**Systemic Neural Stem Cell-Based Therapeutic Interventions for Inflammatory CNS Disorders**

Matteo Donegà, Elena Giusto, Chiara Cossetti and Stefano Pluchino

Additional information is available at the end of the chapter

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

#### **1. Introduction**

rived cells to a rat model of Parkinson's disease: effect of *in vitro* differentiation on

[62] Hargus G, Cooper O, Deleidi M, Levy A, Lee K, Marlow E, Yow A, Soldner F, Hocke‐ meyer D, Hallett PJ, Osborn T, Jaenisch R, Isacson O. Differentiated Parkinson pa‐ tient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proceedings of the National Academy

[63] Kikuchi T, Morizane A, Doi D, Onoe H, Hayashi T, Kawasaki T, Saiki H, Miyamoto S, Takahashi J. Survival of human induced pluripotent stem cell-derived midbrain dopaminergic neurons in the brain of a primate model of Parkinson's disease. Journal

[64] Okano H, Imai T, Okabe M. *Musashi*: a translational regulator of cell fate. Journal of

[65] Higuchi S, Hayashi T, Tarui H, Nishimura O, Nishimura K, Shibata N, Sakamoto H, Agata K. Expression and functional analysis of *musashi*-like genes in planarian CNS

[66] Agata K, Nakajima E, Funayama N, Shibata N, Saito Y, Umesono Y. Two different evolutionary origins of stem cell systems and their molecular basis. Seminars in Cell

[67] Sharif J, Endoh M, Koseki H. Epigenetic memory meets G2/M: to remember or to for‐

[68] Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S, Kawasaki H, Wata‐ nabe Y, Mizuseki K, Sasai Y. Directed differentiation of telencephalic precursors from

[69] Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, Wataya T, Nishiyama A, Muguruma K, Sasai Y. Self-organized formation of po‐ larized cortical tissues from ESCs and its active manipulation by extrinsic signals.

[70] Kirkeby A, Grealish S, Wolf DA, Nelander J, Wood J, Lundblad M, Lindvall O, Par‐ mar M. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Reports 2012

graft survival and teratoma formation. Stem Cells 2006;24(6) 1433-1440.

of Sciences of the United States of America 2010;107(36) 15921-15926.

regeneration. Mechanisms of Development 2008;125(7) 631-645.

embryonic stem cells. Nature Neuroscience 2005;8(3) 288-296.

of Parkinson's Disease 2011;1 395-412.

286 Neural Stem Cells - New Perspectives

Cell Science 2002;115(Pt 7) 1355-1359.

and Developmental Biology 2006;17(4) 503-509.

get? Developmental Cell 2011;20(1) 5-6.

Cell Stem Cell 2008;3(5) 519-532.

28;1(6) 703-714.

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. © 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.

and likely exerted by undifferentiated NPCs releasing immune regulatory and neuroprotec‐ tive molecules within specific microenvironments in response to local stimuli elicited by inflammatory cells (*therapeutic plasticity*). The molecular and cellular mechanism(s) that sustain the multifaceted therapeutic plasticity of NPCs remain far from being fully characterized [9].

cross talk between somatic NPCs and the dysfunctional microenvironment, both at the outer and inner endothelial sides, and their clinico-pathological impact. Finally, we will discuss the rationale of the most recent explorative trials that are bringing neural stem cell therapies into

Systemic stem cell therapies and brain diseases

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

289

Stem cells (SCs) possess the unique ability to self-renew and differentiate into different cell types in the body. Their contribution is essential during embryonic and early post-natal life, where they regulate morphogenesis and development by properly balancing proliferation and differentiation. Though their number is destined to decrease with time, their presence in adult organisms is still required to ensure *homeostasis* and *repair*. While the regenerating properties of some tissues (e.g., the skin) and organs (e.g., the liver) are undisputed, the brain with its unique organization and complexity was considered for long time an exception. In fact, the dogmatic concept '*no new neurons after birth*' (1913) expressed by Santiago Ramon y Cajal, sustaining the limitation of neurogenesis to prenatal life, has been resonating for decades within the scientific community, finally becoming an established belief. NSCs were thought to be present within the brain only during the developmental stage. It was only in the late 60's, thanks to the availability of new techniques and advanced tools of investigation, that the picture of the brain as an immutable organ started to be reviewed. Altman and colleagues,

H)-thymidine pulses and autoradiographs, first demonstrated the presence of prolif‐

erating neurons in different regions of the post-natal brain in rats [1, 2, 27]. However, the turning point was marked later in 1983 when Goldman and Nottebohm at Rockfeller Univer‐ sity (USA) described newly generated neurons at the level of the hyperstriatum ventrale, pars caudalis (HVc), of the ventricular zone in intact adult female canaries [3] Subsequently, numerous pioneering experiments contributed in demonstrating that specific regions of the mammalian CNS undergo a continuous, though moderate, level of neurogenesis throughout

Today it is widely accepted that in the adult mammalian brain, newly generated cells derive from NSCs residing in two regions [29], the ventricular-subventricular zone (V-SVZ) of the forebrain lateral ventricles [2, 27, 30] and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus [1, 31, 32] (Figure 1). Because of the peculiar cellular organization and exclusive microenvironment, these neurogenic regions are commonly referred to as *germinallike niches* [33, 34]. Although different, these two areas share an extremely organized and specialized microenvironment where NSCs can strategically interact with a rich vascular plexus [35, 36], while communicating with their progeny and neighbouring NSCs as well as with differentiated neural cells trough specialized structures (e.g. primary cilium, basal and apical processes). Altogether, these cellular components provide a unique milieu of extracel‐

the clinic.

using (3

adult life [28].

**2.2. Adult neurogenesis in physiological conditions**

**2. Adult neural stem cells**

**2.1. A change in the dogma**

The transplantation of undifferentiated exogenous NPCs very efficiently protects the CNS from experimental chronic degeneration induced by inflammation both in small rodents (mice and rats) [10-14] as well as in non-human primates [15]. Specific homing of systemically injected NPCs is shown, so far, in experimental models of multiple sclerosis (MS), ischemic/ haemorrhagic stroke, SCI and TBI, and epilepsy. I*n vitro* and *in vivo* data provide extensive evidence of the molecular mechanisms behind the ability of NPCs to cross the blood-brain barrier (BBB) and specifically accumulate at the sites of inflammation/tissue damage [16-18]. After entering the CNS using constitutively functional cell adhesion molecules and inflam‐ matory chemokine receptors, systemically injected NPCs accumulate at the level of perivas‐ cular CNS areas, where they establish *atypical ectopic perivascular niches* [16, 19]. In these areas, a much likely active cell-to-cell communication takes place between transplanted NPCs and the different cells of the *atypical niche*. As consequence of this, transplanted NPCs survive while displaying undifferentiated features, and promote neuroprotection by releasing immune modulatory molecules and neurotrophic factors *in situ*. Further evidence exists about an additional peripheral immune-modulatory effect exerted by NPCs [20, 21]. Systemically injected NPCs, in fact, enter also peripheral organs (e.g. draining lymph nodes and spleen) were they accumulate at the boundaries of blood vessels and interact closely with lymphocytes and professional antigen presenting cells (APCs), impairing their maturation and functional activation [15, 22, 23].

NPC-based therapies have been therefore considered a plausible alternative strategy for the treatment of neurological inflammatory disorders. However, some urgent and still unclear questions have to be solved prior to straightforwardly translate most of these exciting experimental observations into clinical medicines, such as: (i) the ideal stem cell source, whether it has to be derived from pluripotent or multipotent sources; (ii) the ideal route of cell administration, whether it has to be focal or systemic; (iii) the optimal time point for cell administration, depending on the disease characteristics; (iv) the ideal balance between differentiation and persistence of stem cells into the targeted tissue and (v) the ideal mechanism of tissue repair to foster, whether it has to be cell replacement or tissue protection/healing. Further, while some encouraging efforts are being devoted towards the development of guidelines and establishment of explorative phase I clinical trials, still one of the major constraints to the easy translation into human medicines is represented by the immunogenicity of allogeneic stem cells, and the modest expandability of somatic human NPCs *in vitro*. Within this scenario, the emerging figure of induced pluripotent stem (iPS) cells [24], induced neuronal (iN) cells [25] and/or induced neural stem cells (iNSCs) [26] holds a new exciting promise.

In this chapter we will describe the most recent evidence of the remarkable therapeutic plasticity of transplanted NPCs, when injected systemically in inflammation-driven CNS degeneration experimental models. We will first focus on the evidence that inspired the modern stem cell experimental therapies and then elaborate on the mechanisms regulating the cross talk between somatic NPCs and the dysfunctional microenvironment, both at the outer and inner endothelial sides, and their clinico-pathological impact. Finally, we will discuss the rationale of the most recent explorative trials that are bringing neural stem cell therapies into the clinic.

#### **2. Adult neural stem cells**

#### **2.1. A change in the dogma**

and likely exerted by undifferentiated NPCs releasing immune regulatory and neuroprotec‐ tive molecules within specific microenvironments in response to local stimuli elicited by inflammatory cells (*therapeutic plasticity*). The molecular and cellular mechanism(s) that sustain the multifaceted therapeutic plasticity of NPCs remain far from being fully characterized [9].

The transplantation of undifferentiated exogenous NPCs very efficiently protects the CNS from experimental chronic degeneration induced by inflammation both in small rodents (mice and rats) [10-14] as well as in non-human primates [15]. Specific homing of systemically injected NPCs is shown, so far, in experimental models of multiple sclerosis (MS), ischemic/ haemorrhagic stroke, SCI and TBI, and epilepsy. I*n vitro* and *in vivo* data provide extensive evidence of the molecular mechanisms behind the ability of NPCs to cross the blood-brain barrier (BBB) and specifically accumulate at the sites of inflammation/tissue damage [16-18]. After entering the CNS using constitutively functional cell adhesion molecules and inflam‐ matory chemokine receptors, systemically injected NPCs accumulate at the level of perivas‐ cular CNS areas, where they establish *atypical ectopic perivascular niches* [16, 19]. In these areas, a much likely active cell-to-cell communication takes place between transplanted NPCs and the different cells of the *atypical niche*. As consequence of this, transplanted NPCs survive while displaying undifferentiated features, and promote neuroprotection by releasing immune modulatory molecules and neurotrophic factors *in situ*. Further evidence exists about an additional peripheral immune-modulatory effect exerted by NPCs [20, 21]. Systemically injected NPCs, in fact, enter also peripheral organs (e.g. draining lymph nodes and spleen) were they accumulate at the boundaries of blood vessels and interact closely with lymphocytes and professional antigen presenting cells (APCs), impairing their maturation and functional

NPC-based therapies have been therefore considered a plausible alternative strategy for the treatment of neurological inflammatory disorders. However, some urgent and still unclear questions have to be solved prior to straightforwardly translate most of these exciting experimental observations into clinical medicines, such as: (i) the ideal stem cell source, whether it has to be derived from pluripotent or multipotent sources; (ii) the ideal route of cell administration, whether it has to be focal or systemic; (iii) the optimal time point for cell administration, depending on the disease characteristics; (iv) the ideal balance between differentiation and persistence of stem cells into the targeted tissue and (v) the ideal mechanism of tissue repair to foster, whether it has to be cell replacement or tissue protection/healing. Further, while some encouraging efforts are being devoted towards the development of guidelines and establishment of explorative phase I clinical trials, still one of the major constraints to the easy translation into human medicines is represented by the immunogenicity of allogeneic stem cells, and the modest expandability of somatic human NPCs *in vitro*. Within this scenario, the emerging figure of induced pluripotent stem (iPS) cells [24], induced neuronal (iN) cells [25] and/or induced neural stem cells (iNSCs) [26] holds a new exciting promise.

In this chapter we will describe the most recent evidence of the remarkable therapeutic plasticity of transplanted NPCs, when injected systemically in inflammation-driven CNS degeneration experimental models. We will first focus on the evidence that inspired the modern stem cell experimental therapies and then elaborate on the mechanisms regulating the

activation [15, 22, 23].

288 Neural Stem Cells - New Perspectives

Stem cells (SCs) possess the unique ability to self-renew and differentiate into different cell types in the body. Their contribution is essential during embryonic and early post-natal life, where they regulate morphogenesis and development by properly balancing proliferation and differentiation. Though their number is destined to decrease with time, their presence in adult organisms is still required to ensure *homeostasis* and *repair*. While the regenerating properties of some tissues (e.g., the skin) and organs (e.g., the liver) are undisputed, the brain with its unique organization and complexity was considered for long time an exception. In fact, the dogmatic concept '*no new neurons after birth*' (1913) expressed by Santiago Ramon y Cajal, sustaining the limitation of neurogenesis to prenatal life, has been resonating for decades within the scientific community, finally becoming an established belief. NSCs were thought to be present within the brain only during the developmental stage. It was only in the late 60's, thanks to the availability of new techniques and advanced tools of investigation, that the picture of the brain as an immutable organ started to be reviewed. Altman and colleagues, using (3 H)-thymidine pulses and autoradiographs, first demonstrated the presence of prolif‐ erating neurons in different regions of the post-natal brain in rats [1, 2, 27]. However, the turning point was marked later in 1983 when Goldman and Nottebohm at Rockfeller Univer‐ sity (USA) described newly generated neurons at the level of the hyperstriatum ventrale, pars caudalis (HVc), of the ventricular zone in intact adult female canaries [3] Subsequently, numerous pioneering experiments contributed in demonstrating that specific regions of the mammalian CNS undergo a continuous, though moderate, level of neurogenesis throughout adult life [28].

