitro* the NSC niche.

complex cellular network [80].

16 Neural Stem Cells - New Perspectives

possible to highlight specific responses of single versus combination of stimuli in a highthroughput way [97].

to exploit the advantages offered by one method or the other, depending on the goal of our

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

http://dx.doi.org/10.5772/55137

19

Our apologies to all whose studies were not mentioned due to space limitations. We thank Riccardo Rossi for the creative illustrations used in the manuscript. L. Conti is supported by the Italian Ministry of Health; S. Casarosa is supported by the University of Trento and Cassa

and Luciano Conti2

2 Dipartimento di Scienze Farmacologiche & Biomolecolari, Università degli Studi di Milano,

[1] Grabel L. Developmental origin of neural stem cells: the glial cell that could. Stem

[2] Alvarez-Buylla A, Lim DA. For the long run: maintaining germinal niches in the

[3] Conti L, Cattaneo E. Neural stem cell systems: physiological players or in vitro enti‐

[4] Pollard S, Conti L, Smith A. Exploitation of adherent neural stem cells in basic and applied neurobiology. Regenerative medicine. 2006;1(1):111-8. Epub 2007/05/01.

[5] Negri-Cesi P, Colciago A, Pravettoni A, Casati L, Conti L, Celotti F. Sexual differen‐ tiation of the rodent hypothalamus: hormonal and environmental influences. The Journal of steroid biochemistry and molecular biology. 2008;109(3-5):294-9. Epub

[6] Hemmati-Brivanlou A, Melton DA. Inhibition of activin receptor signaling promotes

neuralization in Xenopus. Cell. 1994;77(2):273-81. Epub 1994/04/22.

ties? Nature reviews Neuroscience. 2010;11(3):176-87. Epub 2010/01/29.

1 Centre for Integrative Biology, CIBIO, Via delle Regole, Mattarello (TN), Italy

research.

**Acknowledgements**

**Author details**

Simona Casarosa1

**References**

Via Balzaretti, Milan, Italy

2008/04/12.

di Risparmio di Trento e Rovereto.

, Jacopo Zasso2

\*Address all correspondence to: luciano.conti@unimi.it

Cell Rev. 2012;8(2):577-85. Epub 2012/02/22.

adult brain. Neuron. 2004;41(5):683-6.

These platforms are limited to adherent cells only and do not allow cell fates determination on single cells. The hydrogel microwell array, developed on micrometer-sized cavities, permits to analyze both adherent and nonadherent cells, trapped by gravitational sedimentation. The device has been used to analyze single cell-forming neurospheres, avoiding the usual merging events of neurosphere assay [98] and more recently it has been combined with robotic protein spotting to address the role of biochemical and biophysical factors on single nonadherent neural stem cell self-renewal [99].

#### **6. Conclusions**

Our knowledge of the neural progenitor identity and properties during development has been radically revolutionized by the possibility to isolate and expand NSCs *in vitro*. We have reviewed here the current and most commonly used *in vitro* methodologies to isolate, expand and functionally characterize NSC populations. The real identity and the potential lineage relationships between different types of stem/precursor cells isolated and cultured *in vitro* by these different methodologies represents a field of open and intense investigation.

In light of the complexity of the biological concerns governing stem cell maintenance and differentiation, significant progress will require a close coordination between *in vivo* and *in vitro* approaches. In this scenario, *in vitro* systems of NSCs shall allow a deep analysis at cellular level providing useful information to be further validate in the embryo and adult in order to identify relevance to normal physiology.

Establishment of *in vitro* settings necessarily results in disruption of the three-dimensional tissue structure, loss of specific cell-to cell contacts and modification of the extracellular environment and signaling. This might also lead to alteration of biological and molecular properties and acquisition of stem cell features by committed progenitors. Thus, although the versatility shown by NSC cultures *in vitro* can be envisaged as an advantage, extreme caution is necessary when considering the potential *in vivo* translation to developmental biology.

NSC biology holds tremendous potential for neurological therapy. It should be emphasized that the study of the intrinsic properties of NSCs and understanding the mechanisms of interaction between resident CNS cells and grafted NSCs will be mandatory for the develop‐ ment of new therapies able to slow the progression of neurodegenerative diseases.

Beside the therapeutical applications, NSCs systems present unique opportunities that are starting to be successfully explored for genetic or chemical screens in order to identify and optimize molecules/drugs that may allow a tight control on self-renewal and lineage specifi‐ cation of NSCs as well as their functional maturation, thus moving forward NSCs-based therapies.

We can anticipate that a rigorous characterization of the functional features of the NSC populations isolated and propagated by means of different cell culture systems shall allow us to exploit the advantages offered by one method or the other, depending on the goal of our research.

#### **Acknowledgements**

possible to highlight specific responses of single versus combination of stimuli in a high-

These platforms are limited to adherent cells only and do not allow cell fates determination on single cells. The hydrogel microwell array, developed on micrometer-sized cavities, permits to analyze both adherent and nonadherent cells, trapped by gravitational sedimentation. The device has been used to analyze single cell-forming neurospheres, avoiding the usual merging events of neurosphere assay [98] and more recently it has been combined with robotic protein spotting to address the role of biochemical and biophysical factors on single nonadherent

Our knowledge of the neural progenitor identity and properties during development has been radically revolutionized by the possibility to isolate and expand NSCs *in vitro*. We have reviewed here the current and most commonly used *in vitro* methodologies to isolate, expand and functionally characterize NSC populations. The real identity and the potential lineage relationships between different types of stem/precursor cells isolated and cultured *in vitro* by

In light of the complexity of the biological concerns governing stem cell maintenance and differentiation, significant progress will require a close coordination between *in vivo* and *in vitro* approaches. In this scenario, *in vitro* systems of NSCs shall allow a deep analysis at cellular level providing useful information to be further validate in the embryo and adult in order to

Establishment of *in vitro* settings necessarily results in disruption of the three-dimensional tissue structure, loss of specific cell-to cell contacts and modification of the extracellular environment and signaling. This might also lead to alteration of biological and molecular properties and acquisition of stem cell features by committed progenitors. Thus, although the versatility shown by NSC cultures *in vitro* can be envisaged as an advantage, extreme caution is necessary when considering the potential *in vivo* translation to developmental biology.

NSC biology holds tremendous potential for neurological therapy. It should be emphasized that the study of the intrinsic properties of NSCs and understanding the mechanisms of interaction between resident CNS cells and grafted NSCs will be mandatory for the develop‐

Beside the therapeutical applications, NSCs systems present unique opportunities that are starting to be successfully explored for genetic or chemical screens in order to identify and optimize molecules/drugs that may allow a tight control on self-renewal and lineage specifi‐ cation of NSCs as well as their functional maturation, thus moving forward NSCs-based

We can anticipate that a rigorous characterization of the functional features of the NSC populations isolated and propagated by means of different cell culture systems shall allow us

ment of new therapies able to slow the progression of neurodegenerative diseases.

these different methodologies represents a field of open and intense investigation.

throughput way [97].

18 Neural Stem Cells - New Perspectives

**6. Conclusions**

therapies.

neural stem cell self-renewal [99].

identify relevance to normal physiology.

Our apologies to all whose studies were not mentioned due to space limitations. We thank Riccardo Rossi for the creative illustrations used in the manuscript. L. Conti is supported by the Italian Ministry of Health; S. Casarosa is supported by the University of Trento and Cassa di Risparmio di Trento e Rovereto.

#### **Author details**

Simona Casarosa1 , Jacopo Zasso2 and Luciano Conti2

\*Address all correspondence to: luciano.conti@unimi.it

1 Centre for Integrative Biology, CIBIO, Via delle Regole, Mattarello (TN), Italy

2 Dipartimento di Scienze Farmacologiche & Biomolecolari, Università degli Studi di Milano, Via Balzaretti, Milan, Italy

#### **References**


[7] Sasai Y, Lu B, Steinbeisser H, De Robertis EM. Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature. 1995;377(6551): 757. Epub 1995/10/26.

[20] Kiecker C, Niehrs C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development. 2001;128(21):4189-201.

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

http://dx.doi.org/10.5772/55137

21

[21] Kudoh T, Wilson SW, Dawid IB. Distinct roles for Fgf, Wnt and retinoic acid in poste‐ riorizing the neural ectoderm. Development. 2002;129(18):4335-46. Epub 2002/08/17.

[22] Wurst W, Bally-Cuif L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nature reviews Neuroscience. 2001;2(2):99-108. Epub 2001/03/17.

[23] Anderson RM, Lawrence AR, Stottmann RW, Bachiller D, Klingensmith J. Chordin and noggin promote organizing centers of forebrain development in the mouse. De‐

[24] Maden M. Retinoic acid and limb regeneration--a personal view. Int J Dev Biol.

[25] Nordstrom U, Jessell TM, Edlund T. Progressive induction of caudal neural character by graded Wnt signaling. Nature neuroscience. 2002;5(6):525-32. Epub 2002/05/15. [26] Hoch RV, Rubenstein JL, Pleasure S. Genes and signaling events that establish re‐ gional patterning of the mammalian forebrain. Semin Cell Dev Biol. 2009;20(4):

[27] Bertacchi M, Pandolfini L, Murenu E, Viegi A, Capsoni S, Cellerino A, et al. The posi‐ tional identity of mouse ES cell-generated neurons is affected by BMP signaling. Cell

[28] Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, et al. Self-organized formation of polarized cortical tissues from ESCs and its ac‐ tive manipulation by extrinsic signals. Cell stem cell. 2008;3(5):519-32. Epub

[29] Koch P, Opitz T, Steinbeck JA, Ladewig J, Brustle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proceedings of the National Academy of Sciences of the United

[30] Pollard SM, Conti L. Investigating radial glia in vitro. Progress in neurobiology.

[31] Malatesta P, Hartfuss E, Gotz M. Isolation of radial glial cells by fluorescent-activated cell sorting reveals a neuronal lineage. Development. 2000;127(24):5253-63.

[32] Malatesta P, Hack MA, Hartfuss E, Kettenmann H, Klinkert W, Kirchhoff F, et al. Neuronal or glial progeny: regional differences in radial glia fate. Neuron. 2003;37(5):

[33] Pinto L, Gotz M. Radial glial cell heterogeneity--the source of diverse progeny in the

CNS. Progress in neurobiology. 2007;83(1):2-23.

velopment. 2002;129(21):4975-87. Epub 2002/10/25.

2002;46(7):883-6. Epub 2002/11/29.

378-86. Epub 2009/06/30.

Mol Life Sci. 2012 Oct 16.

States of America. 2009.

2007;83(1):53-67.

751-64.

2008/11/06.

Epub 2001/10/31.


[20] Kiecker C, Niehrs C. A morphogen gradient of Wnt/beta-catenin signalling regulates anteroposterior neural patterning in Xenopus. Development. 2001;128(21):4189-201. Epub 2001/10/31.

[7] Sasai Y, Lu B, Steinbeisser H, De Robertis EM. Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature. 1995;377(6551):

[8] Levine AJ, Brivanlou AH. Proposal of a model of mammalian neural induction. Dev

[9] Hemmati-Brivanlou A, Melton D. Vertebrate neural induction. Annu Rev Neurosci.

[10] Stern CD. Neural induction: 10 years on since the 'default model'. Curr Opin Cell Bi‐

[11] Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol.

[12] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell.

[13] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐ ic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-76.

[14] Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol.

[15] Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S, Kawasaki H, et al. Di‐ rected differentiation of telencephalic precursors from embryonic stem cells. Nature

[16] Smukler SR, Runciman SB, Xu S, van der Kooy D. Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences. J Cell Biol.

[17] Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embry‐ onic stem cells from self-renewal to lineage commitment. Development. 2007;134(16):

[18] Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of

[19] Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage.

SMAD signaling. Nat Biotechnol. 2009;27(3):275-80. Epub 2009/03/03.

757. Epub 1995/10/26.

20 Neural Stem Cells - New Perspectives

2001;17:435-62.

2007;131(5):861-72.

2003;21(2):183-6.

Biol. 2007;308(2):247-56. Epub 2007/06/26.

1997;20:43-60. Epub 1997/01/01.

ol. 2006;18(6):692-7. Epub 2006/10/19.

neuroscience. 2005;8(3):288-96. Epub 2005/02/08.

2006;172(1):79-90. Epub 2006/01/05.

2895-902. Epub 2007/07/31.

Genes Dev. 2008;22(2):152-65.


[34] Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M, et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nature neuroscience. 2004;7(9):1003-9.

