**Parenchymal Neuro-Glio-Genesis Versus Germinal Layer-Derived Neurogenesis: Two Faces of Central Nervous System Structural Plasticity**

Luca Bonfanti, Giovanna Ponti, Federico Luzzati, Paola Crociara, Roberta Parolisi and Maria Armentano

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56100

#### **1. Introduction**

The discovery of neural stem cells (NSCs) at the beginning of the nineties led many people to consider definitively broken the dogma of a static central nervous system (CNS) made up of non-renewable elements [1-3]. In parallel, the occurrence and characterization of adult neurogenesis in the olfactory bulb and hippocampus [3-5] triggered new hopes for brain repair. Twenty years after, the dream of regenerative medicine applied to brain/spinal cord injuries and neurodegenerative diseases is still very far [6,7]. As a matter of fact, adult neurogenesis in mammals occurs mainly within two restricted areas known as 'neurogenic sites' [3,8]: the forebrain subventricular zone (SVZ); reviewed in [9] and the hippocampal dentate gyrus (subgranular zone, SGZ); reviewed in [10]. As a direct consequence of such topographical localization, most of the CNS parenchyma out of the two 'classic' neurogenic sites remains substantially a non-renewable tissue. Actually, most of the traumatic/vascular injuries and neurodegenerative diseases do occur in 'non-neurogenic' regions and no efficacious therapies capable of restoring CNS structure and functions through cell replacement are at present available. Thus, two decades after the discovery of NSCs and the reaching of a satisfactory characterization of adult neurogenic sites, a gap remains between the occurrence of stem/ progenitor cells in the CNS of adult mammals and their effective capability to serve in brain repair. Several aspects do converge in explaining this gap [11], partially accounting for the heterogeneity of CNS structural plasticity in mammals (summarized in Table 1)

© 2013 Bonfanti 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.

**Figure 1.** Heterogeneity of postnatal ⁄ adult neurogenic processes in different mammals by considering different as‐ pects and mammalian species. B, Schematic summary of the main sources (progenitor cells) of adult mammalian neu‐ rogenesis, its outcome in vivo ⁄ in culture system, and its possible activation after lesion. In the case of many parenchymal regions, some of these steps are still obscure. BLBP, brain lipid-binding protein; EGL, external germinal layer; GABA, c-aminobutyric acid; Ng2, nerve ⁄ glial antigen 2 proteoglycan; NPY, Neuropeptide Y; SGZ, subgranular zone; SPL, subpial layer; SVZ, subventricular zone; VMM, ventral migratory mass; VMS, ventrocaudal migratory stream; MMS, medial migratory stream; DMS, dorsal migratory stream. Adapted from Ref [30].

In this chapter the neurogenic/gliogenic potential of the mammalian brain parenchyma *in vivo* will be analyzed with particular reference to variables involved in its heterogeneity (e.g., animal species, age, CNS regions; see Figure 1 and Table 1). In particular, these variables do determine the tissue environment in which stem/progenitor cells are immersed, what seems to be extremely important for their activity and outcome. In addition, the origin and nature of stem/progenitor cells would also contribute to their neurogenic/gliogenic potential. It is now well known that cells may have a broader potential than they normally exhibit in vivo when

**Figure 2.** Schematic summary of the features and location of different neurogenic/gliogenic processes occurring spontaneously in the CNS of postnatal and adult mammals. Red dots indicate newlyborn cells. SVZ, subventricular zone; SGZ, subgranular zone; EGL, external germinal layer; SPL, subpial layer (rabbit); PSA, PSA-NCAM; Map5, microtu‐ bule-associated protein 1B; P23, postnatal day 23. Question marks indicate lack of knowledge about the origin, late differentiative steps, and final integration of newly generated parenchymal neurons. Adapted from Ref [32].

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**Figure 2.** Schematic summary of the features and location of different neurogenic/gliogenic processes occurring spontaneously in the CNS of postnatal and adult mammals. Red dots indicate newlyborn cells. SVZ, subventricular zone; SGZ, subgranular zone; EGL, external germinal layer; SPL, subpial layer (rabbit); PSA, PSA-NCAM; Map5, microtu‐ bule-associated protein 1B; P23, postnatal day 23. Question marks indicate lack of knowledge about the origin, late differentiative steps, and final integration of newly generated parenchymal neurons. Adapted from Ref [32].

In this chapter the neurogenic/gliogenic potential of the mammalian brain parenchyma *in vivo* will be analyzed with particular reference to variables involved in its heterogeneity (e.g., animal species, age, CNS regions; see Figure 1 and Table 1). In particular, these variables do determine the tissue environment in which stem/progenitor cells are immersed, what seems to be extremely important for their activity and outcome. In addition, the origin and nature of stem/progenitor cells would also contribute to their neurogenic/gliogenic potential. It is now well known that cells may have a broader potential than they normally exhibit in vivo when

MMS, medial migratory stream; DMS, dorsal migratory stream. Adapted from Ref [30].

242 Neural Stem Cells - New Perspectives

**Figure 1.** Heterogeneity of postnatal ⁄ adult neurogenic processes in different mammals by considering different as‐ pects and mammalian species. B, Schematic summary of the main sources (progenitor cells) of adult mammalian neu‐ rogenesis, its outcome in vivo ⁄ in culture system, and its possible activation after lesion. In the case of many parenchymal regions, some of these steps are still obscure. BLBP, brain lipid-binding protein; EGL, external germinal layer; GABA, c-aminobutyric acid; Ng2, nerve ⁄ glial antigen 2 proteoglycan; NPY, Neuropeptide Y; SGZ, subgranular zone; SPL, subpial layer; SVZ, subventricular zone; VMM, ventral migratory mass; VMS, ventrocaudal migratory stream;

exposed to a different environment, either in vitro or in vivo [29]. Hence, in order to avoid one of the most common misunderstandings, namely the confusion between occurrence of *de novo* cell proliferation in the CNS tissue and existence of true gliogenic/neurogenic processes, here the attention will be focused on the outcome(s) of the newly generated progeny [30].

Since developmental changes also account for loss of CNS reparative/regenerative capacities and neuro-glio-genic potential, a paragraph will be devoted to the progenitor cell develop‐ mental origin. Then, a brief summary of comparative adult neurogenesis will be given. Evolutionary explanations can provide an understanding of the logic followed (or not) by neurogenic processes through phylogeny, also accounting for the failure in mammalian CNS repair/regeneration and scarce usefulness of adult neurogenesis as a possible solution for brain

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**2. Developmental origin of adult neurogenic/gliogenic processes**

What makes it possible the remarkable neurogenesis occurring in neurogenic sites is their direct origin from embryonic germinal layers which retain stem/progenitor cells along with the 'niche' environment allowing their activity [10,33]. The SVZ and SGZ actually are remnants of their embryonic counterpart, from which they maintain several cellular and molecular aspects [9] in parallel with an adaptation to the changing anatomy of the postnatal and adult

During development, the CNS originates from the neuroepithelium, pseudostratified epithe‐ lial cells that maintain contact with both the ventricular and pial surfaces. As brain thickness increases, neuroepitheial cells transform into radial glia [33,36]. Beside their classic role as scaffolding for migrating neurons during embryogenesis and their subsequent transformation into parenchymal astrocytes of the mature CNS [37,38], radial glia cells behave as stem cells, leading to the genesis of astrocytes, neurons [39,40], and, to a lesser extent, oligodendrocytes [41]. Thus radial glial cells not only serve as progenitors for many neurons and glial cells soon after birth, but also give rise to adult SVZ stem cells that continue to produce neurons throughout adult life [41]. The origin of astrocytes that function as neural progenitors in the adult hippocampus has not been determined experimentally. A connection to radial glial cells, has been suggested even in the hippocampal SGZ [42,43]. The relationship of adult NSCs to their developmental precursors offers clues to the unique characteristics that distinguish these germinal astrocytes from other astroglial cells in the brain parenchyma [33]. Indeed, paren‐ chymal astrocytes lose very early their stem cell potential (around postnatal day 10 in mice [44]), although they can still proliferate in the severe gliosis induced after lesion [45], and

On the other hand, gliogenesis persists throughout the CNS in the form of parenchymal cell genesis capable of creating new oligodendrocytes and, to a lesser extent, astrocytes, throughout life [12,15]. Most of this gliogenic activity is attributed to synantocytes/polydendrocytes (Ng2+ cells; see below) which are widespread in the CNS tissue and whose origin is still partially obscure. Oligodendrocytes originate from migratory and mitotic embryonic precursors which progressively mature into postmitotic myelin-producing cells. The sequential expression of developmental markers defines distinct phenotypic stages in the oligodendrocyte lineage, characterized by proliferative capacities, migratory abilities and changes in morphology. Most knowledge on this issue comes from studies on the rodent embryonic spinal cord. The first

repair [31,32].

brain [34,35].

resume multipotentiality *in vitro* [46].


#### **B. Main differences between cell genesis in adult neurogenic sites and in the parenchyma**


**Table 1.** Heterogeneity of adult neuro-glio-genesis

Since developmental changes also account for loss of CNS reparative/regenerative capacities and neuro-glio-genic potential, a paragraph will be devoted to the progenitor cell develop‐ mental origin. Then, a brief summary of comparative adult neurogenesis will be given. Evolutionary explanations can provide an understanding of the logic followed (or not) by neurogenic processes through phylogeny, also accounting for the failure in mammalian CNS repair/regeneration and scarce usefulness of adult neurogenesis as a possible solution for brain repair [31,32].

#### **2. Developmental origin of adult neurogenic/gliogenic processes**

exposed to a different environment, either in vitro or in vivo [29]. Hence, in order to avoid one of the most common misunderstandings, namely the confusion between occurrence of *de novo* cell proliferation in the CNS tissue and existence of true gliogenic/neurogenic processes, here the attention will be focused on the outcome(s) of the newly generated progeny [30].

**Animal species (animal world)** *General plasticity and persistent neurogenesis are usually reduced across phylogeny; in parallel, the reparative/regenerative potential is also reduced*

*extension of adult neurogenesis do exist among mammals*

*(postnatal/protracted neurogenesis) whereas others persist throughout life (persistent*

*neurogenesis). All neurogenic processes are progressively reduced with age*

*SGZ), whereas a niche has not been characterized in parenchymal neurogenesis*

*Neurogenic sites (SVZ, SGZ) directly derive from persistence and modification of preexisting, embryonic germinal layers, whereas for parenchymal cell genesis such direct*

*the two previous points; in parenchymal neurogenesis many variations are linked to*

*In repair: linked to the species; in invertebrates and non-mammalian vertebrates the physiological function is associated with function in repair, whereas in birds and mammals it is only linked to physiology/homeostasis of specific systems*

> *Mainly glial cells (some neurons)*

**Animal species (mammals)** *Unlike previous belief and current bias, remarkable differences in the location and*

**Age** *Some neurogenic processes are extensions of delayed developmental programs*

**Microenvironment (niche)** *A well defined neural stem cell niche sustains neurogenesis in neurogenic sites (SVZ,*

**Location in the CNS** *Location either within a germinal layer-derived niche or in the parenchyma redirects to*

*Neurogenic sites Parenchyma*

**Origin** *Germinal layer-derived No direct link with germinal layers*

*(some astrocytes and oligodendrocytes)*

*local cues of the different CNS regions involved* **Function** *In physiology: linked to the different ecological niches of the animals (present in all*

**B. Main differences between cell genesis in adult neurogenic sites and in the parenchyma**

**Location** *Restricted Widespread* **Primary progenitor cells** *Stem cells Progenitors* **Microenvironment** *Stem cell niche Mature neuropil*

**Fate (process)** *Complete Incomplete*

**A. Variables affecting the nature and features of adult neurogenesis**

*link is not clear*

*animals)*

**Fate (progeny)** *Mainly neurons*

**Table 1.** Heterogeneity of adult neuro-glio-genesis

**Origin of stem/progenitor**

244 Neural Stem Cells - New Perspectives

**cells**

What makes it possible the remarkable neurogenesis occurring in neurogenic sites is their direct origin from embryonic germinal layers which retain stem/progenitor cells along with the 'niche' environment allowing their activity [10,33]. The SVZ and SGZ actually are remnants of their embryonic counterpart, from which they maintain several cellular and molecular aspects [9] in parallel with an adaptation to the changing anatomy of the postnatal and adult brain [34,35].

During development, the CNS originates from the neuroepithelium, pseudostratified epithe‐ lial cells that maintain contact with both the ventricular and pial surfaces. As brain thickness increases, neuroepitheial cells transform into radial glia [33,36]. Beside their classic role as scaffolding for migrating neurons during embryogenesis and their subsequent transformation into parenchymal astrocytes of the mature CNS [37,38], radial glia cells behave as stem cells, leading to the genesis of astrocytes, neurons [39,40], and, to a lesser extent, oligodendrocytes [41]. Thus radial glial cells not only serve as progenitors for many neurons and glial cells soon after birth, but also give rise to adult SVZ stem cells that continue to produce neurons throughout adult life [41]. The origin of astrocytes that function as neural progenitors in the adult hippocampus has not been determined experimentally. A connection to radial glial cells, has been suggested even in the hippocampal SGZ [42,43]. The relationship of adult NSCs to their developmental precursors offers clues to the unique characteristics that distinguish these germinal astrocytes from other astroglial cells in the brain parenchyma [33]. Indeed, paren‐ chymal astrocytes lose very early their stem cell potential (around postnatal day 10 in mice [44]), although they can still proliferate in the severe gliosis induced after lesion [45], and resume multipotentiality *in vitro* [46].

On the other hand, gliogenesis persists throughout the CNS in the form of parenchymal cell genesis capable of creating new oligodendrocytes and, to a lesser extent, astrocytes, throughout life [12,15]. Most of this gliogenic activity is attributed to synantocytes/polydendrocytes (Ng2+ cells; see below) which are widespread in the CNS tissue and whose origin is still partially obscure. Oligodendrocytes originate from migratory and mitotic embryonic precursors which progressively mature into postmitotic myelin-producing cells. The sequential expression of developmental markers defines distinct phenotypic stages in the oligodendrocyte lineage, characterized by proliferative capacities, migratory abilities and changes in morphology. Most knowledge on this issue comes from studies on the rodent embryonic spinal cord. The first oligodendrocyte-committed cell appears at embryonic day 12 (E12) in two columns in the ventral ventricular zone of the motor neuron progenitor domain [47], which is defined by the expression of Olig2 [48]. The embryonic oligodendrocyte precursors are identified by their expression of platelet-derived growth factor alpha receptor (PDGFRα) [49]. The appearence of the oligodendrocyte lineage-associated markers Olig2 (essential for oligodendrocyte specification and differentiation) and PDGFRα (which permits the expansion of the original precursor population) is dependent on the concentrations of Sonic hedgehog (Shh) [50,51]. One or two days after their appearance, PDGFRα+ cells exit the ventricular zone and expand by local proliferation and migration first in the ventral spinal cord region and then dorsally [52]. Finally, they occupy the entire parenchyma by the time of birth [49]. A dorsal source of oligodendrocyte precursors was also shown to contribute to oligodendrogenesis in the spinal cord and hindbrain [53,54]. Fate mapping experiments revealed a double source of oligoden‐ drocyte precursors in the forebrain: cells expressing oligodendrocyte lineage markers, such as Olig1, Olig2, Sox10 and PDGFRα, first appear ventrally, in the neuroepithelium of the medial ganglionic eminence, and then migrate laterally and dorsally into all parts of the developing forebrain by E16 to birth [55]. However, several studies have provided evidence for a dorsal and later source of oligodendrocyte precursors in the lateral and/or caudal ganglionic emi‐ nence(s), which constitute a second wave of cells invading the cortex only by E18 [54,56]. Nevertheless, adult oligodendrocyte derive only by dorsal precursors, since medial ganglionic eminence-derived precursors were demonstrated to completely disappear after birth [56]. On the whole, it is thought that a unique oligodendrocyte population can derive from progenitor domains defined by different signaling molecules, in contrast to what has been established for neuronal specification during embryonic development, where different parts of the ventricular zone generate distinct types of neurons. In the rodent CNS, once PDGFRα+ cells have left the ventricular zone, they start to be termed 'oligodendrocyte progenitor cells' and acquire their most typical marker: an integral membrane chondroitin sulphate proteoglycan named Ng2 (nerve/glial antigen 2). Ng2 expression becomes detectable only at E14 [57], thus, from E17 to adulthood all PDGFRα+ cells are Ng2+, and, conversely, all the parenchymal (non-vascular) Ng2+ cells are PDGFRα+ [57,58]. Early embryonic Ng2+/PDGFαR+ OPCs are small, undiffer‐ entiated, proliferative and motile cells [59]. During embryogenesis, their morphology changes rapidly from a simple oval or polygonal cell body with few unbranched processes to a more differentiated and branched shape with a smaller cell soma [57,60].