#### **2.2. Adult neurogenesis in physiological conditions**

Today it is widely accepted that in the adult mammalian brain, newly generated cells derive from NSCs residing in two regions [29], the ventricular-subventricular zone (V-SVZ) of the forebrain lateral ventricles [2, 27, 30] and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus [1, 31, 32] (Figure 1). Because of the peculiar cellular organization and exclusive microenvironment, these neurogenic regions are commonly referred to as *germinallike niches* [33, 34]. Although different, these two areas share an extremely organized and specialized microenvironment where NSCs can strategically interact with a rich vascular plexus [35, 36], while communicating with their progeny and neighbouring NSCs as well as with differentiated neural cells trough specialized structures (e.g. primary cilium, basal and apical processes). Altogether, these cellular components provide a unique milieu of extracel‐ lular matrix proteins and growth factors other than electrical stimuli, which define the dynamic characteristic of the adult brain stem cell *niches*. In here, a strictly regulated balance between proliferation and differentiation of NSCs ensure the maintenance of a constant, though quantitatively modest, pool of progenitor cells throughout lifetime [37].

cells. These cells give rise to intermediate progenitor cells (IPCs) or type C cells, which lose GFAP immunoreactivity and acquire the expression of the distal-less homeobox (Dlx)-2. These cells finally give origin to a pool of neuroblasts (type A cells) expressing the polysia‐ lylated form of neural cell adhesion molecule (PSA-NCAM) and the early neuronal marker doublecourtin (DCX) [38]. Within rodent's brain these neuroblasts form chains of migra‐ tion along the rostral migratory stream (RMS) to reach the olfactory bulb (OB), where they terminally differentiate into at least six different subtypes of OB interneurons, depending on their origin along the axes of the V-SVZ [39-41]. The V-SVZ niche (Figure 1 A-B) can be divided in three differently organized domains where self-renewing B1 cells receive different signals: *proximal* (or apical, I), *intermediate* (II) and *distal* (or basal, III) [37]. Type B1 cells retain the typical apical-basal bi-polarity of their embryonic predecessors (radial glia) [42] extend‐ ing their processes along the three different domains and spanning the cerebrospinal fluid (CSF) and the blood stream. In the proximal domain (composed by VZ and part of the SVZ) Type B1 cells are enclosed within a cluster of ependymal (type E) cells, which sense the CSF by means of motile cilia and create an appropriate gradient of molecules within the VZ [43]. Type B1 cells are therefore physically separated from the ventricles. Nevertheless, their contact with the CSF is still made possible by a single apical primary cilium extruding in the centre of a rosette of type E cells. Typically, these apical end-foot terminations cluster together to finally arise in the middle of a layer of E cells forming a characteristic *pinwheel structure* resembling the embryonic forebrain germinal zone [36, 42]. Recently, it has been shown that the expression of the adhesion and signalling molecule vascular cell adhesion molecule (VCAM)-1 is critical for the correct positioning of these protrusions and the preservation of this complex structure [44]. The small apical surface of B1 cells gives them the chance to sense the CSF which contains soluble factors, such as insulin-like growth factor (IGF)-2, bone morphogenetic proteins (BMPs) and Noggin, Wnts, Sonic hedgehog (Shh) and retinoic acid, able to modulate NSCs behaviour[45]. At the same time a long basal process from the opposite pole (distal domain), bridges B1 cells to the surrounding vascular plexus that runs in the parenchymal side of the V-SVZ. Here, with a specialized end-foot termination, type B1 cells contact endothelial cells (ECs) of the blood vessels, thus being influenced from soluble factors released from ECs and/or possibly by molecules produced far away from the niche and released in the blood stream. The intermediate domain (composed by the SVZ) contains B1 cell progeny, such as IPCs and neuroblasts, which participate in the maintenance of the niche equilibrium perhaps trough mechanisms of direct feedback on NSCs providing informa‐ tion about the number of new neurons already generated. This balance, seems to be regulated on one side by canonical Notch signalling trough ligands released or expressed by both IPCs and neighbouring B1 cells [46, 47] and on the other side by neurotransmitters [e.g. gammaaminobutyric acid (GABA)] secreted by neuroblasts [48]. Importantly, while many studies have focussed on the role of the microenvironment on the functionality of NSCs [49], much less is known about the role that NSCs themselves exert on the definition of the niche. Recently it has been shown that NSCs in the germinal niches do secrete a multitude of factors, among which some with immune modulatory potentials that may influence the behaviour of the surrounding cells, including microglia [50]. In parallel to the rodent CNS, the lateral wall of the lateral ventricles (and the hippocampus) of the human brain contains NSCs that generate

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

291

**Figure 1. Schematic representations of the adult V-SVZ and SGZ neurogenic compartments.A** and **C**, coronal sec‐ tions of the adult mouse brain showing the localization of the V-SVZ and SGZ of the hippocampus. **B** and **D**, cytoarchi‐ tecture of the V-SVZ (**B**), and of the SGZ of the DG of the hippocampus (**D**) in the adult mammalian brain. **B**, Composition of the B1 cell domain into the V-SVZ. NSCs or type B1 cells (blue) extend from the proximal domain (do‐ main I, dark grey) to the distal domain (domain III, light grey). At the level of the ventricles, B1 cells contact the CSF with their primary cilium extruding in the centre of a rosette of multi-ciliated ependymal cells (yellow), forming the typical pinwheel-like structures on the ventricular surface. Here, NSCs can sense different signals circulating into the CSF. In the distal domain, type B1 cells contact the blood vessels (red) with their specialized end-foot terminations. In the intermediate domain (or domain II) type B1 cells give rise to IPCs (or type C cells, green), which are transit-amplify‐ ing cells generating neuroblasts (or type A cells, red). In this domain they are also in contact with their progeny, neigh‐ bouring cells and neuronal terminations. **D**, Composition of the RA domain at the level of the DG of the SGZ. RAs (or type 1 cells, blue) extend from the hilus of the hippocampus (domain I, dark gray) to the IML (distal domain or domain III, light gray). At the level of domain I, RAs sense the hilus microenvironment with their primary cilium and contact other RAs, IPCs and blood vessels (red). RAs extend, trough their main shaft, into the distal domain where their arbori‐ sations receive signals from glial cells and neuronal terminations. RAs give rise to IPCs that mature (trough blue IPC1 or type 2a cells, and light green IPC2 or type 2b cells) and differentiate into immature granule cells (IGC, red). During their maturation, IPCs move from the proximal domain to the intermediate domain (or domain II, composed by SGZ and GCL), where RAs receive signals from the progeny, neighbouring NSCs, interneurons (purple) and microglia (grey). Finally IGC differentiate into mature GC (green), which extend their axons into the hilus and arbores dendrites into the distal domain. Only few new-born neurons survive and become a long-lasting GC (pink).

#### *2.2.1. Defining the cellular composition of the V-SVZ*

The V-SVZ is situated in proximity of the lateral ventricles and contains slow-cycling SCs with astroglial properties that express glial-fibrillary acidic protein (GFAP), called type B1 cells. These cells give rise to intermediate progenitor cells (IPCs) or type C cells, which lose GFAP immunoreactivity and acquire the expression of the distal-less homeobox (Dlx)-2. These cells finally give origin to a pool of neuroblasts (type A cells) expressing the polysia‐ lylated form of neural cell adhesion molecule (PSA-NCAM) and the early neuronal marker doublecourtin (DCX) [38]. Within rodent's brain these neuroblasts form chains of migra‐ tion along the rostral migratory stream (RMS) to reach the olfactory bulb (OB), where they terminally differentiate into at least six different subtypes of OB interneurons, depending on their origin along the axes of the V-SVZ [39-41]. The V-SVZ niche (Figure 1 A-B) can be divided in three differently organized domains where self-renewing B1 cells receive different signals: *proximal* (or apical, I), *intermediate* (II) and *distal* (or basal, III) [37]. Type B1 cells retain the typical apical-basal bi-polarity of their embryonic predecessors (radial glia) [42] extend‐ ing their processes along the three different domains and spanning the cerebrospinal fluid (CSF) and the blood stream. In the proximal domain (composed by VZ and part of the SVZ) Type B1 cells are enclosed within a cluster of ependymal (type E) cells, which sense the CSF by means of motile cilia and create an appropriate gradient of molecules within the VZ [43]. Type B1 cells are therefore physically separated from the ventricles. Nevertheless, their contact with the CSF is still made possible by a single apical primary cilium extruding in the centre of a rosette of type E cells. Typically, these apical end-foot terminations cluster together to finally arise in the middle of a layer of E cells forming a characteristic *pinwheel structure* resembling the embryonic forebrain germinal zone [36, 42]. Recently, it has been shown that the expression of the adhesion and signalling molecule vascular cell adhesion molecule (VCAM)-1 is critical for the correct positioning of these protrusions and the preservation of this complex structure [44]. The small apical surface of B1 cells gives them the chance to sense the CSF which contains soluble factors, such as insulin-like growth factor (IGF)-2, bone morphogenetic proteins (BMPs) and Noggin, Wnts, Sonic hedgehog (Shh) and retinoic acid, able to modulate NSCs behaviour[45]. At the same time a long basal process from the opposite pole (distal domain), bridges B1 cells to the surrounding vascular plexus that runs in the parenchymal side of the V-SVZ. Here, with a specialized end-foot termination, type B1 cells contact endothelial cells (ECs) of the blood vessels, thus being influenced from soluble factors released from ECs and/or possibly by molecules produced far away from the niche and released in the blood stream. The intermediate domain (composed by the SVZ) contains B1 cell progeny, such as IPCs and neuroblasts, which participate in the maintenance of the niche equilibrium perhaps trough mechanisms of direct feedback on NSCs providing informa‐ tion about the number of new neurons already generated. This balance, seems to be regulated on one side by canonical Notch signalling trough ligands released or expressed by both IPCs and neighbouring B1 cells [46, 47] and on the other side by neurotransmitters [e.g. gammaaminobutyric acid (GABA)] secreted by neuroblasts [48]. Importantly, while many studies have focussed on the role of the microenvironment on the functionality of NSCs [49], much less is known about the role that NSCs themselves exert on the definition of the niche. Recently it has been shown that NSCs in the germinal niches do secrete a multitude of factors, among which some with immune modulatory potentials that may influence the behaviour of the surrounding cells, including microglia [50]. In parallel to the rodent CNS, the lateral wall of the lateral ventricles (and the hippocampus) of the human brain contains NSCs that generate

lular matrix proteins and growth factors other than electrical stimuli, which define the dynamic characteristic of the adult brain stem cell *niches*. In here, a strictly regulated balance between proliferation and differentiation of NSCs ensure the maintenance of a constant, though

**Figure 1. Schematic representations of the adult V-SVZ and SGZ neurogenic compartments.A** and **C**, coronal sec‐ tions of the adult mouse brain showing the localization of the V-SVZ and SGZ of the hippocampus. **B** and **D**, cytoarchi‐ tecture of the V-SVZ (**B**), and of the SGZ of the DG of the hippocampus (**D**) in the adult mammalian brain. **B**, Composition of the B1 cell domain into the V-SVZ. NSCs or type B1 cells (blue) extend from the proximal domain (do‐ main I, dark grey) to the distal domain (domain III, light grey). At the level of the ventricles, B1 cells contact the CSF with their primary cilium extruding in the centre of a rosette of multi-ciliated ependymal cells (yellow), forming the typical pinwheel-like structures on the ventricular surface. Here, NSCs can sense different signals circulating into the CSF. In the distal domain, type B1 cells contact the blood vessels (red) with their specialized end-foot terminations. In the intermediate domain (or domain II) type B1 cells give rise to IPCs (or type C cells, green), which are transit-amplify‐ ing cells generating neuroblasts (or type A cells, red). In this domain they are also in contact with their progeny, neigh‐ bouring cells and neuronal terminations. **D**, Composition of the RA domain at the level of the DG of the SGZ. RAs (or type 1 cells, blue) extend from the hilus of the hippocampus (domain I, dark gray) to the IML (distal domain or domain III, light gray). At the level of domain I, RAs sense the hilus microenvironment with their primary cilium and contact other RAs, IPCs and blood vessels (red). RAs extend, trough their main shaft, into the distal domain where their arbori‐ sations receive signals from glial cells and neuronal terminations. RAs give rise to IPCs that mature (trough blue IPC1 or type 2a cells, and light green IPC2 or type 2b cells) and differentiate into immature granule cells (IGC, red). During their maturation, IPCs move from the proximal domain to the intermediate domain (or domain II, composed by SGZ and GCL), where RAs receive signals from the progeny, neighbouring NSCs, interneurons (purple) and microglia (grey). Finally IGC differentiate into mature GC (green), which extend their axons into the hilus and arbores dendrites into the

The V-SVZ is situated in proximity of the lateral ventricles and contains slow-cycling SCs with astroglial properties that express glial-fibrillary acidic protein (GFAP), called type B1

distal domain. Only few new-born neurons survive and become a long-lasting GC (pink).

*2.2.1. Defining the cellular composition of the V-SVZ*

quantitatively modest, pool of progenitor cells throughout lifetime [37].

290 Neural Stem Cells - New Perspectives

new neurons throughout adult life [51-53]. A total of four layers have been observed forming the human lateral ventricular wall, which comprise a monolayer of ependymal cells, a hypocellular gap, a ribbon of astrocytes, and a transitional zone into the brain parenchyma [52, 54]. Unlike the rodent and non-human primate brain [55], SVZ astrocytes of the human brain are separated from the ependyma by a hypocellular gap [52]. The presence of promi‐ nent neurogenesis in the V-SVZ as well as of a RMS of migrating neuroblasts in the human brain has been, however, intensively debated (for a preview, see [56]). Initially it was reported the existence of a ribbon of astrocytes in the adult human V-SVZ that function as multipo‐ tent NSCs in culture although, only few proliferating cells and no evidence of chains of migratory (β-III tubulin positive) immature neurons were observed [52]. In contrast, a later report evidenced a robust cell proliferation in adult human V-SVZ and the presence of a RMS of neuroblasts along a lateral ventricular extension that connects the lateral ventricle to the OB [51]. Finally, two recent studies have provided evidence of a small ventricular lumen connecting the lateral ventricles to the OB that is observed only in the foetal [57], but not adult, human brain [55, 58]. Interestingly, the absence of this ventricular extension has been confirmed even in the postnatal infant human brain [58], whereas a new medial migratory stream (MMS) targeting the prefrontal cortex has been observed. Altogether these findings suggest a dynamic evolution in human SVZ neurogenesis throughout life; with the infant human SVZ, RMS and MMS activity, undergoing a progressive extinction at ages older than 18 months post-natal [58].

the intermediate domain. In this area astrocytes receive inputs from their progeny, including immature and mature granule neurons, IPCs and different neuronal and glial (e.g. microglia) cell types. Type 2a cells expressing Achaete-scute homolog (Ascl)-1 (also known as Mash-1) a transcription factor important for neuronal commitment - are likely to originate in the proximal domain and then rapidly migrate into the intermediate one, where they divide once before differentiating into type 2b cells that will express DCX [62]. Similarly to V-SVZ, feedback mechanisms from the progeny, such as canonical and non-canonical Notch signalling are responsible for the quiescence of RAs or their transition to IPCs [63, 64]. In the IML, RAs terminate with an elaborate and branched structure contacting glial cells, neuronal processes and synapses. Although the contacts taking place in this area are still not completely under‐ stood, it seems probable that the GABAergic and glutamatergic inputs coming from inter‐ neurons and mossy cells, are important for the regulation of NSCs [65]. These astrocyte-like cells of CNS germinal areas work as real pacemakers of adult neurogenesis, as they receive internal and external inputs from their main shaft as well as from the end-foot of their radial processes that contact ECs in the V-SVZ [42], or embedded into the molecular layer in the SGZ [41]. However, despite the relatively high rate of neurogenesis, only a minority of new born cells eventually survive, mature and integrate within the existing circuitries at the level of the GCL of the hippocampus [66]. In parallel, postnatal SGZ neurogenesis in the human brain has been demonstrated to occur across the lifespan [55]. Although the role of new born neurons generating in the SGZ is not yet fully understood, increasing evidence suggest a possible role

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**3. CNS inflammation effects on endogenous adult NSC niches**

**3.1. Switching from an immune-privileged to an immune-specialized state**

(microglia and monocyte-derived macrophages) and the adaptive (mainly CD4+

system are present within the brain parenchyma and exert beneficial effects on adult brain plasticity and neurogenesis, as well as on the spontaneous attempt of the CNS to self-repair

cells) immune

Protection and homeostasis are fundamental keystones for the proper maintenance of the CNS. Hence, brain and spinal cord must be kept under an extreme security state to ensure their fully functionality and, ultimately, the survival of an organism. However, in the past the CNS has been often regarded as an immune *privileged* site, where immune cells were not supposed to enter and interact with cells of the nervous system. This common belief was strongly supported by observations showing lack of lymphatic vessels, absence of parenchymal APCs, low expression of constitutive major histocompatibility complex (MHC) class I and II molecules within the brain parenchyma, as well as poor rejection of transplanted allo- or xeno-graft. In the last decades this historical concept has been extensively revised, and there is now con‐ vincing evidence that the CNS is instead an immune *specialized* site, where a complex regimen of immune surveillance does occur under physiological as well as pathological conditions and is essential to guarantee its optimal functionality [67]. It is now clear that cells of both the innate

in learning and memory function [55].

following an injury [68].