[45] De Filippis L, Lamorte G, Snyder EY, Malgaroli A, Vescovi AL. A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oli‐

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

http://dx.doi.org/10.5772/55137

23

[46] Reynolds BA, Tetzlaff W, Weiss S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. The Journal of neuroscience : the of‐

[47] Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707-10.

[48] Pastrana E, Silva-Vargas V, Doetsch F. Eyes wide open: a critical review of sphereformation as an assay for stem cells. Cell stem cell. 2011;8(5):486-98. Epub 2011/05/10.

[49] Chojnacki AK, Mak GK, Weiss S. Identity crisis for adult periventricular neural stem cells: subventricular zone astrocytes, ependymal cells or both? Nature reviews Neu‐

[50] Chojnacki A, Weiss S. Production of neurons, astrocytes and oligodendrocytes from

[51] Ciccolini F. Identification of two distinct types of multipotent neural precursors that appear sequentially during CNS development. Molecular and cellular neurosciences.

[52] Machon O, Backman M, Krauss S, Kozmik Z. The cellular fate of cortical progenitors is not maintained in neurosphere cultures. Molecular and cellular neurosciences.

[53] Singec I, Knoth R, Meyer RP, Maciaczyk J, Volk B, Nikkhah G, et al. Defining the ac‐ tual sensitivity and specificity of the neurosphere assay in stem cell biology. Nature

[54] Jessberger S, Clemenson GD, Jr., Gage FH. Spontaneous fusion and nonclonal

[55] Campos LS. Neurospheres: insights into neural stem cell biology. Journal of neuro‐

[56] Bez A, Corsini E, Curti D, Biggiogera M, Colombo A, Nicosia RF, et al. Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization.

[57] Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD. Single factors di‐ rect the differentiation of stem cells from the fetal and adult central nervous system.

[58] Palmer TD, Takahashi J, Gage FH. The adult rat hippocampus contains primordial neural stem cells. Molecular and cellular neurosciences. 1997;8(6):389-404.

growth of adult neural stem cells. Stem Cells. 2007;25(4):871-4.

ficial journal of the Society for Neuroscience. 1992;12(11):4565-74.

mammalian CNS stem cells. Nat Protoc. 2008;3(6):935-40.

godendrocytes. Stem Cells. 2007;25(9):2312-21.

roscience. 2009;10(2):153-63.

2001;17(5):895-907.

2005;30(3):388-97.

methods. 2006;3(10):801-6.

science research. 2004;78(6):761-9.

Brain research. 2003;993(1-2):18-29.

Genes Dev. 1996;10(24):3129-40.


[45] De Filippis L, Lamorte G, Snyder EY, Malgaroli A, Vescovi AL. A novel, immortal, and multipotent human neural stem cell line generating functional neurons and oli‐ godendrocytes. Stem Cells. 2007;25(9):2312-21.

[34] Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M, et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nature neuroscience.

[35] Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS biology.

[36] Goffredo D, Conti L, Di Febo F, Biella G, Tosoni A, Vago G, et al. Setting the condi‐ tions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells. Cell death and differentia‐

[37] Spiliotopoulos D, Goffredo D, Conti L, Di Febo F, Biella G, Toselli M, et al. An opti‐ mized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal popula‐

[38] Biella G, Di Febo F, Goffredo D, Moiana A, Taglietti V, Conti L, et al. Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials. Neuro‐

[39] Haubensak W, Attardo A, Denk W, Huttner WB. Neurons arise in the basal neuroe‐ pithelium of the early mammalian telencephalon: a major site of neurogenesis. Pro‐ ceedings of the National Academy of Sciences of the United States of America.

[40] Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, Ogawa M. Asymmetric pro‐ duction of surface-dividing and non-surface-dividing cortical progenitor cells. Devel‐

[41] Pinto L, Mader MT, Irmler M, Gentilini M, Santoni F, Drechsel D, et al. Prospective isolation of functionally distinct radial glial subtypes--lineage and transcriptome

[42] Martinez-Serrano A, Bjorklund A. Immortalized neural progenitor cells for CNS gene

[43] Cacci E, Villa A, Parmar M, Cavallaro M, Mandahl N, Lindvall O, et al. Generation of human cortical neurons from a new immortal fetal neural stem cell line. Exp Cell

[44] Cattaneo E, Conti L. Generation and characterization of embryonic striatal condition‐ ally immortalized ST14A cells. Journal of neuroscience research. 1998;53(2):223-34.

analysis. Molecular and cellular neurosciences. 2008;38(1):15-42.

transfer and repair. Trends Neurosci. 1997;20(11):530-8.

tion. Neurobiology of disease. 2009;34(2):320-31. Epub 2009/02/25.

2004;7(9):1003-9.

22 Neural Stem Cells - New Perspectives

2005;3(9):e283.

tion. 2008;15(12):1847-56.

science. 2007;149(1):38-52.

2004;101(9):3196-201.

opment. 2004;131(13):3133-45.

Res. 2007;313(3):588-601.

Epub 1998/07/22.


[59] Castiglioni V, Onorati M, Rochon C, Cattaneo E. Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing al‐ terations in the lysosomal pathway. Neurobiology of disease. 2012;46(1):30-40. Epub 2012/01/10.

embryonic stem cell differentiation. Molecular and cellular neurosciences. 2008;38(3):

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

http://dx.doi.org/10.5772/55137

25

[70] Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem

[71] Gabay L, Lowell S, Rubin LL, Anderson DJ. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron.

[72] Hack MA, Sugimori M, Lundberg C, Nakafuku M, Gotz M. Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Molecular and cellular neu‐

[73] Parish CL, Castelo-Branco G, Rawal N, Tonnesen J, Sorensen AT, Salto C, et al. Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement thera‐

[74] Bithell A, Finch SE, Hornby MF, Williams BP. Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes

[75] Suh H, Consiglio A, Ray J, Sawai T, D'Amour KA, Gage FH. In vivo fate analysis re‐ veals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the

[76] Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neuro‐

[77] Lutolf MP, Blau HM. Artificial stem cell niches. Adv Mater. 2009;21(32-33):3255-68.

[78] Cimetta E, Figallo E, Cannizzaro C, Elvassore N, Vunjak-Novakovic G. Micro-bio‐ reactor arrays for controlling cellular environments: design principles for human em‐

[79] Burdick JA, Vunjak-Novakovic G. Engineered microenvironments for controlled stem cell differentiation. Tissue engineering Part A. 2009;15(2):205-19. Epub

[80] Park JY, Kim SK, Woo DH, Lee EJ, Kim JH, Lee SH. Differentiation of neural progeni‐ tor cells in a microfluidic chip-generated cytokine gradient. Stem Cells. 2009;27(11):

[81] Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables

[82] Hunt NC, Grover LM. Cell encapsulation using biopolymer gels for regenerative

and applications. Biomaterials. 2003;24(24):4337-51. Epub 2003/08/19.

medicine. Biotechnology letters. 2010;32(6):733-42. Epub 2010/02/16.

bryonic stem cell applications. Methods. 2009;47(2):81-9. Epub 2008/10/28.

py in parkinsonian mice. J Clin Invest. 2008;118(1):149-60.

genesis. Cell. 2008;132(4):645-60. Epub 2008/02/26.

their neuronal subtype specification. Stem Cells. 2008;26(6):1565-74.

adult hippocampus. Cell stem cell. 2007;1(5):515-28. Epub 2008/03/29.

393-403.

2003;40(3):485-99.

Epub 2010/10/01.

2008/08/13.

2646-54. Epub 2009/08/28.

cells. Neuron. 2002;36(6):1021-34.

rosciences. 2004;25(4):664-78.


embryonic stem cell differentiation. Molecular and cellular neurosciences. 2008;38(3): 393-403.

[70] Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron. 2002;36(6):1021-34.

[59] Castiglioni V, Onorati M, Rochon C, Cattaneo E. Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing al‐ terations in the lysosomal pathway. Neurobiology of disease. 2012;46(1):30-40. Epub

[60] Onorati M, Binetti M, Conti L, Camnasio S, Calabrese G, Albieri I, et al. Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments. Cellular and molecular life sciences : CMLS.

[61] Pollard SM, Conti L, Sun Y, Goffredo D, Smith A. Adherent neural stem (NS) cells

[62] Sun Y, Pollard S, Conti L, Toselli M, Biella G, Parkin G, et al. Long-term tripotent dif‐ ferentiation capacity of human neural stem (NS) cells in adherent culture. Molecular

[63] Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell stem cell. 2009;4(6):568-80. Epub

[64] Conti L, Crisafulli L, Caldera V, Tortoreto M, Brilli E, Conforti P, et al. REST controls self-renewal and tumorigenic competence of human glioblastoma cells. PloS one.

[65] Falk A, Koch P, Kesavan J, Takashima Y, Ladewig J, Alexander M, et al. Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons. PloS one. 2012;7(1):e29597. Epub

[66] Dromard C, Bartolami S, Deleyrolle L, Takebayashi H, Ripoll C, Simonneau L, et al. NG2 and Olig2 expression provides evidence for phenotypic deregulation of cul‐ tured central nervous system and peripheral nervous system neural precursor cells.

[67] Ciccolini F, Svendsen CN. Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth factor (EGF) responsiveness in mouse striatal precursor cells: iden‐ tification of neural precursors responding to both EGF and FGF-2. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1998;18(19):

[68] Ligon KL, Huillard E, Mehta S, Kesari S, Liu H, Alberta JA, et al. Olig2-regulated lin‐ eage-restricted pathway controls replication competence in neural stem cells and ma‐

[69] Pollard SM, Wallbank R, Tomlinson S, Grotewold L, Smith A. Fibroblast growth fac‐ tor induces a neural stem cell phenotype in foetal forebrain progenitors and during

from fetal and adult forebrain. Cereb Cortex. 2006;16 Suppl 1:i112-20.

2012/01/10.

24 Neural Stem Cells - New Perspectives

2009/06/06.

2012/01/25.

7869-80.

2011;68(10):1769-83. Epub 2010/10/29.

2012;7(6):e38486. Epub 2012/06/16.

Stem Cells. 2007;25(2):340-53.

lignant glioma. Neuron. 2007;53(4):503-17.

and cellular neurosciences. 2008;38(2):245-58.


[83] Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical reviews. 2001;101(7):1869-79. Epub 2001/11/17.

[95] Brafman DA, Shah KD, Fellner T, Chien S, Willert K. Defining long-term mainte‐ nance conditions of human embryonic stem cells with arrayed cellular microenviron‐ ment technology. Stem cells and development. 2009;18(8):1141-54. Epub 2009/03/31.

Systems for *ex–vivo* Isolation and Culturing of Neural Stem Cells

http://dx.doi.org/10.5772/55137

27

[96] LaBarge MA, Nelson CM, Villadsen R, Fridriksdottir A, Ruth JR, Stampfer MR, et al. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments. Integrative biology : quantitative biosciences from

[97] Soen Y, Mori A, Palmer TD, Brown PO. Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments. Molecu‐

[98] Cordey M, Limacher M, Kobel S, Taylor V, Lutolf MP. Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays. Stem Cells.

[99] Gobaa S, Hoehnel S, Roccio M, Negro A, Kobel S, Lutolf MP. Artificial niche microar‐ rays for probing single stem cell fate in high throughput. Nature methods. 2011;8(11):

nano to macro. 2009;1(1):70-9. Epub 2009/12/22.

lar systems biology. 2006;2:37. Epub 2006/07/06.

2008;26(10):2586-94.

949-55. Epub 2011/10/11.


[95] Brafman DA, Shah KD, Fellner T, Chien S, Willert K. Defining long-term mainte‐ nance conditions of human embryonic stem cells with arrayed cellular microenviron‐ ment technology. Stem cells and development. 2009;18(8):1141-54. Epub 2009/03/31.

[83] Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical reviews.