consequence of the architecture of the developing parenchyma overlying the ventricular stem cell zone of the embryo [9]. This common pattern across animal species, along with data reported above on the origin of cycling glial progenitors in mammals, indirectly suggests that adult parenchymal neuro-glio-genesis ultimately derives from embryonic germinal layers, yet

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Unlike mammals, other classes of vertebrates including fish, amphibians, and reptiles, harbor a more widespread adult neurogenesis in the parenchyma. In these animals, stem and progenitor cells, in addition to their role in physiological plasticity, also participate in brain repair and regeneration. Failure in mammalian brain repair after traumatic, vascu‐ lar, and neurodegenerative injuries is due to: i) a strong reduction in the extension of neurogenic regions within the whole CNS; ii) a substantial lack of CNS reparative/regener‐ ative capacity; iii) the fact that adult neurogenic sites subserve specific physiological functions rather than brain repair; for review, see [11,62,63]. It is important to note that although the occurrence of good neurogenic potentials would generally favor brain repair (at least by making available stem/progenitor cells) there is not a direct, linear relation‐ ship between occurrence of stem/progenitor cells and repair/regeneration, the latter processes strongly depending on the tissue environment and/or tissue reactions; for selected

Neurogenic processes are detectable in wide regions of the CNS in invertebrates and nonmammalian vertebrates [61,65,66], whereas in mammals they are restricted to two privileged areas (neurogenic sites) and the remaining CNS is largely made up of non-renewable tissue [30,67,68]. The state of substantial 'general plasticity' and cell renewal existing in the oldest living metazoans, so that all cell types, including neurons, are balanced in their production and loss [69,70], is progressively reduced in vertebrates, although fish and amphibians still maintain remarkable regenerative capacities [71,72]. Then, in birds and mammals a transition between regeneration permissive and non-permissive stages occurs soon after birth, and highly-restricted spots of adult neurogenesis subserve homeostatic functions in specific neural circuits [73,74]. The decrease in neurogenic abilities occurs in parallel with topographical/ numerical restriction of germinal layer-derived stem cell niches, whereas the decrease in regenerative abilities occurs in parallel with other aspects: the impossibility to re-access to embryonic developmental programs during adulthood [75], the lack of differentiated cells capable of dedifferentiation [76], the development of a strong immune surveillance [77] and the consequent tissue reactions, most of which detrimental (reviewed in [11,64]). In some cases, the stem cells found in the CNS of non-mammalian vertebrates are deployed for postnatal development of parts of the brain until the final structure is reached. In other cases, postnatal neurogenesis continues into adulthood leading to a net increase of the number of neurons with age. Finally, in other cases, stem cells fuel neuronal turnover. An example is the protracted development of the cerebellar granular layer in mammals, which in adult teleosts actually

being able to persist independently in some cases.

examples of neurogenesis and regeneration see [64].

**3. Comparative adult neurogenesis and brain repair**

Coming back to adult neurogenesis, non mammalian vertebrates including fish, amphibians, and reptiles harbor a more widespread genesis of neurons in the parenchyma. Such processes, due to their location, are apparently independent from the primitive germinal layers. Never‐ theless, recent studies which analysed in more detail the origin of adult neurogenesis in fish show that all neurogenic processes likely originate from remnants of the germinal layers; reviewed in [61]. Teleost proliferation zones reflect a general proliferation pattern along the ventricular walls of the brain, distinctly localized in all its subdivisions along the rostrocaudal axis. Between 12 and 16 distinct proliferation zones have been recognized in different teleost species [61]. Hence, across different animal classes, most stem cell populations retain contact to the ventricular system, and they appear as neuroepithelial cells, radial glial or astroglial cell types. The different shapes of these progenitors have been suggested to be a secondary consequence of the architecture of the developing parenchyma overlying the ventricular stem cell zone of the embryo [9]. This common pattern across animal species, along with data reported above on the origin of cycling glial progenitors in mammals, indirectly suggests that adult parenchymal neuro-glio-genesis ultimately derives from embryonic germinal layers, yet being able to persist independently in some cases.

#### **3. Comparative adult neurogenesis and brain repair**

oligodendrocyte-committed cell appears at embryonic day 12 (E12) in two columns in the ventral ventricular zone of the motor neuron progenitor domain [47], which is defined by the expression of Olig2 [48]. The embryonic oligodendrocyte precursors are identified by their expression of platelet-derived growth factor alpha receptor (PDGFRα) [49]. The appearence of the oligodendrocyte lineage-associated markers Olig2 (essential for oligodendrocyte specification and differentiation) and PDGFRα (which permits the expansion of the original precursor population) is dependent on the concentrations of Sonic hedgehog (Shh) [50,51]. One or two days after their appearance, PDGFRα+ cells exit the ventricular zone and expand by local proliferation and migration first in the ventral spinal cord region and then dorsally [52]. Finally, they occupy the entire parenchyma by the time of birth [49]. A dorsal source of oligodendrocyte precursors was also shown to contribute to oligodendrogenesis in the spinal cord and hindbrain [53,54]. Fate mapping experiments revealed a double source of oligoden‐ drocyte precursors in the forebrain: cells expressing oligodendrocyte lineage markers, such as Olig1, Olig2, Sox10 and PDGFRα, first appear ventrally, in the neuroepithelium of the medial ganglionic eminence, and then migrate laterally and dorsally into all parts of the developing forebrain by E16 to birth [55]. However, several studies have provided evidence for a dorsal and later source of oligodendrocyte precursors in the lateral and/or caudal ganglionic emi‐ nence(s), which constitute a second wave of cells invading the cortex only by E18 [54,56]. Nevertheless, adult oligodendrocyte derive only by dorsal precursors, since medial ganglionic eminence-derived precursors were demonstrated to completely disappear after birth [56]. On the whole, it is thought that a unique oligodendrocyte population can derive from progenitor domains defined by different signaling molecules, in contrast to what has been established for neuronal specification during embryonic development, where different parts of the ventricular zone generate distinct types of neurons. In the rodent CNS, once PDGFRα+ cells have left the ventricular zone, they start to be termed 'oligodendrocyte progenitor cells' and acquire their most typical marker: an integral membrane chondroitin sulphate proteoglycan named Ng2 (nerve/glial antigen 2). Ng2 expression becomes detectable only at E14 [57], thus, from E17 to adulthood all PDGFRα+ cells are Ng2+, and, conversely, all the parenchymal (non-vascular) Ng2+ cells are PDGFRα+ [57,58]. Early embryonic Ng2+/PDGFαR+ OPCs are small, undiffer‐ entiated, proliferative and motile cells [59]. During embryogenesis, their morphology changes rapidly from a simple oval or polygonal cell body with few unbranched processes to a more

246 Neural Stem Cells - New Perspectives

differentiated and branched shape with a smaller cell soma [57,60].

Coming back to adult neurogenesis, non mammalian vertebrates including fish, amphibians, and reptiles harbor a more widespread genesis of neurons in the parenchyma. Such processes, due to their location, are apparently independent from the primitive germinal layers. Never‐ theless, recent studies which analysed in more detail the origin of adult neurogenesis in fish show that all neurogenic processes likely originate from remnants of the germinal layers; reviewed in [61]. Teleost proliferation zones reflect a general proliferation pattern along the ventricular walls of the brain, distinctly localized in all its subdivisions along the rostrocaudal axis. Between 12 and 16 distinct proliferation zones have been recognized in different teleost species [61]. Hence, across different animal classes, most stem cell populations retain contact to the ventricular system, and they appear as neuroepithelial cells, radial glial or astroglial cell types. The different shapes of these progenitors have been suggested to be a secondary

Unlike mammals, other classes of vertebrates including fish, amphibians, and reptiles, harbor a more widespread adult neurogenesis in the parenchyma. In these animals, stem and progenitor cells, in addition to their role in physiological plasticity, also participate in brain repair and regeneration. Failure in mammalian brain repair after traumatic, vascu‐ lar, and neurodegenerative injuries is due to: i) a strong reduction in the extension of neurogenic regions within the whole CNS; ii) a substantial lack of CNS reparative/regener‐ ative capacity; iii) the fact that adult neurogenic sites subserve specific physiological functions rather than brain repair; for review, see [11,62,63]. It is important to note that although the occurrence of good neurogenic potentials would generally favor brain repair (at least by making available stem/progenitor cells) there is not a direct, linear relation‐ ship between occurrence of stem/progenitor cells and repair/regeneration, the latter processes strongly depending on the tissue environment and/or tissue reactions; for selected examples of neurogenesis and regeneration see [64].

Neurogenic processes are detectable in wide regions of the CNS in invertebrates and nonmammalian vertebrates [61,65,66], whereas in mammals they are restricted to two privileged areas (neurogenic sites) and the remaining CNS is largely made up of non-renewable tissue [30,67,68]. The state of substantial 'general plasticity' and cell renewal existing in the oldest living metazoans, so that all cell types, including neurons, are balanced in their production and loss [69,70], is progressively reduced in vertebrates, although fish and amphibians still maintain remarkable regenerative capacities [71,72]. Then, in birds and mammals a transition between regeneration permissive and non-permissive stages occurs soon after birth, and highly-restricted spots of adult neurogenesis subserve homeostatic functions in specific neural circuits [73,74]. The decrease in neurogenic abilities occurs in parallel with topographical/ numerical restriction of germinal layer-derived stem cell niches, whereas the decrease in regenerative abilities occurs in parallel with other aspects: the impossibility to re-access to embryonic developmental programs during adulthood [75], the lack of differentiated cells capable of dedifferentiation [76], the development of a strong immune surveillance [77] and the consequent tissue reactions, most of which detrimental (reviewed in [11,64]). In some cases, the stem cells found in the CNS of non-mammalian vertebrates are deployed for postnatal development of parts of the brain until the final structure is reached. In other cases, postnatal neurogenesis continues into adulthood leading to a net increase of the number of neurons with age. Finally, in other cases, stem cells fuel neuronal turnover. An example is the protracted development of the cerebellar granular layer in mammals, which in adult teleosts actually becomes a persistent neurogenesis, where the granular layer continuously grows and no definite adult cerebellar size is reached [61].

on several variables (see Table 1). The main elements of heterogeneity can be summarized as follows: i) the location of progenitors (gathered within restricted neurogenic sites or widely spread out in the parenchyma); ii) the nature of the progenitors (*bona fide* NSCs versus different types of progenitors); iii) the genetic and molecular features of the progenitors (cell lineage: neuronal-like versus glial-like; identification of differentiative stages dependent on the available markers); iv) the existence or not of well characterized neurogenic niches (absence of niches or occurrence of atypical/non-identified niches in the parenchyma?); v) the extension in time after birth (protracted, transient persistent neurogenesis); vi) the ultimate fate of the progeny in terms of cell lineage (neuronal versus glial; astrocytic versus oligodendrocytic); vii) the ultimate fate of the progeny in terms of cell integration into circuits (complete versus incomplete neurogenesis); viii) the spontaneous occurrence of the process versus its injuryinduced appearance. This latter point could be considered a further step beyond the so-called 'constitutive' neurogenesis, namely the spontaneous, continuous genesis of new neurons as

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Due to the multifaceted aspects of the above mentioned processes, some problems of termi‐ nology can also be raised (see Refs. [30,32]). A common misunderstanding consists of a different use of the word 'neurogenesis', which can be intended either as 'genesis of neurons' or as 'genesis of neural cells', i.e. neurons and glia. Embryonic neurogenesis, namely the process of building up the whole CNS, involves both neuro- and glio-genesis, occurring in largely overlapping and strictly intermingled phases, whereas neurogenesis and gliogenesis can occur separately in the adult. The landscape is even more complex, since research on adult neurogenesis brought developmental neuroscience within the mature brain, and the intermix of structurally plastic changes involving cell genesis/differentiation with the fully assembled adult tissue is accompanied by a previously unexpected intermix of cell lineages (e.g., newly formed neuroblasts arising from astrocytic-like stem cells *in vivo*). For this reason, in this review article, when not speaking of well characterized cell lineages, the notion of 'cell genesis' instead of 'neurogenesis' will be used, since in most 'neurogenic' processes different cell types can be considered among the progenitors, and different progenies can be generated. Hence, apart from detailed knowledge gathered around the activity of SVZ and SGZ neurogenic sites, many aspects of parenchymal cell genesis remain obscure and/or unesplored, as a consequence of the heterogeneity depicted above. In the last few years, parenchymal neuro-glio-genesis was among the most studied, yet less known, issues, due to the widespread location of the progenitor cells and to the substantial lack of markers which specifically identify their real origin as well as the stage-specific steps of their differentiation. As a consequence, the presence/ absence of neurogenic processes within different CNS parenchymal regions in different mammalian species is still quite controversial and debatable. In most cases, parenchymal cell genesis occurs at low levels, at the limit of technical detection. Furthermore, in some cases it is very difficult to show its final outcome(s), most of the parenchymal neurogenesis appearing 'incomplete' as to the final differentiation/integration of the progeny [30] (Figure 2). Finally, to correctly classify both germinal layer-derived and parenchymal neurogenesis some other aspects should be taken into account, such as the temporal extension of 'protracted'/'transi‐ ent'developmental neurogenic processes with respect to a 'constitutive'/'persistent' neuro‐ genesis [30]. A further aspect is that of lesion-induced neuro-glio-genesis, namely the genesis

part of a physiologic, homeostatic process [85].

In addition, when considering mammals, the failure in CNS repair is a result of evolutionary constraints in which the injured tissue would not favor a strategy of regeneration but rather one of minimizing further damage (e.g., gliotic reaction [78]). Hence, as a consequence of multiple, converging aspects, CNS regenerative capacity in mammals could have reached a point of non-return, in parallel with the persistence of some neurogenic processes which remain mainly focused on physiological functions (e.g., cell renewal/addition in selective neural circuits linked to learning/memory tasks [73,74]).

An increased consciousness that the scarce reparative capacity of the mammalian CNS depends on multiple aspects should indicate that it is very unlike the finding of a single molecular factor or pharmacological treatment capable of eliciting repair/regeneration. Comparative results from vertebrate species of different classes have demonstrated that adult neurogenesis is widespread among vertebrates but is employed by different species in different functional contexts [74,79,80], and a growing number of reports show a remarkable heterogeneity even among mammals [17-19]. This variability concerns both the organization/ extension/function of the two neurogenic sites and many examples of parenchymal neuro‐ genesis; reviewed in [30] (see below). This fact, along with our still incomplete knowledge of adult neurogenesis in humans (especially within the parenchyma), partially hampers the reaching of well established 'common rules' which might be used in the translation of experimental preclinical data to human medicine. Thus, dealing with mammalian CNS structural plasticity, high levels of heterogeneity involving different 'types' of neurogenic processes should be taken into account.

#### **4. Heterogeneity of cell genesis in the mammalian CNS**

We now know that 'classic' neurogenic sites are consistently present in all mammals studied, although with some differences, particularly when the outcome(s) of the neurogenic process are involved [30]. The occurrence of a rostral migratory stream which is active throughout life in rodents but temporally restricted to the postnatal period in humans [81] is a prototypical example of variability among mammals. Indeed, in humans this neurogenic process seems to fall in a delayed developmental process rather than adult neurogenesis (see below).