#### *2.2.2. Defining the cellular composition of the SGZ*

The second putative progenitor cell compartment is located in the SGZ of the DG of the hippocampus (Figure 1C-D), namely the region of the brain involved in learning and memory [1, 31, 32]. In this area, NSCs residing at the interface of the hilus and dentate gyrus are called type-1 progenitors or radial astrocytes (RAs) [59] and they mainly correspond to astroglial cells [60]. They mature in dentate granule cells and migrate towards the granule cell layer (GCL) to finally integrate into hippocampal circuitry [59]. RAs, unlike B1 cells of the V-SVZ, are found deeper into the brain parenchyma, surrounded by neurons, neighbouring RAs and other glial cells but without any chance to contact the CSF [37]. However, B1 cells and RAs share some key features: they both express astroglial markers, have ultrastructural characteristics of astrocytes [41] and possess long processes reaching different compartments of the niche far away from where the cell bodies reside [37]. RAs function as the primary precursors for the generation of new dentate granule neurons, either directly or via the generation of IPC1 (type 2a cells) and IPC2 (type 2b cells) [61]. Similarly to the V-SVZ, also the SGZ can be subdivided in a proximal, intermediate and distal domain along which RAs, with their polarized structure (apical-basal), span from the hilus interface (proximal domain) to the inner molecular layer (IML, distal domain) [37]. The proximal domain contains the primary cilium (important for Sonic hedgehog (Shh) signalling), which sense the hilus microenvironment, and lateral processes contacting other RAs and IPCs and, importantly, blood vessels. Here ECs release vascular endothelial growth factor (VEGF), IGF and brain-derived neurotrophic factor (BDNF) responsible for the regulation of the balance between proliferation and differentiation. RAs have their cell bodies in the SGZ and extend their main shaft along the GCL, which compose the intermediate domain. In this area astrocytes receive inputs from their progeny, including immature and mature granule neurons, IPCs and different neuronal and glial (e.g. microglia) cell types. Type 2a cells expressing Achaete-scute homolog (Ascl)-1 (also known as Mash-1) a transcription factor important for neuronal commitment - are likely to originate in the proximal domain and then rapidly migrate into the intermediate one, where they divide once before differentiating into type 2b cells that will express DCX [62]. Similarly to V-SVZ, feedback mechanisms from the progeny, such as canonical and non-canonical Notch signalling are responsible for the quiescence of RAs or their transition to IPCs [63, 64]. In the IML, RAs terminate with an elaborate and branched structure contacting glial cells, neuronal processes and synapses. Although the contacts taking place in this area are still not completely under‐ stood, it seems probable that the GABAergic and glutamatergic inputs coming from inter‐ neurons and mossy cells, are important for the regulation of NSCs [65]. These astrocyte-like cells of CNS germinal areas work as real pacemakers of adult neurogenesis, as they receive internal and external inputs from their main shaft as well as from the end-foot of their radial processes that contact ECs in the V-SVZ [42], or embedded into the molecular layer in the SGZ [41]. However, despite the relatively high rate of neurogenesis, only a minority of new born cells eventually survive, mature and integrate within the existing circuitries at the level of the GCL of the hippocampus [66]. In parallel, postnatal SGZ neurogenesis in the human brain has been demonstrated to occur across the lifespan [55]. Although the role of new born neurons generating in the SGZ is not yet fully understood, increasing evidence suggest a possible role in learning and memory function [55].

new neurons throughout adult life [51-53]. A total of four layers have been observed forming the human lateral ventricular wall, which comprise a monolayer of ependymal cells, a hypocellular gap, a ribbon of astrocytes, and a transitional zone into the brain parenchyma [52, 54]. Unlike the rodent and non-human primate brain [55], SVZ astrocytes of the human brain are separated from the ependyma by a hypocellular gap [52]. The presence of promi‐ nent neurogenesis in the V-SVZ as well as of a RMS of migrating neuroblasts in the human brain has been, however, intensively debated (for a preview, see [56]). Initially it was reported the existence of a ribbon of astrocytes in the adult human V-SVZ that function as multipo‐ tent NSCs in culture although, only few proliferating cells and no evidence of chains of migratory (β-III tubulin positive) immature neurons were observed [52]. In contrast, a later report evidenced a robust cell proliferation in adult human V-SVZ and the presence of a RMS of neuroblasts along a lateral ventricular extension that connects the lateral ventricle to the OB [51]. Finally, two recent studies have provided evidence of a small ventricular lumen connecting the lateral ventricles to the OB that is observed only in the foetal [57], but not adult, human brain [55, 58]. Interestingly, the absence of this ventricular extension has been confirmed even in the postnatal infant human brain [58], whereas a new medial migratory stream (MMS) targeting the prefrontal cortex has been observed. Altogether these findings suggest a dynamic evolution in human SVZ neurogenesis throughout life; with the infant human SVZ, RMS and MMS activity, undergoing a progressive extinction at ages older than

The second putative progenitor cell compartment is located in the SGZ of the DG of the hippocampus (Figure 1C-D), namely the region of the brain involved in learning and memory [1, 31, 32]. In this area, NSCs residing at the interface of the hilus and dentate gyrus are called type-1 progenitors or radial astrocytes (RAs) [59] and they mainly correspond to astroglial cells [60]. They mature in dentate granule cells and migrate towards the granule cell layer (GCL) to finally integrate into hippocampal circuitry [59]. RAs, unlike B1 cells of the V-SVZ, are found deeper into the brain parenchyma, surrounded by neurons, neighbouring RAs and other glial cells but without any chance to contact the CSF [37]. However, B1 cells and RAs share some key features: they both express astroglial markers, have ultrastructural characteristics of astrocytes [41] and possess long processes reaching different compartments of the niche far away from where the cell bodies reside [37]. RAs function as the primary precursors for the generation of new dentate granule neurons, either directly or via the generation of IPC1 (type 2a cells) and IPC2 (type 2b cells) [61]. Similarly to the V-SVZ, also the SGZ can be subdivided in a proximal, intermediate and distal domain along which RAs, with their polarized structure (apical-basal), span from the hilus interface (proximal domain) to the inner molecular layer (IML, distal domain) [37]. The proximal domain contains the primary cilium (important for Sonic hedgehog (Shh) signalling), which sense the hilus microenvironment, and lateral processes contacting other RAs and IPCs and, importantly, blood vessels. Here ECs release vascular endothelial growth factor (VEGF), IGF and brain-derived neurotrophic factor (BDNF) responsible for the regulation of the balance between proliferation and differentiation. RAs have their cell bodies in the SGZ and extend their main shaft along the GCL, which compose

18 months post-natal [58].

292 Neural Stem Cells - New Perspectives

*2.2.2. Defining the cellular composition of the SGZ*

#### **3. CNS inflammation effects on endogenous adult NSC niches**

#### **3.1. Switching from an immune-privileged to an immune-specialized state**

Protection and homeostasis are fundamental keystones for the proper maintenance of the CNS. Hence, brain and spinal cord must be kept under an extreme security state to ensure their fully functionality and, ultimately, the survival of an organism. However, in the past the CNS has been often regarded as an immune *privileged* site, where immune cells were not supposed to enter and interact with cells of the nervous system. This common belief was strongly supported by observations showing lack of lymphatic vessels, absence of parenchymal APCs, low expression of constitutive major histocompatibility complex (MHC) class I and II molecules within the brain parenchyma, as well as poor rejection of transplanted allo- or xeno-graft. In the last decades this historical concept has been extensively revised, and there is now con‐ vincing evidence that the CNS is instead an immune *specialized* site, where a complex regimen of immune surveillance does occur under physiological as well as pathological conditions and is essential to guarantee its optimal functionality [67]. It is now clear that cells of both the innate (microglia and monocyte-derived macrophages) and the adaptive (mainly CD4+ cells) immune system are present within the brain parenchyma and exert beneficial effects on adult brain plasticity and neurogenesis, as well as on the spontaneous attempt of the CNS to self-repair following an injury [68].

#### **3.2. Effects of inflammation on neurogenesis**

Studies conducted over the last decade have extensively proved that the immune and nervous systems interact by engaging an active bidirectional crosstalk. Indeed, the expression of receptors able to recognize inflammatory mediators released by activated immune cells allows endogenous progenitor cells to increase their proliferation rate and specifically home to the site of inflammation after a trauma. As a consequence, both acute [69, 70] and chronic CNS inflammation [6, 71] has been shown to perturb the anatomical architecture and functional activity of adult germinal niches.

pre-immunized with a sub-clinical amount of myelin peptides, allowed to better analyse the time course effect of auto-immune inflammation in the neurogenic areas. In this experimen‐ tal model a decreased proliferation in the proximity of the V-SVZ was observed at 3 days, followed by an increase at 7 days after the injection of the cytokines, suggesting a regenera‐ tive attempt at the level of the V-SVZ area. Interestingly, the concomitant death of neuro‐ blasts, the decreased type C cell proliferation, and the reduction of type A migrating cells, during the initial phase, might explain the impaired long-term olfactory memory observed by means of behavioural analysis [75]. Altogether, these findings suggest the existence of a compensatory mechanism of the injured brain in its attempt to counteract neuronal injury and disturbed conductivity resulting from T cell attack to the myelin sheaths wrapping the

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In agreement with what described in animal models, SVZ activation and expansion have been found at the level of periventricular active and chronic active lesions in MS patients, thus suggesting that the repetitive exposure to inflammatory insults does not completely exhaust the proliferative potential of the SVZ [77]. V-SVZ from post-mortem brains shows an altered balance between neurogenesis and gliogenesis, likely related to these inflammation effects within the neurogenic niche of MS patients [78]. Interestingly, the majority of MS patients show deficits in attention, information processing capacity and long-term memory, thus suggesting that neuronal damage in MS can result not only in motor and sensory deficits but also cognitive impairment. In support of these MRI techniques revealed structural alterations in the hippo‐

Acute events, occurring in non-autoimmune diseases such as stroke, have been similarly proved of giving rise to increased proliferation of endogenous NSCs in the V-SVZ. These cells migrate from the neurogenic niche towards the ischemic boundary regions of the striatum and cerebral cortex, where they differentiate into mature striatal neurons [79-81]. During this (injury-reactive) site-specific homing, newly generated neuroblasts form chain-like structures in association with reactive astrocytes and blood vessels in the striatum, a reminiscence of the embryonic migration of type A cells along the RMS [82, 83]. Initially, this potential self-repair mechanism was supposed to happen only during the acute post-stroke phase. However, subsequent studies showed that stroke-induced neurogenesis is an extensive and long-lasting (up to 2 weeks) event, with continuous production of mature striatal neurons for several months after the insult [84]. Unfortunately, the vast majority of migrating new born neurons die within few weeks after the ischemia, and only few damaged cells (about 0.1%) are replaced by newly generated neurons [85]. Similar evidence of stroke-induced neurogenesis has been reported in post-mortem brains, where new born neurons are present in the ischemic penum‐ bra surrounding cerebral cortical infarcts, preferentially localized in the vicinity of blood vessels [80]. The identification of those factors able to influence NSCs proliferation, homing and survival after stroke may have a great therapeutic impact. Several cytokines and growth factors that may be released by injured cells are thought to play a substantial role in promoting the observed neurogenic response after stroke. Among these, ciliary neurotrophic factor (CNTF) [86], transforming growth factor (TGF)-α [87], VEGF [88], fibroblast growth factor

axons, which is among the most accepted causes of EAE and MS [76].

campus, evidencing marked hippocampal atrophy [73].

(FGF)-2 [89] and erythropoietin (Epo) [90] have been proposed.

Work on EAE mice, the most widely accepted model of MS, has shown that chronic CNS inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-immunized mice causes a transient decrease in the proliferation rate of both C and B1 type cells and a contemporary increased accumulation of neuroblasts within the V-SVZ [6]. This effect, observed during the peak of the disease, was attributed to cell non-autonomous factors, such as pro-inflammato‐ ry (Th1) cytokines [e.g. interferon (IFN)- and its intracellular effector Stat-1]. However, these data contrast with other studies showing how inflammatory demyelination in MOG+/- mice immunized with purified mouse myelin increased proliferation and mobilization of neural progenitor cells from the V-SVZ of adult mice. Surprisingly, while new born cells generat‐ ed at the level of V-SVZ commonly intended to differentiate into neurons, in response to EAE, these cells were able to generate astrocytes and oligodendrocytes as well, thus suggesting that inflammation can diverge (at least partially) their intrinsic nature [4]. Increased proliferation, measured in terms of BrdU-positive cells, has been found also at the level of the hippocampus both during the acute and chronic phases of the disease in MOG35-55 immunized mice. Similarly to the observed accumulation of neuroblasts in the V-SVZ, autoimmune inflammation leads to increased numbers of immature DCX-positive cells in the DG of the hippocampus [72]. Even though some alterations in the Notch, Wnt/βcatenin, Shh, and BDNF signalling pathways have been observed, their real contribution to the deregulation of hippocampal neurogenesis in the course of chronic autoimmune neuroinflammation needs to be further confirmed [72]. In addition, magnetic resonance imaging (MRI) techniques revealed structural alterations in the hippocampus, evidencing marked hippocampal atrophy [73], which may correlate with deficits in attention, informa‐ tion processing capacity and long-term memory observed in the majority of MS patients. Enhanced proliferation during the acute phase of the disease has been observed in proteoli‐ pid protein (PLP)139-151-induced relapsing EAE in SJL mice. However, during both the relapsing and chronic phase of the disease, the number of SVZ progenitors cells decreased, without changes in the ultrastructural features of the type B, C or A cells, but accompa‐ nied by an impaired maturation of oligodendrocyte progenitor cells (OPCs). This suggests that the chronic activation of glial cells (namely microglia and astrocytes) might be deleteri‐ ous for the repair potential of endogenous brain stem/progenitor cells. Indeed, minocyclineinduced inactivation of microglia during the chronic phase in relapsing-remitting EAE mice was associated with an improvement in the number of proliferating Sox2/Bromodeoxyuri‐ dine (BrdU)+ neural stem cells [74]. Finally, models of targeted focal EAE, obtained by stereotactic injection of cytokines [e.g. tumor necrosis factor (TNF)-α and INF-γ] in rodents pre-immunized with a sub-clinical amount of myelin peptides, allowed to better analyse the time course effect of auto-immune inflammation in the neurogenic areas. In this experimen‐ tal model a decreased proliferation in the proximity of the V-SVZ was observed at 3 days, followed by an increase at 7 days after the injection of the cytokines, suggesting a regenera‐ tive attempt at the level of the V-SVZ area. Interestingly, the concomitant death of neuro‐ blasts, the decreased type C cell proliferation, and the reduction of type A migrating cells, during the initial phase, might explain the impaired long-term olfactory memory observed by means of behavioural analysis [75]. Altogether, these findings suggest the existence of a compensatory mechanism of the injured brain in its attempt to counteract neuronal injury and disturbed conductivity resulting from T cell attack to the myelin sheaths wrapping the axons, which is among the most accepted causes of EAE and MS [76].

**3.2. Effects of inflammation on neurogenesis**

activity of adult germinal niches.