[84] Mather ML, Tomlins PE. Hydrogels in regenerative medicine: towards understand‐ ing structure-function relationships. Regenerative medicine. 2010;5(5):809-21. Epub

[85] Nuttelman CR, Rice MA, Rydholm AE, Salinas CN, Shah DN, Anseth KS. Macromo‐ lecular Monomers for the Synthesis of Hydrogel Niches and Their Application in Cell Encapsulation and Tissue Engineering. Progress in polymer science. 2008;33(2):

[86] Peppas NA, Hilt JZ, Khademhosseini A, Langer R. Hydrogels in biology and medi‐ cine: From molecular principles to bionanotechnology. Adv Mater. 2006;18(11):

[87] Sanchis J, Canal F, Lucas R, Vicent MJ. Polymer-drug conjugates for novel molecular

[88] Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of a self-complemen‐ tary oligopeptide to form a stable macroscopic membrane. Proceedings of the Na‐ tional Academy of Sciences of the United States of America. 1993;90(8):3334-8. Epub

[89] Holmes TC, de Lacalle S, Su X, Liu G, Rich A, Zhang S. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(12):

[90] Cunha C, Panseri S, Villa O, Silva D, Gelain F. 3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds. International journal of

[91] Liedmann A, Rolfs A, Frech MJ. Cultivation of human neural progenitor cells in a 3 dimensional self-assembling peptide hydrogel. Journal of visualized experiments :

[92] Ortinau S, Schmich J, Block S, Liedmann A, Jonas L, Weiss DG, et al. Effect of 3Dscaffold formation on differentiation and survival in human neural progenitor cells.

[93] Yla-Outinen L, Joki T, Varjola M, Skottman H, Narkilahti S. Three-dimensional growth matrix for human embryonic stem cell-derived neuronal cells. Journal of tis‐

[94] Nakaji-Hirabayashi T, Kato K, Iwata H. Improvement of neural stem cell survival in collagen hydrogels by incorporating laminin-derived cell adhesive polypeptides. Bio‐

targets. Nanomedicine (Lond). 2010;5(6):915-35. Epub 2010/08/26.

2001;101(7):1869-79. Epub 2001/11/17.

2010/09/28.

26 Neural Stem Cells - New Perspectives

1345-60.

1993/04/15.

167-79. Epub 2008/02/01.

6728-33. Epub 2000/06/07.

nanomedicine. 2011;6:943-55. Epub 2011/07/02.

Biomedical engineering online. 2010;9:70. Epub 2010/11/13.

conjugate chemistry. 2012;23(2):212-21. Epub 2012/01/11.

sue engineering and regenerative medicine. 2012. Epub 2012/05/23.

JoVE. 2012(59):e3830. Epub 2012/01/20.


**Chapter 2**

**Neural Stem Cell Heterogeneity**

Additional information is available at the end of the chapter

contribute of tissue homeostasis and regeneration.

**2. Neurogenesis in the subventricular zone**

The concept of neurogenic neural stem cells in the brains of adult mammals including humans is now widely accepted. In rodents these cells have been studied extensively both in vitro and in vivo. Of the two primary neurogenic regions in the rodent brain, the subventricular zone of the lateral ventricle wall generates the most neurons of multiple phenotypes. The newly generated neurons in the subventricular zone migrate to the olfactory bulb replenishing neurons and reconstituting the local circuitry responsible for olfaction. The dentate gyrus of the hippocampus generates a single neuron type, glutamatergic granule cells. These newborn granule cells contribute to specific forms of memory by integrating into existent circuits (Shors et al., 2001; Clelland et al., 2009; Garthe et al., 2009). Over the last few years, what was once considered to be a homogeneous population of astrocytic stem cells in both neurogenic brain regions is now turning out to be a more complex mixture of cells. Heterogeneous populations of cells with stem cell properties are being discovered in both the subventricular zone and dentate gyrus. This heterogeneity combined with potential diversity in signals forming the local niches could provide a situation where these multiple neural stem cell subpopulations

The lateral walls of the forebrain ventricles contain stem cells that generate neuronal subpo‐ pulations of the olfactory bulb throughout life (Reynolds and Weiss, 1992; Morshead et al., 1994; Doetsch et al., 1999b; Gage, 2000; Mirzadeh et al., 2008). Although much remains to be learnt about the neurogenic process and the fate determinants controlling maintenance, proliferation and differentiation of stem and progenitors cells in the subventricular zone, morphological, immunological and lineage tracing has recently uncovered a striking hetero‐

> © 2013 Taylor; 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,

© 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,

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

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

Verdon Taylor

**1. Introduction**

http://dx.doi.org/10.5772/55676

#### **Chapter 2**

## **Neural Stem Cell Heterogeneity**

Verdon Taylor

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55676

#### **1. Introduction**

The concept of neurogenic neural stem cells in the brains of adult mammals including humans is now widely accepted. In rodents these cells have been studied extensively both in vitro and in vivo. Of the two primary neurogenic regions in the rodent brain, the subventricular zone of the lateral ventricle wall generates the most neurons of multiple phenotypes. The newly generated neurons in the subventricular zone migrate to the olfactory bulb replenishing neurons and reconstituting the local circuitry responsible for olfaction. The dentate gyrus of the hippocampus generates a single neuron type, glutamatergic granule cells. These newborn granule cells contribute to specific forms of memory by integrating into existent circuits (Shors et al., 2001; Clelland et al., 2009; Garthe et al., 2009). Over the last few years, what was once considered to be a homogeneous population of astrocytic stem cells in both neurogenic brain regions is now turning out to be a more complex mixture of cells. Heterogeneous populations of cells with stem cell properties are being discovered in both the subventricular zone and dentate gyrus. This heterogeneity combined with potential diversity in signals forming the local niches could provide a situation where these multiple neural stem cell subpopulations contribute of tissue homeostasis and regeneration.

#### **2. Neurogenesis in the subventricular zone**

The lateral walls of the forebrain ventricles contain stem cells that generate neuronal subpo‐ pulations of the olfactory bulb throughout life (Reynolds and Weiss, 1992; Morshead et al., 1994; Doetsch et al., 1999b; Gage, 2000; Mirzadeh et al., 2008). Although much remains to be learnt about the neurogenic process and the fate determinants controlling maintenance, proliferation and differentiation of stem and progenitors cells in the subventricular zone, morphological, immunological and lineage tracing has recently uncovered a striking hetero‐

© 2013 Taylor; 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.

geneity in the putative stem cell pool. In the first sections of this chapter I will look at some of the key findings and experiments identifying the stem cells and following their fate. I will also ask the question of whether single neural stem cells are multipotent in vivo and look at some for the experimental data addressing this and also cover emerging experimental data showing heterogeneity within the stem cell pool.

whichthedifferentiationpotentialoftheneural stemcells is controlled(BasakandTaylor, 2009). Assuming that all stem cells with the subventricular zone have the same potential, local differences within the niche or signals interpreted by committed progenitors *en route* to their final destination would be responsible for determining the multiple neuronal fates. The ectopic grafting of stem cells into the subventricular zone indicates some degree of plasticity within the neural stem cell population and suggest niche specific signals as fate determinants (Suhonen et al., 1996). However, even with the same niche, some neural stem cells seem to have autono‐ mous fates and be heterogeneous in their potential (Kohwi et al., 2007; Merkle et al., 2007). By using homochronic/heterochronic transplantation experiments it has been shown that progen‐ itor cells at different ontogenetic stages are intrinsically directed toward specific lineages (De Marchis et al., 2007). In addition, neuroblasts in the rostral migratory stream are also heteroge‐ neous and may be committed to specific neuronal fates even before reaching the olfactory bulb (Hack et al., 2005;Kohwi et al., 2005).Thus,ratherthanbeinguniversallyplastic,theneural stem cell pool may be made up of many stem cells with restricted potentials. This is also supported by region specific, viral-mediated genetic labeling of the subventricular neural stem cells in juvenile mice which show diversity in neuronal progeny generated rather than generating all neuron types (Merkle et al., 2007). Granule cells, the major neuron subtypes to be generated during adulthood, are produced from all anteroposterior and dorsoventral locations in the subventricular zone. However, most granule cells are generated from the dorsal and ventral most aspects of the subventricular zone (Merkle et al., 2007). Within this regionalization, the granule neurons generated from the dorsal subventricular zone migrate to a more superficial location in the granule cell layer of the olfactory bulb while those generated ventrally settle deeperinthegranulecelllayer(Merkleetal.,2007).Thisregionalspecificationcanalsobemapped tothelocationofthestemcellsduringearlypostnataldevelopmentindicatingnotonlyaregional but also a developmentally-regulated fate specification (Merkle et al., 2007). Similarly, periglo‐ merular neurons that migrate to the outer layer of the olfactory bulb also show a regionspecific origin.Dorsalregions ofthe subventricular zone generate the majority ofthe thymidine hydroxylase-positive neurons whereas Calbindin-positive periglomerular neurons are generated preferentially from the ventral subventricular zone (Merkle et al., 2007). Calretininpositive periglomerular and granule cells are generated from the medial wall of the lateral ventricle. As this region produces proportionally fewer granule cells in total this suggests that the niche of the medial wall directs the fate of neural stem cells towards Calretinin neuron generation. Although these findings do not rule out niche specific programming of multipo‐ tent cell fate, heterotopic transplantation strongly suggests that stem cells retain their differen‐

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 31

tial potential when grafted into a different axial location (Merkle et al., 2007).

For many years mitotic inactivity or quiescence has been viewed as a primary stem cell trait. However, recent data in many systems including the intestine and blood suggest that stem cell may not need to be quiescent and some can divide frequently to drive the generation of new cells (Wilson et al., 2008; Essers et al., 2009; Fuchs, 2009; Li and Clevers, 2010). These active

**5. Mitotically active or quiescent neural stem cells**

#### **3. The subventricular zone and its progenitors**

Continued neurogenesis from cells within the subependymal layer of the lateral ventricle wall implies stem cells as a driving force and a regulatory niche. Ultrastructural electronmicroscopic analysis has been instrumental in defining the morphological differences among cells within the subependymal layer of the ventricle wall (Doetsch et al., 1997; Doetsch et al., 1999a; Mirzadeh et al., 2008). Combining electromicroscopy with functional regeneration of the neurogenic niche, astrocytes have been shown to be primary progenitors of the subventricular zone (Doetsch et al., 1999b; Doetsch et al., 1999a; Doetsch et al., 2002). The subventricular zone astrocytes are defined as B-cells. B-cells have a polarized morphology extending an apical process and sensory cilium that projects between the ependymal call (E-cells) lining the lateral ventricle. These B-cell projections organize the E-cells into characteristic pinwheel structure (Mirzadeh et al., 2008). This is likely to be an important structural and signaling center in the stem cell niche. Based on their ultrastructural characteristics and location the B-cell population can be divided into two. B1-cells have their cell body between the chains of neuroblasts (Acells) and the ependymal lining. B1-cells are quiescent and, based on thymidine incorporation assays and electronmicroscopic analysis, they rarely divide. B2-cells are more displaced towards the parenchyma of the underlying striatum and unsheathe the migrating chains of neuroblasts on route to the olfactory bulb (Doetsch et al., 1997). Unlike the structurally related B1-cells, B2-cells divide more prevalently. C-cells are the committed progeny of the B-cells, likely generated by asymmetric cell division, and they are mitotically highly active but undergo a limited number of divisions before differentiating. The progeny of the transient amplifying C-cells, the A-cells, migrate in chains through tubes formed by B-cells to the olfactory bulb. In adulthood, interneurons of the granule cell layer are the major newborn neuron type in the olfactory bulb, and together with periglomerular neurons, reform local circuits. In addition to neurons of the olfactory bulb, oligodendrocytes are also continuously generated in the subventricular zone and migrate to the corpus callosum. These oligodendrocytes are the product of Olig2-positive transient amplifying cells (a second type of C-cell). The relationship between the neurogenic C-cells and those that generate oligodendrocytes is hotly debated, as is whether they are the products of the same multipotent neural stem cells in the subventricular zone.