In addition to neurogenic sites, studies carried out during the last two decades revealed the presence of local, parenchymal progenitors which retain some proliferative capacity in most of the mature mammalian CNS [12,14,15,17-19,82] (Figure 1). This fact suggests that structural plasticity involving *de novo* cell genesis in the CNS could be more widespread than previously thought. As a consequence of the increasing number of reports investigating adult neurogen‐ esis in mammals, our perception of this biological process has gained new perspectives and nuances; for deeper analysis see [30,66,83,84]. What was previously thought as "the genesis of new neurons in restricted brain areas endowed with NSCs", can now be intended as a highly heterogeneous phenomenon (summarized in Figures 1 and 2), whose heterogeneity depends on several variables (see Table 1). The main elements of heterogeneity can be summarized as follows: i) the location of progenitors (gathered within restricted neurogenic sites or widely spread out in the parenchyma); ii) the nature of the progenitors (*bona fide* NSCs versus different types of progenitors); iii) the genetic and molecular features of the progenitors (cell lineage: neuronal-like versus glial-like; identification of differentiative stages dependent on the available markers); iv) the existence or not of well characterized neurogenic niches (absence of niches or occurrence of atypical/non-identified niches in the parenchyma?); v) the extension in time after birth (protracted, transient persistent neurogenesis); vi) the ultimate fate of the progeny in terms of cell lineage (neuronal versus glial; astrocytic versus oligodendrocytic); vii) the ultimate fate of the progeny in terms of cell integration into circuits (complete versus incomplete neurogenesis); viii) the spontaneous occurrence of the process versus its injuryinduced appearance. This latter point could be considered a further step beyond the so-called 'constitutive' neurogenesis, namely the spontaneous, continuous genesis of new neurons as part of a physiologic, homeostatic process [85].

becomes a persistent neurogenesis, where the granular layer continuously grows and no

In addition, when considering mammals, the failure in CNS repair is a result of evolutionary constraints in which the injured tissue would not favor a strategy of regeneration but rather one of minimizing further damage (e.g., gliotic reaction [78]). Hence, as a consequence of multiple, converging aspects, CNS regenerative capacity in mammals could have reached a point of non-return, in parallel with the persistence of some neurogenic processes which remain mainly focused on physiological functions (e.g., cell renewal/addition in selective

An increased consciousness that the scarce reparative capacity of the mammalian CNS depends on multiple aspects should indicate that it is very unlike the finding of a single molecular factor or pharmacological treatment capable of eliciting repair/regeneration. Comparative results from vertebrate species of different classes have demonstrated that adult neurogenesis is widespread among vertebrates but is employed by different species in different functional contexts [74,79,80], and a growing number of reports show a remarkable heterogeneity even among mammals [17-19]. This variability concerns both the organization/ extension/function of the two neurogenic sites and many examples of parenchymal neuro‐ genesis; reviewed in [30] (see below). This fact, along with our still incomplete knowledge of adult neurogenesis in humans (especially within the parenchyma), partially hampers the reaching of well established 'common rules' which might be used in the translation of experimental preclinical data to human medicine. Thus, dealing with mammalian CNS structural plasticity, high levels of heterogeneity involving different 'types' of neurogenic

We now know that 'classic' neurogenic sites are consistently present in all mammals studied, although with some differences, particularly when the outcome(s) of the neurogenic process are involved [30]. The occurrence of a rostral migratory stream which is active throughout life in rodents but temporally restricted to the postnatal period in humans [81] is a prototypical example of variability among mammals. Indeed, in humans this neurogenic process seems to

In addition to neurogenic sites, studies carried out during the last two decades revealed the presence of local, parenchymal progenitors which retain some proliferative capacity in most of the mature mammalian CNS [12,14,15,17-19,82] (Figure 1). This fact suggests that structural plasticity involving *de novo* cell genesis in the CNS could be more widespread than previously thought. As a consequence of the increasing number of reports investigating adult neurogen‐ esis in mammals, our perception of this biological process has gained new perspectives and nuances; for deeper analysis see [30,66,83,84]. What was previously thought as "the genesis of new neurons in restricted brain areas endowed with NSCs", can now be intended as a highly heterogeneous phenomenon (summarized in Figures 1 and 2), whose heterogeneity depends

fall in a delayed developmental process rather than adult neurogenesis (see below).

definite adult cerebellar size is reached [61].

248 Neural Stem Cells - New Perspectives

processes should be taken into account.

**4. Heterogeneity of cell genesis in the mammalian CNS**

neural circuits linked to learning/memory tasks [73,74]).

Due to the multifaceted aspects of the above mentioned processes, some problems of termi‐ nology can also be raised (see Refs. [30,32]). A common misunderstanding consists of a different use of the word 'neurogenesis', which can be intended either as 'genesis of neurons' or as 'genesis of neural cells', i.e. neurons and glia. Embryonic neurogenesis, namely the process of building up the whole CNS, involves both neuro- and glio-genesis, occurring in largely overlapping and strictly intermingled phases, whereas neurogenesis and gliogenesis can occur separately in the adult. The landscape is even more complex, since research on adult neurogenesis brought developmental neuroscience within the mature brain, and the intermix of structurally plastic changes involving cell genesis/differentiation with the fully assembled adult tissue is accompanied by a previously unexpected intermix of cell lineages (e.g., newly formed neuroblasts arising from astrocytic-like stem cells *in vivo*). For this reason, in this review article, when not speaking of well characterized cell lineages, the notion of 'cell genesis' instead of 'neurogenesis' will be used, since in most 'neurogenic' processes different cell types can be considered among the progenitors, and different progenies can be generated. Hence, apart from detailed knowledge gathered around the activity of SVZ and SGZ neurogenic sites, many aspects of parenchymal cell genesis remain obscure and/or unesplored, as a consequence of the heterogeneity depicted above. In the last few years, parenchymal neuro-glio-genesis was among the most studied, yet less known, issues, due to the widespread location of the progenitor cells and to the substantial lack of markers which specifically identify their real origin as well as the stage-specific steps of their differentiation. As a consequence, the presence/ absence of neurogenic processes within different CNS parenchymal regions in different mammalian species is still quite controversial and debatable. In most cases, parenchymal cell genesis occurs at low levels, at the limit of technical detection. Furthermore, in some cases it is very difficult to show its final outcome(s), most of the parenchymal neurogenesis appearing 'incomplete' as to the final differentiation/integration of the progeny [30] (Figure 2). Finally, to correctly classify both germinal layer-derived and parenchymal neurogenesis some other aspects should be taken into account, such as the temporal extension of 'protracted'/'transi‐ ent'developmental neurogenic processes with respect to a 'constitutive'/'persistent' neuro‐ genesis [30]. A further aspect is that of lesion-induced neuro-glio-genesis, namely the genesis of new cells as a consequence of different types of CNS injury [18,25,26,86] or altered homeo‐ stasis [87]. This is an important point since many lines of research in the field of neural repair directed to manipulate stem cells in the perspective of intracerebral transplantation did not produced substantial therapeutic innovations. As an alternative, another approach might be that of stimulating/modulating the endogenous sources of cell progenitors present both in germinal layer-derived stem cell niches (SVZ and hippocampus) and in the parenchyma.

independently from the remnants of germinal layers, yet their final outcome and their role in

Parenchymal Neuro-Glio-Genesis Versus Germinal Layer-Derived Neurogenesis: Two Faces of …

http://dx.doi.org/10.5772/56100

251

The heterogeneity in parenchymal neurogenesis adds to that described for neurogenic processes occurring in adult neurogenic sites, which have been related to adaptation to ecological pressures [80]. At present, this is one of the most satisfactory functional explanations for adult neurogenesis in the entire phylogenetic tree, along with multiple, geneticallydetermined variables spanning from the brain anatomy/developmental history to the animal lifespan [93]. This range of possibilities can also be increased by non-genetic variables, such as

Among the unsolved issues of parenchymal neurogenesis are the numerous reports which have not been confirmed by further studies or by other laboratories [22,23,26,94-96], along with a series of data which have been denied in studies trying to reproduce the same results [24,97-99]. Without entering in the scientific and technical discussion about these controversies, it is evident that we still not grasp the real limits of parenchymal neurogenesis and that further studies are required before finally accept or deny the existence of some neurogenic processes.

A case placed in between the spontaneous and experimentally-induced neurogenesis, is that of the hypothalamus. Several publications based on experiments carried out on rodents have been reporting data on this brain region as a new site for adult constitutive neurogenesis in mammals (for review see [100]). Under physiological conditions, both in rats [101] and mice [102,103], proliferative activity does occur in the ependymal layer of the third ventricle and within the surrounding parenchyma. In rats, Xu and collaborators using electron microscopy and immunohistochemistry showed that tanycytes lining the 3rd ventricle proliferate and express molecules usually found in glial, stem-like progenitor cells, such as BLBP and nestin. The presence of putative neural progenitors was further supported by the isolation of cells able to give rise to neurospheres from the hypothalamus. One month after BrdU injection, proliferating cells, some of which expressing Hu protein, were detected in the surrounding parenchyma. Similar results were obtained in mice [102], yet in both rodent species no clear evidence has supported constitutive and complete hypothalamic adult neurogenesis under physiological conditions. A significant increase in hypothalamic proliferating cells can be obtained by performing i.v. delivery of BrdU (350% more positive nuclei, in comparison to i.p. treated animals), nevertheless, in spite of such cell proliferation the level of neurogenesis in the intact hypothalamus seems to be arrested at a very premature stage. On the other hand, growth factor infusion [82,101,104] or certain experimental conditions/models, such as prolonged heat exposure [105] and the mutant mice investigated by Pierce and Xu (2010), seem to increase neurogenesis in the hypothalamus. Intracerebroventricular infusion of insulin growth factor I in rats [104] triggered an intense proliferation along the 3rd periventricular area and in the parenchyma of the caudal hypothalamus. As concerns the genesis of new neurons, after i.v. treatment with bFGF in rats [101], and CNTF in mice [82], it was shown that prolif‐ eration induced by growth factors can be followed by genesis of newborn neurons. Detailed morphological and molecular analyses of the 3rd periventricular region of these animals showed interesting architectural similarities with the SVZ neurogenic niche (e.g., proliferating astroglial cells contacting the ventricle by an apical process bearing a single cilium), with

the adult neural circuits remains obscure; reviewed in [30].

experience-dependent cues [79,80].

#### **5. Parenchymal neurogenesis**

Spontaneous (constitutive) parenchymal neurogenesis can be considered as a very rare phenomenon in mammals, and its regional location has been shown to be dependent on the animal species, age, and physiological⁄pathological states [30]. Different examples of neuro‐ genesis occurring outside the two neurogenic sites have been described in rodents [17,82], rabbits [18,19] and monkeys [22,88]. Remarkable differences can be observed between closely related orders (e.g., rodents and lagomorphs [18,19]), between species (e.g., rat and mouse [17,23,89,90]), and even different strains [91,92].

Most parenchymal neurogenesis described in adult rodents seems to occur spontaneously at very low levels, rather being elicited/enhanced after specific physiological or pathological conditions [17,82,86,87] (see below). Dayer and colleagues [17] showed the occurrence of new neurons in the deep layers of the rat cerebral cortex. By labelling newlyborn cells with multiple intra-peritoneal injections of BrdU and using markers of both immature and mature neurons to characterize the new cells through a detailed confocal analysis at different survival times, they demonstrated genesis of new GABAergic interneurons in both neocortex and striatum. At 4-5 weeks survival time, the 0.4 +/- 0.13% of the BrdU+ cells were mature NeuN+ neurons in the neocortex. Morphologic and phenotypic analyses assert these cells belong to different categories of cortical interneurons. Interestingly, although several BrdU+/DCX+/Tuc4+ neuroblasts were identified close to the SVZ periventricular region, the great majority of cortical BrdU+ cells were positive for Ng2. From these data the Authors suggested that adult cortical newborn interneurons might originate from *in situ* progenitors. Other examples of spontaneous parenchymal neurogenesis have been described in lagomorphs. In rabbits, newly generated neurons are spontaneously produced in other regions of the adult brain starting from local, parenchymal progenitors. In the caudate nucleus, newly formed neuroblasts form longitudinally-arranged, doublecortin (DCX) and PSA-NCAM immunoreactive striatal chains similar to the SVZ chains [18]. These neuroblasts are generated from clusters of proliferating cells which express the astroglial marker brain lipid binding protein (BLBP), and about 1/6 of surviving cells differentiate into calretinin striatal interneurons. Always in rabbits, in sharp contrast with our common knowledge concerning the CNS of other mammals studied so far, a remarkable genesis of cells is detectable in the peripuberal, and to a lesser extent, adult cerebellar cortex [19]. Systemically-administered BrdU detected at different post-injection survival times (up to two months) reveals newly generated PSA-NCAM+/DCX+/Pax2+ interneurons of neuroepithelial origin homogeneously distributed in the cerebellar cortex. Thus, in the striatal and cerebellar parenchyma of lagomorphs new neurons are generated independently from the remnants of germinal layers, yet their final outcome and their role in the adult neural circuits remains obscure; reviewed in [30].

of new cells as a consequence of different types of CNS injury [18,25,26,86] or altered homeo‐ stasis [87]. This is an important point since many lines of research in the field of neural repair directed to manipulate stem cells in the perspective of intracerebral transplantation did not produced substantial therapeutic innovations. As an alternative, another approach might be that of stimulating/modulating the endogenous sources of cell progenitors present both in germinal layer-derived stem cell niches (SVZ and hippocampus) and in the parenchyma.

Spontaneous (constitutive) parenchymal neurogenesis can be considered as a very rare phenomenon in mammals, and its regional location has been shown to be dependent on the animal species, age, and physiological⁄pathological states [30]. Different examples of neuro‐ genesis occurring outside the two neurogenic sites have been described in rodents [17,82], rabbits [18,19] and monkeys [22,88]. Remarkable differences can be observed between closely related orders (e.g., rodents and lagomorphs [18,19]), between species (e.g., rat and mouse

Most parenchymal neurogenesis described in adult rodents seems to occur spontaneously at very low levels, rather being elicited/enhanced after specific physiological or pathological conditions [17,82,86,87] (see below). Dayer and colleagues [17] showed the occurrence of new neurons in the deep layers of the rat cerebral cortex. By labelling newlyborn cells with multiple intra-peritoneal injections of BrdU and using markers of both immature and mature neurons to characterize the new cells through a detailed confocal analysis at different survival times, they demonstrated genesis of new GABAergic interneurons in both neocortex and striatum. At 4-5 weeks survival time, the 0.4 +/- 0.13% of the BrdU+ cells were mature NeuN+ neurons in the neocortex. Morphologic and phenotypic analyses assert these cells belong to different categories of cortical interneurons. Interestingly, although several BrdU+/DCX+/Tuc4+ neuroblasts were identified close to the SVZ periventricular region, the great majority of cortical BrdU+ cells were positive for Ng2. From these data the Authors suggested that adult cortical newborn interneurons might originate from *in situ* progenitors. Other examples of spontaneous parenchymal neurogenesis have been described in lagomorphs. In rabbits, newly generated neurons are spontaneously produced in other regions of the adult brain starting from local, parenchymal progenitors. In the caudate nucleus, newly formed neuroblasts form longitudinally-arranged, doublecortin (DCX) and PSA-NCAM immunoreactive striatal chains similar to the SVZ chains [18]. These neuroblasts are generated from clusters of proliferating cells which express the astroglial marker brain lipid binding protein (BLBP), and about 1/6 of surviving cells differentiate into calretinin striatal interneurons. Always in rabbits, in sharp contrast with our common knowledge concerning the CNS of other mammals studied so far, a remarkable genesis of cells is detectable in the peripuberal, and to a lesser extent, adult cerebellar cortex [19]. Systemically-administered BrdU detected at different post-injection survival times (up to two months) reveals newly generated PSA-NCAM+/DCX+/Pax2+ interneurons of neuroepithelial origin homogeneously distributed in the cerebellar cortex. Thus, in the striatal and cerebellar parenchyma of lagomorphs new neurons are generated

**5. Parenchymal neurogenesis**

250 Neural Stem Cells - New Perspectives

[17,23,89,90]), and even different strains [91,92].

The heterogeneity in parenchymal neurogenesis adds to that described for neurogenic processes occurring in adult neurogenic sites, which have been related to adaptation to ecological pressures [80]. At present, this is one of the most satisfactory functional explanations for adult neurogenesis in the entire phylogenetic tree, along with multiple, geneticallydetermined variables spanning from the brain anatomy/developmental history to the animal lifespan [93]. This range of possibilities can also be increased by non-genetic variables, such as experience-dependent cues [79,80].