294 Neural Stem Cells - New Perspectives

Studies conducted over the last decade have extensively proved that the immune and nervous systems interact by engaging an active bidirectional crosstalk. Indeed, the expression of receptors able to recognize inflammatory mediators released by activated immune cells allows endogenous progenitor cells to increase their proliferation rate and specifically home to the site of inflammation after a trauma. As a consequence, both acute [69, 70] and chronic CNS inflammation [6, 71] has been shown to perturb the anatomical architecture and functional

Work on EAE mice, the most widely accepted model of MS, has shown that chronic CNS inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-immunized mice causes a transient decrease in the proliferation rate of both C and B1 type cells and a contemporary increased accumulation of neuroblasts within the V-SVZ [6]. This effect, observed during the peak of the disease, was attributed to cell non-autonomous factors, such as pro-inflammato‐ ry (Th1) cytokines [e.g. interferon (IFN)- and its intracellular effector Stat-1]. However, these data contrast with other studies showing how inflammatory demyelination in MOG+/- mice immunized with purified mouse myelin increased proliferation and mobilization of neural progenitor cells from the V-SVZ of adult mice. Surprisingly, while new born cells generat‐ ed at the level of V-SVZ commonly intended to differentiate into neurons, in response to EAE, these cells were able to generate astrocytes and oligodendrocytes as well, thus suggesting that inflammation can diverge (at least partially) their intrinsic nature [4]. Increased proliferation, measured in terms of BrdU-positive cells, has been found also at the level of the hippocampus both during the acute and chronic phases of the disease in MOG35-55 immunized mice. Similarly to the observed accumulation of neuroblasts in the V-SVZ, autoimmune inflammation leads to increased numbers of immature DCX-positive cells in the DG of the hippocampus [72]. Even though some alterations in the Notch, Wnt/βcatenin, Shh, and BDNF signalling pathways have been observed, their real contribution to the deregulation of hippocampal neurogenesis in the course of chronic autoimmune neuroinflammation needs to be further confirmed [72]. In addition, magnetic resonance imaging (MRI) techniques revealed structural alterations in the hippocampus, evidencing marked hippocampal atrophy [73], which may correlate with deficits in attention, informa‐ tion processing capacity and long-term memory observed in the majority of MS patients. Enhanced proliferation during the acute phase of the disease has been observed in proteoli‐ pid protein (PLP)139-151-induced relapsing EAE in SJL mice. However, during both the relapsing and chronic phase of the disease, the number of SVZ progenitors cells decreased, without changes in the ultrastructural features of the type B, C or A cells, but accompa‐ nied by an impaired maturation of oligodendrocyte progenitor cells (OPCs). This suggests that the chronic activation of glial cells (namely microglia and astrocytes) might be deleteri‐ ous for the repair potential of endogenous brain stem/progenitor cells. Indeed, minocyclineinduced inactivation of microglia during the chronic phase in relapsing-remitting EAE mice was associated with an improvement in the number of proliferating Sox2/Bromodeoxyuri‐ dine (BrdU)+ neural stem cells [74]. Finally, models of targeted focal EAE, obtained by stereotactic injection of cytokines [e.g. tumor necrosis factor (TNF)-α and INF-γ] in rodents

In agreement with what described in animal models, SVZ activation and expansion have been found at the level of periventricular active and chronic active lesions in MS patients, thus suggesting that the repetitive exposure to inflammatory insults does not completely exhaust the proliferative potential of the SVZ [77]. V-SVZ from post-mortem brains shows an altered balance between neurogenesis and gliogenesis, likely related to these inflammation effects within the neurogenic niche of MS patients [78]. Interestingly, the majority of MS patients show deficits in attention, information processing capacity and long-term memory, thus suggesting that neuronal damage in MS can result not only in motor and sensory deficits but also cognitive impairment. In support of these MRI techniques revealed structural alterations in the hippo‐ campus, evidencing marked hippocampal atrophy [73].

Acute events, occurring in non-autoimmune diseases such as stroke, have been similarly proved of giving rise to increased proliferation of endogenous NSCs in the V-SVZ. These cells migrate from the neurogenic niche towards the ischemic boundary regions of the striatum and cerebral cortex, where they differentiate into mature striatal neurons [79-81]. During this (injury-reactive) site-specific homing, newly generated neuroblasts form chain-like structures in association with reactive astrocytes and blood vessels in the striatum, a reminiscence of the embryonic migration of type A cells along the RMS [82, 83]. Initially, this potential self-repair mechanism was supposed to happen only during the acute post-stroke phase. However, subsequent studies showed that stroke-induced neurogenesis is an extensive and long-lasting (up to 2 weeks) event, with continuous production of mature striatal neurons for several months after the insult [84]. Unfortunately, the vast majority of migrating new born neurons die within few weeks after the ischemia, and only few damaged cells (about 0.1%) are replaced by newly generated neurons [85]. Similar evidence of stroke-induced neurogenesis has been reported in post-mortem brains, where new born neurons are present in the ischemic penum‐ bra surrounding cerebral cortical infarcts, preferentially localized in the vicinity of blood vessels [80]. The identification of those factors able to influence NSCs proliferation, homing and survival after stroke may have a great therapeutic impact. Several cytokines and growth factors that may be released by injured cells are thought to play a substantial role in promoting the observed neurogenic response after stroke. Among these, ciliary neurotrophic factor (CNTF) [86], transforming growth factor (TGF)-α [87], VEGF [88], fibroblast growth factor (FGF)-2 [89] and erythropoietin (Epo) [90] have been proposed.

Much less is known about the presence of adult neurogenesis after SCI. Most likely, this can be ascribed to a more diffuse scepticism concerning the existence of stem cells within the spinal cord. Indeed, even if the spinal cord is generally considered a non-neurogenic tissue, multi‐ potent precursors can be isolated and propagated *in vitro* [91, 92]. In addition, spinal neuro‐ genesis has been shown to occur to a limited extent in response to several types of trauma [93-95]. In a very recent study, the modulation of neurogenesis in the more canonical niches of the adult brain has been investigated following SCI in rats. Interestingly, BrdU+ positive cells were found to be significantly decreased both at the level of the V-SVZ and the SGZ in subacute [15 days post injury (dpi)] condition. However, while V-SVZ proliferation returns to normal levels at 90 dpi, this does not happen at the hippocampal level. This could be equally explained by either a higher plasticity in the V-SVZ or a higher sensibility of the SGZ [96]. Alterations in adult neurogenesis have been extensively observed in a multitude of models of other neurodegenerative diseases. Rats suffering by pilocarpine-induced temporal lobe epilepsy (TLE), exhibit increased neurogenesis in the V-SVZ [97] as well as in the SGZ [98] after a period of latency and then it lasts for several weeks following prolonged seizures activity. Further, status epilepticus (SE) seems to accelerate the maturation and integration of adult new born DG cells [99]. However, chronic TLE induces a decrease of neurogenesis, as children affected by frequent seizures show decreased numbers of newly generated neurons and proliferating cells [100]. Impaired (cell type specific) proliferation of V-SVZ as well as SGZ progenitors has been observed also in experimental models of Alzheimer's disease (AD) [101]. While it is not clear whether this is reflected in an increased [102] or decreased neurogenesis [103], mainly because of the high number of different models used [104, 105], a recent study suggested that abnormalities at the level of both the neurogenic niches might precede the onset of amyloid deposition and memory impairments [106]. Interestingly, in post-mortem brains of patients with AD, it has been observed an increase of neurogenesis into the SGZ accompa‐ nied by depletion in the V-SVZ [107].

The early (acute) post-traumatic phase of a neuroinflammatory process involves the action of resident microglial cells. These cells of myeloid origin are usually present in a resting but dynamic state, ready to shift their activity and undergo morphological and functional trans‐ formations in response to any kind of brain damage or injury [111, 112]. While on one side the selective ablation of microglia has been shown to exacerbate the ischemic injury in a mouse model of focal cerebral ischemia [113], on the other side mounting evidence indicates that chronic microglial activation may also contribute to the development and progression of neurodegenerative disorders, mainly through the release of pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and TNF-α [114]. As an example, infiltrating blood-derived macro‐ phages have been shown to exert a beneficial role in an experimental model of SCI, where they contribute to limit the action of activated resident microglia, whose prolonged presence would finally lead to detrimental consequences [115]. Indeed, when the activity of microglia is not properly contained, their action may lead to a prolonged (chronic) inflammation eventually

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Endogenous T cells are key components of the protective immunity process. As such, phys‐ iological trafficking of lymphocytes through the CNS is required to support the essential function of immune surveillance [116]. Cellular composition analysis of the CSF of healthy patients has revealed that up to 80% of the total number of cells is represented by central memory and effector memory T-cells. This atypical composition, which is very different from the one of the blood, also suggests a major role for the CSF in the defence of the CNS [117]. Indeed, the CSF drains the interstitial fluid of the CNS and brings CNS antigens to the cervical lymph nodes, thus supplying for the absence of a proper lymphatic drainage [118, 119]. Following the seminal observation that self-specific T-cells recognizing myelin basic protein were able to protect injured CNS neurons from secondary degeneration in a rat model of optic nerve crash injury [120], several studies further supported the idea that T cell-dependent autoimmunity might promote recovery from CNS injuries [121, 122]. These studies finally culminated with the idea that boosting T-cell response to CNS antigens by means of immuni‐ zation with CNS myelin-associated self-antigens could have enhanced this therapeutic potential [123-125]. Also, myelin-reactive T-cells possess neuroprotective effects, which may be essentially attributed to their ability to release neurotrophic factors such as BDNF, nerve growth factor (NGF) and CNTF [126]. Importantly though, auto-reactive T-cells showing this protective effect may turn out to be harmful if escaping the control exerted by the immune system, finally resulting into the development of autoimmune diseases such as MS. Therefore, a strict control is required to finely tune the balance between the *good* and the *bad* [76].

To further complicate the scene, several inflammatory mediators, such as TNF-α, TGF-β, IL-1, IL-6, IL-10 and IL-12 may have contrasting effects (e.g neuroprotective *vs.* neurotoxic) de‐ pending on the overall context. As an example, the role of IL-6 is crucial for the induction of EAE [127], and its overexpression exacerbates tissue injury in experimental models of SCI [128, 129]. Also, high levels of this inflammatory marker in the blood of patients undergoing inflammatory response after stroke correlates with the disease severity and poor clinical outcome [130]. Accordingly, the use of monoclonal antibody directed towards IL-6 proved to be beneficial in the treatment of acute SCI and MOG35-55-induced EAE [131, 132]. However,

culminating in the formation of fibrotic tissue.

#### **3.3. The double face of inflammation**

According to what described, it is suggestive that the CNS is able to start a beneficial, though limited, process of self-repair. However, most of the new born cells generated following injury are destined to die within few weeks, maybe due to a failure in their integration or due to the inflammatory milieu. Even if these cells have been shown to fully differentiate into mature neurons [82], the very low rate of occurrence imposes logical concerns regarding the thera‐ peutic value of this regenerative response to brain injury [81, 108]. Because of the rearrange‐ ments occurring in the neurogenic niches after an inflammatory event, the immune system has been accredited as one of the major responsible of this failure. This assumption is further corroborated by the observation that the increasing complexity of the immune system over the evolutionary process has been accompanied by a concomitant loss of regenerative capacity [109]. Also, several findings link inflammation to the pathogenesis of neurodegenerative disorders and anti-inflammatory drugs seem to be promising candidates for their treatment. Recent studies suggest that inflammation may indeed have a neuroprotective effect [110]. Nevertheless, the real effect of inflammation in several of these pathologies still needs to be completely clarified.

The early (acute) post-traumatic phase of a neuroinflammatory process involves the action of resident microglial cells. These cells of myeloid origin are usually present in a resting but dynamic state, ready to shift their activity and undergo morphological and functional trans‐ formations in response to any kind of brain damage or injury [111, 112]. While on one side the selective ablation of microglia has been shown to exacerbate the ischemic injury in a mouse model of focal cerebral ischemia [113], on the other side mounting evidence indicates that chronic microglial activation may also contribute to the development and progression of neurodegenerative disorders, mainly through the release of pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and TNF-α [114]. As an example, infiltrating blood-derived macro‐ phages have been shown to exert a beneficial role in an experimental model of SCI, where they contribute to limit the action of activated resident microglia, whose prolonged presence would finally lead to detrimental consequences [115]. Indeed, when the activity of microglia is not properly contained, their action may lead to a prolonged (chronic) inflammation eventually culminating in the formation of fibrotic tissue.

Much less is known about the presence of adult neurogenesis after SCI. Most likely, this can be ascribed to a more diffuse scepticism concerning the existence of stem cells within the spinal cord. Indeed, even if the spinal cord is generally considered a non-neurogenic tissue, multi‐ potent precursors can be isolated and propagated *in vitro* [91, 92]. In addition, spinal neuro‐ genesis has been shown to occur to a limited extent in response to several types of trauma [93-95]. In a very recent study, the modulation of neurogenesis in the more canonical niches of the adult brain has been investigated following SCI in rats. Interestingly, BrdU+ positive cells were found to be significantly decreased both at the level of the V-SVZ and the SGZ in subacute [15 days post injury (dpi)] condition. However, while V-SVZ proliferation returns to normal levels at 90 dpi, this does not happen at the hippocampal level. This could be equally explained by either a higher plasticity in the V-SVZ or a higher sensibility of the SGZ [96]. Alterations in adult neurogenesis have been extensively observed in a multitude of models of other neurodegenerative diseases. Rats suffering by pilocarpine-induced temporal lobe epilepsy (TLE), exhibit increased neurogenesis in the V-SVZ [97] as well as in the SGZ [98] after a period of latency and then it lasts for several weeks following prolonged seizures activity. Further, status epilepticus (SE) seems to accelerate the maturation and integration of adult new born DG cells [99]. However, chronic TLE induces a decrease of neurogenesis, as children affected by frequent seizures show decreased numbers of newly generated neurons and proliferating cells [100]. Impaired (cell type specific) proliferation of V-SVZ as well as SGZ progenitors has been observed also in experimental models of Alzheimer's disease (AD) [101]. While it is not clear whether this is reflected in an increased [102] or decreased neurogenesis [103], mainly because of the high number of different models used [104, 105], a recent study suggested that abnormalities at the level of both the neurogenic niches might precede the onset of amyloid deposition and memory impairments [106]. Interestingly, in post-mortem brains of patients with AD, it has been observed an increase of neurogenesis into the SGZ accompa‐

According to what described, it is suggestive that the CNS is able to start a beneficial, though limited, process of self-repair. However, most of the new born cells generated following injury are destined to die within few weeks, maybe due to a failure in their integration or due to the inflammatory milieu. Even if these cells have been shown to fully differentiate into mature neurons [82], the very low rate of occurrence imposes logical concerns regarding the thera‐ peutic value of this regenerative response to brain injury [81, 108]. Because of the rearrange‐ ments occurring in the neurogenic niches after an inflammatory event, the immune system has been accredited as one of the major responsible of this failure. This assumption is further corroborated by the observation that the increasing complexity of the immune system over the evolutionary process has been accompanied by a concomitant loss of regenerative capacity [109]. Also, several findings link inflammation to the pathogenesis of neurodegenerative disorders and anti-inflammatory drugs seem to be promising candidates for their treatment. Recent studies suggest that inflammation may indeed have a neuroprotective effect [110]. Nevertheless, the real effect of inflammation in several of these pathologies still needs to be

nied by depletion in the V-SVZ [107].

296 Neural Stem Cells - New Perspectives

**3.3. The double face of inflammation**

completely clarified.