#### **4. Heterogeneity within the subventricular zone neural stem cell pool**

The mechanisms controlling the fate of progenitors in the subventricular zone remain unclear. The niche and its local interactions, morphogens and growth factors are one potential mode by whichthedifferentiationpotentialoftheneural stemcells is controlled(BasakandTaylor, 2009). Assuming that all stem cells with the subventricular zone have the same potential, local differences within the niche or signals interpreted by committed progenitors *en route* to their final destination would be responsible for determining the multiple neuronal fates. The ectopic grafting of stem cells into the subventricular zone indicates some degree of plasticity within the neural stem cell population and suggest niche specific signals as fate determinants (Suhonen et al., 1996). However, even with the same niche, some neural stem cells seem to have autono‐ mous fates and be heterogeneous in their potential (Kohwi et al., 2007; Merkle et al., 2007). By using homochronic/heterochronic transplantation experiments it has been shown that progen‐ itor cells at different ontogenetic stages are intrinsically directed toward specific lineages (De Marchis et al., 2007). In addition, neuroblasts in the rostral migratory stream are also heteroge‐ neous and may be committed to specific neuronal fates even before reaching the olfactory bulb (Hacket al., 2005;Kohwi et al., 2005).Thus,ratherthanbeinguniversallyplastic,theneural stem cell pool may be made up of many stem cells with restricted potentials. This is also supported by region specific, viral-mediated genetic labeling of the subventricular neural stem cells in juvenile mice which show diversity in neuronal progeny generated rather than generating all neuron types (Merkle et al., 2007). Granule cells, the major neuron subtypes to be generated during adulthood, are produced from all anteroposterior and dorsoventral locations in the subventricular zone. However, most granule cells are generated from the dorsal and ventral most aspects of the subventricular zone (Merkle et al., 2007). Within this regionalization, the granule neurons generated from the dorsal subventricular zone migrate to a more superficial location in the granule cell layer of the olfactory bulb while those generated ventrally settle deeperinthegranulecelllayer(Merkleetal.,2007).Thisregionalspecificationcanalsobemapped tothelocationofthestemcellsduringearlypostnataldevelopmentindicatingnotonlyaregional but also a developmentally-regulated fate specification (Merkle et al., 2007). Similarly, periglo‐ merular neurons that migrate to the outer layer of the olfactory bulb also show a regionspecific origin.Dorsalregions ofthe subventricular zone generate the majority ofthe thymidine hydroxylase-positive neurons whereas Calbindin-positive periglomerular neurons are generated preferentially from the ventral subventricular zone (Merkle et al., 2007). Calretininpositive periglomerular and granule cells are generated from the medial wall of the lateral ventricle. As this region produces proportionally fewer granule cells in total this suggests that the niche of the medial wall directs the fate of neural stem cells towards Calretinin neuron generation. Although these findings do not rule out niche specific programming of multipo‐ tent cell fate, heterotopic transplantation strongly suggests that stem cells retain their differen‐ tial potential when grafted into a different axial location (Merkle et al., 2007).

#### **5. Mitotically active or quiescent neural stem cells**

geneity in the putative stem cell pool. In the first sections of this chapter I will look at some of the key findings and experiments identifying the stem cells and following their fate. I will also ask the question of whether single neural stem cells are multipotent in vivo and look at some for the experimental data addressing this and also cover emerging experimental data showing

Continued neurogenesis from cells within the subependymal layer of the lateral ventricle wall implies stem cells as a driving force and a regulatory niche. Ultrastructural electronmicroscopic analysis has been instrumental in defining the morphological differences among cells within the subependymal layer of the ventricle wall (Doetsch et al., 1997; Doetsch et al., 1999a; Mirzadeh et al., 2008). Combining electromicroscopy with functional regeneration of the neurogenic niche, astrocytes have been shown to be primary progenitors of the subventricular zone (Doetsch et al., 1999b; Doetsch et al., 1999a; Doetsch et al., 2002). The subventricular zone astrocytes are defined as B-cells. B-cells have a polarized morphology extending an apical process and sensory cilium that projects between the ependymal call (E-cells) lining the lateral ventricle. These B-cell projections organize the E-cells into characteristic pinwheel structure (Mirzadeh et al., 2008). This is likely to be an important structural and signaling center in the stem cell niche. Based on their ultrastructural characteristics and location the B-cell population can be divided into two. B1-cells have their cell body between the chains of neuroblasts (Acells) and the ependymal lining. B1-cells are quiescent and, based on thymidine incorporation assays and electronmicroscopic analysis, they rarely divide. B2-cells are more displaced towards the parenchyma of the underlying striatum and unsheathe the migrating chains of neuroblasts on route to the olfactory bulb (Doetsch et al., 1997). Unlike the structurally related B1-cells, B2-cells divide more prevalently. C-cells are the committed progeny of the B-cells, likely generated by asymmetric cell division, and they are mitotically highly active but undergo a limited number of divisions before differentiating. The progeny of the transient amplifying C-cells, the A-cells, migrate in chains through tubes formed by B-cells to the olfactory bulb. In adulthood, interneurons of the granule cell layer are the major newborn neuron type in the olfactory bulb, and together with periglomerular neurons, reform local circuits. In addition to neurons of the olfactory bulb, oligodendrocytes are also continuously generated in the subventricular zone and migrate to the corpus callosum. These oligodendrocytes are the product of Olig2-positive transient amplifying cells (a second type of C-cell). The relationship between the neurogenic C-cells and those that generate oligodendrocytes is hotly debated, as is whether they are the products of the same multipotent neural stem cells in the subventricular

**4. Heterogeneity within the subventricular zone neural stem cell pool**

The mechanisms controlling the fate of progenitors in the subventricular zone remain unclear. The niche and its local interactions, morphogens and growth factors are one potential mode by

heterogeneity within the stem cell pool.

30 Neural Stem Cells - New Perspectives

zone.

**3. The subventricular zone and its progenitors**

For many years mitotic inactivity or quiescence has been viewed as a primary stem cell trait. However, recent data in many systems including the intestine and blood suggest that stem cell may not need to be quiescent and some can divide frequently to drive the generation of new cells (Wilson et al., 2008; Essers et al., 2009; Fuchs, 2009; Li and Clevers, 2010). These active stem cells are the force behind tissue homeostasis and may reside side-by-side with quiescent stem cells that rarely if ever divide but that could be responsible for tissue regeneration. Ultrastructural cellular analysis of the subventricular zone implied that even within the B-cell compartment, B1 cells rarely if ever divide whereas B2 cells are detected in cell cycle (Doetsch et al., 1997). This raised the possibility that in the adult brain stem cells may also either be able to adopt different fates or, different neural stem cells exist which show strikingly different mitotic potential. More recently, mitotically active cells in the subventricular zone were show to be in close proximity to blood vessels suggesting a mitotic influence of the endothelium or blood-born factors (Shen et al., 2008; Tavazoie et al., 2008). This is particularly intriguing as endothelial cells express the Notch ligand Jagged1 and can active neural stem cells regulating maintenance and proliferation both in vitro and in vivo thus implying that activated neural stem cells my have a vascular contribution to their niche (Shen et al., 2004; Nyfeler et al., 2005).

maintenance in the subventricular zone by repressing neuronal commitment of the stem cell but also suppresses mitotic activity of B1 cells. In addition, canonical Notch signaling is implicated in repressing the mitotic activity in ependymal cells lining the lateral ventricle during ischemic lesions (Carlen et al., 2009). Although the role of ependymal cells as stem cells is highly controversial, it remains possible that, under some degenerative/regenerative conditions, even these differentiated cells may be able to dedifferentiate or transdifferentiate to generate neuroblasts. How this regulation of proliferation function is controlled by Notch is unclear. However, analysis of Notch1 function in the subventricular zone suggests differ‐ ential receptor usage by neural stem cell in different mitotic states. The Notch gene family contains four genes encoding highly related receptors. These receptors are able to bind all five canonical ligands. At least three Notchs, Notch1, Notch2 and Notch3 are expressed in the subventriuclar zone (Stump et al., 2002; Basak et al., 2012). Notch1, Notch2 and Notch3 are expressed by B-cells whereas Notch1 is also expressed by C-cells, A-cells and E-cells (Nyfeler et al., 2005; Carlen et al., 2009; Imayoshi et al., 2010; Basak et al., 2012). Genetic conditional inactivation of Notch1 from B-cells induces a loss of self-renewal during homeostatic neuro‐ genesis. Notch1-deficient active stem cells fail to self-renew and spontaneously differentiate – similar to ablation of the canonical DNA-binding component of the pathway RBP-J in these cells. However, unlike when RBP-J is deleted, Notch1-deficiency in B1-cells does not result in spontaneous mitotic activity (Basak et al., 2012). The regulation of cell proliferation by Notch signaling has also been implicated in vitro where cultured neural stem cells lacking Notch1 fail to self-renew and differentiate and in the adult zebrafish quiescent progenitors proliferate when treated with the gamma-secretase inhibitor DAPT, which blocks Notch (Nyfeler et al., 2005; Chapouton et al., 2010). Conversely, B1-cells, although they express Notch1, do not seem to depend upon it for a quiescence signal. Thus, it is likely that molecular compensation or signal diversity between the Notch receptors is responsible for the quiescence of B1-cells. This

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 33

It has been difficult to identify and study active neural stem cell in the adult mouse subven‐ tricular zone due to an absence of selective markers. Most transgenes used to label neurogenic stem cells utilize the *Nestin*, *GLAST* or *Hes5* promoters (Mori et al., 2006; Balordi and Fishell, 2007; Lagace et al., 2007; Giachino and Taylor, 2009; Imayoshi et al., 2010; Bonaguidi et al., 2011; Basak et al., 2012). These promoters are all expressed by both quiescent and mitotic stem cells. However, combinations of transgenic reporter and surface expression of the Prominin1 associated glycoepitope CD133 and binding of the mitogen epidermal growth factor is able to select active from quiescent stem cells, C-cells and neuroblasts (Pastrana et al., 2009). Con‐ versely, Inhibitor of DNA binding protein 1 (Id1) a target of transforming growth factor-β signaling, is expressed predominantly by quiescent B1-cells. Transgenic mice expressing green fluorescent protein under the control of the Id1 promoter label quiescent B1-cells in the subventricular zone (Nam and Benezra, 2009). These Id1-positive GFAP-positive B1 cells are relatively rare and divide infrequently to generate neuroblasts likely by asymmetric cell division. Interestingly, mitotic activity of subventricular zone neural stem cells requires Id proteins with loss of function resulting in a loss of self-renewal and neurogenesis (Nam and Benezra, 2009). It remains to be shown whether and how the quiescent and active stem cells in the subventricular zone are related to each other or whether they fulfill distinct functions

remains to be examined in detail.

In summary of current and past data, the heterogeneous mitotic activity among neural stem cells suggests at least two potential scenarios. Either individual cells are able to transit between a quiescent and an activated state, or, that there are different stem cells, some which are quiescent and rarely divide, and others that are more mitotically active, dividing frequently and driving the production of new neurons destined for the olfactory bulb. A similar situation of active and dormant stem cells is present in the crypts of the large intestine where previously identified slow or rarely dividing stem cells in the +4 position seem to be the cells responsible for regenerating the epithelial lining of the gut. Conversely, mitotically active cells that are interdigitated with paneth cells at the base of the crypt replenish the epithelial cells lining the villi (Li and Clevers, 2010).

#### **6. Active and quiescent stem cells show differences in Notch signaling**

Notch signaling regulates cell fate in many cell systems and across species (Artavanis-Tsakonas et al., 1999; Louvi and Artavanis-Tsakonas, 2006). Lateral signaling between neighboring cells presenting Notch ligands and expressing receptors classically results in binary fate decisions, often in cells undergoing cell division. Notch signaling is active in the subventricular zone and multiple ligands are present on B, C and E cells providing the potential for lateral signaling (Stump et al., 2002; Nyfeler et al., 2005; Imayoshi et al., 2010). Genetic ablation of Notch signaling in stem cells of the subventricular zone results in precocious differentiation and neurogenesis (Imayoshi et al., 2010; Basak et al., 2012). This in turn results in a loss of neural stem cells and a subsequent long-term suppression of neurogenesis. This is a "classical" role for Notch in the regulation of cell fate, whereby loss of Notch signaling during what should be an asymmetric neural stem cell division results in both daughter cells adopting a differentiated cell fate and a concomitant loss of stem cell self-renewal. However, the ablation of Notch from B-cells also results in quiescent B1-cells entering the cell cycle and the active neurogenic pool. This activation of cells that are normally in a mitotically inactive state contributes to a pulse of increased neuroblast production before extinction of the stem cells pool following inactivation of canonical Notch signaling (Imayoshi et al., 2010; Basak et al., 2012). Hence, Notch signaling through its canonical pathway not only regulates stem cell maintenance in the subventricular zone by repressing neuronal commitment of the stem cell but also suppresses mitotic activity of B1 cells. In addition, canonical Notch signaling is implicated in repressing the mitotic activity in ependymal cells lining the lateral ventricle during ischemic lesions (Carlen et al., 2009). Although the role of ependymal cells as stem cells is highly controversial, it remains possible that, under some degenerative/regenerative conditions, even these differentiated cells may be able to dedifferentiate or transdifferentiate to generate neuroblasts. How this regulation of proliferation function is controlled by Notch is unclear. However, analysis of Notch1 function in the subventricular zone suggests differ‐ ential receptor usage by neural stem cell in different mitotic states. The Notch gene family contains four genes encoding highly related receptors. These receptors are able to bind all five canonical ligands. At least three Notchs, Notch1, Notch2 and Notch3 are expressed in the subventriuclar zone (Stump et al., 2002; Basak et al., 2012). Notch1, Notch2 and Notch3 are expressed by B-cells whereas Notch1 is also expressed by C-cells, A-cells and E-cells (Nyfeler et al., 2005; Carlen et al., 2009; Imayoshi et al., 2010; Basak et al., 2012). Genetic conditional inactivation of Notch1 from B-cells induces a loss of self-renewal during homeostatic neuro‐ genesis. Notch1-deficient active stem cells fail to self-renew and spontaneously differentiate – similar to ablation of the canonical DNA-binding component of the pathway RBP-J in these cells. However, unlike when RBP-J is deleted, Notch1-deficiency in B1-cells does not result in spontaneous mitotic activity (Basak et al., 2012). The regulation of cell proliferation by Notch signaling has also been implicated in vitro where cultured neural stem cells lacking Notch1 fail to self-renew and differentiate and in the adult zebrafish quiescent progenitors proliferate when treated with the gamma-secretase inhibitor DAPT, which blocks Notch (Nyfeler et al., 2005; Chapouton et al., 2010). Conversely, B1-cells, although they express Notch1, do not seem to depend upon it for a quiescence signal. Thus, it is likely that molecular compensation or signal diversity between the Notch receptors is responsible for the quiescence of B1-cells. This remains to be examined in detail.