Among the unsolved issues of parenchymal neurogenesis are the numerous reports which have not been confirmed by further studies or by other laboratories [22,23,26,94-96], along with a series of data which have been denied in studies trying to reproduce the same results [24,97-99]. Without entering in the scientific and technical discussion about these controversies, it is evident that we still not grasp the real limits of parenchymal neurogenesis and that further studies are required before finally accept or deny the existence of some neurogenic processes.

A case placed in between the spontaneous and experimentally-induced neurogenesis, is that of the hypothalamus. Several publications based on experiments carried out on rodents have been reporting data on this brain region as a new site for adult constitutive neurogenesis in mammals (for review see [100]). Under physiological conditions, both in rats [101] and mice [102,103], proliferative activity does occur in the ependymal layer of the third ventricle and within the surrounding parenchyma. In rats, Xu and collaborators using electron microscopy and immunohistochemistry showed that tanycytes lining the 3rd ventricle proliferate and express molecules usually found in glial, stem-like progenitor cells, such as BLBP and nestin. The presence of putative neural progenitors was further supported by the isolation of cells able to give rise to neurospheres from the hypothalamus. One month after BrdU injection, proliferating cells, some of which expressing Hu protein, were detected in the surrounding parenchyma. Similar results were obtained in mice [102], yet in both rodent species no clear evidence has supported constitutive and complete hypothalamic adult neurogenesis under physiological conditions. A significant increase in hypothalamic proliferating cells can be obtained by performing i.v. delivery of BrdU (350% more positive nuclei, in comparison to i.p. treated animals), nevertheless, in spite of such cell proliferation the level of neurogenesis in the intact hypothalamus seems to be arrested at a very premature stage. On the other hand, growth factor infusion [82,101,104] or certain experimental conditions/models, such as prolonged heat exposure [105] and the mutant mice investigated by Pierce and Xu (2010), seem to increase neurogenesis in the hypothalamus. Intracerebroventricular infusion of insulin growth factor I in rats [104] triggered an intense proliferation along the 3rd periventricular area and in the parenchyma of the caudal hypothalamus. As concerns the genesis of new neurons, after i.v. treatment with bFGF in rats [101], and CNTF in mice [82], it was shown that prolif‐ eration induced by growth factors can be followed by genesis of newborn neurons. Detailed morphological and molecular analyses of the 3rd periventricular region of these animals showed interesting architectural similarities with the SVZ neurogenic niche (e.g., proliferating astroglial cells contacting the ventricle by an apical process bearing a single cilium), with tanycytes as primary proliferating elements lining the 3rd ventricle [104]. Yet, additional studies are necessary to clearly demonstrate/confirm whether hypothalamic newborn neurons generated after physiological/pathological stimulation actually become part of the pre-existing circuits playing a role in energy-balance mechanisms.

Initial studies in both cortex and striatum reported that a tiny fraction of lesion-induced neurons may differentiate into projection neurons, suggesting that endogenous neuronal progenitors may have the potential to replace degenerated neurons [23,25,115]. However, these results have not been confirmed by others [28,116]. Moreover, it is now clear that most of the lesion-induced neurons have a transient existence and, at least in the striatum, they do not express markers of projection neurons nor transcription factors involved in their specification [28,116]. Several attempts have been made to increase the survival of these cells, with little success [117]. An intriguing possibility to be explored is that lesion-induced neuroblasts occurring in multiple forms of brain injury are committed to transient neuronal types, which contribute to restorative rather than replacement mechanisms [28,63]. This idea is further supported by data showing that a transient existence often characterizes also cortical and

Parenchymal Neuro-Glio-Genesis Versus Germinal Layer-Derived Neurogenesis: Two Faces of …

http://dx.doi.org/10.5772/56100

253

Neuronal progenitors in the SVZ and SGZ have been shown to respond to injuries by strongly increasing their proliferation, in the SVZ, also migrating towards damaged regions [25,115]. In parallel, recent reports have showed that in the degenerated neocortex and striatum, new neurons can also be produced locally from parenchymal neuronal progenitors [28,83,113]. In the neocortex, Ohira et al. [86] showed that mild ischemia might stimulate the generation of newborn GABAergic interneurons from progenitors residing in cortical layer I. These cells were not quiescent in normal conditions as they expressed the endogeneous marker of cell proliferation Ki67 and they could be labeled with retroviral vectors. Ohira and coworkers could not define the exact nature of the parenchymal progenitors, which, intriguingly, are very close to the leptomeninges, from which neuronal progenitors have been recently isolated [118,119].

More specific lineage tracing study will be necessary to confirm the real origin of neural progenitors activated after lesion. Lineage tracing has shown that reactive astrocytes isolated from the adult neocortex can give rise to neurospheres *in vitro* [46,120]. To date, the only *in vivo* evidence that neocortical astrocytes can be neurogenic has been obtained in early postnatal mice after hypoxia/Ischemia [113]. A recent study showed that even if the neural stem cells derived from adult neocortical astrocytes maintain the capacity for self-renew when transplanted in the SVZ, they were still unable to produce neurons [121]. This observation casted some doubts over the actual role of these cells as neuronal progenitors *in vivo*. None‐ theless, this result may only indicate that the neurogenic potential of cortical and SVZ

Another example of the *in vivo* genesis of new neurons within the lesioned brain parenchyma has been obtained in the striatum in a mice model of progressive striatal degeneration, the Creb1CamkcreCrem-/- mutant mice (CBCM) [28,122]. In this model the SVZ acts as a source of postmitotic neuroblasts that enter the striatum from a specific subcallosal migratory stream, as individual elements. Luzzati and coworkers [28] showed that the striatum of CBCM mice contains also tightly clustered neuroblasts which originate locally from parenchymal prolif‐ erating progenitors. These cells showed features of intermediate neuronal progenitors of the SVZ and SGZ such as clustering and co-expression of glial (Sox2, Sox9, BLBP) and neuronal markers (Dlx, Sp8, DCX), and the expression of the EGFr [123-126]. This study clearly shows that the mature parenchyma can be permissive to neuronal genesis, although Luzzati and

striatal neurons generated in normal conditions [18,89].

progenitors rely on distinct factors.

Taking into account the multifaceted aspects dealing with parenchymal neurogenesis, difficulties encountered in such type of research are not only technical. They are also linked to the occurrence of processes placed in the middle between two well characterized extremes of structural plasticity, such as synaptic plasticity, and 'complete' adult neurogenesis. In a recent review article [30] five levels have been dissected in the neurogenic processes in order to critically evaluate/compare different parenchymal neurogenic events (see also Figure 2). The subsequent steps span from cell division to possible integration of specified/differentiated elements into the CNS tissue, and according to this view, only when any of the five steps are filled the neurogenic process should be classified as 'complete'. As a result, all the parenchymal neurogenic processes described until now can actually be considered as incomplete. This could explain why many claims of neurogenic processes were subsequently refuted because not sustained by experimental evidence. The piriform cortex is one of those regions in which results reported by different researchers are quite controversial; see for example [88,106-108]. Since long time, this cortical region is known to harbor a population of neurons immunoreactive for PSA-NCAM and DCX [108-110], which are two markers highly expressed in newly generated neurons but also present in non newly generated cells [110]. Indeed, deeper investigations have shown that the piriform cortex contains a population of immature, non-newly generated neurons which display very few (or no) synapses and are frequently ensheathed by glial lamellae [108]. These cells, by remaining in an immature state for indeterminate time, can represent a 'reservoir' of neurons that could possibly be recruited into the preexisting neural circuits although not generated *ex novo* [111].

In conclusion, alternative and multiple forms of plasticity involving neurons can overlap within the so-called non-neurogenic tissue, affecting preexisting cells/circuits and increasing the complexity of the whole picture of brain structural remodeling.

#### **6. Lesion-induced (reactive) neurogenesis**

Brain lesions have been shown to stimulate neurogenesis in normally non-neurogenic regions such as the neocortex and the striatum. In the neocortex these responses are limited to specific conditions such as targeted apoptosis or mild ischemia [23,86,112,113]. By contrast, several lesion paradigms, associated to both strong or mild degeneration and inflammation, have been shown to induce neurogenesis in the striatum [25,28,114]. It is unknown if lesioned neocortex and striatum have distinct needs for immature neurons or if the neocortical tissue response is more detrimental for neurogenesis. This fundamental point reveals our very poor knowledge of lesion-induced neurogenesis. Indeed, despite an intense research, we have only little information regarding the nature, fate and potential of the progenitors stimulated by brain lesions, the mechanisms that trigger their activation and eventually their functional role.

Initial studies in both cortex and striatum reported that a tiny fraction of lesion-induced neurons may differentiate into projection neurons, suggesting that endogenous neuronal progenitors may have the potential to replace degenerated neurons [23,25,115]. However, these results have not been confirmed by others [28,116]. Moreover, it is now clear that most of the lesion-induced neurons have a transient existence and, at least in the striatum, they do not express markers of projection neurons nor transcription factors involved in their specification [28,116]. Several attempts have been made to increase the survival of these cells, with little success [117]. An intriguing possibility to be explored is that lesion-induced neuroblasts occurring in multiple forms of brain injury are committed to transient neuronal types, which contribute to restorative rather than replacement mechanisms [28,63]. This idea is further supported by data showing that a transient existence often characterizes also cortical and striatal neurons generated in normal conditions [18,89].

tanycytes as primary proliferating elements lining the 3rd ventricle [104]. Yet, additional studies are necessary to clearly demonstrate/confirm whether hypothalamic newborn neurons generated after physiological/pathological stimulation actually become part of the pre-existing

Taking into account the multifaceted aspects dealing with parenchymal neurogenesis, difficulties encountered in such type of research are not only technical. They are also linked to the occurrence of processes placed in the middle between two well characterized extremes of structural plasticity, such as synaptic plasticity, and 'complete' adult neurogenesis. In a recent review article [30] five levels have been dissected in the neurogenic processes in order to critically evaluate/compare different parenchymal neurogenic events (see also Figure 2). The subsequent steps span from cell division to possible integration of specified/differentiated elements into the CNS tissue, and according to this view, only when any of the five steps are filled the neurogenic process should be classified as 'complete'. As a result, all the parenchymal neurogenic processes described until now can actually be considered as incomplete. This could explain why many claims of neurogenic processes were subsequently refuted because not sustained by experimental evidence. The piriform cortex is one of those regions in which results reported by different researchers are quite controversial; see for example [88,106-108]. Since long time, this cortical region is known to harbor a population of neurons immunoreactive for PSA-NCAM and DCX [108-110], which are two markers highly expressed in newly generated neurons but also present in non newly generated cells [110]. Indeed, deeper investigations have shown that the piriform cortex contains a population of immature, non-newly generated neurons which display very few (or no) synapses and are frequently ensheathed by glial lamellae [108]. These cells, by remaining in an immature state for indeterminate time, can represent a 'reservoir' of neurons that could possibly be recruited into the preexisting neural

In conclusion, alternative and multiple forms of plasticity involving neurons can overlap within the so-called non-neurogenic tissue, affecting preexisting cells/circuits and increasing

Brain lesions have been shown to stimulate neurogenesis in normally non-neurogenic regions such as the neocortex and the striatum. In the neocortex these responses are limited to specific conditions such as targeted apoptosis or mild ischemia [23,86,112,113]. By contrast, several lesion paradigms, associated to both strong or mild degeneration and inflammation, have been shown to induce neurogenesis in the striatum [25,28,114]. It is unknown if lesioned neocortex and striatum have distinct needs for immature neurons or if the neocortical tissue response is more detrimental for neurogenesis. This fundamental point reveals our very poor knowledge of lesion-induced neurogenesis. Indeed, despite an intense research, we have only little information regarding the nature, fate and potential of the progenitors stimulated by brain lesions, the mechanisms that trigger their activation and eventually their functional role.

circuits playing a role in energy-balance mechanisms.

252 Neural Stem Cells - New Perspectives

circuits although not generated *ex novo* [111].

**6. Lesion-induced (reactive) neurogenesis**

the complexity of the whole picture of brain structural remodeling.

Neuronal progenitors in the SVZ and SGZ have been shown to respond to injuries by strongly increasing their proliferation, in the SVZ, also migrating towards damaged regions [25,115]. In parallel, recent reports have showed that in the degenerated neocortex and striatum, new neurons can also be produced locally from parenchymal neuronal progenitors [28,83,113]. In the neocortex, Ohira et al. [86] showed that mild ischemia might stimulate the generation of newborn GABAergic interneurons from progenitors residing in cortical layer I. These cells were not quiescent in normal conditions as they expressed the endogeneous marker of cell proliferation Ki67 and they could be labeled with retroviral vectors. Ohira and coworkers could not define the exact nature of the parenchymal progenitors, which, intriguingly, are very close to the leptomeninges, from which neuronal progenitors have been recently isolated [118,119].

More specific lineage tracing study will be necessary to confirm the real origin of neural progenitors activated after lesion. Lineage tracing has shown that reactive astrocytes isolated from the adult neocortex can give rise to neurospheres *in vitro* [46,120]. To date, the only *in vivo* evidence that neocortical astrocytes can be neurogenic has been obtained in early postnatal mice after hypoxia/Ischemia [113]. A recent study showed that even if the neural stem cells derived from adult neocortical astrocytes maintain the capacity for self-renew when transplanted in the SVZ, they were still unable to produce neurons [121]. This observation casted some doubts over the actual role of these cells as neuronal progenitors *in vivo*. None‐ theless, this result may only indicate that the neurogenic potential of cortical and SVZ progenitors rely on distinct factors.

Another example of the *in vivo* genesis of new neurons within the lesioned brain parenchyma has been obtained in the striatum in a mice model of progressive striatal degeneration, the Creb1CamkcreCrem-/- mutant mice (CBCM) [28,122]. In this model the SVZ acts as a source of postmitotic neuroblasts that enter the striatum from a specific subcallosal migratory stream, as individual elements. Luzzati and coworkers [28] showed that the striatum of CBCM mice contains also tightly clustered neuroblasts which originate locally from parenchymal prolif‐ erating progenitors. These cells showed features of intermediate neuronal progenitors of the SVZ and SGZ such as clustering and co-expression of glial (Sox2, Sox9, BLBP) and neuronal markers (Dlx, Sp8, DCX), and the expression of the EGFr [123-126]. This study clearly shows that the mature parenchyma can be permissive to neuronal genesis, although Luzzati and coworkers could not trace the origin of the observed striatal parenchymal neuronal progeni‐ tors. Nonetheless, two possibilities can be considered: i) striatal neuronal progenitors could derive from the displacement of primary/intermediate progenitors from the SVZ; ii) they could represent local cells becoming neurogenic in response to neurodegeneration.

drocyte antigens. Some observations provide circumstantial evidences for the oligodendro‐ glial fate of polydendrocytes *in vivo*. For instance, they co-express the PDGFRα, and during the first postnatal week, in the corpus callosum and cortex, they start expressing the immature oligodendrocyte antigen O4 [133]. Polydendrocytes also express the basic helix-loop-helix (bHLH) transcription factors Olig1 and Olig2, which are required for oligodendrocyte specification and differentiation [132,134] as well as Sox9 and Sox10 transcription factors. Moreover, pulse-chase labelling of proliferating cells using 5-bromo-2′-deoxyuridine (BrdU) revealed that the number of BrdU+Ng2+ cells decreases while that of BrdU+ oligodendrocytes increases over time [12,135]. Cell grafting experiments have shown that polydendrocytes give rise to myelinating cells when they are transplanted into an environment free of endogenous myelinating cells [136]. Recently, more direct evidence for the oligodendroglial fate of polydendrocytes was obtained from cell fate-mapping experiments using transgenic mice that express Cre recombinase (Cre) in Ng2-expressing cells or that express inducible Cre (CreeR), under the regulation of the Cspg4, PDGFRα or Olig2 genes, which enable determination of the fate of polydendrocytes at a given time during development [95,137,138]. These studies

Parenchymal Neuro-Glio-Genesis Versus Germinal Layer-Derived Neurogenesis: Two Faces of …

http://dx.doi.org/10.5772/56100

255

showed that oligodendrocytes continue to be generated in the mature brain.

astroglial fate in the normal *in vivo* environment [128].

of polydendrocytes (Crociara et al., in preparation; Figure 2).