Endogenous T cells are key components of the protective immunity process. As such, phys‐ iological trafficking of lymphocytes through the CNS is required to support the essential function of immune surveillance [116]. Cellular composition analysis of the CSF of healthy patients has revealed that up to 80% of the total number of cells is represented by central memory and effector memory T-cells. This atypical composition, which is very different from the one of the blood, also suggests a major role for the CSF in the defence of the CNS [117]. Indeed, the CSF drains the interstitial fluid of the CNS and brings CNS antigens to the cervical lymph nodes, thus supplying for the absence of a proper lymphatic drainage [118, 119]. Following the seminal observation that self-specific T-cells recognizing myelin basic protein were able to protect injured CNS neurons from secondary degeneration in a rat model of optic nerve crash injury [120], several studies further supported the idea that T cell-dependent autoimmunity might promote recovery from CNS injuries [121, 122]. These studies finally culminated with the idea that boosting T-cell response to CNS antigens by means of immuni‐ zation with CNS myelin-associated self-antigens could have enhanced this therapeutic potential [123-125]. Also, myelin-reactive T-cells possess neuroprotective effects, which may be essentially attributed to their ability to release neurotrophic factors such as BDNF, nerve growth factor (NGF) and CNTF [126]. Importantly though, auto-reactive T-cells showing this protective effect may turn out to be harmful if escaping the control exerted by the immune system, finally resulting into the development of autoimmune diseases such as MS. Therefore, a strict control is required to finely tune the balance between the *good* and the *bad* [76].

To further complicate the scene, several inflammatory mediators, such as TNF-α, TGF-β, IL-1, IL-6, IL-10 and IL-12 may have contrasting effects (e.g neuroprotective *vs.* neurotoxic) de‐ pending on the overall context. As an example, the role of IL-6 is crucial for the induction of EAE [127], and its overexpression exacerbates tissue injury in experimental models of SCI [128, 129]. Also, high levels of this inflammatory marker in the blood of patients undergoing inflammatory response after stroke correlates with the disease severity and poor clinical outcome [130]. Accordingly, the use of monoclonal antibody directed towards IL-6 proved to be beneficial in the treatment of acute SCI and MOG35-55-induced EAE [131, 132]. However, IL-6 knockout mice showed significantly increased of chronic (but not acute) lesion volumes and worse long-term functional outcome after stroke [133]. This may imply the need of a finely tuned regulation, most likely depending on their precise timing and location other than on the specific nature of the disease. Indeed, while some pathologies such as MS, SCI, stroke, TBI and are characterized by an acute inflammatory event followed by secondary neurodegeneration, others, such as epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), and Hunting‐ ton's disease (HD) are instead caused by primary neurodegeneration subsequently leading to secondary reactive inflammation [19].

mice receiving either mouse neurospheres (i.c.v) or single cell NPCs (i.v.), at 6 and 8 dpi

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299

The therapeutic efficacy of systemically administered NPCs has been later observed in a different pathological context, such as stroke. Mouse somatic NPCs, systemically transplanted 3 days after middle cerebral artery occlusion (MCAo), resulted in a better recovery, signifi‐ cantly improving the neurological severity score starting from 18 days post transplantation (dpt) until the end of the follow-up (30 dpt) [140]. Similar neurological improvements were observed in rats subjected to MCAo and common carotid artery occlusion (CCAo) after i.t. transplantation (7 days after stroke) of rat NPCs [141]. Significant locomotor recovery was also observed after acute systemic NPC transplant in mice suffering from contusion SCI [13].

Similarly to rodent cells, human NPCs have been proved to be therapeutically efficacious. Foetal NPCs administered either i.v. or i.t. at the disease onset, reduced the severity of MOG1-125-induced chronic EAE in common marmosets [15]. Further, human embryonic stem cell (ESC)-derived NPCs have been shown to reduce disease severity of chronic EAE mice [142]. Human immortalized NPCs have been widely enrolled in stroke models. The systemic injection of the HB1.F3 NPC line resulted in neurological improvements of rats treated 1 day after MCAo or intracerebral hemorrhage (ICH) stroke models [12, 137, 138]. The same line resulted therapeutically effective also in quinolinic acid-induced experimental HD in rats, where NPCs administered intravenously at 7 days after disease induction, significantly

All these evidences showing behavioural recovery upon systemic injection of NPCs in different CNS inflammatory models, led to the investigation of the molecular mechanisms standing behind, since this capacity bears the hope of developing less invasive surgical techniques to implant therapeutic adult human stem cells into patients affected by highly debilitating CNS

Brain and spinal cord are protected by a complex control system, composed by tight barriers shielding the action of a unique troop of immune cells. Indeed, to access the brain and spinal cord parenchyma, circulating cells have to breach through all the barriers that closely seal the CNS from the surrounding environment. Namely, these are the blood-brain barrier (BBB) at the level of parenchymal capillaries and post-capillary venules, the blood-cerebrospinal fluid barrier (BCSFB) at the level of the choroid plexus in the brain ventricles, and the bloodleptomeningeal barrier (BLMB) at the level of the leptomeningeal/subarachnoid space. The main role of these barriers is to maintain the chemical composition of the CNS microenviron‐ ment, thus ensuring the proper functionality of neuronal circuits, synaptic transmission and remodelling, angiogenesis, and neurogenesis in the adult brain, while their rupture is involved in many neuroinflammatory disorders [143]. Because of the existence of such barriers, the

access of systemically injected NPCs to the CNS parenchyma seems quite unlikely.

Structurally, the main component of the BBB is represented by specialized ECs characterized by the absence of fenestrae, low pinocytotic activity and by the presence of intercellular tight

respectively also showed significant clinical amelioration [20, 139].

ameliorated the behavioural outcome [14].

disorders, such as MS, stroke, SCI, epilepsy, PD, AD and HD.

**4.2. Homing capacity: NPCs breach the CNS barriers**

As described in this paragraph, the CNS is able to regulate the proliferation rate within the adult neurogenic niches as an extreme attempt to respond to damages in both primary and secondary inflammatory neurodegenerative diseases. Nevertheless, this process is not robust enough to effectively re-establish the complex functionality of the CNS. Therefore, protocols aiming at pharmacological manipulation of endogenous precursors from germinal niche(s), *in vivo,* might be therapeutically inefficacious in inflammatory CNS disorders. Thank to the development of protocols allowing *in vitro* growth and large scale-up of brain-derived NPCs [134], innovative therapies, for both acute and chronic CNS inflammatory disorders, based on stem cell transplants have been proposed [7]. Transplantation of adult exogenous NPCs represents, in fact, an alternative, and possibly more efficacious, therapeutic approach that might overcome the limited endogenous repair. Motivated by the ambitious expectation to achieve CNS repair (and/or regeneration) via functional neural cell replacement, many different preclinical studies have evidenced a potential benefit of NPC-based treatments in experimental animal models of several neurological diseases [8].

#### **4. Exogenous NPC-based therapies: The systemic administration**

#### **4.1. Systemic injection and functional recovery**

Following the seminal observation that systemically delivered NPCs were able to target an intracranial tumour in rodent (both mice and rats) model of experimental brain tumours [135], numerous studies started to investigate the validity of this administration route in a variety of CNS disorders. Over the last decade, data have been provided on the feasibility of systemic NPC transplants via either intravenous (i.v.) cell injection into the blood stream, or interacer‐ ebroventricular (i.c.v.)/intrathecal (i.t.) into the CSF, in experimental CNS disease models [10-13, 16, 136-138]. Adult somatic mouse NPCs, administered 22 days post immunization (dpi) greatly reduced the functional impairment observed in chronic MOG35-55-immunized EAE mice [11]. Also, rat NPC neurospheres, administered i.c.v. or i.t. in rats affected by acute EAE, attenuate the clinical symptoms when administered at the same day of disease induction (0 dpi) [136]. Intravenous injected mouse NPCs were proved to be efficacious in PLP139-151 immunized relapsing EAE mice [16]. Indeed, mice, treated with NPCs at the disease onset or at the time of the first relapse, recovered faster and showed a decrease in the relapse rate compared to controls. At the end of the follow-up (90 dpi) both treatments resulted in a lower relapsing remitting EAE cumulative score [16]. Finally, MOG35-55-immunized chronic EAE mice receiving either mouse neurospheres (i.c.v) or single cell NPCs (i.v.), at 6 and 8 dpi respectively also showed significant clinical amelioration [20, 139].

The therapeutic efficacy of systemically administered NPCs has been later observed in a different pathological context, such as stroke. Mouse somatic NPCs, systemically transplanted 3 days after middle cerebral artery occlusion (MCAo), resulted in a better recovery, signifi‐ cantly improving the neurological severity score starting from 18 days post transplantation (dpt) until the end of the follow-up (30 dpt) [140]. Similar neurological improvements were observed in rats subjected to MCAo and common carotid artery occlusion (CCAo) after i.t. transplantation (7 days after stroke) of rat NPCs [141]. Significant locomotor recovery was also observed after acute systemic NPC transplant in mice suffering from contusion SCI [13].

Similarly to rodent cells, human NPCs have been proved to be therapeutically efficacious. Foetal NPCs administered either i.v. or i.t. at the disease onset, reduced the severity of MOG1-125-induced chronic EAE in common marmosets [15]. Further, human embryonic stem cell (ESC)-derived NPCs have been shown to reduce disease severity of chronic EAE mice [142]. Human immortalized NPCs have been widely enrolled in stroke models. The systemic injection of the HB1.F3 NPC line resulted in neurological improvements of rats treated 1 day after MCAo or intracerebral hemorrhage (ICH) stroke models [12, 137, 138]. The same line resulted therapeutically effective also in quinolinic acid-induced experimental HD in rats, where NPCs administered intravenously at 7 days after disease induction, significantly ameliorated the behavioural outcome [14].

All these evidences showing behavioural recovery upon systemic injection of NPCs in different CNS inflammatory models, led to the investigation of the molecular mechanisms standing behind, since this capacity bears the hope of developing less invasive surgical techniques to implant therapeutic adult human stem cells into patients affected by highly debilitating CNS disorders, such as MS, stroke, SCI, epilepsy, PD, AD and HD.

#### **4.2. Homing capacity: NPCs breach the CNS barriers**

IL-6 knockout mice showed significantly increased of chronic (but not acute) lesion volumes and worse long-term functional outcome after stroke [133]. This may imply the need of a finely tuned regulation, most likely depending on their precise timing and location other than on the specific nature of the disease. Indeed, while some pathologies such as MS, SCI, stroke, TBI and are characterized by an acute inflammatory event followed by secondary neurodegeneration, others, such as epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), and Hunting‐ ton's disease (HD) are instead caused by primary neurodegeneration subsequently leading to

As described in this paragraph, the CNS is able to regulate the proliferation rate within the adult neurogenic niches as an extreme attempt to respond to damages in both primary and secondary inflammatory neurodegenerative diseases. Nevertheless, this process is not robust enough to effectively re-establish the complex functionality of the CNS. Therefore, protocols aiming at pharmacological manipulation of endogenous precursors from germinal niche(s), *in vivo,* might be therapeutically inefficacious in inflammatory CNS disorders. Thank to the development of protocols allowing *in vitro* growth and large scale-up of brain-derived NPCs [134], innovative therapies, for both acute and chronic CNS inflammatory disorders, based on stem cell transplants have been proposed [7]. Transplantation of adult exogenous NPCs represents, in fact, an alternative, and possibly more efficacious, therapeutic approach that might overcome the limited endogenous repair. Motivated by the ambitious expectation to achieve CNS repair (and/or regeneration) via functional neural cell replacement, many different preclinical studies have evidenced a potential benefit of NPC-based treatments in

experimental animal models of several neurological diseases [8].

**4.1. Systemic injection and functional recovery**

**4. Exogenous NPC-based therapies: The systemic administration**

Following the seminal observation that systemically delivered NPCs were able to target an intracranial tumour in rodent (both mice and rats) model of experimental brain tumours [135], numerous studies started to investigate the validity of this administration route in a variety of CNS disorders. Over the last decade, data have been provided on the feasibility of systemic NPC transplants via either intravenous (i.v.) cell injection into the blood stream, or interacer‐ ebroventricular (i.c.v.)/intrathecal (i.t.) into the CSF, in experimental CNS disease models [10-13, 16, 136-138]. Adult somatic mouse NPCs, administered 22 days post immunization (dpi) greatly reduced the functional impairment observed in chronic MOG35-55-immunized EAE mice [11]. Also, rat NPC neurospheres, administered i.c.v. or i.t. in rats affected by acute EAE, attenuate the clinical symptoms when administered at the same day of disease induction (0 dpi) [136]. Intravenous injected mouse NPCs were proved to be efficacious in PLP139-151 immunized relapsing EAE mice [16]. Indeed, mice, treated with NPCs at the disease onset or at the time of the first relapse, recovered faster and showed a decrease in the relapse rate compared to controls. At the end of the follow-up (90 dpi) both treatments resulted in a lower relapsing remitting EAE cumulative score [16]. Finally, MOG35-55-immunized chronic EAE

secondary reactive inflammation [19].

298 Neural Stem Cells - New Perspectives

Brain and spinal cord are protected by a complex control system, composed by tight barriers shielding the action of a unique troop of immune cells. Indeed, to access the brain and spinal cord parenchyma, circulating cells have to breach through all the barriers that closely seal the CNS from the surrounding environment. Namely, these are the blood-brain barrier (BBB) at the level of parenchymal capillaries and post-capillary venules, the blood-cerebrospinal fluid barrier (BCSFB) at the level of the choroid plexus in the brain ventricles, and the bloodleptomeningeal barrier (BLMB) at the level of the leptomeningeal/subarachnoid space. The main role of these barriers is to maintain the chemical composition of the CNS microenviron‐ ment, thus ensuring the proper functionality of neuronal circuits, synaptic transmission and remodelling, angiogenesis, and neurogenesis in the adult brain, while their rupture is involved in many neuroinflammatory disorders [143]. Because of the existence of such barriers, the access of systemically injected NPCs to the CNS parenchyma seems quite unlikely.