stem cells are the force behind tissue homeostasis and may reside side-by-side with quiescent stem cells that rarely if ever divide but that could be responsible for tissue regeneration. Ultrastructural cellular analysis of the subventricular zone implied that even within the B-cell compartment, B1 cells rarely if ever divide whereas B2 cells are detected in cell cycle (Doetsch et al., 1997). This raised the possibility that in the adult brain stem cells may also either be able to adopt different fates or, different neural stem cells exist which show strikingly different mitotic potential. More recently, mitotically active cells in the subventricular zone were show to be in close proximity to blood vessels suggesting a mitotic influence of the endothelium or blood-born factors (Shen et al., 2008; Tavazoie et al., 2008). This is particularly intriguing as endothelial cells express the Notch ligand Jagged1 and can active neural stem cells regulating maintenance and proliferation both in vitro and in vivo thus implying that activated neural stem cells my have a vascular contribution to their niche (Shen et al., 2004; Nyfeler et al., 2005). In summary of current and past data, the heterogeneous mitotic activity among neural stem cells suggests at least two potential scenarios. Either individual cells are able to transit between a quiescent and an activated state, or, that there are different stem cells, some which are quiescent and rarely divide, and others that are more mitotically active, dividing frequently and driving the production of new neurons destined for the olfactory bulb. A similar situation of active and dormant stem cells is present in the crypts of the large intestine where previously identified slow or rarely dividing stem cells in the +4 position seem to be the cells responsible for regenerating the epithelial lining of the gut. Conversely, mitotically active cells that are interdigitated with paneth cells at the base of the crypt replenish the epithelial cells lining the

**6. Active and quiescent stem cells show differences in Notch signaling**

Notch signaling regulates cell fate in many cell systems and across species (Artavanis-Tsakonas et al., 1999; Louvi and Artavanis-Tsakonas, 2006). Lateral signaling between neighboring cells presenting Notch ligands and expressing receptors classically results in binary fate decisions, often in cells undergoing cell division. Notch signaling is active in the subventricular zone and multiple ligands are present on B, C and E cells providing the potential for lateral signaling (Stump et al., 2002; Nyfeler et al., 2005; Imayoshi et al., 2010). Genetic ablation of Notch signaling in stem cells of the subventricular zone results in precocious differentiation and neurogenesis (Imayoshi et al., 2010; Basak et al., 2012). This in turn results in a loss of neural stem cells and a subsequent long-term suppression of neurogenesis. This is a "classical" role for Notch in the regulation of cell fate, whereby loss of Notch signaling during what should be an asymmetric neural stem cell division results in both daughter cells adopting a differentiated cell fate and a concomitant loss of stem cell self-renewal. However, the ablation of Notch from B-cells also results in quiescent B1-cells entering the cell cycle and the active neurogenic pool. This activation of cells that are normally in a mitotically inactive state contributes to a pulse of increased neuroblast production before extinction of the stem cells pool following inactivation of canonical Notch signaling (Imayoshi et al., 2010; Basak et al., 2012). Hence, Notch signaling through its canonical pathway not only regulates stem cell

villi (Li and Clevers, 2010).

32 Neural Stem Cells - New Perspectives

It has been difficult to identify and study active neural stem cell in the adult mouse subven‐ tricular zone due to an absence of selective markers. Most transgenes used to label neurogenic stem cells utilize the *Nestin*, *GLAST* or *Hes5* promoters (Mori et al., 2006; Balordi and Fishell, 2007; Lagace et al., 2007; Giachino and Taylor, 2009; Imayoshi et al., 2010; Bonaguidi et al., 2011; Basak et al., 2012). These promoters are all expressed by both quiescent and mitotic stem cells. However, combinations of transgenic reporter and surface expression of the Prominin1 associated glycoepitope CD133 and binding of the mitogen epidermal growth factor is able to select active from quiescent stem cells, C-cells and neuroblasts (Pastrana et al., 2009). Con‐ versely, Inhibitor of DNA binding protein 1 (Id1) a target of transforming growth factor-β signaling, is expressed predominantly by quiescent B1-cells. Transgenic mice expressing green fluorescent protein under the control of the Id1 promoter label quiescent B1-cells in the subventricular zone (Nam and Benezra, 2009). These Id1-positive GFAP-positive B1 cells are relatively rare and divide infrequently to generate neuroblasts likely by asymmetric cell division. Interestingly, mitotic activity of subventricular zone neural stem cells requires Id proteins with loss of function resulting in a loss of self-renewal and neurogenesis (Nam and Benezra, 2009). It remains to be shown whether and how the quiescent and active stem cells in the subventricular zone are related to each other or whether they fulfill distinct functions for example homeostatic neurogenesis and regeneration. It is likely that the elucidation of the diverse neural stem cells in the subventricular zone is going to require the combination of different markers and genetic tools (Beckervordersandforth et al., 2010).

**9. Multiple stem cell populations in the dentate gyrus**

(Lugert et al., 2010; Venere et al., 2012).

**external cues**

The classical view of stem cells in the adult dentate gyrus implicates the quiescent radial glial like Type-1 cells as the primary progenitor. However, retroviral labeling is common‐ ly used to examine neurogenesis in the dentate and to label cells that continue to gener‐ ate multiple neurons over time (Seri et al., 2001; Suh et al., 2007). As retroviral integration and thus viral gene expression are dependent upon cells passing through the cell cycle, some long-term neurogenic stem cells in the dentate must be mitotically active. Radial Type-1 cells are rarely labeled in these retroviral experiments suggesting that other cells that lack a radial process must also display self-renewing and long-term neurogenic stem cell potential (Suh et al., 2007). This is also supported by lentiviral labeling experiments driving reporter expression from the *Sox2* promoter (Suh et al., 2007). Expression of the transcription factor Sox2 is associated with progenitor cells of the brain and required for their maintenance by regulating Notch, Sonic Hedgehog expression and Wnt activity (Steiner et al., 2008; Favaro et al., 2009; Kuwabara et al., 2009). A population of none radial stem cells with horizontally orientated processes has been identified by Cre-recombinase mediated lineage tracing (Suh et al., 2007). These horizontal cells display stem cell characteristics but are clearly distinct from the previously described Type-1 and Type-2 cells. Horizontal Type-1 neural stem cells like radial Type-1 stem cells in the dentate gyrus have active Notch signaling and are labeled with a Notch signal reporter allele *Hes5::GFP* (Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010). However, although they express Nestin they do not express the astrocytic protein GFAP. Hence, there remains some debate and despite their similarity in morphology to Type-2 cells, horizontal Type-1 stem cells do not express classic Type-2 cell markers including the proneural transcription factor Ascl1 – a Notch repressed target gene – Tbr2 or Doublecortin (Steiner et al., 2006; Lugert et al., 2010). In addition, horizontal Type-1 cells are more mitotically active than their radial counterparts (Lugert et al., 2010). Therefore, the horizontal *Hes5*-positive cells likely represent the Sox2-positive population of stem cells and those stem cells commonly traced and analyzed by retroviral labeling. Although the relationship between the radial and horizontal stem cells is not clear, horizontal cells rarely generate radial Type-1 cells in viral lineage tracing experiments. Interestingly, activated neurogenic stem cells in the dentate gyrus express Sox1, which, like Sox2 and Sox3, is a member of the SoxB1 family. *Sox1*, like *Hes5*, is expressed by radial and horizontal Type-1 cells (Venere et al., 2012). Lineage tracing shows that Sox1-positive Type-1 cells include the active neural stem cells and support that neurogenic stem cells in the dentate gyrus may switch between active and inactive states

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 35

**10. Radial and horizontal hippocampal stem cells respond selectively to**

The classical view is that radial Type-1 stem cells divide infrequently to generate transient amplifying progenitors through asymmetric cell divisions. However, as described above there

### **7. The hippocampus continually generates neurons which participate in memory formation**

In contrast to the subventricular zone where proliferation and neurogenesis are eradicat‐ ed soon after birth in humans, the dentate gyrus of the human hippocampus, like in rodents, continues to generate neurons from mitotically active progenitors cells all the way into adulthood. The cellular composition of the neurogenic niche in the dentate gyrus has been studied extensively (Seri et al., 2001; Kempermann et al., 2004; Steiner et al., 2006; Steiner et al., 2008). However, the identity and regulation of neural stem cells in the dentate gyrus remains unclear.

#### **8. The progenitor pool in the dentate gyrus is morphologically and functionally heterogeneous**

Self-renewing neural stem cells in the subgranular zone of the adult hippocampal den‐ tate gyrus (also referred to as Type-1 cells) produce intermediate progenitor cells (IPs, Type-2a cells), NeuroD1 and Doublecortin-positive neuroblasts (Type-2b) and subsequent‐ ly granule neurons (Seri et al., 2001; Kempermann et al., 2004; Steiner et al., 2006). Type-1 neural stem cells have their cell bodies in the subgranular zone and extend a long process through the granule cell layer to the overlaying molecular layer. Type-2 cells are transi‐ ent intermediate progenitors. They also have their cell body in the subgranular zone but lack a long radial process and have a more rounded morphology with short stubby processes (Seri et al., 2001; Steiner et al., 2006). Neuroblasts by contrast extend a leading process and migrate into the granule cell layer. Whereas radial Type-1 cells are quies‐ cent, Type-2 cells divide readily expanding the progenitor pool. Previous Bromodeoxyuri‐ dine labeling experiments suggested that Type-2a cells, which express proneural transcription factors, are the major proliferative progenitor in the adult dentate gyrus (Steiner et al., 2006). In addition, retroviral-labeling experiments showed that neuroblasts that have extended a radial process, exit cell cycle, and only go through one or two cell divisions (Seri et al., 2001). However, recent genetic labeling and lineage tracing of stem cells in the dentate gyrus revealed that Ascl1-positive Type-2a cells do not undergo symmetric cell divisions but generate an addition intermediate cell type, Tbr2-positive Type-2 cells (recently referred to as Type-2ab cells) (Bonaguidi et al., 2012; Lugert et al., 2012). The Tbr2-positive cells divide frequently to amplify the progenitor pool and increase the number of neurons generated from each stem cell division (Lugert et al., 2012).