**8. Conclusion and future perspectives**

Early cell-culture studies showed that OPCs purified from rat optic nerves differentiate not only into oligodendrocytes but also into process-bearing 'type-2 astrocytes' in the presence of serum factors, which led to the concept of bipotential oligodendrocyte type-2-astrocyte (O-2A) progenitor cells [139]. There are now controversial observations suggesting that bipotentiality of polydendrocytes might be real or an *in vitro* artifact [136,140,141], and most likely these cells are inherently capable of differentiating into astrocytes but are prevented from fulfilling their

On the whole, while all of these studies consistently support the oligodendrocyte lineage of the Ng2+ cells, the genesis of astrocytes from Ng2+ cells is confirmed only during postnatal ages. All these different and sometimes controversial results may be explained by some methodological/technical differences, but may also reflect heterogeneity in progenitor cell populations⁄subpopulations (mostly not yet identified), which is far to be elucidated [98]. In this context, we have recently identified a population of multipolar glial cells immunoreactive for the microtubule associated protein 5 (Map5) [142], which share features but also differences with Ng2+ progenitor cells [19]. These multipolar, Map5+ cells are newly generated, paren‐ chymal elements of the oligodendroglial lineage, which represent a stage-specific population

The CNS of mammals, in spite of having lost most of its regenerative/repair capacity with respect to other phyla, is endowed with remarkable plasticity. This property is heterogene‐ ously distributed in different regions and can manifest in different ways. A better knowledge of the various forms of spontaneous and lesion-induced structural plasticity, of their mutual relationships and of the relative underlying mechanisms is fundamental in order to figure out

Together, these data suggests that specific degenerative conditions can stimulate the produc‐ tion of new neurons not only in the neurogenic niches but also in the mature brain parenchyma. This tissue has been classically considered non-permissive for neuronal progenitors, an idea mainly derived from the observation that SVZ and SGZ neural stem cells differentiate only into glial cells when transplanted into the brain parenchyma (for review, see [127]). In light on the accumulating evidence for parenchymal neurogenesis, the classical concept that the mature brain parenchyma is not permissive for the genesis of new neurons should be restricted to SVZ and SGZ progenitors. Yet, future studies should better analyze whether factors modulating the lesion-induced parenchymal neurogenic potential may differ from those acting on 'classic' neurogenic site progenitors.

#### **7. Parenchymal gliogenesis**

In the past, neurogenesis and gliogenesis had always been kept separate, the latter being considered less important than the former. In recent years, adult gliogenesis has been reevaluated as many populations of progenitor cells with glial-like features and proliferative capacity have been shown to exist in the mature mammalian CNS [13,15]. Actually, paren‐ chymal cell genesis in the so-called non-neurogenic regions is mainly gliogenic. In most regions of the CNS, parenchymal progenitors assure a slow process of 'constitutive' gliogenesis leading to renewal of oligodendrocytes and, to a lesser extent, astrocytes [12,15,128]. In rodents, the major population of cycling progenitors located outside the germinal niches are Ng2+ cells morphologically, antigenically, functionally distinct from mature astrocytes, oligodendrocytes and microglia [12-15]. These cells are also called 'polydendrocytes' to highlight their stellate morphology and lineal relationship to oligodendrocytes [15], 'synantocytes' [14] for their contiguity to neurons, or 'oligodendrocyte progenitor cells' (OPCs) because found able of generating myelinating oligodendrocytes [12,129,130]. Nevertheless, many polydendrocytes remain as a resident cell population of Ng2-expressing cells in the mature white and grey matter after oligodendrocytes are generated. Thus it is widely accepted they represent the fourth CNS major glial population [15], representing 2-9% of total cells [13]. In the last decade, Ng2+ cells have generated a lot of interest among neuroscientists, because they show a series of features quite unusual in OPCs. These include: i) an almost uniform distribution in both grey and white matter areas; ii) a stellate morphology; iii) an intimate association with neurons from which they receive synapses [13,14]; iv) proliferative capacity in the adult brain [13,131, 132], and v) a potential for giving rise to astrocytes and neurons that may be recruited to areas of lesion in the context of brain injury or pathology [128]. At present, it is generally accepted that polydendrocytes are OPCs, even if the demonstration that polydendrocytes differentiate into mature myelinating oligodendrocytes *in vivo* is challenging, because Ng2 expression is lost before the terminal differentiation of these cells and the appearance of mature oligoden‐ drocyte antigens. Some observations provide circumstantial evidences for the oligodendro‐ glial fate of polydendrocytes *in vivo*. For instance, they co-express the PDGFRα, and during the first postnatal week, in the corpus callosum and cortex, they start expressing the immature oligodendrocyte antigen O4 [133]. Polydendrocytes also express the basic helix-loop-helix (bHLH) transcription factors Olig1 and Olig2, which are required for oligodendrocyte specification and differentiation [132,134] as well as Sox9 and Sox10 transcription factors. Moreover, pulse-chase labelling of proliferating cells using 5-bromo-2′-deoxyuridine (BrdU) revealed that the number of BrdU+Ng2+ cells decreases while that of BrdU+ oligodendrocytes increases over time [12,135]. Cell grafting experiments have shown that polydendrocytes give rise to myelinating cells when they are transplanted into an environment free of endogenous myelinating cells [136]. Recently, more direct evidence for the oligodendroglial fate of polydendrocytes was obtained from cell fate-mapping experiments using transgenic mice that express Cre recombinase (Cre) in Ng2-expressing cells or that express inducible Cre (CreeR), under the regulation of the Cspg4, PDGFRα or Olig2 genes, which enable determination of the fate of polydendrocytes at a given time during development [95,137,138]. These studies showed that oligodendrocytes continue to be generated in the mature brain.

Early cell-culture studies showed that OPCs purified from rat optic nerves differentiate not only into oligodendrocytes but also into process-bearing 'type-2 astrocytes' in the presence of serum factors, which led to the concept of bipotential oligodendrocyte type-2-astrocyte (O-2A) progenitor cells [139]. There are now controversial observations suggesting that bipotentiality of polydendrocytes might be real or an *in vitro* artifact [136,140,141], and most likely these cells are inherently capable of differentiating into astrocytes but are prevented from fulfilling their astroglial fate in the normal *in vivo* environment [128].

On the whole, while all of these studies consistently support the oligodendrocyte lineage of the Ng2+ cells, the genesis of astrocytes from Ng2+ cells is confirmed only during postnatal ages. All these different and sometimes controversial results may be explained by some methodological/technical differences, but may also reflect heterogeneity in progenitor cell populations⁄subpopulations (mostly not yet identified), which is far to be elucidated [98]. In this context, we have recently identified a population of multipolar glial cells immunoreactive for the microtubule associated protein 5 (Map5) [142], which share features but also differences with Ng2+ progenitor cells [19]. These multipolar, Map5+ cells are newly generated, paren‐ chymal elements of the oligodendroglial lineage, which represent a stage-specific population of polydendrocytes (Crociara et al., in preparation; Figure 2).

#### **8. Conclusion and future perspectives**

coworkers could not trace the origin of the observed striatal parenchymal neuronal progeni‐ tors. Nonetheless, two possibilities can be considered: i) striatal neuronal progenitors could derive from the displacement of primary/intermediate progenitors from the SVZ; ii) they could

Together, these data suggests that specific degenerative conditions can stimulate the produc‐ tion of new neurons not only in the neurogenic niches but also in the mature brain parenchyma. This tissue has been classically considered non-permissive for neuronal progenitors, an idea mainly derived from the observation that SVZ and SGZ neural stem cells differentiate only into glial cells when transplanted into the brain parenchyma (for review, see [127]). In light on the accumulating evidence for parenchymal neurogenesis, the classical concept that the mature brain parenchyma is not permissive for the genesis of new neurons should be restricted to SVZ and SGZ progenitors. Yet, future studies should better analyze whether factors modulating the lesion-induced parenchymal neurogenic potential may differ from those acting on 'classic'

In the past, neurogenesis and gliogenesis had always been kept separate, the latter being considered less important than the former. In recent years, adult gliogenesis has been reevaluated as many populations of progenitor cells with glial-like features and proliferative capacity have been shown to exist in the mature mammalian CNS [13,15]. Actually, paren‐ chymal cell genesis in the so-called non-neurogenic regions is mainly gliogenic. In most regions of the CNS, parenchymal progenitors assure a slow process of 'constitutive' gliogenesis leading to renewal of oligodendrocytes and, to a lesser extent, astrocytes [12,15,128]. In rodents, the major population of cycling progenitors located outside the germinal niches are Ng2+ cells morphologically, antigenically, functionally distinct from mature astrocytes, oligodendrocytes and microglia [12-15]. These cells are also called 'polydendrocytes' to highlight their stellate morphology and lineal relationship to oligodendrocytes [15], 'synantocytes' [14] for their contiguity to neurons, or 'oligodendrocyte progenitor cells' (OPCs) because found able of generating myelinating oligodendrocytes [12,129,130]. Nevertheless, many polydendrocytes remain as a resident cell population of Ng2-expressing cells in the mature white and grey matter after oligodendrocytes are generated. Thus it is widely accepted they represent the fourth CNS major glial population [15], representing 2-9% of total cells [13]. In the last decade, Ng2+ cells have generated a lot of interest among neuroscientists, because they show a series of features quite unusual in OPCs. These include: i) an almost uniform distribution in both grey and white matter areas; ii) a stellate morphology; iii) an intimate association with neurons from which they receive synapses [13,14]; iv) proliferative capacity in the adult brain [13,131, 132], and v) a potential for giving rise to astrocytes and neurons that may be recruited to areas of lesion in the context of brain injury or pathology [128]. At present, it is generally accepted that polydendrocytes are OPCs, even if the demonstration that polydendrocytes differentiate into mature myelinating oligodendrocytes *in vivo* is challenging, because Ng2 expression is lost before the terminal differentiation of these cells and the appearance of mature oligoden‐

represent local cells becoming neurogenic in response to neurodegeneration.

neurogenic site progenitors.

254 Neural Stem Cells - New Perspectives

**7. Parenchymal gliogenesis**

The CNS of mammals, in spite of having lost most of its regenerative/repair capacity with respect to other phyla, is endowed with remarkable plasticity. This property is heterogene‐ ously distributed in different regions and can manifest in different ways. A better knowledge of the various forms of spontaneous and lesion-induced structural plasticity, of their mutual relationships and of the relative underlying mechanisms is fundamental in order to figure out new efficacious therapeutic perspectives for brain repair. During the last two decades, the discovery of neural stem cells and the studies on adult neurogenesis have opened the intri‐ guing possibility of cell replacement-aimed therapeutic strategies. Under pressure of this perspective, studies on CNS stem cells and progenitors have increased exponentially, some‐ times leading to excessive emphasis about theoretical correlations between neuro-glio-genic processes and brain repair. In this context, focusing on the 'real' neurogenic/gliogenic potential of the mammalian CNS should avoid to turn an exciting biological discovery into a therapeutic illusion. Indeed, the approach of regenerative medicine applied to the CNS is still hampered by overwhelming problems concerning the final integration of both transplanted and endo‐ genously-induced cells [6]. The reason of this failure might be mostly due to evolutionary constraints [78], and to the fact that cell renewal typical of adult constitutive neurogenesis is primarily involved in tissue homeostasis of highly restricted regions, being hardly useful in response to external injury and neurodegenerative brain damage affecting the parenchyma [11,62]. On the other hand, the parenchymal cell genesis might represent a new plastic potential to be explored within wide regions of the CNS, including those areas affected by different neurodegenerative diseases and traumatic injuries. With respect to classic SVZ and SGZ neurogenesis, parenchymal neuro-glio-genesis does constitute an alternative source of progenitors, although with different outcomes [30]. Indeed, a vast number of reports currently published in this domain, although accurate and carried out with multiple technical ap‐ proaches, do suggest that in most cases newly formed elements barely survive and do not fully integrate. In addition, the extreme heterogeneity of parenchymal neuro-glio-genesis makes the brain parenchyma a harsh territory, in which many questions remain unanswered and new ones are opened (see Box 1). For instance, beside a deep knowledge on the cell cycle and early cell lineage in neurogenic sites (see for example [143,144]), such information is starting to be gathered only in specific perenchymal regions and or situations [145,146]. Hence, further studies of parenchymal stem/progenitor cells, on their origin and their different fates and outcomes, should grant new challenges in the multifaceted field of CNS structural plasticity and repair.

**Author details**

Maria Armentano

\*Address all correspondence to: luca.bonfanti@unito.it

Neurosciences, vol. 1, pp. 67-73, 2000.

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Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin, Italy

, Giovanna Ponti, Federico Luzzati, Paola Crociara, Roberta Parolisi and

Parenchymal Neuro-Glio-Genesis Versus Germinal Layer-Derived Neurogenesis: Two Faces of …

http://dx.doi.org/10.5772/56100

257

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Luca Bonfanti\*

**References**

249-266, 1998.

2011.


• Which are the real stemness properties of different parenchymal progenitors?


• Which are the factors leading to the progressive decrease of neurogenic and gliogenic activity with increasing age, both in neurogenic sites and parenchyma?

#### **Author details**

new efficacious therapeutic perspectives for brain repair. During the last two decades, the discovery of neural stem cells and the studies on adult neurogenesis have opened the intri‐ guing possibility of cell replacement-aimed therapeutic strategies. Under pressure of this perspective, studies on CNS stem cells and progenitors have increased exponentially, some‐ times leading to excessive emphasis about theoretical correlations between neuro-glio-genic processes and brain repair. In this context, focusing on the 'real' neurogenic/gliogenic potential of the mammalian CNS should avoid to turn an exciting biological discovery into a therapeutic illusion. Indeed, the approach of regenerative medicine applied to the CNS is still hampered by overwhelming problems concerning the final integration of both transplanted and endo‐ genously-induced cells [6]. The reason of this failure might be mostly due to evolutionary constraints [78], and to the fact that cell renewal typical of adult constitutive neurogenesis is primarily involved in tissue homeostasis of highly restricted regions, being hardly useful in response to external injury and neurodegenerative brain damage affecting the parenchyma [11,62]. On the other hand, the parenchymal cell genesis might represent a new plastic potential to be explored within wide regions of the CNS, including those areas affected by different neurodegenerative diseases and traumatic injuries. With respect to classic SVZ and SGZ neurogenesis, parenchymal neuro-glio-genesis does constitute an alternative source of progenitors, although with different outcomes [30]. Indeed, a vast number of reports currently published in this domain, although accurate and carried out with multiple technical ap‐ proaches, do suggest that in most cases newly formed elements barely survive and do not fully integrate. In addition, the extreme heterogeneity of parenchymal neuro-glio-genesis makes the brain parenchyma a harsh territory, in which many questions remain unanswered and new ones are opened (see Box 1). For instance, beside a deep knowledge on the cell cycle and early cell lineage in neurogenic sites (see for example [143,144]), such information is starting to be gathered only in specific perenchymal regions and or situations [145,146]. Hence, further studies of parenchymal stem/progenitor cells, on their origin and their different fates and outcomes, should grant new challenges in the multifaceted field of CNS structural plasticity

• Which is the real extension of parenchymal cell genesis in the CNS of different mammals and in humans?

• Which stem/progenitor cells do contribute to postnatal neurogenesis but become depleted as their progeny

• Which are the specific stimuli that can trigger quiescent parenchymal progenitor cell division and differentiation? • Can the fate of parenchymal progenitors be altered by microenvironmental cues or it is predetermined? To which

• Which are the factors leading to the progressive decrease of neurogenic and gliogenic activity with increasing age,

• Can distinct parenchymal stem/progenitor cells be forced to produce unusual progeny if needed?

and repair.

256 Neural Stem Cells - New Perspectives

• Do parenchymal progenitors divide asymmetrically?

extent these changes do depend on regional localization?

both in neurogenic sites and parenchyma?

**Box 1.** Some open questions

• Which are the real stemness properties of different parenchymal progenitors?

differentiates, and which continue to replenish the stem/progenitor cell reservoir?

• Which is the origin of the different types of parenchymal progenitors? • What is(are) the ultimate fate(s) of parenchymal neuro-glio-genesis?