Structurally, the main component of the BBB is represented by specialized ECs characterized by the absence of fenestrae, low pinocytotic activity and by the presence of intercellular tight junctions (TJs) [144]. This clutched arrangement prevents the free passage of molecules, while the transport of nutrients into the CNS and the discard of toxic molecules into the circulation is ensured by active mechanisms, thus guarantying a proper neuronal activity [145]. Moreover, the BBB is an essential constituent of the so-called *neurovascular unit*, a boundary zone defined on one side by the *endothelium basement membrane* (located in the abluminal side of the vasculature) and on the other by the *parenchymal basement membrane*, which establishes the ultimate border between the perivascular space and the CNS parenchyma. In post-capillary venules these two membranes lay in close proximity, leaving just a narrow perivascular space in between, which becomes more significant at the level of arteries and veins. In this area, occasional APCs (leptomeningeal mesothelial cells) reside and play a major role in the immunosurveilance program of the CNS. Finally, the inner and outer sides of the parenchymal basement membrane are juxtaposed to the *glia limitans*, whose crossing seems to be crucial for the effective triggering of a neuroinflammation process [146, 147]. The functionality of the BBB in clinical situations, such as those occurring in some neurodegenerative disorders like MS, ischemic stroke, sub-arachnoid haemorrhage, TBI and AD is markedly reduced, leading to an increased permeability and trafficking of immune cells into the CNS parenchyma [148-152].

regulated and normal T cell expressed and secreted (RANTES)/CCL5 [162], EBI1 ligand chemokine (ELC)/CCL19, secondary lymphoid-tissue chemokine (SLC)/CCL21 released by the inflamed endothelia and CCR7 [163]. By binding to their G-coupled receptor (e.g.: C-X-C chemokine receptor -CXCR- type 4 for SDF-1 and CCR2 for MCP-1), these chemokines transmit an inside out signalling to T-cell surface integrins, which undergo dramatic conformational modifications thus increasing their *avidity* (specificity for the ligand). Once engaged in such a firm adhesion, T-cells need to make their way through the endothelium. To this purpose they start probing the vasculature to find the optimal site to breach the wall. Following adhesion to blood vessel walls, leukocytes undergo a series of actin rearrangements that eventually mark their transition to a more flatten and polarized shape [164]. Finally, T-cells cross the border either by paracellular or transcellular diapedesis or by creating pores through the cells *transcellular diapedesis* -. While the former require the disassembly of the intercellular junction structure, the latter involves the formation of "cell-in-cell" interactions through the arrange‐ ment of docking structures or *transmigratory cups* enriched in ICAM-1 and VCAM-1, which

Systemic stem cell therapies and brain diseases

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

301

A very similar sequential process has been shown being recapitulated when systemically injected NPCs specifically home to the site of damage. In fact, NPCs posses the ability to reach the cerebral parenchyma where they eventually induce recovery in animal models of neuro‐ degenerative diseases such as EAE [10, 11] stroke [12, 166], SCI [167, 168], epilepsy [138, 169], HD [14], other than glioblastoma [135, 170]. The first studies showing the extravasation capacity of NPCs [10-12] clearly demonstrated that this capacity was strictly related to the activation of the ECs by an inflammation process occurring within the brain. NPCs adminis‐ tered either i.c.v. or i.v. to healthy animals were, in fact, never observed inside the CNS, while mainly accumulating in peripheral organs, or remaining confined in the ventricles or subar‐ achnoid space. Only after activation of endothelial cells, exogenous NPCs were observed to accumulate into the CNS. Systemically injected NPCs are, in fact, able to follow a gradient of chemoattractants (e.g. pro-inflammatory cytokines and chemokines) released by the inflam‐ matory lesions into the blood stream and CSF. Following these signals, NPCs rapidly reach the source of pro-inflammatory molecules within and interact with the activated endothelial/ ependymal cells around inflamed CNS tissues. At this level, NPCs and endothelial cells start an organized sequence of events resembling those described for T cell extravasation that allow the selective entrance and specific *homing* of transplanted cells in multifocal inflammatory CNS areas [16]. Interestingly enough, only small percentages (between 1-5%) of the systemically administered NPCs actually infiltrate and integrate within the CNS [11, 13, 140]. Mouse SVZderived adult NPCs transplanted in a subacute model of brain inflammation were shown to adhere to the CD49d counterligand VCAM-1 [16]. Further *in vivo* experiments showed that migration of mouse NPCs towards the site of damage is dependent on the CXCR4-SDF-1α signalling in mouse models of MS and brain tumour [17, 171]. In stroke models, the upregulation of VCAM-1 on the surface of endothelial cells facilitates the targeting and the subsequent extravasation of VLA-4 expressing NPCs to the site of injury [18]. In line with this, mouse NPCs sorted via FACS for the presence of VLA-4 revealed a more efficient transendo‐ thelial migration in a mouse model of stroke after intracarotid injection [18]. More recently it

partially embrace migrating leukocytes [165].

Most of the knowledge about the mechanisms that allow circulating cells to breach the barrier(s) [117, 153, 154] and move into the CNS parenchyma comes from observations conducted with models of CNS inflammation. Initial studies showed how intravenously injected radioactively labelled encephalitogenic T cells were able to cross the BBB of healthy recipients [153]. It was also shown that, while activation is mandatory for T-cells to cross the endothelial barrier and reach perivascular spaces, antigen specificity is dispensable to further cross the glia limitans and invade the CNS parenchyma after having encountered the appro‐ priate APCs [155].

The extravasation of specific T cells requires a multistep process [156]. The first step, known as *capture* (in non-inflamed endothelia) or *tethering* (in inflamed endothelia) *and rolling* is represented by an initial, transient contact promoted by the specific interaction between members of the selectin and integrin families expressed by the activated endothelium with their respective ligands on circulating immune cells. It has been shown how the recruit‐ ment of inflammatory cells across the BBB involves α4-integrin and its ligands of the immunoglobulin (Ig) superfamily, namely vascular cell adhesion molecule (VCAM)-1 and mucosal addressin cell adhesion molecule (MAdCAM)-1 [147]. Upon this initial contact, circulating cells decrease their initial speed and resist the shear stress created by the blood flow, mainly through endothelial intercellular adhesion molecule (ICAM)-1 and VCAM-1, but not ICAM-2 [157]. Elegant studies have consistently shown that the inhibition of the dimeric α4β1-integrin and its cognate receptor VCAM-1 on the activated endothelium prevented the accumulation of leukocytes in the CNS and the development of EAE [158]. Interestingly, when the inflammatory process is started, α4β1-integrin is no more dispensa‐ ble for T-cell capture or rolling [159].

The following step requires the *firm adhesion* and *crawling* of T-cells along the vascular wall. This is orchestrated by some chemokines and chemoattractants, such as stromal cell-derived factor (SDF)-1α/CXCL12 [160], monocyte chemoattractant protein (MCP)-1/CCL2 [161], regulated and normal T cell expressed and secreted (RANTES)/CCL5 [162], EBI1 ligand chemokine (ELC)/CCL19, secondary lymphoid-tissue chemokine (SLC)/CCL21 released by the inflamed endothelia and CCR7 [163]. By binding to their G-coupled receptor (e.g.: C-X-C chemokine receptor -CXCR- type 4 for SDF-1 and CCR2 for MCP-1), these chemokines transmit an inside out signalling to T-cell surface integrins, which undergo dramatic conformational modifications thus increasing their *avidity* (specificity for the ligand). Once engaged in such a firm adhesion, T-cells need to make their way through the endothelium. To this purpose they start probing the vasculature to find the optimal site to breach the wall. Following adhesion to blood vessel walls, leukocytes undergo a series of actin rearrangements that eventually mark their transition to a more flatten and polarized shape [164]. Finally, T-cells cross the border either by paracellular or transcellular diapedesis or by creating pores through the cells *transcellular diapedesis* -. While the former require the disassembly of the intercellular junction structure, the latter involves the formation of "cell-in-cell" interactions through the arrange‐ ment of docking structures or *transmigratory cups* enriched in ICAM-1 and VCAM-1, which partially embrace migrating leukocytes [165].

junctions (TJs) [144]. This clutched arrangement prevents the free passage of molecules, while the transport of nutrients into the CNS and the discard of toxic molecules into the circulation is ensured by active mechanisms, thus guarantying a proper neuronal activity [145]. Moreover, the BBB is an essential constituent of the so-called *neurovascular unit*, a boundary zone defined on one side by the *endothelium basement membrane* (located in the abluminal side of the vasculature) and on the other by the *parenchymal basement membrane*, which establishes the ultimate border between the perivascular space and the CNS parenchyma. In post-capillary venules these two membranes lay in close proximity, leaving just a narrow perivascular space in between, which becomes more significant at the level of arteries and veins. In this area, occasional APCs (leptomeningeal mesothelial cells) reside and play a major role in the immunosurveilance program of the CNS. Finally, the inner and outer sides of the parenchymal basement membrane are juxtaposed to the *glia limitans*, whose crossing seems to be crucial for the effective triggering of a neuroinflammation process [146, 147]. The functionality of the BBB in clinical situations, such as those occurring in some neurodegenerative disorders like MS, ischemic stroke, sub-arachnoid haemorrhage, TBI and AD is markedly reduced, leading to an increased permeability and trafficking of immune cells into the CNS parenchyma [148-152].

Most of the knowledge about the mechanisms that allow circulating cells to breach the barrier(s) [117, 153, 154] and move into the CNS parenchyma comes from observations conducted with models of CNS inflammation. Initial studies showed how intravenously injected radioactively labelled encephalitogenic T cells were able to cross the BBB of healthy recipients [153]. It was also shown that, while activation is mandatory for T-cells to cross the endothelial barrier and reach perivascular spaces, antigen specificity is dispensable to further cross the glia limitans and invade the CNS parenchyma after having encountered the appro‐

The extravasation of specific T cells requires a multistep process [156]. The first step, known as *capture* (in non-inflamed endothelia) or *tethering* (in inflamed endothelia) *and rolling* is represented by an initial, transient contact promoted by the specific interaction between members of the selectin and integrin families expressed by the activated endothelium with their respective ligands on circulating immune cells. It has been shown how the recruit‐ ment of inflammatory cells across the BBB involves α4-integrin and its ligands of the immunoglobulin (Ig) superfamily, namely vascular cell adhesion molecule (VCAM)-1 and mucosal addressin cell adhesion molecule (MAdCAM)-1 [147]. Upon this initial contact, circulating cells decrease their initial speed and resist the shear stress created by the blood flow, mainly through endothelial intercellular adhesion molecule (ICAM)-1 and VCAM-1, but not ICAM-2 [157]. Elegant studies have consistently shown that the inhibition of the dimeric α4β1-integrin and its cognate receptor VCAM-1 on the activated endothelium prevented the accumulation of leukocytes in the CNS and the development of EAE [158]. Interestingly, when the inflammatory process is started, α4β1-integrin is no more dispensa‐

The following step requires the *firm adhesion* and *crawling* of T-cells along the vascular wall. This is orchestrated by some chemokines and chemoattractants, such as stromal cell-derived factor (SDF)-1α/CXCL12 [160], monocyte chemoattractant protein (MCP)-1/CCL2 [161],

priate APCs [155].

300 Neural Stem Cells - New Perspectives

ble for T-cell capture or rolling [159].

A very similar sequential process has been shown being recapitulated when systemically injected NPCs specifically home to the site of damage. In fact, NPCs posses the ability to reach the cerebral parenchyma where they eventually induce recovery in animal models of neuro‐ degenerative diseases such as EAE [10, 11] stroke [12, 166], SCI [167, 168], epilepsy [138, 169], HD [14], other than glioblastoma [135, 170]. The first studies showing the extravasation capacity of NPCs [10-12] clearly demonstrated that this capacity was strictly related to the activation of the ECs by an inflammation process occurring within the brain. NPCs adminis‐ tered either i.c.v. or i.v. to healthy animals were, in fact, never observed inside the CNS, while mainly accumulating in peripheral organs, or remaining confined in the ventricles or subar‐ achnoid space. Only after activation of endothelial cells, exogenous NPCs were observed to accumulate into the CNS. Systemically injected NPCs are, in fact, able to follow a gradient of chemoattractants (e.g. pro-inflammatory cytokines and chemokines) released by the inflam‐ matory lesions into the blood stream and CSF. Following these signals, NPCs rapidly reach the source of pro-inflammatory molecules within and interact with the activated endothelial/ ependymal cells around inflamed CNS tissues. At this level, NPCs and endothelial cells start an organized sequence of events resembling those described for T cell extravasation that allow the selective entrance and specific *homing* of transplanted cells in multifocal inflammatory CNS areas [16]. Interestingly enough, only small percentages (between 1-5%) of the systemically administered NPCs actually infiltrate and integrate within the CNS [11, 13, 140]. Mouse SVZderived adult NPCs transplanted in a subacute model of brain inflammation were shown to adhere to the CD49d counterligand VCAM-1 [16]. Further *in vivo* experiments showed that migration of mouse NPCs towards the site of damage is dependent on the CXCR4-SDF-1α signalling in mouse models of MS and brain tumour [17, 171]. In stroke models, the upregulation of VCAM-1 on the surface of endothelial cells facilitates the targeting and the subsequent extravasation of VLA-4 expressing NPCs to the site of injury [18]. In line with this, mouse NPCs sorted via FACS for the presence of VLA-4 revealed a more efficient transendo‐ thelial migration in a mouse model of stroke after intracarotid injection [18]. More recently it

was shown that also the CCR2/CCL2 interaction is substantially involved in the recruitment of systemically delivered NPCs in a mouse model of stroke [172].

with host cells [179]. Possible mechanisms of communication include also secretion of growth factors, hormones, cytokines, chemokines and small molecular mediators [180], cell-to-cell interactions via tunnelling nanotubes [181] and secretion of circular membrane vesicles [182],

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Correlative evidence suggest that, depending on local inflammatory milieu, transplanted NPCs may either remain in the niche while maintaining an undifferentiated state, or move out from the niche, finally acquiring a terminally differentiated phenotype [16]. When systemically injected in chronic EAE mice, syngenic NPCs were found almost exclusively in areas of CNS damage, mainly within the submeningeal space in close proximity to subpial inflammatory foci (after i.t. stem cell injection), or around post-capillary venules (after i.v. stem cell injection) [11]. Ten days after transplantation, relatively few cells were found in the CNS parenchyma and at 30 dpi many of the surviving donor cells were localized deeply within the brain parenchyma and displayed a marked distribution pattern: most of them were confined within areas of demyelination and axonal loss, and only very few cells were found within regions where the myelin architecture was preserved [11]. Similar results were obtained after i.c.v NPC injection at the peak of EAE in rats: cells entered into the brain or spinal cord parenchyma and mostly accumulated at sites of inflamed white matter but not into adjacent grey matter regions. In line with the previous study, after 2 weeks cells had migrated into distant white matter tracts but, on the contrary, most of them had acquired specific markers of the astroglial and oligo‐ dendroglial lineages [184]. Mouse NPC transplants in rodents affected by EAE are also associated with significantly reduced glial scar formation [11] and an increased survival and recruitment of endogenous neural cells participating to the naturally occurring brain repara‐

Human NPCs have shown a higher rate of cell integration after being administered in different animal models. In particular, the HB1.F3 immortalized cell line, i.v. injected in a model of ischemic stroke, were shown to enter the ischemic area and differentiate into neurons and astrocytes, similarly to what observed with focal injected cells [12, 187]. Transplanted cells seemed to adapt their fate accordingly to the region of engraftment, showing the appropriate neuronal and glial markers. NeuN+ and NF+ cells were identi‐ fied primarily in the CA1 area of the hippocampus and in the dentate gyrus, mixed with GFAP+ cells. Vimentin, GFAP and NF markers showed a progressive expression during the first 2-3 weeks after transplantation, suggesting a step-by-step maturation of the cells [187]. The very same line of cells, injected in a rat model of ICH [137], was observed to infil‐ trate the brain, survive and migrate towards the peri-hematomal areas. The cells were found mainly differentiating into GFAP+ and NeuN+ cells. However, the rapid behavioural recovery observed in ICH rats as soon as 2 weeks after transplantation, suggested that the NPC therapeutic effect was mainly related to neuroprotection, rather than to integration into neuronal circuitry [137, 187], since the latter would require longer time to produce clinical ameliorations. A similar trend towards human NPC differentiation has been observed in animal models of SCI, SE and HD. HB1.F3 hNPCs administered in mice subjected to compression SCI, were observed to differentiate into βIII-tubulin+ neurons at 21 days after transplantation [167]. GABA-immunoreactive interneurons were, instead,

other than cell-to-cell contacts [183].

tive response upon myelin damage [10, 15, 16, 185, 186].

*In vitro* experiments confirmed that mouse NPCs express many functional receptors on theirs surfaces, among which the α4 subunit of the integrin VLA-4 [16], the SDF-1α receptor CXCR4 [173] and CD44, a cell-surface glycoprotein that binds to hyaluronic acid (HA) and is expressed also in activated T cells [174, 175]. Interestingly, NPCs led to the formation of transmigratory cups, enriched in multiple adhesion molecules such as ICAM-1 and VCAM-1, on the surface of endothelial cells [175] as previously shown for T lymphocytes diapedesis [176].