#### **9. Multiple stem cell populations in the dentate gyrus**

for example homeostatic neurogenesis and regeneration. It is likely that the elucidation of the diverse neural stem cells in the subventricular zone is going to require the combination of

**7. The hippocampus continually generates neurons which participate in**

In contrast to the subventricular zone where proliferation and neurogenesis are eradicat‐ ed soon after birth in humans, the dentate gyrus of the human hippocampus, like in rodents, continues to generate neurons from mitotically active progenitors cells all the way into adulthood. The cellular composition of the neurogenic niche in the dentate gyrus has been studied extensively (Seri et al., 2001; Kempermann et al., 2004; Steiner et al., 2006; Steiner et al., 2008). However, the identity and regulation of neural stem cells in the dentate

**8. The progenitor pool in the dentate gyrus is morphologically and**

Self-renewing neural stem cells in the subgranular zone of the adult hippocampal den‐ tate gyrus (also referred to as Type-1 cells) produce intermediate progenitor cells (IPs, Type-2a cells), NeuroD1 and Doublecortin-positive neuroblasts (Type-2b) and subsequent‐ ly granule neurons (Seri et al., 2001; Kempermann et al., 2004; Steiner et al., 2006). Type-1 neural stem cells have their cell bodies in the subgranular zone and extend a long process through the granule cell layer to the overlaying molecular layer. Type-2 cells are transi‐ ent intermediate progenitors. They also have their cell body in the subgranular zone but lack a long radial process and have a more rounded morphology with short stubby processes (Seri et al., 2001; Steiner et al., 2006). Neuroblasts by contrast extend a leading process and migrate into the granule cell layer. Whereas radial Type-1 cells are quies‐ cent, Type-2 cells divide readily expanding the progenitor pool. Previous Bromodeoxyuri‐ dine labeling experiments suggested that Type-2a cells, which express proneural transcription factors, are the major proliferative progenitor in the adult dentate gyrus (Steiner et al., 2006). In addition, retroviral-labeling experiments showed that neuroblasts that have extended a radial process, exit cell cycle, and only go through one or two cell divisions (Seri et al., 2001). However, recent genetic labeling and lineage tracing of stem cells in the dentate gyrus revealed that Ascl1-positive Type-2a cells do not undergo symmetric cell divisions but generate an addition intermediate cell type, Tbr2-positive Type-2 cells (recently referred to as Type-2ab cells) (Bonaguidi et al., 2012; Lugert et al., 2012). The Tbr2-positive cells divide frequently to amplify the progenitor pool and increase the number of neurons generated from each stem cell division (Lugert et al., 2012).

different markers and genetic tools (Beckervordersandforth et al., 2010).

**memory formation**

34 Neural Stem Cells - New Perspectives

gyrus remains unclear.

**functionally heterogeneous**

The classical view of stem cells in the adult dentate gyrus implicates the quiescent radial glial like Type-1 cells as the primary progenitor. However, retroviral labeling is common‐ ly used to examine neurogenesis in the dentate and to label cells that continue to gener‐ ate multiple neurons over time (Seri et al., 2001; Suh et al., 2007). As retroviral integration and thus viral gene expression are dependent upon cells passing through the cell cycle, some long-term neurogenic stem cells in the dentate must be mitotically active. Radial Type-1 cells are rarely labeled in these retroviral experiments suggesting that other cells that lack a radial process must also display self-renewing and long-term neurogenic stem cell potential (Suh et al., 2007). This is also supported by lentiviral labeling experiments driving reporter expression from the *Sox2* promoter (Suh et al., 2007). Expression of the transcription factor Sox2 is associated with progenitor cells of the brain and required for their maintenance by regulating Notch, Sonic Hedgehog expression and Wnt activity (Steiner et al., 2008; Favaro et al., 2009; Kuwabara et al., 2009). A population of none radial stem cells with horizontally orientated processes has been identified by Cre-recombinase mediated lineage tracing (Suh et al., 2007). These horizontal cells display stem cell characteristics but are clearly distinct from the previously described Type-1 and Type-2 cells. Horizontal Type-1 neural stem cells like radial Type-1 stem cells in the dentate gyrus have active Notch signaling and are labeled with a Notch signal reporter allele *Hes5::GFP* (Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010). However, although they express Nestin they do not express the astrocytic protein GFAP. Hence, there remains some debate and despite their similarity in morphology to Type-2 cells, horizontal Type-1 stem cells do not express classic Type-2 cell markers including the proneural transcription factor Ascl1 – a Notch repressed target gene – Tbr2 or Doublecortin (Steiner et al., 2006; Lugert et al., 2010). In addition, horizontal Type-1 cells are more mitotically active than their radial counterparts (Lugert et al., 2010). Therefore, the horizontal *Hes5*-positive cells likely represent the Sox2-positive population of stem cells and those stem cells commonly traced and analyzed by retroviral labeling. Although the relationship between the radial and horizontal stem cells is not clear, horizontal cells rarely generate radial Type-1 cells in viral lineage tracing experiments. Interestingly, activated neurogenic stem cells in the dentate gyrus express Sox1, which, like Sox2 and Sox3, is a member of the SoxB1 family. *Sox1*, like *Hes5*, is expressed by radial and horizontal Type-1 cells (Venere et al., 2012). Lineage tracing shows that Sox1-positive Type-1 cells include the active neural stem cells and support that neurogenic stem cells in the dentate gyrus may switch between active and inactive states (Lugert et al., 2010; Venere et al., 2012).

#### **10. Radial and horizontal hippocampal stem cells respond selectively to external cues**

The classical view is that radial Type-1 stem cells divide infrequently to generate transient amplifying progenitors through asymmetric cell divisions. However, as described above there are additional progenitors in the hippocampal dentate gyrus that can function as stem cells. Hence, the question arises what are the functions of these multiple putative neural stem cells? Do they both contribute to neurogenesis in the adult hippocampus and are they in a lineage relationship with each other? Genetic labeling experiments suggest that both radial and horizontal stem cells may be functionally distinct or at least they respond differently to different pathophysiological cues (Lugert et al., 2010).

the stem cell pool and differentiation into astrocytes (Encinas et al., 2011; Encinas and Sierra, 2012), others suggest that the stem cells are not lost but become dormant with age (Lugert et al., 2010; Bonaguidi et al., 2011; Venere et al., 2012). Hence, the reason for the substantial reduction in neuron production remains unclear but may be caused by a

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 37

Genetic lineage tracing of *Nestin* expressing cells revealed that, parallel to the reduced number of neurons generated from the labeled stem cells, radial Type-1 cells in the aged mouse brain enter cell cycle and, following a few cell divisions, differentiate into polymor‐ phic astrocytes that lose radial morphology and presumably stem cell potential (Encinas et al., 2011). This "deforestation" or expenditure of the stem cells likely contributes to the reduction in mitotic progenitors and neurons (Encinas and Sierra, 2012). Surprisingly, in a parallel study using the same genetic tools, clonal analysis indicated that *Nestin* express‐ ing stem cells within the subgranular layer can undergo prolonged neurogenesis. In addition, these clonal experiments revealed an additional degree of heterogeneity within the stem cell population of the dentate gyrus. Some labeled Type-1 cells remained quiescent over many months and failed to generate any viable offspring. Other Type-1 cells divided and generated clones of cells that included progenitors, neurons and astrocytes indicat‐ ing multipotency (Bonaguidi et al., 2011). Partially supporting the proposal that some *Nestin*-expressing Type-1 cells may exit the stem cell pool, clones were found that contained only differentiated cells. Taken together these data indicate heterogeneity within the stem cells pools and it seems that a combination of entry of stem cells into a dormant state coupled with a partial loss of some progenitors may contribute to the age related decline

In contrast, neural stem cells in the dentate gyrus labeled by Notch activity and Sox2 expression remain in the aged dentate gyrus (Lugert et al., 2010; Bonaguidi et al., 2011; Lugert et al., 2012). Interestingly however, the proportion of the cells that are mitotically active, which is predominantly the horizontal population, are lost. Hence, even in aged mice the number of stem cells remains relatively constant but their mitotic activity reduces and actively prolifer‐ ating cells are lost, become quiescent, or dormant (Lugert et al., 2010; Bonaguidi et al., 2011; Lugert et al., 2012). This is similar to findings that *Sox1*–positive stem cells remain long-term

A loss of stem cells in the dentate gyrus would suggest that the neurogenic process cannot be rescued or reversed in aged animals. However, physical exercise and pathological stimulation both stimulate proliferation, neural stem cell activation and under some conditions increased numbers of newly generated neurons (Rao et al., 2005; van Praag et al., 2005; Hattiangady et al., 2008; Jessberger and Gage, 2008; Rao et al., 2008; Zhao et al., 2008). Hence, although loss of stem cells could contribute to the age-related decline in neuron production, some cells with stem cell potential remain even in the dentate gyrus of old mice and these can be activated to proliferate and generate new cells (Lugert et al., 2010; Venere et al., 2012). It still remains unclear whether radial Type-1 cells in old mice enter the cell cycle during physical exercise or whether the few remaining horizontal cells could reactivate in the aged brain or whether a

neurogenic and can enter and exit the active stem cells pools (Venere et al., 2012).

culmination of physiological changes.

in neurogenesis (Bonaguidi et al., 2011; Encinas et al., 2011).

Analysis of hippocampal neurogenesis has shown it to be a dynamic process that diminishes with age but can be stimulated and modulated by physiology and pathology (Kuhn et al., 1996; Kempermann et al., 1998; Ben Abdallah et al., 2008; Fabel and Kempermann, 2008; Parent and Murphy, 2008; Steiner et al., 2008; Zhao et al., 2008). Voluntary physical exercise induces increased proliferation and generation of immature neurons. These neurons do not readily integrate into the dentate gyrus but the increased proliferation of the stem cells is significant (Fabel and Kempermann, 2008). Notch signaling also controls neural stem cell maintenance and differentiation within the dentate gyrus (Breunig et al., 2007; Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010; Lugert et al., 2012). Loss of Notch activity results in the loss of neural stem cells and their precocious differentiation culminating in a loss of neuron production (Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010). Genetic labeling of neural stem cells in the dentate that display Notch signaling has uncovered diversity in stem cell responses to pathophysiology (Lugert et al., 2010). Physical exercise stimulates proliferation of the radial type1 cells but not the horizontal stem cells (Lugert et al., 2010). Running induces the radial cells to enter the active stem cell pool without expanding the total stem cell population. This suggests that radial cells in physically active animals undergo asymmetric cell divisions to generate committed progenitors that increase the number of newborn neurons whilst main‐ taining the Type-1 stem cell pool through self-renewal. This also implies that radial stem cells respond to stimuli generated by increased physical activity that are not seen or are not interpreted in the same way by the horizontal stem cells. These findings seem, at first glance, to contradict previous experiments where Nestin expressing progenitors were labeled and suggested that radial Type-1 cells do not proliferate significantly in running mice (Steiner et al., 2008). It is likely that the differences in result reflect the different experimental paradigms used to identify the stem cells of the dentate gyrus. Where as *Hes5* expression identifies a smaller population of cells more restricted to the stem cell pools in the subgranular cell layer, the *Nestin* promoter is expressed by stem cells and more committed progenitors (Bonaguidi et al., 2012). Hence, it remains possible that the different labeling techniques and the extent of cell labeling could effect the quantification and interpretation.

#### **11. Selective loss of active stem cells in the hippocampus of aged mice**

Neurogenesis in the mammalian brain diminishes dramatically after birth, even in the dentate gyrus where neurons are continuously generated throughout life. This reduced neurogenesis is associated with a loss of mitotic cells (Kuhn et al., 1996; Kempermann et al., 1998; Ben Abdallah et al., 2008; Steiner et al., 2008). Whereas some reports have suggested an irreversible loss of neural stem cells in the dentate gyrus due to exit from the stem cell pool and differentiation into astrocytes (Encinas et al., 2011; Encinas and Sierra, 2012), others suggest that the stem cells are not lost but become dormant with age (Lugert et al., 2010; Bonaguidi et al., 2011; Venere et al., 2012). Hence, the reason for the substantial reduction in neuron production remains unclear but may be caused by a culmination of physiological changes.

are additional progenitors in the hippocampal dentate gyrus that can function as stem cells. Hence, the question arises what are the functions of these multiple putative neural stem cells? Do they both contribute to neurogenesis in the adult hippocampus and are they in a lineage relationship with each other? Genetic labeling experiments suggest that both radial and horizontal stem cells may be functionally distinct or at least they respond differently to

Analysis of hippocampal neurogenesis has shown it to be a dynamic process that diminishes with age but can be stimulated and modulated by physiology and pathology (Kuhn et al., 1996; Kempermann et al., 1998; Ben Abdallah et al., 2008; Fabel and Kempermann, 2008; Parent and Murphy, 2008; Steiner et al., 2008; Zhao et al., 2008). Voluntary physical exercise induces increased proliferation and generation of immature neurons. These neurons do not readily integrate into the dentate gyrus but the increased proliferation of the stem cells is significant (Fabel and Kempermann, 2008). Notch signaling also controls neural stem cell maintenance and differentiation within the dentate gyrus (Breunig et al., 2007; Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010; Lugert et al., 2012). Loss of Notch activity results in the loss of neural stem cells and their precocious differentiation culminating in a loss of neuron production (Ables et al., 2010; Ehm et al., 2010; Lugert et al., 2010). Genetic labeling of neural stem cells in the dentate that display Notch signaling has uncovered diversity in stem cell responses to pathophysiology (Lugert et al., 2010). Physical exercise stimulates proliferation of the radial type1 cells but not the horizontal stem cells (Lugert et al., 2010). Running induces the radial cells to enter the active stem cell pool without expanding the total stem cell population. This suggests that radial cells in physically active animals undergo asymmetric cell divisions to generate committed progenitors that increase the number of newborn neurons whilst main‐ taining the Type-1 stem cell pool through self-renewal. This also implies that radial stem cells respond to stimuli generated by increased physical activity that are not seen or are not interpreted in the same way by the horizontal stem cells. These findings seem, at first glance, to contradict previous experiments where Nestin expressing progenitors were labeled and suggested that radial Type-1 cells do not proliferate significantly in running mice (Steiner et al., 2008). It is likely that the differences in result reflect the different experimental paradigms used to identify the stem cells of the dentate gyrus. Where as *Hes5* expression identifies a smaller population of cells more restricted to the stem cell pools in the subgranular cell layer, the *Nestin* promoter is expressed by stem cells and more committed progenitors (Bonaguidi et al., 2012). Hence, it remains possible that the different labeling techniques and the extent of

different pathophysiological cues (Lugert et al., 2010).