Luca Bonfanti\* , Giovanna Ponti, Federico Luzzati, Paola Crociara, Roberta Parolisi and Maria Armentano

\*Address all correspondence to: luca.bonfanti@unito.it

Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin, Italy

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**Section 3**

**Neural Stem Cells and Regenerative Medicine**


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**Chapter 10**

**A Survey of the Molecular Basis for the Generation of**

**Functional Dopaminergic Neurons from Pluripotent**

**Stem Cells: Insights from Regenerative Biology and**

Animals that possess regenerative abilities are widespread in the animal kingdom [1]. Hydra, planarian, zebrafish, newt and axolotl are known prominent species, and the cellular aspects of the stem cell system for regeneration are well elucidated [2]. However, few animals can be used to investigate the molecular basis of neuronal regeneration, in spite of the presence of prominent regenerative animals, as mentioned above. Planarians, for instance, can regenerate a functional brain after amputation in a few days, even from non-brain tissue [3,4]. Newts can regenerate several tissues and organs (*i.e.,* lens, limbs, jaws, hearts and tails) with recovery of function and physiology after injury or tissue removal [5-8]. These animals achieve regenera‐ tion of missing nervous system utilizing stem cells. However, it is difficult to regenerate nervous system in mammalians, including human beings, although these animals possess neural stem cells. Therefore, regenerative animals provide unique opportunities to investigate the generation and utilization of stem cells to repair lost or injured tissue in non-regenerative animals. On the other hand, the successful derivation of neural cells from human embryonic stem cells (ESCs) [9] and induced pluripotent stem cells (iPSCs) [10,11] under *in vitro* conditions provides a new experimental strategy for clinical translation. In other words, although human beings lack regenerative abilities, the new clinical strategy of "regenerative medicine," including cell-transplantation therapy, has been developed to recover lost neural functions by using stem cells. This research field has become a greatly advancing scientific field worldwide.

> © 2013 Nishimura 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.

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,

**Regenerative Medicine**

Kaneyasu Nishimura, Yoshihisa Kitamura,

Additional information is available at the end of the chapter

Kiyokazu Agata and Jun Takahashi

http://dx.doi.org/10.5772/55442

**1. Introduction**

**A Survey of the Molecular Basis for the Generation of Functional Dopaminergic Neurons from Pluripotent Stem Cells: Insights from Regenerative Biology and Regenerative Medicine**

Kaneyasu Nishimura, Yoshihisa Kitamura, Kiyokazu Agata and Jun Takahashi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55442

#### **1. Introduction**

Animals that possess regenerative abilities are widespread in the animal kingdom [1]. Hydra, planarian, zebrafish, newt and axolotl are known prominent species, and the cellular aspects of the stem cell system for regeneration are well elucidated [2]. However, few animals can be used to investigate the molecular basis of neuronal regeneration, in spite of the presence of prominent regenerative animals, as mentioned above. Planarians, for instance, can regenerate a functional brain after amputation in a few days, even from non-brain tissue [3,4]. Newts can regenerate several tissues and organs (*i.e.,* lens, limbs, jaws, hearts and tails) with recovery of function and physiology after injury or tissue removal [5-8]. These animals achieve regenera‐ tion of missing nervous system utilizing stem cells. However, it is difficult to regenerate nervous system in mammalians, including human beings, although these animals possess neural stem cells. Therefore, regenerative animals provide unique opportunities to investigate the generation and utilization of stem cells to repair lost or injured tissue in non-regenerative animals. On the other hand, the successful derivation of neural cells from human embryonic stem cells (ESCs) [9] and induced pluripotent stem cells (iPSCs) [10,11] under *in vitro* conditions provides a new experimental strategy for clinical translation. In other words, although human beings lack regenerative abilities, the new clinical strategy of "regenerative medicine," including cell-transplantation therapy, has been developed to recover lost neural functions by using stem cells. This research field has become a greatly advancing scientific field worldwide.

© 2013 Nishimura 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.

In this chapter, we focus on the molecular systems of generation of functional dopaminergic (DA) neurons *in vivo* and/or *in vitro* in regenerative and non-regenerative animals. The first topic investigates how regenerative animals recruit new DA neurons from stem cells after injury. The second topic explores how to generate DA neurons from mammalian ESCs and iPSCs under *in vitro* conditions. The third topic evaluates clinical applications for human neural disease, especially Parkinson's disease.

pluripotent stem cells) is formed by mitogen-activated protein (MAP) kinase activation 24 hours after amputation [25,26]. It has been revealed that blastema cells are supplied from the post-blastema region via mitosis from G2-phase pluripotent stem cells. Activation of c-Jun-Nterminal kinase (JNK) after amputation induces G2/M transition and supplies blastema cells. Subsequently, activation of extracellular signal-related kinase (ERK) signalling is required for blastema cells to exit the undifferentiated state and enter the differentiation state in order to form the brain rudiment [27]. Similarly, activation of ERK signalling facilitates exit from selfrenewal and regulates differentiation signals, as in mouse ESCs [28]. These findings indicate that planarian stem cells and mouse ESCs may possess a similar molecular basis for cell fate

A Survey of the Molecular Basis for the Generation of Functional Dopaminergic Neurons from…

http://dx.doi.org/10.5772/55442

273

After the formation of the brain rudiment, the wnt and bone morphogenic protein (BMP) signaling pathways regulate brain polarity along the anterior-posterior axis [29,30] and the dorso-ventral axis [31,32], respectively. Therefore, pluripotent stem cells are regulated by various signals in spacio and temporal manners to form the brain. Similarly, wnt and BMP signaling pathways also regulate polarity of neural tube formation in mammalian early

During head regeneration, DA neurons begin to appear in future brain regions starting three days after amputation. On day 5, the number of new DA neurons increases and axons start to extend. On day 7, brain regeneration is complete along with complete reconstruction of DA neurons (Figure 1A) [15]. Other neurotransmitter-synthesizing neurons, such as GABAergic, octopaminergic (OA) and cholinergic neurons are also regenerated in a similar manner from pluripotent stem cells during brain regeneration [17-19]. Recently, we revealed that the numbers of each type of different brain neurons are maintained in a constant ratio that is dependent on body size in intact planarians [21]. For instance, the ratio of DA neurons to OA neurons is 2:1 in intact planarian brains. Interestingly, in the early stage of brain regeneration, the ratio is larger than 2:1; however, it is gradually restored to 2:1 during brain regeneration. The ratio among different neuronal cell types fluctuates in the early stages of regeneration and is gradually restored to the original ratio. These data suggested that non-cell-autonomous

*2.1.2. The stem cell system of DA neuroregeneration in the head regeneration process*

mechanisms utilized to adjust the ratio among different types of brain neurons [21].

We recently established an experimental model to investigate cell-type specific regeneration following selective degeneration by 6-hydroxydopamine (6-OHDA), a DA neurotoxin [22]. Accordingtoourobservations,DAneurons are completelydegeneratedwithin24hours after 6- OHDA-administration. Newly generated DA neurons begin to appear in the brain four days after 6-OHDA-induced lesions. Thereafter, the number and the axons of DA neurons gradual‐ ly recover over a period of several days. Finally, DA neurons are completely recovered within 14daysafter6-OHDA-inducedlesions(Figure1B).Bromodeoxyuridine(BrdU)-pulseandchase experiments indicatethatnewlygeneratedDAneuronsarederivedfromproliferativestemcells that enter the S-phase in the trunk region and migrate to the head region from the trunk region

**2.2. DA neuronal regeneration after selective DA lesions**

determination.

development [33,34].

#### **2. DA neuronal regeneration in regenerative animals**

Freshwater planarians, *Plathelminthes*, have a primitive central nervous system (CNS) that is composed of a well-organized brain and a pair of ventral nerve cords [12]. Large-scale expression analyses have revealed that many neural genes involved in vertebrate brain development and function are also expressed in distinct domains of the planarian CNS [13,14]. These results indicate that the planarian CNS is functionally regionalized according to a discrete expression of neural-specific genes.

We recently discovered that the planarian CNS contains dopamine, serotonin, γ-aminobutyric acid (GABA) and acetylcholine, which are known to be present in mammalians, and that the planarian nervous system constitutes particular neural networks and functions [15-19]. Since planarians possess pluripotent stem cells throughout their entire bodies, their CNS can be completelyregeneratedalongwithrecoveryofmorphologyandfunctionafter amputation[20].

Recently, two different processes of DA neuroregeneration in the planarian *Dugesia japonica* have been described. One involves DA neuroregeneration accompanied by brain regeneration after artificial amputation [15,21]. The other involves DA neuronal regeneration after selective degeneration of DA neurons by the DA neurotoxin 6-hydroxydopamine (6-OHDA) [22]. Although both of these processes are achieved by manipulating pluripotent stem cells, the systems of the processes are different between these regenerative processes [23]. In this section, we focus on these two processes of DA neuroregeneration in planarians, one of prominent regenerative animals.

#### **2.1. Brain regeneration and DA neuroregeneration in planarians**

Planarians can regenerate a functional brain within 7-10 days after amputation. This regener‐ ative process can be divided into at least five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation and (5) functional recovery. Each step is defined by sequential gene expression alterations that are similar to those observed in mammalian brain development [20].

#### *2.1.1. The early stage of brain regeneration after amputation*

The first step of head regeneration after amputation is wound closure, which involves adhesion of the dorsal and ventral tissues, thereby inducing activation of *noggin-like gene A* (*DjnlgA*) at the edge of the amputated site [24]. Subsequently, a blastema (a mass of cells derived from pluripotent stem cells) is formed by mitogen-activated protein (MAP) kinase activation 24 hours after amputation [25,26]. It has been revealed that blastema cells are supplied from the post-blastema region via mitosis from G2-phase pluripotent stem cells. Activation of c-Jun-Nterminal kinase (JNK) after amputation induces G2/M transition and supplies blastema cells. Subsequently, activation of extracellular signal-related kinase (ERK) signalling is required for blastema cells to exit the undifferentiated state and enter the differentiation state in order to form the brain rudiment [27]. Similarly, activation of ERK signalling facilitates exit from selfrenewal and regulates differentiation signals, as in mouse ESCs [28]. These findings indicate that planarian stem cells and mouse ESCs may possess a similar molecular basis for cell fate determination.

In this chapter, we focus on the molecular systems of generation of functional dopaminergic (DA) neurons *in vivo* and/or *in vitro* in regenerative and non-regenerative animals. The first topic investigates how regenerative animals recruit new DA neurons from stem cells after injury. The second topic explores how to generate DA neurons from mammalian ESCs and iPSCs under *in vitro* conditions. The third topic evaluates clinical applications for human neural

Freshwater planarians, *Plathelminthes*, have a primitive central nervous system (CNS) that is composed of a well-organized brain and a pair of ventral nerve cords [12]. Large-scale expression analyses have revealed that many neural genes involved in vertebrate brain development and function are also expressed in distinct domains of the planarian CNS [13,14]. These results indicate that the planarian CNS is functionally regionalized according to a

We recently discovered that the planarian CNS contains dopamine, serotonin, γ-aminobutyric acid (GABA) and acetylcholine, which are known to be present in mammalians, and that the planarian nervous system constitutes particular neural networks and functions [15-19]. Since planarians possess pluripotent stem cells throughout their entire bodies, their CNS can be completelyregeneratedalongwithrecoveryofmorphologyandfunctionafter amputation[20].

Recently, two different processes of DA neuroregeneration in the planarian *Dugesia japonica* have been described. One involves DA neuroregeneration accompanied by brain regeneration after artificial amputation [15,21]. The other involves DA neuronal regeneration after selective degeneration of DA neurons by the DA neurotoxin 6-hydroxydopamine (6-OHDA) [22]. Although both of these processes are achieved by manipulating pluripotent stem cells, the systems of the processes are different between these regenerative processes [23]. In this section, we focus on these two processes of DA neuroregeneration in planarians, one of prominent

Planarians can regenerate a functional brain within 7-10 days after amputation. This regener‐ ative process can be divided into at least five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation and (5) functional recovery. Each step is defined by sequential gene expression alterations that are similar to those

The first step of head regeneration after amputation is wound closure, which involves adhesion of the dorsal and ventral tissues, thereby inducing activation of *noggin-like gene A* (*DjnlgA*) at the edge of the amputated site [24]. Subsequently, a blastema (a mass of cells derived from

disease, especially Parkinson's disease.

272 Neural Stem Cells - New Perspectives

discrete expression of neural-specific genes.

regenerative animals.

**2. DA neuronal regeneration in regenerative animals**

**2.1. Brain regeneration and DA neuroregeneration in planarians**

observed in mammalian brain development [20].

*2.1.1. The early stage of brain regeneration after amputation*

After the formation of the brain rudiment, the wnt and bone morphogenic protein (BMP) signaling pathways regulate brain polarity along the anterior-posterior axis [29,30] and the dorso-ventral axis [31,32], respectively. Therefore, pluripotent stem cells are regulated by various signals in spacio and temporal manners to form the brain. Similarly, wnt and BMP signaling pathways also regulate polarity of neural tube formation in mammalian early development [33,34].

#### *2.1.2. The stem cell system of DA neuroregeneration in the head regeneration process*

During head regeneration, DA neurons begin to appear in future brain regions starting three days after amputation. On day 5, the number of new DA neurons increases and axons start to extend. On day 7, brain regeneration is complete along with complete reconstruction of DA neurons (Figure 1A) [15]. Other neurotransmitter-synthesizing neurons, such as GABAergic, octopaminergic (OA) and cholinergic neurons are also regenerated in a similar manner from pluripotent stem cells during brain regeneration [17-19]. Recently, we revealed that the numbers of each type of different brain neurons are maintained in a constant ratio that is dependent on body size in intact planarians [21]. For instance, the ratio of DA neurons to OA neurons is 2:1 in intact planarian brains. Interestingly, in the early stage of brain regeneration, the ratio is larger than 2:1; however, it is gradually restored to 2:1 during brain regeneration. The ratio among different neuronal cell types fluctuates in the early stages of regeneration and is gradually restored to the original ratio. These data suggested that non-cell-autonomous mechanisms utilized to adjust the ratio among different types of brain neurons [21].

#### **2.2. DA neuronal regeneration after selective DA lesions**

We recently established an experimental model to investigate cell-type specific regeneration following selective degeneration by 6-hydroxydopamine (6-OHDA), a DA neurotoxin [22]. Accordingtoourobservations,DAneurons are completelydegeneratedwithin24hours after 6- OHDA-administration. Newly generated DA neurons begin to appear in the brain four days after 6-OHDA-induced lesions. Thereafter, the number and the axons of DA neurons gradual‐ ly recover over a period of several days. Finally, DA neurons are completely recovered within 14daysafter6-OHDA-inducedlesions(Figure1B).Bromodeoxyuridine(BrdU)-pulseandchase experiments indicatethatnewlygeneratedDAneuronsarederivedfromproliferativestemcells that enter the S-phase in the trunk region and migrate to the head region from the trunk region withoutenteringtheM-phase(*i.e.*,theyremainintheG2-phase)andthengiverisetoDAneurons in the head region (Figure 1C). In addition, histological analyses support the hypothesis that pluripotent stem cells may directly give rise to differentiated DA neurons in planarians (Figure 1D, E). This observation suggests that G2-phase proliferating stem cells can respond to degener‐ ation of DA neurons and are committed to DA neurons in planarians [22].

Although most vertebrates show low regenerative capabilities, newts have powerful regen‐ erative abilities among adult vertebrates. Surprisingly, adult newts regenerate brain tissue after partial brainectomy in spite of having a complex brain structure [35,36]. Recent reports indicate that newts maintain the neurogenic potential to repair lost midbrain DA neurons, even in the adult state [37]. Although proliferative abilities are essentially quiescent in the midbrain of adult newts, quiescent ependymoglia cells are activated by the degeneration of midbrain DA neurons. Additionally, activated ependymoglia cells start to proliferate and differentiate to DA neurons to repair lost DA neurons. This neurogenic potential is activated under conditions of injury-responsive cell-replacement and not under homeostatic conditions [38,39]. These reports indicated that these regenerative animals possess unique stem cells system to regenerative missing DA neurons.

#### **3. Strategies for generation of DA neurons in non-regenerative animals**

Regenerative animals easily regenerate lost brain and neural tissues by maturating stem cells. Regenerative animals provide us unique ideas to generate neural tissue from mammalian pluripotent stem cells such as ESCs and iPSCs.