Similarly, also immortalized human NPC lines express CD44 [175] and CXCR4 [173]. However, in a recent study, human NPCs were shown to interact with activated ECs through integrins α2, α6 and β1 rather than CXCR4 [177]. Further, human NPCs express the receptors CXCR1 and CXCR5, which mediate their *in vitro* migration across a monolayer of human brain ECs in response to IL-8/CXCL8 and B lymphocyte chemoattractant (BLC)/CXCL13, chemokines previously known to favour the trans-endothelial migration of immune cells [178].

All these evidences suggest that systemically injected mouse and human NPCs share the expression of a variety of functional immune-like receptors, such as functional cell adhesion molecules (e.g. CD44 and VLA-4) and inflammatory chemokine receptors (e.g. CCR2, CCR5 and CXCR4), giving them a unique leukocyte-like molecular signature. This characteristic, allowing NPC interaction with activated endothelial and ependymal cells, represents an essential requirement in the therapeutic paradigm of systemic delivery. Therefore, the discovery of the specific homing ability of NPCs across the BBB opened new frontiers for the treatment of CNS diseases, in particular for those diseases characterized by disseminated damage.

#### **4.3. NPC interaction with the dysfunctional CNS microenvironment: The establishment of ectopic niches**

Consistent data exists reporting the ability of i.v. injected NPCs to cross the BBB and accumu‐ late into the CNS. Here, exogenous NPCs co-exist with different host components, such as ECs, infiltrating inflammatory cells, activated macrophages/microglia and reactive astrocytes [19]. In this context, the intimate association with ECs, the physical proximity to the vasculature and the enhanced expression of stem cell regulators and growth factors involved both in angiogenesis and neurogenesis has been described to play a major role in defining a molecular architecture reminiscent of prototypical germinal stem cell niches [16]. Within these *atypical ectopic perivascular niches*, in addition to hierarchical (mother-to-daughter) communication, a sophisticated level of cell-to-cell horizontal communication takes place between transplanted NPCs and resident cells. Recent evidences confirm that NPCs are able to communicate with host cells via cellular contacts. For instance, functional gap junction formation has been shown to allow exogenous NPCs to rescue host neurons and their projections in animal models of Purkinje neurodegeneration. Gap junctions permitted the trans-cellular delivery of homeo‐ stasis-modulating molecules, as well as directly influenced the coordinated activity of the host network via Ca++ waves. Moreover, hypoxic preconditioning of NPCs before their *in vitro* engraftment increased Connexin 43 expression and improved subsequent communication with host cells [179]. Possible mechanisms of communication include also secretion of growth factors, hormones, cytokines, chemokines and small molecular mediators [180], cell-to-cell interactions via tunnelling nanotubes [181] and secretion of circular membrane vesicles [182], other than cell-to-cell contacts [183].

was shown that also the CCR2/CCL2 interaction is substantially involved in the recruitment

*In vitro* experiments confirmed that mouse NPCs express many functional receptors on theirs surfaces, among which the α4 subunit of the integrin VLA-4 [16], the SDF-1α receptor CXCR4 [173] and CD44, a cell-surface glycoprotein that binds to hyaluronic acid (HA) and is expressed also in activated T cells [174, 175]. Interestingly, NPCs led to the formation of transmigratory cups, enriched in multiple adhesion molecules such as ICAM-1 and VCAM-1, on the surface

Similarly, also immortalized human NPC lines express CD44 [175] and CXCR4 [173]. However, in a recent study, human NPCs were shown to interact with activated ECs through integrins α2, α6 and β1 rather than CXCR4 [177]. Further, human NPCs express the receptors CXCR1 and CXCR5, which mediate their *in vitro* migration across a monolayer of human brain ECs in response to IL-8/CXCL8 and B lymphocyte chemoattractant (BLC)/CXCL13, chemokines

All these evidences suggest that systemically injected mouse and human NPCs share the expression of a variety of functional immune-like receptors, such as functional cell adhesion molecules (e.g. CD44 and VLA-4) and inflammatory chemokine receptors (e.g. CCR2, CCR5 and CXCR4), giving them a unique leukocyte-like molecular signature. This characteristic, allowing NPC interaction with activated endothelial and ependymal cells, represents an essential requirement in the therapeutic paradigm of systemic delivery. Therefore, the discovery of the specific homing ability of NPCs across the BBB opened new frontiers for the treatment of CNS diseases, in particular for those diseases characterized by disseminated

**4.3. NPC interaction with the dysfunctional CNS microenvironment: The establishment of**

Consistent data exists reporting the ability of i.v. injected NPCs to cross the BBB and accumu‐ late into the CNS. Here, exogenous NPCs co-exist with different host components, such as ECs, infiltrating inflammatory cells, activated macrophages/microglia and reactive astrocytes [19]. In this context, the intimate association with ECs, the physical proximity to the vasculature and the enhanced expression of stem cell regulators and growth factors involved both in angiogenesis and neurogenesis has been described to play a major role in defining a molecular architecture reminiscent of prototypical germinal stem cell niches [16]. Within these *atypical ectopic perivascular niches*, in addition to hierarchical (mother-to-daughter) communication, a sophisticated level of cell-to-cell horizontal communication takes place between transplanted NPCs and resident cells. Recent evidences confirm that NPCs are able to communicate with host cells via cellular contacts. For instance, functional gap junction formation has been shown to allow exogenous NPCs to rescue host neurons and their projections in animal models of Purkinje neurodegeneration. Gap junctions permitted the trans-cellular delivery of homeo‐ stasis-modulating molecules, as well as directly influenced the coordinated activity of the host network via Ca++ waves. Moreover, hypoxic preconditioning of NPCs before their *in vitro* engraftment increased Connexin 43 expression and improved subsequent communication

of endothelial cells [175] as previously shown for T lymphocytes diapedesis [176].

previously known to favour the trans-endothelial migration of immune cells [178].

of systemically delivered NPCs in a mouse model of stroke [172].

damage.

**ectopic niches**

302 Neural Stem Cells - New Perspectives

Correlative evidence suggest that, depending on local inflammatory milieu, transplanted NPCs may either remain in the niche while maintaining an undifferentiated state, or move out from the niche, finally acquiring a terminally differentiated phenotype [16]. When systemically injected in chronic EAE mice, syngenic NPCs were found almost exclusively in areas of CNS damage, mainly within the submeningeal space in close proximity to subpial inflammatory foci (after i.t. stem cell injection), or around post-capillary venules (after i.v. stem cell injection) [11]. Ten days after transplantation, relatively few cells were found in the CNS parenchyma and at 30 dpi many of the surviving donor cells were localized deeply within the brain parenchyma and displayed a marked distribution pattern: most of them were confined within areas of demyelination and axonal loss, and only very few cells were found within regions where the myelin architecture was preserved [11]. Similar results were obtained after i.c.v NPC injection at the peak of EAE in rats: cells entered into the brain or spinal cord parenchyma and mostly accumulated at sites of inflamed white matter but not into adjacent grey matter regions. In line with the previous study, after 2 weeks cells had migrated into distant white matter tracts but, on the contrary, most of them had acquired specific markers of the astroglial and oligo‐ dendroglial lineages [184]. Mouse NPC transplants in rodents affected by EAE are also associated with significantly reduced glial scar formation [11] and an increased survival and recruitment of endogenous neural cells participating to the naturally occurring brain repara‐ tive response upon myelin damage [10, 15, 16, 185, 186].

Human NPCs have shown a higher rate of cell integration after being administered in different animal models. In particular, the HB1.F3 immortalized cell line, i.v. injected in a model of ischemic stroke, were shown to enter the ischemic area and differentiate into neurons and astrocytes, similarly to what observed with focal injected cells [12, 187]. Transplanted cells seemed to adapt their fate accordingly to the region of engraftment, showing the appropriate neuronal and glial markers. NeuN+ and NF+ cells were identi‐ fied primarily in the CA1 area of the hippocampus and in the dentate gyrus, mixed with GFAP+ cells. Vimentin, GFAP and NF markers showed a progressive expression during the first 2-3 weeks after transplantation, suggesting a step-by-step maturation of the cells [187]. The very same line of cells, injected in a rat model of ICH [137], was observed to infil‐ trate the brain, survive and migrate towards the peri-hematomal areas. The cells were found mainly differentiating into GFAP+ and NeuN+ cells. However, the rapid behavioural recovery observed in ICH rats as soon as 2 weeks after transplantation, suggested that the NPC therapeutic effect was mainly related to neuroprotection, rather than to integration into neuronal circuitry [137, 187], since the latter would require longer time to produce clinical ameliorations. A similar trend towards human NPC differentiation has been observed in animal models of SCI, SE and HD. HB1.F3 hNPCs administered in mice subjected to compression SCI, were observed to differentiate into βIII-tubulin+ neurons at 21 days after transplantation [167]. GABA-immunoreactive interneurons were, instead, observed originating from HB1.F3 when systemically administered the day after lithiumpilocarpine induction of experimental SE in rats [138]. Further, HB1.F3 cells injected 7 days after unilateral quinolinic acid (QA)-induced model of HD in rats were found to stay confined around blood vessels, mostly in the damaged hemisphere and only partially differentiating in GFAP+ and NeuN+ cells at 3 weeks post transplantation [14]*.*

More recently, adult mouse NPCs systemically injected in mice (3 injections few hours after the injury) suffering from acute contusion SCI, showed an undifferentiated morphology (similarly to what observed in EAE) at the level of the damaged CNS. *Ex vivo* RT-PCR analysis showed NPC-driven up regulation of BDNF, NT-3, NGF, leukemia inhibitory factor (LIF) and TNF- α only at 48h after treatments, while no differences were observed neither at 24h or 7 days after transplantation [13]. Similarly to what observed by indirect evidence *in vivo*, realtime PCR gene expression analysis directly revealed high levels of NGF, BDNF and NT-3 and glial cell line-derived neurotrophic factor (GDNF) in the transcripts of cultured rat NPCs [141, 191]. In addition, in line with the observed pro-angiogeneic effect *in vivo* after transplantation of mouse NPCs in stroke models [18, 190], human NPCs were proved to secrete VEGF *in*

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All these evidences suggested that the underlying molecular mechanisms by which trans‐ planted NPCs instruct tissue protection effects are partly related to increased *in vivo* bioavailability of major neurotrophins [11, 138, 139, 193] able to modulate the host environ‐ ment resulting more permissive to regeneration. Neurotrophins exert important roles as mediators in cell cycle regulation, cell survival, and differentiation during development and adulthood. The delivery of diffusible proteins to the CNS has been seen as a possible therapeutic weapon for neurological diseases. However, because the CNS is likely impene‐ trable for many of these diffusible proteins, NPCs might be envisaged as carrier of neurotro‐ phic factors. To this aim, NPCs have been genetically modified to act as *Trojan horses* to deliverthe desired diffusible molecules at the site of injury, thus fostering theirinnate capacity to secrete neurotrophic and growth factors [194]. Among all the candidate neurotrophic factors to be delivered, GDNF has shown a potent neuroprotective effect on a variety of neuronal inflammatory models, such as stroke and PD [195-197]. However, its effects are generally transient and need consecutive administrations to obtain long-standing results. NPCs over-expressing GDNF can instead provide durable neuroprotective effects, as shown with mouse NPCs, transplanted i.c.v in rats 3 days after MCAo [198]. The exogenous cells resulted in an overall increase of cell survival of endogenous cells after the insult, which in turn was associated to a partial functional recovery. Interestingly, treated rats also dis‐ played a significant increase of the synaptic proteins synaptophysin and post-synaptic density protein (PSD)-95, suggesting an enhanced neuronal function and a possible reconstruction of endogenous neural circuitries after the grafting [198]. Finally, a recent study showed that the intravenous administration HB1.F3 human NPCs transduced with INF-β and cytosine deaminase (CD), was able to interfere with toll-like-receptor (TLR)-4 (up-regulated into the site of injury) suppressing the SCI-induced proliferation of reactive astrocytes and promot‐ ing functional recovery [199]. Other neurotrophic factors, such as BDNF and VEGF, have been over-expressed in NPCs and mainly tested upon intraparenchymal injection in models of SCI [200] or ICH [201, 202]. Taken together, these data suggest that the clinical ameliora‐ tion observed in CNS disease animal models are, at least in part, mediated by a multilay‐

*vitro* [192].

ered NPC neurotrophic signature.

Despite these evidences showing the ability of exogenous NPCs to survive and differentiate into multiple derivatives according to local cues, the majority of the data provided has substantially failed to show convincing relevant differentiation and integration of transplanted NPCs *in vivo*. It is now quite evident that NPCs (and more generally somatic adult SCs) might protect the CNS trough mechanisms alternative to direct cell replacement, which imply the interaction of NPCs with both resident neural and immune cells [7, 188]. Cell replacement is therefore only one of the multiple ways by which transplanted NPCs can promote tissue repair, and a much more complex therapeutic scenario should be foreseen. The concept of *stem cell therapeutic plasticity* (or *functional multipotency*) has therefore emerged, describing the different way(s) NPCs use to interact with tissue-resident *vs.* infiltrating immune cells, at the level of the inflammatory tissue context in which they are either transplanted or migrate to after transplantation. These bystander effects, are mainly represented by *neuroprotection,* which might occur both through secretion of soluble factors and cell-to-cell contact interactions and *immunomodulation,* intended as the capacity of NPCs to influence the activity of the immune system in the CNS and/or in the periphery, at the level of secondary lymphoid organs [5, 19].

#### *4.3.1. Tissue trophic effects*

NPCs may exert their neuroprotective effect by increasing *in situ* bioavailability of several molecules, such as neurotrophins, growth factors and developmental stem cell regulators, thus promoting the survival and function of endogenous glial and neuronal progenitors that escaped the primary insults [19].