36 Neural Stem Cells - New Perspectives

cell labeling could effect the quantification and interpretation.

**11. Selective loss of active stem cells in the hippocampus of aged mice**

Neurogenesis in the mammalian brain diminishes dramatically after birth, even in the dentate gyrus where neurons are continuously generated throughout life. This reduced neurogenesis is associated with a loss of mitotic cells (Kuhn et al., 1996; Kempermann et al., 1998; Ben Abdallah et al., 2008; Steiner et al., 2008). Whereas some reports have suggested an irreversible loss of neural stem cells in the dentate gyrus due to exit from Genetic lineage tracing of *Nestin* expressing cells revealed that, parallel to the reduced number of neurons generated from the labeled stem cells, radial Type-1 cells in the aged mouse brain enter cell cycle and, following a few cell divisions, differentiate into polymor‐ phic astrocytes that lose radial morphology and presumably stem cell potential (Encinas et al., 2011). This "deforestation" or expenditure of the stem cells likely contributes to the reduction in mitotic progenitors and neurons (Encinas and Sierra, 2012). Surprisingly, in a parallel study using the same genetic tools, clonal analysis indicated that *Nestin* express‐ ing stem cells within the subgranular layer can undergo prolonged neurogenesis. In addition, these clonal experiments revealed an additional degree of heterogeneity within the stem cell population of the dentate gyrus. Some labeled Type-1 cells remained quiescent over many months and failed to generate any viable offspring. Other Type-1 cells divided and generated clones of cells that included progenitors, neurons and astrocytes indicat‐ ing multipotency (Bonaguidi et al., 2011). Partially supporting the proposal that some *Nestin*-expressing Type-1 cells may exit the stem cell pool, clones were found that contained only differentiated cells. Taken together these data indicate heterogeneity within the stem cells pools and it seems that a combination of entry of stem cells into a dormant state coupled with a partial loss of some progenitors may contribute to the age related decline in neurogenesis (Bonaguidi et al., 2011; Encinas et al., 2011).

In contrast, neural stem cells in the dentate gyrus labeled by Notch activity and Sox2 expression remain in the aged dentate gyrus (Lugert et al., 2010; Bonaguidi et al., 2011; Lugert et al., 2012). Interestingly however, the proportion of the cells that are mitotically active, which is predominantly the horizontal population, are lost. Hence, even in aged mice the number of stem cells remains relatively constant but their mitotic activity reduces and actively prolifer‐ ating cells are lost, become quiescent, or dormant (Lugert et al., 2010; Bonaguidi et al., 2011; Lugert et al., 2012). This is similar to findings that *Sox1*–positive stem cells remain long-term neurogenic and can enter and exit the active stem cells pools (Venere et al., 2012).

A loss of stem cells in the dentate gyrus would suggest that the neurogenic process cannot be rescued or reversed in aged animals. However, physical exercise and pathological stimulation both stimulate proliferation, neural stem cell activation and under some conditions increased numbers of newly generated neurons (Rao et al., 2005; van Praag et al., 2005; Hattiangady et al., 2008; Jessberger and Gage, 2008; Rao et al., 2008; Zhao et al., 2008). Hence, although loss of stem cells could contribute to the age-related decline in neuron production, some cells with stem cell potential remain even in the dentate gyrus of old mice and these can be activated to proliferate and generate new cells (Lugert et al., 2010; Venere et al., 2012). It still remains unclear whether radial Type-1 cells in old mice enter the cell cycle during physical exercise or whether the few remaining horizontal cells could reactivate in the aged brain or whether a distinct cell population, previously not studied or labeled with the tools and techniques current available, replenishes the neural stem cell pools.

**References**

[1] Ables, J. L., Decarolis, N. A., Johnson, M. A., Rivera, P. D., Gao, Z., Cooper, D. C., Radtke, F., Hsieh, J. and Eisch, A. J. (2010). Notch1 is required for maintenance of the

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 39

[2] Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate

[3] Balordi, F. and Fishell, G. (2007). Mosaic removal of hedgehog signaling in the adult SVZ reveals that the residual wild-type stem cells have a limited capacity for self-re‐

[4] Basak, O. and Taylor, V. (2009). Stem cells of the adult mammalian brain and their

[5] Basak, O, Giachino, C, & Fiorini, E. MacDonald, H. R. and Taylor, V. (2012). Neuro‐ genic subventricular zone stem/progenitor cells are Notch1-dependent in their active

[6] Beckervordersandforth, R., Tripathi, P., Ninkovic, J., Bayam, E., Lepier, A., Stempf‐ huber, B., Kirchhoff, F., Hirrlinger, J., Haslinger, A., Lie, D. C., Beckers, J., Yoder, B., Irmler, M. and Gotz, M. (2010). In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells. Cell Stem Cell

[7] Ben Abdallah N. M., Slomianka, L., Vyssotski, A. L. and Lipp, H. (2010). Early agerelated changes in adult hippocampal neurogenesis in C57 mice. Neurobiol Aging.

[8] Bonaguidi, M. A., Song, J., Ming, G. L. and Song, H. (2012). A unifying hypothesis on mammalian neural stem cell properties in the adult hippocampus. Curr Opin Neuro‐

[9] Bonaguidi, M. A., Wheeler, M. A., Shapiro, J. S., Stadel, R. P., Sun, G. J., Ming, G. L. and Song, H. (2011). In vivo clonal analysis reveals self-renewing and multipotent

[10] Breunig, J. J., Silbereis, J., Vaccarino, F. M., Sestan, N. and Rakic, P. (2007). Notch reg‐ ulates cell fate and dendrite morphology of newborn neurons in the postnatal den‐

[11] Carlen, M., Meletis, K., Goritz, C., Darsalia, V., Evergren, E., Tanigaki, K., Amendola, M., Barnabe-Heider, F., Yeung, M. S., Naldini, L., Honjo, T., Kokaia, Z., Shupliakov, O., Cassidy, R. M., Lindvall, O. and Frisen, J. (2009). Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke. Nat Neurosci.

biol. 22, 754-761. doi: 710.1016/j.conb.2012.1003.1013. Epub 2012 Apr 1013.

adult neural stem cell characteristics. Cell. 145, 1142-1155.

tate gyrus. Proc Natl Acad Sci U S A 104, 20558-20563.

reservoir of adult hippocampal stem cells. J Neurosci. 30, 10484-10492.

control and signal integration in development. Science 284, 770-776.

newal. J Neurosci 27, 14248-14259.

7, 744-758.

31, 151-161.

12, 259-267.

niche. Cell Mol Life Sci. 66, 1057-1072.

but not quiescent state J Neurosci., 32, 5654-5666.

#### **12. Seizures induce neural stem cell proliferation in the hippocampus**

Chronic temporal lobe epilepsy is associated with an increase production of neurons in the dentate gyrus (Parent, 2007; Scharfman and Gray, 2007). Conversely, acute seizures dramati‐ cally induce abnormal production of neurons in the dentate gyrus, which may contribute to chronic epilepsy. Whether generation of new neurons in the hippocampus of patients with epilepsy is a result of the disease or contributes to the cause is not clear. In mice, experimentally induced seizures effect neuron production at multiple levels and not least by disproportion‐ ately increasing the number of neuroblasts (Type-3 cells) (Jessberger et al., 2005). Both the radial Type-1 and the horizontal stem cells are activated in response to experimentally induced seizures (Huttmann et al., 2003; Lugert et al., 2012; Venere et al., 2012). However, the proportion of radial cells that enter cell cycle is rather modest and the population is not expanded suggesting that their divisions generate more committed progenitors. The horizontal stem cells respond more homogeneously to seizures. The majority of them enter the cell cycle and the total number increases significantly (Lugert et al., 2010). The increase in horizontal cells could be the result of symmetric cell division but also generation of horizontal cells from the radial stem cell pool. Although current tools and techniques have not been able to address the mechanism, the increase in mitotically active stem cells following chronic seize and the differential response of the different stem cell pools has important implications for the cause and progression of temporal lobe epilepsy in humans.

#### **13. Future perspectives**

In the future it will be a major challenge to elucidate the heterogeneity within the stem cells pools and to address there cellular function. This will include understanding how these different populations and cell states are regulated and whether their functions are controlled by distinct niche signals or genetic and epigenetic mechanisms. Only the detailed analysis of neural stem cells in the adult brain could uncover their functions in homeostasis, aging and disease. This would raise the exciting possibility that specific neural stem cell subtypes could be directly targeted for therapy.

#### **Author details**

Verdon Taylor

Embryology and Stem Cell Biology, Department of Biomedicine, University of Basel, Basel, Switzerland

#### **References**

distinct cell population, previously not studied or labeled with the tools and techniques current

**12. Seizures induce neural stem cell proliferation in the hippocampus**

Chronic temporal lobe epilepsy is associated with an increase production of neurons in the dentate gyrus (Parent, 2007; Scharfman and Gray, 2007). Conversely, acute seizures dramati‐ cally induce abnormal production of neurons in the dentate gyrus, which may contribute to chronic epilepsy. Whether generation of new neurons in the hippocampus of patients with epilepsy is a result of the disease or contributes to the cause is not clear. In mice, experimentally induced seizures effect neuron production at multiple levels and not least by disproportion‐ ately increasing the number of neuroblasts (Type-3 cells) (Jessberger et al., 2005). Both the radial Type-1 and the horizontal stem cells are activated in response to experimentally induced seizures (Huttmann et al., 2003; Lugert et al., 2012; Venere et al., 2012). However, the proportion of radial cells that enter cell cycle is rather modest and the population is not expanded suggesting that their divisions generate more committed progenitors. The horizontal stem cells respond more homogeneously to seizures. The majority of them enter the cell cycle and the total number increases significantly (Lugert et al., 2010). The increase in horizontal cells could be the result of symmetric cell division but also generation of horizontal cells from the radial stem cell pool. Although current tools and techniques have not been able to address the mechanism, the increase in mitotically active stem cells following chronic seize and the differential response of the different stem cell pools has important implications for the cause

In the future it will be a major challenge to elucidate the heterogeneity within the stem cells pools and to address there cellular function. This will include understanding how these different populations and cell states are regulated and whether their functions are controlled by distinct niche signals or genetic and epigenetic mechanisms. Only the detailed analysis of neural stem cells in the adult brain could uncover their functions in homeostasis, aging and disease. This would raise the exciting possibility that specific neural stem cell subtypes could

Embryology and Stem Cell Biology, Department of Biomedicine, University of Basel, Basel,

available, replenishes the neural stem cell pools.

38 Neural Stem Cells - New Perspectives

and progression of temporal lobe epilepsy in humans.

**13. Future perspectives**

be directly targeted for therapy.

**Author details**

Verdon Taylor

Switzerland


[12] Chapouton, P., Skupien, P., Hesl, B., Coolen, M., Moore, J. C., Madelaine, R., Kremm‐ er, E., Faus-Kessler, T., Blader, P., Lawson, N. D. and Bally-Cuif, L. (2010). Notch ac‐ tivity levels control the balance between quiescence and recruitment of adult neural stem cells. J Neurosci. 30, 7961-7974.

[22] Essers, M. A., Offner, S., Blanco-Bose, W. E., Waibler, Z., Kalinke, U., Duchosal, M. A. and Trumpp, A. (2009). IFNalpha activates dormant haematopoietic stem cells in

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 41

[23] Fabel, K. and Kempermann, G. (2008). Physical activity and the regulation of neuro‐

[24] Favaro, R., Valotta, M., Ferri, A. L., Latorre, E., Mariani, J., Giachino, C., Lancini, C., Tosetti, V., Ottolenghi, S., Taylor, V. and Nicolis, S. K. (2009). Hippocampal develop‐ ment and neural stem cell maintenance require Sox2-dependent regulation of Shh.