Although the capacity for brain formation is present during the developmental stage among animals, including human beings, it is difficult to regenerate missing neurons and brain tissue in the adult state. It has been reported that neurogenesis homeostatically occurs in the restricted regions of the adult mammalian brain such as hippocampus and subventricular zone [40,41]. However, it remains controversial whether dopaminergic neurogenesis/neuroregeneration occurs in the adult mammalian midbrain [42,43] and whether neurogenic potential is "lost" or "quiescent" in the adult mammalian brain. Regardless, this potential is not adequate to recover missing neurons and brain tissue in the adult mammalian brain. If human beings had an adequate regenerative potential, some types of neural disorders and brain injuries might be self-curable. However, it is difficult to self-repair neurodegenerative disorders. Therefore, it is strongly expected that neuronal differentiation techniques will contribute for therapeutic applications, such as cell-transplantation therapy using ESCs/iPSCs.

**3.1. Induction of midbrain DA neurons** *in vitro* **from ESCs/iPSCs**

orange color represents ventral nerve cords (VNCs). The green cells are DA neurons.

new fields for both basic neuroscience research and therapeutic applications.

In 2000, it was reported that stromal cell-derived inducing activity (SDIA) strongly promotes neural induction in mouse ESCs co-cultured with mouse PA6 stromal cells under serum-free conditions without growth factors [44]. SDIA-induced neurons contain high amounts of DA neurons and are integrated into the 6-OHDA-lesioned mouse striatum after transplantation. Additionally, it has been proven that functional DA neurons can be differentiated from primate and human ESCs using SDIA [45,46]. Therefore, the establishment of the SDIA method opened

**Figure 1. A**) The regeneration process of DA neurons after decapitation. Immunofluorescence of brain DA neurons one day, three days, five days and seven days after decapitation. (**B**) The process of DA neuronal regeneration in the brain after 6-OHDA-induced-lesions. Immunofluorescence of brain DA neurons in an intact brain three days, seven days and 14 days after 6-OHDA-administration. (**C**) BrdU-incorporation is detected in newly generated DA neurons five days after 6-OHDA-administration. Double-immunofluorescence of tyrosine hydroxylase (TH; green) and BrdU (magenta). The arrowheads indicate BrdU/TH-double positive neurons. (**D, E**) Double-fluorescence of *TH mRNA* (green) and PiwiA proteins (a marker for pluripotent stem cells; magenta) seven days after 6-OHDA-administration. Scale bars: 200 µm (**A-D**), 50 µm (high magnification image in C) and 50 µm (**E**). (**F**) A schematic drawing of the pla‐ narian brain nervous system and an image of the distribution of DA neurons. The blue color represents the brain. The

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275

Parkinson's disease (PD) is a candidate disease for the expected application of cell-transplan‐ tation therapy. PD is an intractable neurodegenerative disorder that arises from the progres‐ sive death of DA neurons in the substantia nigra pars compacta. Although human beings lack adequate abilities to regenerate DA neurons, techniques to generate midbrain DA neurons from ESCs/iPSCs *in vitro* have been developed and are being improved for use in celltransplantation therapy (Figure 2).

withoutenteringtheM-phase(*i.e.*,theyremainintheG2-phase)andthengiverisetoDAneurons in the head region (Figure 1C). In addition, histological analyses support the hypothesis that pluripotent stem cells may directly give rise to differentiated DA neurons in planarians (Figure 1D, E). This observation suggests that G2-phase proliferating stem cells can respond to degener‐

Although most vertebrates show low regenerative capabilities, newts have powerful regen‐ erative abilities among adult vertebrates. Surprisingly, adult newts regenerate brain tissue after partial brainectomy in spite of having a complex brain structure [35,36]. Recent reports indicate that newts maintain the neurogenic potential to repair lost midbrain DA neurons, even in the adult state [37]. Although proliferative abilities are essentially quiescent in the midbrain of adult newts, quiescent ependymoglia cells are activated by the degeneration of midbrain DA neurons. Additionally, activated ependymoglia cells start to proliferate and differentiate to DA neurons to repair lost DA neurons. This neurogenic potential is activated under conditions of injury-responsive cell-replacement and not under homeostatic conditions [38,39]. These reports indicated that these regenerative animals possess unique stem cells

**3. Strategies for generation of DA neurons in non-regenerative animals**

Regenerative animals easily regenerate lost brain and neural tissues by maturating stem cells. Regenerative animals provide us unique ideas to generate neural tissue from mammalian

Although the capacity for brain formation is present during the developmental stage among animals, including human beings, it is difficult to regenerate missing neurons and brain tissue in the adult state. It has been reported that neurogenesis homeostatically occurs in the restricted regions of the adult mammalian brain such as hippocampus and subventricular zone [40,41]. However, it remains controversial whether dopaminergic neurogenesis/neuroregeneration occurs in the adult mammalian midbrain [42,43] and whether neurogenic potential is "lost" or "quiescent" in the adult mammalian brain. Regardless, this potential is not adequate to recover missing neurons and brain tissue in the adult mammalian brain. If human beings had an adequate regenerative potential, some types of neural disorders and brain injuries might be self-curable. However, it is difficult to self-repair neurodegenerative disorders. Therefore, it is strongly expected that neuronal differentiation techniques will contribute for therapeutic

Parkinson's disease (PD) is a candidate disease for the expected application of cell-transplan‐ tation therapy. PD is an intractable neurodegenerative disorder that arises from the progres‐ sive death of DA neurons in the substantia nigra pars compacta. Although human beings lack adequate abilities to regenerate DA neurons, techniques to generate midbrain DA neurons from ESCs/iPSCs *in vitro* have been developed and are being improved for use in cell-

ation of DA neurons and are committed to DA neurons in planarians [22].

system to regenerative missing DA neurons.

274 Neural Stem Cells - New Perspectives

pluripotent stem cells such as ESCs and iPSCs.

transplantation therapy (Figure 2).

applications, such as cell-transplantation therapy using ESCs/iPSCs.

**Figure 1. A**) The regeneration process of DA neurons after decapitation. Immunofluorescence of brain DA neurons one day, three days, five days and seven days after decapitation. (**B**) The process of DA neuronal regeneration in the brain after 6-OHDA-induced-lesions. Immunofluorescence of brain DA neurons in an intact brain three days, seven days and 14 days after 6-OHDA-administration. (**C**) BrdU-incorporation is detected in newly generated DA neurons five days after 6-OHDA-administration. Double-immunofluorescence of tyrosine hydroxylase (TH; green) and BrdU (magenta). The arrowheads indicate BrdU/TH-double positive neurons. (**D, E**) Double-fluorescence of *TH mRNA* (green) and PiwiA proteins (a marker for pluripotent stem cells; magenta) seven days after 6-OHDA-administration. Scale bars: 200 µm (**A-D**), 50 µm (high magnification image in C) and 50 µm (**E**). (**F**) A schematic drawing of the pla‐ narian brain nervous system and an image of the distribution of DA neurons. The blue color represents the brain. The orange color represents ventral nerve cords (VNCs). The green cells are DA neurons.

#### **3.1. Induction of midbrain DA neurons** *in vitro* **from ESCs/iPSCs**

In 2000, it was reported that stromal cell-derived inducing activity (SDIA) strongly promotes neural induction in mouse ESCs co-cultured with mouse PA6 stromal cells under serum-free conditions without growth factors [44]. SDIA-induced neurons contain high amounts of DA neurons and are integrated into the 6-OHDA-lesioned mouse striatum after transplantation. Additionally, it has been proven that functional DA neurons can be differentiated from primate and human ESCs using SDIA [45,46]. Therefore, the establishment of the SDIA method opened new fields for both basic neuroscience research and therapeutic applications.

**4. Towards the clinical application of ESCs/iPSC-derived DA neurons in**

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277

Clinically, PD is estimated to affect approximately 1% of the population over 65 years of age, and PD patients often exhibit muscle rigidity, tremors, bradykinesia and akinesia. Currently, the primary clinical treatment for PD is dopamine replacement therapy using L-dihydroxy‐ phenylalanine (L-DOPA) and/or dopamine receptor agonists. Although pharmacotherapy temporarily improves parkinsonian symptoms, the efficacy of pharmacotherapy is gradually lost over long-term treatment, and the wearing-off phenomenon, the on-off phenomenon and drug-induced dyskinesia develop. In addition, the progression of the degeneration of midbrain DA neurons cannot be delayed. Therefore, new strategies such as cell transplantation therapy

The first clinical trial of cell transplantation using human fetal ventral midbrain in PD patients was performed in the latter half of the 1980's [59]. Currently, over 400 PD patients have been evaluated in this clinical trial. Some PD patients who underwent grafting have exhibited drastic improvements in movement symptoms. However, strict ethical problems remain regarding the use of human fetal cells for the treatment of human disease. Therefore, efficient methods need to be developed for differentiating DA neurons from ESCs/iPSCs, instead of using human fetal cells, as described above. In addition, the efficiency and safety of ESCs/ iPSCs-derived DA neurons should be evaluated *in vivo* experiments using animal models before conducting human trials. In general, DA neurotoxins such as rotenone, 6-OHDA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are used to create parkinsonian animal models with rodents and non-human primates. In particular, since the pathological symptoms and brain anatomy of monkeys are similar to those of humans, the outcomes of monkey trials

In 2005, it was reported that intra-striatal primate ESC-derived DA neurons survive and function in the putamen in MPTP-lesioned primate parkinsonian models [60]. In that study, positron emission tomography imaging revealed that [18F]-F-DOPA uptake increased in grafted monkeys 14 weeks after transplantation. In addition, the neurological scores of the grafted monkeys improved in comparison with that observed in sham-operated monkeys starting from 10 weeks after transplantation. This study is the first report to indicate the functional efficiency of grafted DA neurons derived from primate ESCs and has opened up

Meanwhile, human ESC/iPSC-derive DA neurons have the potential to improve motor function in PD model rats after intra-striatal grafting [56,61,62]. However, some problems, such as tumorigenicity, remain in clinical trials. We recently reported that long-term neural maturation (> 28 days) of human ESCs reduces tumorigenicity after grafting in primate parkinsonian models. In addition, motor symptoms are also improved by grafting human ESCderived DA neurons that have maturated over a long term (42 days). These results suggest that human ESC-derived DA neurons that are differentiated for appropriate terms strongly

**Parkinson's disease**

are expected to recover lost DA neurons.

strongly contribute to realizing human trials.

**4.1. Preclinical trials of human ESCs/iPSC-derived DA neurons for PD**

the possibility for transplantation therapy using ESC-derived DA neurons.

**Figure 2. A**) A phase contrast image of colonies of undifferentiated human iPSCs on SNL feeder cells. (**B**) DA neurons derived from human iPSCs differentiated using the SFEB method *in vitro*. Double-immunofluorescence of ßIII-tubulin (TubßIII), a neuronal marker (green), and TH, a marker of DA neurons (magenta). Scale bars: 500 µm (**A**) and 50 µm (**B**).

Recently, the molecular mechanisms of mammalian brain development have become better understood. The expression and secretion of patterning factors facilitate neural induction and define anterior-posterior and dorso-ventral patterning of the mammalian brain. For instance, mammalian midbrain development is governed by fibroblast growth factor 8 (FGF8) and Sonic hedgehog (Shh), which are locally expressed at the midbrain-hindbrain boundary and the ventral neural tube, respectively [47]. The floor plate is located along the ventral midline of the neural tube and is known to function as a signaling center during brain development and a source of midbrain DA neurons [48,49]. Midbrain DA neuronal specification is regulated by several transcription factors, including Lmx1a, FoxA2, Nurr1 and Pitx3 [50-53]. Therefore, cellular aspects of brain development provide ideas for improving the differentiation methods of authentic neural identity and subtype specification, such as differentiating DA neurons from ESCs/iPSCs *in vitro*.

Recently, neural lineage commitment has been improved with dual inhibition of SMAD signaling by bone morphogenic protein (BMP) inhibitor (noggin and dorsomorphine) and transforming growth factor-ß (TGF-ß)/activin/nodal inhibitor (SB431542). These combinations promote efficient neural induction of both human ESCs and iPSCs in serum-free, floating cultures of embryoid body-like aggregates (SFEB) and stromal (PA6) feeder co-cultures [54,55]. In addition, GSK3ß inhibitor (CHIR99021) strongly activates wnt signaling and induces the Lmx1a expression in FoxA2-positive floor plate precursors and the neurogenic conversion of human ESC-derived midbrain floor plates towards DA neurons [56]. This differentiation method mimics mammalian brain development.

Recently, human ESCs/iPSCs maintenance and differentiation have been advanced in order to achieve clinical translation, which results in chemically defined conditions and the elimination of animal-derived components and the need for feeder cells [57,58].

### **4. Towards the clinical application of ESCs/iPSC-derived DA neurons in Parkinson's disease**

Clinically, PD is estimated to affect approximately 1% of the population over 65 years of age, and PD patients often exhibit muscle rigidity, tremors, bradykinesia and akinesia. Currently, the primary clinical treatment for PD is dopamine replacement therapy using L-dihydroxy‐ phenylalanine (L-DOPA) and/or dopamine receptor agonists. Although pharmacotherapy temporarily improves parkinsonian symptoms, the efficacy of pharmacotherapy is gradually lost over long-term treatment, and the wearing-off phenomenon, the on-off phenomenon and drug-induced dyskinesia develop. In addition, the progression of the degeneration of midbrain DA neurons cannot be delayed. Therefore, new strategies such as cell transplantation therapy are expected to recover lost DA neurons.

The first clinical trial of cell transplantation using human fetal ventral midbrain in PD patients was performed in the latter half of the 1980's [59]. Currently, over 400 PD patients have been evaluated in this clinical trial. Some PD patients who underwent grafting have exhibited drastic improvements in movement symptoms. However, strict ethical problems remain regarding the use of human fetal cells for the treatment of human disease. Therefore, efficient methods need to be developed for differentiating DA neurons from ESCs/iPSCs, instead of using human fetal cells, as described above. In addition, the efficiency and safety of ESCs/ iPSCs-derived DA neurons should be evaluated *in vivo* experiments using animal models before conducting human trials. In general, DA neurotoxins such as rotenone, 6-OHDA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are used to create parkinsonian animal models with rodents and non-human primates. In particular, since the pathological symptoms and brain anatomy of monkeys are similar to those of humans, the outcomes of monkey trials strongly contribute to realizing human trials.

#### **4.1. Preclinical trials of human ESCs/iPSC-derived DA neurons for PD**

Recently, the molecular mechanisms of mammalian brain development have become better understood. The expression and secretion of patterning factors facilitate neural induction and define anterior-posterior and dorso-ventral patterning of the mammalian brain. For instance, mammalian midbrain development is governed by fibroblast growth factor 8 (FGF8) and Sonic hedgehog (Shh), which are locally expressed at the midbrain-hindbrain boundary and the ventral neural tube, respectively [47]. The floor plate is located along the ventral midline of the neural tube and is known to function as a signaling center during brain development and a source of midbrain DA neurons [48,49]. Midbrain DA neuronal specification is regulated by several transcription factors, including Lmx1a, FoxA2, Nurr1 and Pitx3 [50-53]. Therefore, cellular aspects of brain development provide ideas for improving the differentiation methods of authentic neural identity and subtype specification, such as differentiating DA neurons from

**Figure 2. A**) A phase contrast image of colonies of undifferentiated human iPSCs on SNL feeder cells. (**B**) DA neurons derived from human iPSCs differentiated using the SFEB method *in vitro*. Double-immunofluorescence of ßIII-tubulin (TubßIII), a neuronal marker (green), and TH, a marker of DA neurons (magenta). Scale bars: 500 µm (**A**) and 50 µm

Recently, neural lineage commitment has been improved with dual inhibition of SMAD signaling by bone morphogenic protein (BMP) inhibitor (noggin and dorsomorphine) and transforming growth factor-ß (TGF-ß)/activin/nodal inhibitor (SB431542). These combinations promote efficient neural induction of both human ESCs and iPSCs in serum-free, floating cultures of embryoid body-like aggregates (SFEB) and stromal (PA6) feeder co-cultures [54,55]. In addition, GSK3ß inhibitor (CHIR99021) strongly activates wnt signaling and induces the Lmx1a expression in FoxA2-positive floor plate precursors and the neurogenic conversion of human ESC-derived midbrain floor plates towards DA neurons [56]. This differentiation

Recently, human ESCs/iPSCs maintenance and differentiation have been advanced in order to achieve clinical translation, which results in chemically defined conditions and the elimination

ESCs/iPSCs *in vitro*.

276 Neural Stem Cells - New Perspectives

(**B**).

method mimics mammalian brain development.

of animal-derived components and the need for feeder cells [57,58].

In 2005, it was reported that intra-striatal primate ESC-derived DA neurons survive and function in the putamen in MPTP-lesioned primate parkinsonian models [60]. In that study, positron emission tomography imaging revealed that [18F]-F-DOPA uptake increased in grafted monkeys 14 weeks after transplantation. In addition, the neurological scores of the grafted monkeys improved in comparison with that observed in sham-operated monkeys starting from 10 weeks after transplantation. This study is the first report to indicate the functional efficiency of grafted DA neurons derived from primate ESCs and has opened up the possibility for transplantation therapy using ESC-derived DA neurons.

Meanwhile, human ESC/iPSC-derive DA neurons have the potential to improve motor function in PD model rats after intra-striatal grafting [56,61,62]. However, some problems, such as tumorigenicity, remain in clinical trials. We recently reported that long-term neural maturation (> 28 days) of human ESCs reduces tumorigenicity after grafting in primate parkinsonian models. In addition, motor symptoms are also improved by grafting human ESCderived DA neurons that have maturated over a long term (42 days). These results suggest that human ESC-derived DA neurons that are differentiated for appropriate terms strongly contribute to both reducing the risk of tumor genesis and improving parkinsonian motor dysfunction [46]. Recently, we first reported that human iPSC-derived DA neurons that are differentiated under feeder-free and serum-free conditions survived in an MPTP-lesioned primate parkinsonian model for six months [63]. This report may support the therapeutic potential of human iPSCs for future clinical trials.

signal molecules contribute to the recruitment of G2-phase stem cells to DA neurons in

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279

In the case of newts, another regenerative animals, quiescent ependymoglia cells can sense degeneration of DA neurons, and re-enter into the cell cycle to restore lost DA neurons [39]. In this case, DA receptor-expressing surrounding cells (*i.e*., ependymoglia cells) respond to the degeneration of DA neurons via DA receptor signaling, and contribute to regenerate DA neurons. Therefore, the responsibility of stem cells to extrinsic environment is important for

The findings from regenerative animals may provide any idea to generate DA neurons from ESCs/iPSCs of non-regenerative animals. Recent strategies for DA neuronal induction from ESCs/iPSCs have based on mimicking midbrain development, that is, the activation of morphogenic factors and transcriptional regulation. In addition, the establishment of 3 dimensional (3D) culture systems strongly contributes to mimicking the complicated organo‐ genesis and lead to the acquisition of sub-regional identities because the cells respond to

Recently, Kirkeby *et al.* developed an induction method for human ESC-derived DA neurons using dual-SMAD inhibition with embryoid body formation. The gene expression profile and transplantation aspects of human ESC-derived DA neurons can be recaptured in the human fetal midbrain [70], suggesting that 3D cultures are suitable for mimicking brain organogenetic

In addition, our findings in planarians may contribute to improving strategies of celltransplantation therapy. That is, how to integrate ESCs/iPSC-derived grafting neurons into the host brain. We found that commitment to DA neurons in stem cells occurs during the G2-phase of the cell cycle. Which state of committed cells can be easily incorporated into lesioned regions and whether the location of commitment activates important factor(s) in the incorporation of committed cells into appropriate positions should be considered. Additionally, both planarians and newts showed drastic behavioral recovery according to DA neuronal regeneration [22,37]. This suggested that regenerated DA neurons contribut‐ ed to functional recover by integrating to the existing neural circuit. We speculate that this phenomenon may provide to the any ideas how to integrate ESCs/iPSC-derived neurons into existing neural circuit after grafting and contribute to recover the motor function. We will attempt to answer several important questions in our future works. A deeper under‐ standing of the answers to the above questions may provide unique clues regarding not only how to commit DA neurons in vitro, but also how to optimize cell-transplantation

Stem cell research is an interesting field in basic science due to its potential therapeutic applications. It is now extensively studied to investigate that the cellular and molecular

DA neuronal regeneration in both planarians and newts.

processes in authentic midbrain DA neurons.

planarians.

extrinsic signals [57, 69].

therapy in the future.

**6. Conclusion**

#### **5. Stem cell utilization** *in vivo* **and** *in vitro* **for non-regenerative animals: Lessons from regenerative animals**

Although the number of regenerative animals used to investigate DA neuronal regenera‐ tion is limited, the cellular mechanisms of DA neuronal regeneration are starting to be understood [22,39]. The cellular and molecular mechanisms for DA neuronal regeneration in regenerative animals provide new ideas for generation of DA neurons in non-regenera‐ tive animals. Our histological analysis of regeneration indicates that pluripotent stem cells may directly give rise to differentiated DA neurons in the planarian head region (Figure 1D, E) [22]. In addition, we have not yet obtained direct evidence for the presence of neural stem cells in planarians. First, we have not observed proliferating cells in the brain, either during regeneration or in intact brains. Second, the expression of the planarian *musashi* family of genes supports the above hypothesis. *Musashi*, an RNA binding protein, is expressed in neural stem cells in various animals[64]. Although three *musashi-*like genes (*DjmlgA*-*C*) have been isolated from planarians, the expressions of these genes are detect‐ ed in the planarian CNS and are not eliminated by X-ray irradiation [65]. These results indicate that planarian *musashi-*like genes are expressed in differentiated cells, not in proliferative stem cells. Based on these observations, we speculate that the neural stem cell system most likely evolved at a later stage of evolution [66].

In the case of brain regeneration, the brain rudiment is formed inside of the blastema. The cells that participate in blastema formation already exist from the proliferative state, and a portion of these cells start to form the brain rudiment [25,26]. Therefore, the commitment of DA neurons may immediately occur after brain rudiment and brain pattern formation.

In the case of DA neuronal regeneration after 6-OHDA-induced lesions, G2-phase stem cells are recruited into DA neurons in the brain. Since the presence of neural stem cells were not clarified in planarians, the key roles for differentiation into DA neurons from pluripotent stem cells would function in G2-phase in the cell cycle. The neurons remaining in the brain after 6- OHDA-induced lesions may play an important role in sensing the loss of DA neurons and recruiting G2-phase stem cells into DA neurons [22]. Recent findings using eukaryote cells indicate that cell fate determination of either self-renewal or differentiation occurs during the G2/M phase in the cell cycle [67]. Therefore, the results of our study concerning the cellular system of DA neuronal regeneration are supported by these observations. Therefore, G2-phase stem cell strongly contributed to DA neuronal regeneration by response to extrinsic environ‐ ment in planarians. However, we have not yet understand a clear answer regarding which signal molecules contribute to the recruitment of G2-phase stem cells to DA neurons in planarians.

In the case of newts, another regenerative animals, quiescent ependymoglia cells can sense degeneration of DA neurons, and re-enter into the cell cycle to restore lost DA neurons [39]. In this case, DA receptor-expressing surrounding cells (*i.e*., ependymoglia cells) respond to the degeneration of DA neurons via DA receptor signaling, and contribute to regenerate DA neurons. Therefore, the responsibility of stem cells to extrinsic environment is important for DA neuronal regeneration in both planarians and newts.

The findings from regenerative animals may provide any idea to generate DA neurons from ESCs/iPSCs of non-regenerative animals. Recent strategies for DA neuronal induction from ESCs/iPSCs have based on mimicking midbrain development, that is, the activation of morphogenic factors and transcriptional regulation. In addition, the establishment of 3 dimensional (3D) culture systems strongly contributes to mimicking the complicated organo‐ genesis and lead to the acquisition of sub-regional identities because the cells respond to extrinsic signals [57, 69].

Recently, Kirkeby *et al.* developed an induction method for human ESC-derived DA neurons using dual-SMAD inhibition with embryoid body formation. The gene expression profile and transplantation aspects of human ESC-derived DA neurons can be recaptured in the human fetal midbrain [70], suggesting that 3D cultures are suitable for mimicking brain organogenetic processes in authentic midbrain DA neurons.

In addition, our findings in planarians may contribute to improving strategies of celltransplantation therapy. That is, how to integrate ESCs/iPSC-derived grafting neurons into the host brain. We found that commitment to DA neurons in stem cells occurs during the G2-phase of the cell cycle. Which state of committed cells can be easily incorporated into lesioned regions and whether the location of commitment activates important factor(s) in the incorporation of committed cells into appropriate positions should be considered. Additionally, both planarians and newts showed drastic behavioral recovery according to DA neuronal regeneration [22,37]. This suggested that regenerated DA neurons contribut‐ ed to functional recover by integrating to the existing neural circuit. We speculate that this phenomenon may provide to the any ideas how to integrate ESCs/iPSC-derived neurons into existing neural circuit after grafting and contribute to recover the motor function. We will attempt to answer several important questions in our future works. A deeper under‐ standing of the answers to the above questions may provide unique clues regarding not only how to commit DA neurons in vitro, but also how to optimize cell-transplantation therapy in the future.

#### **6. Conclusion**

contribute to both reducing the risk of tumor genesis and improving parkinsonian motor dysfunction [46]. Recently, we first reported that human iPSC-derived DA neurons that are differentiated under feeder-free and serum-free conditions survived in an MPTP-lesioned primate parkinsonian model for six months [63]. This report may support the therapeutic

**5. Stem cell utilization** *in vivo* **and** *in vitro* **for non-regenerative animals:**

Although the number of regenerative animals used to investigate DA neuronal regenera‐ tion is limited, the cellular mechanisms of DA neuronal regeneration are starting to be understood [22,39]. The cellular and molecular mechanisms for DA neuronal regeneration in regenerative animals provide new ideas for generation of DA neurons in non-regenera‐ tive animals. Our histological analysis of regeneration indicates that pluripotent stem cells may directly give rise to differentiated DA neurons in the planarian head region (Figure 1D, E) [22]. In addition, we have not yet obtained direct evidence for the presence of neural stem cells in planarians. First, we have not observed proliferating cells in the brain, either during regeneration or in intact brains. Second, the expression of the planarian *musashi* family of genes supports the above hypothesis. *Musashi*, an RNA binding protein, is expressed in neural stem cells in various animals[64]. Although three *musashi-*like genes (*DjmlgA*-*C*) have been isolated from planarians, the expressions of these genes are detect‐ ed in the planarian CNS and are not eliminated by X-ray irradiation [65]. These results indicate that planarian *musashi-*like genes are expressed in differentiated cells, not in proliferative stem cells. Based on these observations, we speculate that the neural stem cell

In the case of brain regeneration, the brain rudiment is formed inside of the blastema. The cells that participate in blastema formation already exist from the proliferative state, and a portion of these cells start to form the brain rudiment [25,26]. Therefore, the commitment of DA neurons may immediately occur after brain rudiment and brain pattern formation.

In the case of DA neuronal regeneration after 6-OHDA-induced lesions, G2-phase stem cells are recruited into DA neurons in the brain. Since the presence of neural stem cells were not clarified in planarians, the key roles for differentiation into DA neurons from pluripotent stem cells would function in G2-phase in the cell cycle. The neurons remaining in the brain after 6- OHDA-induced lesions may play an important role in sensing the loss of DA neurons and recruiting G2-phase stem cells into DA neurons [22]. Recent findings using eukaryote cells indicate that cell fate determination of either self-renewal or differentiation occurs during the G2/M phase in the cell cycle [67]. Therefore, the results of our study concerning the cellular system of DA neuronal regeneration are supported by these observations. Therefore, G2-phase stem cell strongly contributed to DA neuronal regeneration by response to extrinsic environ‐ ment in planarians. However, we have not yet understand a clear answer regarding which

potential of human iPSCs for future clinical trials.

278 Neural Stem Cells - New Perspectives

**Lessons from regenerative animals**

system most likely evolved at a later stage of evolution [66].

Stem cell research is an interesting field in basic science due to its potential therapeutic applications. It is now extensively studied to investigate that the cellular and molecular mechanisms of DA neuronal regeneration in many regenerative animals *in vivo* have been described over the last several years, although numerous unclear issues remain. Important aspects learned from regenerative animals suggest that the cell sources and stem cell systems of DA neuronal regeneration may reflect several concepts in the achievement of regeneration, even among regenerative animals. Therefore, it is difficult to find a common rule for DA neuronal regeneration among animals. However, this trend in regenerative biology will be more emphasized by discoveries made in future studies of regenerative animals, and will provide hints about more efficient utilization of stem cells towards clinical application. Importantly, studying the diversity of regenerative contexts in different animals may contrib‐ ute to highlighting diverse concepts and attractive clues for investigating the generation of DA neurons *in vivo* and *in vitro* in regenerative medicine.

[4] Reddien PW, Sánchez Alvarado A. Fundamentals of planarian regeneration. Annual

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http://dx.doi.org/10.5772/55442

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#### **Acknowledgements**

We thank all members of the laboratory for discussions and support. This work was supported by a Grant-in-Aid for a Grant-in-Aid for Creative Research to K.A. and a Grant-in-Aid for Global COE Program A06.

#### **Author details**

Kaneyasu Nishimura1 , Yoshihisa Kitamura2 , Kiyokazu Agata3 and Jun Takahashi1

1 Department of Biological Repair, Institute for Frontier Medical Sciences, and Laboratory of Neuronal Regeneration, Department of Clinical Application, Center for iPS cell Research and Application (CiRA), Kyoto University, Japan

2 Department of Neurobiology, Kyoto Pharmaceutical University, Japan

3 Department of Biophysics, Graduate School of Science, Kyoto University, Japan

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We thank all members of the laboratory for discussions and support. This work was supported by a Grant-in-Aid for a Grant-in-Aid for Creative Research to K.A. and a Grant-in-Aid for

1 Department of Biological Repair, Institute for Frontier Medical Sciences, and Laboratory of Neuronal Regeneration, Department of Clinical Application, Center for iPS cell Research

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**Chapter 11**

**Systemic Neural Stem Cell-Based Therapeutic**

**Interventions for Inflammatory CNS Disorders**

Regenerative processes occurring under physiological (*maintenance*) [1-3] and pathological (*reparative*) [4-6] conditions are a fundamental part of life, and vary greatly among different species, individuals, and tissues. Despite the central nervous system (CNS) has been consid‐ ered for years as a perennial tissue, it has recently become clear that both physiological and reparative regeneration occur also within the CNS to sustain tissue homeostasis and repair. Importantly, the proliferation and differentiation of endogenous neural stem cells (NSCs) residing within the healthy CNS, or surviving injury, are considered crucial in sustaining these events. However, these processes are not robust enough to promote a functional and stable recovery of the nervous system architecture. Thus, the development of cell-based therapies designed to promote functional (direct *vs.* indirect) neural cell replacement was anticipated [7]. Nevertheless, most of the experimental cell therapies with neural lineage-committed progen‐ itors have failed to foster substantial repair in disease models where the anatomical and functional damage is widespread and an inflamed and/or degenerative microenvironment coexists. Conversely, the systemic injection of *in vitro* expanded neural stem/precursor cells (NPCs) – both as neurospheres as well as plastic-adherent monolayers - has provided a remarkable amelioration of the clinico-pathological features of rodents affected by experi‐ mental inflammatory CNS disorders that include experimental autoimmune encephalomye‐ litis (EAE), cerebral ischemic/haemorrhagic stroke, spinal cord injury (SCI) and traumatic brain injury (TBI). This has been shown to be dependent on the capacity of transplanted NPCs to engage multiple mechanisms of action within specific microenvironments *in vivo* [8]. Among a wide range of potential therapeutic actions – and in addition to the expected cell replacement – this phenomenon may also occur via several *bystander effects*. These effects are heterogeneous

> © 2013 Donegà 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.

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,

Matteo Donegà, Elena Giusto, Chiara Cossetti and

Additional information is available at the end of the chapter

Stefano Pluchino

**1. Introduction**

http://dx.doi.org/10.5772/55426