[25] Fuchs, E. (2009). The tortoise and the hair: slow-cycling cells in the stem cell race.

[27] Garthe, A., Behr, J. and Kempermann, G. (2009). Adult-generated hippocampal neu‐ rons allow the flexible use of spatially precise learning strategies. PLoS One. 4, e5464.

[28] Giachino, C. and Taylor, V. (2009). Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic

[29] Hack, M. A., Saghatelyan, A., de Chevigny, A., Pfeifer, A., Ashery-Padan, R., Lledo, P. M. and Gotz, M. (2005). Neuronal fate determinants of adult olfactory bulb neuro‐

[30] Hattiangady, B., Rao, M. S. and Shetty, A. K. (2008). Plasticity of hippocampal stem/ progenitor cells to enhance neurogenesis in response to kainate-induced injury is lost

[31] Huttmann, K., Sadgrove, M., Wallraff, A., Hinterkeuser, S., Kirchhoff, F., Steinhauser, C. and Gray, W. P. (2003). Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical

[32] Imayoshi, I., Sakamoto, M., Yamaguchi, M., Mori, K. and Kageyama, R. (2010). Essen‐ tial roles of Notch signaling in maintenance of neural stem cells in developing and

[33] Jessberger, S. and Gage, F. H. (2008). Stem-cell-associated structural and functional

[34] Kempermann, G., Kuhn, H. G. and Gage, F. H. (1998). Experience-induced neurogen‐

[35] Kempermann, G., Jessberger, S., Steiner, B. and Kronenberg, G. (2004). Milestones of neuronal development in the adult hippocampus. Trends Neurosci 27, 447-452.

plasticity in the aging hippocampus. Psychol Aging 23, 684-691.

esis in the senescent dentate gyrus. J Neurosci. 18, 3206-3212.

genesis in the adult and aging brain. Neuromolecular Med 10, 59-66.

[26] Gage, F. H. (2000). Mammalian neural stem cells. Science 287, 1433-1438.

niches in the olfactory bulb. Eur J Neurosci 30, 9-24.

vivo. Nature. 458, 904-908.

Nat Neurosci.

Cell. 137, 811-819.

Epub 2009 May 5467.

genesis. Nat Neurosci. 8, 865-872.

by middle age. Aging Cell 7, 207-224.

analysis. Eur J Neurosci 18, 2769-2778.

adult brains. J Neurosci 30, 3489-3498.


[22] Essers, M. A., Offner, S., Blanco-Bose, W. E., Waibler, Z., Kalinke, U., Duchosal, M. A. and Trumpp, A. (2009). IFNalpha activates dormant haematopoietic stem cells in vivo. Nature. 458, 904-908.

[12] Chapouton, P., Skupien, P., Hesl, B., Coolen, M., Moore, J. C., Madelaine, R., Kremm‐ er, E., Faus-Kessler, T., Blader, P., Lawson, N. D. and Bally-Cuif, L. (2010). Notch ac‐ tivity levels control the balance between quiescence and recruitment of adult neural

[13] Clelland, C. D., Choi, M., Romberg, C., Clemenson, G. D., Jr., Fragniere, A., Tyers, P., Jessberger, S., Saksida, L. M., Barker, R. A., Gage, F. H. and Bussey, T. J. (2009). A functional role for adult hippocampal neurogenesis in spatial pattern separation. Sci‐

[14] De Marchis, S., Bovetti, S., Carletti, B., Hsieh, Y. C., Garzotto, D., Peretto, P., Fasolo, A., Puche, A. C. and Rossi, F. (2007). Generation of distinct types of periglomerular olfactory bulb interneurons during development and in adult mice: implication for intrinsic properties of the subventricular zone progenitor population. J Neurosci. 27,

[15] Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1997). Cellular composi‐ tion and three-dimensional organization of the subventricular germinal zone in the

[16] Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999a). Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 96,

[17] Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999b). Subventricular zone astrocytes are neural stem cells in the adult mammalian

[18] Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (2002). EGF converts transit-amplifying neurogenic precursors in the adult brain into

[19] Ehm, O., Goritz, C., Covic, M., Schaffner, I., Schwarz, T. J., Karaca, E., Kempkes, B., Kremmer, E., Pfrieger, F. W., Espinosa, L., Bigas, A., Giachino, C., Taylor, V., Frisen, J. and Lie, D. C. (2010). RBPJkappa-dependent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus. J Neurosci. 30,

[20] Encinas, J. M. and Sierra, A. (2012). Neural stem cell deforestation as the main force driving the age-related decline in adult hippocampal neurogenesis. Behav Brain Res.

[21] Encinas, J. M., Michurina, T. V., Peunova, N., Park, J. H., Tordo, J., Peterson, D. A., Fishell, G., Koulakov, A. and Enikolopov, G. (2011). Division-coupled astrocytic dif‐ ferentiation and age-related depletion of neural stem cells in the adult hippocampus.

stem cells. J Neurosci. 30, 7961-7974.

adult mammalian brain. J Neurosci 17, 5046-5061.

multipotent stem cells. Neuron 36, 1021-1034.

ence. 325, 210-213.

40 Neural Stem Cells - New Perspectives

657-664.

11619-11624.

13794-13807.

227, 433-439.

Cell Stem Cell. 8, 566-579.

brain. Cell 97, 703-716.


[36] Kohwi, M., Osumi, N., Rubenstein, J. L. and Alvarez-Buylla, A. (2005). Pax6 is re‐ quired for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb. J Neurosci. 25, 6997-7003.

mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.

Neural Stem Cell Heterogeneity http://dx.doi.org/10.5772/55676 43

[49] Nam, H. S. and Benezra, R. (2009). High levels of Id1 expression define B1 type adult

[50] Nyfeler, Y., Kirch, R. D., Mantei, N., Leone, D. P., Radtke, F., Suter, U. and Taylor, V. (2005). Jagged1 signals in the postnatal subventricular zone are required for neural

[51] Parent, J. M. and Murphy, G. G. (2008). Mechanisms and functional significance of aberrant seizure-induced hippocampal neurogenesis. Epilepsia 49 Suppl 5, 19-25.

[52] Pastrana, E., Cheng, L. C. and Doetsch, F. (2009). Simultaneous prospective purifica‐ tion of adult subventricular zone neural stem cells and their progeny. Proc Natl Acad

[53] Rao, M. S., Hattiangady, B. and Shetty, A. K. (2008). Status epilepticus during old age is not associated with enhanced hippocampal neurogenesis. Hippocampus 18,

[54] Rao, M. S., Hattiangady, B., Abdel-Rahman, A., Stanley, D. P. and Shetty, A. K. (2005). Newly born cells in the ageing dentate gyrus display normal migration, sur‐ vival and neuronal fate choice but endure retarded early maturation. Eur J Neurosci

[55] Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from iso‐ lated cells of the adult mammalian central nervous system. Science 255, 1707-1710.

[56] Seri, B., Garcia-Verdugo, J. M., McEwen, B. S. and Alvarez-Buylla, A. (2001). Astro‐ cytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21,

[57] Shen, Q., Wang, Y., Kokovay, E., Lin, G., Chuang, S. M., Goderie, S. K., Roysam, B. and Temple, S. (2008). Adult SVZ stem cells lie in a vascular niche: a quantitative

[58] Shen, Q., Goderie, S. K., Jin, L., Karanth, N., Sun, Y., Abramova, N., Vincent, P., Pum‐ iglia, K. and Temple, S. (2004). Endothelial cells stimulate self-renewal and expand

[59] Shors, T. J., Miesegaes, G., Beylin, A., Zhao, M., Rydel, T. and Gould, E. (2001). Neu‐ rogenesis in the adult is involved in the formation of trace memories. Nature 410,

[60] Steiner, B., Zurborg, S., Horster, H., Fabel, K. and Kempermann, G. (2008). Differen‐ tial 24 h responsiveness of Prox1-expressing precursor cells in adult hippocampal neurogenesis to physical activity, environmental enrichment, and kainic acid-in‐

analysis of niche cell-cell interactions. Cell Stem Cell. 3, 289-300.

neurogenesis of neural stem cells. Science 304, 1338-1340.

duced seizures. Neuroscience. 154, 521-529.

Neuron 13, 1071-12082.

Sci U S A. 106, 6387-6392.

931-944.

21, 464-476.

7153-7160.

372-376.

neural stem cells. Cell Stem Cell. 5, 515-526.

stem cell self-renewal. EMBO J 24, 3504-3515.


mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071-12082.

[49] Nam, H. S. and Benezra, R. (2009). High levels of Id1 expression define B1 type adult neural stem cells. Cell Stem Cell. 5, 515-526.

[36] Kohwi, M., Osumi, N., Rubenstein, J. L. and Alvarez-Buylla, A. (2005). Pax6 is re‐ quired for making specific subpopulations of granule and periglomerular neurons in

[37] Kohwi, M., Petryniak, M. A., Long, J. E., Ekker, M., Obata, K., Yanagawa, Y., Ruben‐ stein, J. L. and Alvarez-Buylla, A. (2007). A subpopulation of olfactory bulb GABAer‐ gic interneurons is derived from Emx1- and Dlx5/6-expressing progenitors. J

[38] Kuhn, H. G., Dickinson-Anson, H. and Gage, F. H. (1996). Neurogenesis in the den‐ tate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J

[39] Kuwabara, T., Hsieh, J., Muotri, A., Yeo, G., Warashina, M., Lie, D. C., Moore, L., Na‐ kashima, K., Asashima, M. and Gage, F. H. (2009). Wnt-mediated activation of Neu‐ roD1 and retro-elements during adult neurogenesis. Nat Neurosci 12, 1097-1105. [40] Lagace, D. C., Whitman, M. C., Noonan, M. A., Ables, J. L., DeCarolis, N. A., Arguel‐ lo, A. A., Donovan, M. H., Fischer, S. J., Farnbauch, L. A., Beech, R. D., DiLeone, R. J., Greer, C. A., Mandyam, C. D. and Eisch, A. J. (2007). Dynamic contribution of nestin-

[41] Li, L. and Clevers, H. (2010). Coexistence of quiescent and active adult stem cells in

[42] Louvi, A. and Artavanis-Tsakonas, S. (2006). Notch signalling in vertebrate neural

[43] Lugert, S., Vogt, M., Tchorz, J. S., Muller, M., Giachino, C. and Taylor, V. (2012). Ho‐ meostatic neurogenesis in the adult hippocampus does not involve amplification of

[44] Lugert, S., Basak, O., Knuckles, P., Haussler, U., Fabel, K., Gotz, M., Haas, C. A., Kempermann, G., Taylor, V. and Giachino, C. (2010). Quiescent and active hippo‐ campal neural stem cells with distinct morphologies respond selectively to physio‐

[45] Merkle, F. T., Mirzadeh, Z. and Alvarez-Buylla, A. (2007). Mosaic organization of

[46] Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (2008). Neural stem cells confer unique pinwheel architecture to the ven‐ tricular surface in neurogenic regions of the adult brain. Cell Stem Cell. 3, 265-278. [47] Mori, T., Tanaka, K., Buffo, A., Wurst, W., Kuhn, R. and Gotz, M. (2006). Inducible gene deletion in astroglia and radial glia--a valuable tool for functional and lineage

[48] Morshead, C. M., Reynolds, B. A., Craig, C. G., McBurney, M. W., Staines, W. A., Mo‐ rassutti, D., Weiss, S. and van der Kooy, D. (1994). Neural stem cells in the adult

logical and pathological stimuli and aging. Cell Stem Cell 6, 445-456.

expressing stem cells to adult neurogenesis. J Neurosci. 27, 12623-12629.

the olfactory bulb. J Neurosci. 25, 6997-7003.

Neurosci. 27, 6878-6891.

42 Neural Stem Cells - New Perspectives

Neurosci. 16, 2027-2033.

mammals. Science 327, 542-545.

analysis. Glia 54, 21-34.

development. Nat Rev Neurosci 7, 93-102.

Ascl1(high) intermediate progenitors. Nat Commun., 670.

neural stem cells in the adult brain. Science 317, 381-384.


[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 neurogenesis. Glia. 54, 805-814.

**Chapter 3**

**Diversity of Neural Stem/Progenitor Populations:**

Emília Madarász

**1. Introduction**

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

Additional information is available at the end of the chapter

**Varieties by Age, Regional Origin and Environment**

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,

© 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,

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

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