Mouse NPCs systemically injected in mice affected by middle cerebral artery occlusion (MCAo) were observed to mostly maintain an undifferentiated phenotype, while accumulat‐ ing at the boundaries of the lesioned area [140, 141]. Tissue survival was associated with a down regulation of inflammation, glial scar formation and neuronal apoptotic cell death at both mRNA and protein levels [140]. Increased levels of BDNF, NGF and neurotrophin (NT)-3 were found in the CSF of stroke-affected rats after intra-cisterna magna administration of NPCs. In addition, immunohistochemical analysis of the injured brain revealed an increase of MHC class I levels in treated rats [141]. Interestingly, this neuroprotective effect in the ischemic microenvironment seems to start very soon after the systemic administration of cells. In fact, data have been provided showing an increase in the gene expression levels of IGF-1, VEGF, TGF-1β, BDNF and CXCL12/SDF1-α in the NPC-transplanted MCAo brain, as soon as 24 hours after the acute i.c.v. injection [189]. Further, NPCs have been proved to increase *in vivo* vascularisation when administered after stroke, most likely due to their ability to increase the presence of VEGF, FGF, BDNF and chemoattractant factors (such as SDF-1α), which promote angiogenesis and mobilization of endogenous endothelial progenitors [18, 190]

More recently, adult mouse NPCs systemically injected in mice (3 injections few hours after the injury) suffering from acute contusion SCI, showed an undifferentiated morphology (similarly to what observed in EAE) at the level of the damaged CNS. *Ex vivo* RT-PCR analysis showed NPC-driven up regulation of BDNF, NT-3, NGF, leukemia inhibitory factor (LIF) and TNF- α only at 48h after treatments, while no differences were observed neither at 24h or 7 days after transplantation [13]. Similarly to what observed by indirect evidence *in vivo*, realtime PCR gene expression analysis directly revealed high levels of NGF, BDNF and NT-3 and glial cell line-derived neurotrophic factor (GDNF) in the transcripts of cultured rat NPCs [141, 191]. In addition, in line with the observed pro-angiogeneic effect *in vivo* after transplantation of mouse NPCs in stroke models [18, 190], human NPCs were proved to secrete VEGF *in vitro* [192].

observed originating from HB1.F3 when systemically administered the day after lithiumpilocarpine induction of experimental SE in rats [138]. Further, HB1.F3 cells injected 7 days after unilateral quinolinic acid (QA)-induced model of HD in rats were found to stay confined around blood vessels, mostly in the damaged hemisphere and only partially

Despite these evidences showing the ability of exogenous NPCs to survive and differentiate into multiple derivatives according to local cues, the majority of the data provided has substantially failed to show convincing relevant differentiation and integration of transplanted NPCs *in vivo*. It is now quite evident that NPCs (and more generally somatic adult SCs) might protect the CNS trough mechanisms alternative to direct cell replacement, which imply the interaction of NPCs with both resident neural and immune cells [7, 188]. Cell replacement is therefore only one of the multiple ways by which transplanted NPCs can promote tissue repair, and a much more complex therapeutic scenario should be foreseen. The concept of *stem cell therapeutic plasticity* (or *functional multipotency*) has therefore emerged, describing the different way(s) NPCs use to interact with tissue-resident *vs.* infiltrating immune cells, at the level of the inflammatory tissue context in which they are either transplanted or migrate to after transplantation. These bystander effects, are mainly represented by *neuroprotection,* which might occur both through secretion of soluble factors and cell-to-cell contact interactions and *immunomodulation,* intended as the capacity of NPCs to influence the activity of the immune system in the CNS and/or in the periphery, at the level of secondary lymphoid organs [5, 19].

NPCs may exert their neuroprotective effect by increasing *in situ* bioavailability of several molecules, such as neurotrophins, growth factors and developmental stem cell regulators, thus promoting the survival and function of endogenous glial and neuronal progenitors that

Mouse NPCs systemically injected in mice affected by middle cerebral artery occlusion (MCAo) were observed to mostly maintain an undifferentiated phenotype, while accumulat‐ ing at the boundaries of the lesioned area [140, 141]. Tissue survival was associated with a down regulation of inflammation, glial scar formation and neuronal apoptotic cell death at both mRNA and protein levels [140]. Increased levels of BDNF, NGF and neurotrophin (NT)-3 were found in the CSF of stroke-affected rats after intra-cisterna magna administration of NPCs. In addition, immunohistochemical analysis of the injured brain revealed an increase of MHC class I levels in treated rats [141]. Interestingly, this neuroprotective effect in the ischemic microenvironment seems to start very soon after the systemic administration of cells. In fact, data have been provided showing an increase in the gene expression levels of IGF-1, VEGF, TGF-1β, BDNF and CXCL12/SDF1-α in the NPC-transplanted MCAo brain, as soon as 24 hours after the acute i.c.v. injection [189]. Further, NPCs have been proved to increase *in vivo* vascularisation when administered after stroke, most likely due to their ability to increase the presence of VEGF, FGF, BDNF and chemoattractant factors (such as SDF-1α), which promote

angiogenesis and mobilization of endogenous endothelial progenitors [18, 190]

differentiating in GFAP+ and NeuN+ cells at 3 weeks post transplantation [14]*.*

*4.3.1. Tissue trophic effects*

304 Neural Stem Cells - New Perspectives

escaped the primary insults [19].

All these evidences suggested that the underlying molecular mechanisms by which trans‐ planted NPCs instruct tissue protection effects are partly related to increased *in vivo* bioavailability of major neurotrophins [11, 138, 139, 193] able to modulate the host environ‐ ment resulting more permissive to regeneration. Neurotrophins exert important roles as mediators in cell cycle regulation, cell survival, and differentiation during development and adulthood. The delivery of diffusible proteins to the CNS has been seen as a possible therapeutic weapon for neurological diseases. However, because the CNS is likely impene‐ trable for many of these diffusible proteins, NPCs might be envisaged as carrier of neurotro‐ phic factors. To this aim, NPCs have been genetically modified to act as *Trojan horses* to deliverthe desired diffusible molecules at the site of injury, thus fostering theirinnate capacity to secrete neurotrophic and growth factors [194]. Among all the candidate neurotrophic factors to be delivered, GDNF has shown a potent neuroprotective effect on a variety of neuronal inflammatory models, such as stroke and PD [195-197]. However, its effects are generally transient and need consecutive administrations to obtain long-standing results. NPCs over-expressing GDNF can instead provide durable neuroprotective effects, as shown with mouse NPCs, transplanted i.c.v in rats 3 days after MCAo [198]. The exogenous cells resulted in an overall increase of cell survival of endogenous cells after the insult, which in turn was associated to a partial functional recovery. Interestingly, treated rats also dis‐ played a significant increase of the synaptic proteins synaptophysin and post-synaptic density protein (PSD)-95, suggesting an enhanced neuronal function and a possible reconstruction of endogenous neural circuitries after the grafting [198]. Finally, a recent study showed that the intravenous administration HB1.F3 human NPCs transduced with INF-β and cytosine deaminase (CD), was able to interfere with toll-like-receptor (TLR)-4 (up-regulated into the site of injury) suppressing the SCI-induced proliferation of reactive astrocytes and promot‐ ing functional recovery [199]. Other neurotrophic factors, such as BDNF and VEGF, have been over-expressed in NPCs and mainly tested upon intraparenchymal injection in models of SCI [200] or ICH [201, 202]. Taken together, these data suggest that the clinical ameliora‐ tion observed in CNS disease animal models are, at least in part, mediated by a multilay‐ ered NPC neurotrophic signature.

#### *4.3.2. Regulation of the immune system*

ConsiderableevidenceoftheimmunemodulatorycapacityofNPCshasderivedfromtransplan‐ tation studies throughdifferentroutes in theEAEmodel.Asmentioned,transplanted NPCs are consistentlyfoundaroundinflamedbloodvessels,inclosecontactwithbothendogenousneural cells (e.g. astrocytes and neurons) and CNS-infiltrating blood-borne CD45+ immune cells [185]. Also, i.c.v. administered NPCs were found to attenuate brain inflammation primarily through a reductionofperivascularinfiltrates andCD3+Tcellswitha concomitantincrease ofCD25+ and CD25+ /CD62L+ regulatory T cells [136]. Interestingly, i.v. injection of NPCs also protects against chronic neural tissue loss as well as disease-related disability in EAE, via induction of apopto‐ sis of blood-borne CNS-infiltrating encephalitogenic T cells [185]

tor (TCR)-mediated stimuli (e.g., concanavalin A and anti-CD3/anti-CD28) [136, 214]. NPC/T lymphocyte co-culture experiments suggest that part of the anti-proliferative effect of NPCs might depend on the inhibition of IL-2 and IL-6 signalling on T lymphocytes [214]. In addi‐ tion, NPCs have shown a selective pro-apoptotic effect on Th17 cells *in vitro* via a FasL (CD95L) dependent mechanism, identifying the axis Fas-Birc3 as an additional survival pathway for NPCs [215]. Mouse C17.2 NPCs also suppress T-cell proliferation, at least in part, by reactive productionofthe solublemediatorsnitric oxide (NO) andprostaglandinE2 (PGE2). Highlevels ofNOandPGE2areinfactinducedinTcellswhenco-culturedwithNPCs.Inaddition,inducible NO synthase (iNOS) and microsomal type 1 PGES (mPGES-1) are detected in NPCs in coculture with T-cells, suggesting that NO and PGE2 production in NPCs is induced by expo‐

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Human NPCs have been proved to suppress the proliferation and alter the cytokine secretion profiles of activated T cells on both xenogeneic antigen-specific T cells derived from EAE induced non-human primates (common marmosets), and allogeneic mitogen-activated T cells. Co-culture of human NPCs with T cells, revealed their immune modulatory capacity through both direct cell-to-cell contacts as well as via the release of soluble mediators into the culture medium [15]. Notably, in contrast to the mouse counterpart, human NPCs have a limited cytotoxicity against T cells *in vitro*, given that FasL is only barely detectable on their surface. However, human NPCs exposed to cytokines express high levels of TNF-α, resulting in a higher cytotoxic potential against monocytes and macrophages [217]. In line with this, immortalized human NPCs were also proved, trough direct *in vitro* experiments, to reduce T cell activation and proliferation. Conditioned media collected from human NSCs (HB1.F3 line) *in vitro*, directly suppress the proliferation of activated human T cells through both induction of apoptosis and cell cycle arrest. Nonetheless, human NPC-released mediators alter the cytokine production pattern of T lymphocytes, increasing the expression of IL-4, IL-10, TNF-

α, and IFN-γ and decreasing IL-2, thus affecting the overall activation [200].

**4.4. NPC interactions with the dysfunctional non-CNS microenvironment**

ing in the attenuation of the inflammatory response following EAE, stroke and SCI.

InparalleltotheobservedimmunemodulationandneuroprotectionintotheCNS,other studies have shown that systemically injected cells may act also outside the injured CNS. Different studies, in fact, have reported the capacity of NPCs to target and synergize with immune cells at the level of secondary lymphoid organs (e.g. draining lymph nodes) and the spleen, result‐

It was initially showed *in vitro* that NPCs strongly inhibited the ability of EAE-derived lympho‐ cytes to produce pro-inflammatory Th1 cytokines in response to MOG35-55 stimulation. In addition, specificallyactivatedTcells isolatedfromEAEmice treatedwithNPCs,weredeficient in their ability to adoptively transfer EAE (to a naïve host), thus suggesting a long-lasting inhibitionof encephalitogenicityofTcells [20].Furtherdatahavebeenprovidedabout a specific and almost exclusive targeting of the peripheral immune system in SJL mice with PLP-in‐ duced EAE, in which NPCs had been injected subcutaneously (s.c.) at 3 and 10 dpi [21]. This alternative administration protocol showed a significant clinical improvement in EAE mice despiteinjectedcellswereneverbeenconsistentlyfoundintotheinflamedCNS.Sub-cutaneous‐

sure to activated T cells [216].

NPCs have been shown to possess immune modulatory capacity also in models of stroke, where T cells do not have a major role in the disease pathology. Irrespectively of the route of administration (systemically *vs.* intraparechimally), transplanted NPCs migrate towards the site of infarct in MCAo and ICH models [12, 137, 138, 203-207] and once reached the ischemic boundary zone (IBZ), grafted NPCs interact with the inflammatory environment. The subacute (*delayed*) i.v. injection of mouse NPCs after MCAo in mice, significantly down-regulates multiple RNA species involved in inflammation, including IFN-γ, TNF-α, IL-1β, IL-6 and leptin receptor [140]. Therefore, NPCs may exert an immune modulatory action, causing a profound down regulation of inflammatory *lymphoid* (T cells) and *myeloid* (macrophages) cells within inflamed brain areas.

While the inhibition of the T cell responses by NPCs is a quite established concept [208], the effect of the interaction between transplanted NPCs and microglia/macrophages is still controversial, mainly because of the non-univocal data regarding the role sustained by professional phagocytes under CNS inflammatory conditions. *In vivo* studies have in fact produced opposite evidences that might underline once more the bimodal action of some immune regulators [209]. NPC transplantation promote the infiltration of CD11b+ myeloid cells into the brain of MCAo mice, thus suggesting that myeloid cell activation might be required for transplanted NPCs to exert part of their neuroprotective action [189]. Indeed, MCAo mice in which CD11b+ microglia have been selectively ablated showed exacerbation of the ischemia-dependent brain injury [113]. However, several studies have showed a significant reduction of microglia/macrophages in the brain of mice, with either ischemic or haemorrhagic stroke, together with improved neuronal survival and locomotor functions after NPC trans‐ plantation [22, 140]. Also in this case, NPCs have been engineered in order to increase their immune modulatory capacity. Recently, mouse NPCs were transduced with IL-10, which has been proved to efficiently suppress EAE symptoms and promote survival of neurons and oligodendrocytes [210-212]. Mouse NPCs, transduced with a lentiviral vector encoding IL-10, showed enhanced ability to induce remyelination, neuronal repair and immune suppression after systemic injection in EAE mice compared to control NPCs [213].

*In vitro*, NPCs are shown to increase the apoptosis of PLP139-151-specific Th1 pro-inflammatory (but not Th2 anti-inflammatory) cells through the engagement of death receptors, including FasL,TNF-relatedapoptosis-inducingligand(TRAIL), andAPO3L,onthe surfaceofNPCs [16]. Mouse and rat NPCs also inhibit T cell activation and proliferation in response to T cell recep‐ tor (TCR)-mediated stimuli (e.g., concanavalin A and anti-CD3/anti-CD28) [136, 214]. NPC/T lymphocyte co-culture experiments suggest that part of the anti-proliferative effect of NPCs might depend on the inhibition of IL-2 and IL-6 signalling on T lymphocytes [214]. In addi‐ tion, NPCs have shown a selective pro-apoptotic effect on Th17 cells *in vitro* via a FasL (CD95L) dependent mechanism, identifying the axis Fas-Birc3 as an additional survival pathway for NPCs [215]. Mouse C17.2 NPCs also suppress T-cell proliferation, at least in part, by reactive productionofthe solublemediatorsnitric oxide (NO) andprostaglandinE2 (PGE2). Highlevels ofNOandPGE2areinfactinducedinTcellswhenco-culturedwithNPCs.Inaddition,inducible NO synthase (iNOS) and microsomal type 1 PGES (mPGES-1) are detected in NPCs in coculture with T-cells, suggesting that NO and PGE2 production in NPCs is induced by expo‐ sure to activated T cells [216].

*4.3.2. Regulation of the immune system*

CD25+

/CD62L+

306 Neural Stem Cells - New Perspectives

within inflamed brain areas.

ConsiderableevidenceoftheimmunemodulatorycapacityofNPCshasderivedfromtransplan‐ tation studies throughdifferentroutes in theEAEmodel.Asmentioned,transplanted NPCs are consistentlyfoundaroundinflamedbloodvessels,inclosecontactwithbothendogenousneural

Also, i.c.v. administered NPCs were found to attenuate brain inflammation primarily through a reductionofperivascularinfiltrates andCD3+Tcellswitha concomitantincrease ofCD25+ and

chronic neural tissue loss as well as disease-related disability in EAE, via induction of apopto‐

NPCs have been shown to possess immune modulatory capacity also in models of stroke, where T cells do not have a major role in the disease pathology. Irrespectively of the route of administration (systemically *vs.* intraparechimally), transplanted NPCs migrate towards the site of infarct in MCAo and ICH models [12, 137, 138, 203-207] and once reached the ischemic boundary zone (IBZ), grafted NPCs interact with the inflammatory environment. The subacute (*delayed*) i.v. injection of mouse NPCs after MCAo in mice, significantly down-regulates multiple RNA species involved in inflammation, including IFN-γ, TNF-α, IL-1β, IL-6 and leptin receptor [140]. Therefore, NPCs may exert an immune modulatory action, causing a profound down regulation of inflammatory *lymphoid* (T cells) and *myeloid* (macrophages) cells

While the inhibition of the T cell responses by NPCs is a quite established concept [208], the effect of the interaction between transplanted NPCs and microglia/macrophages is still controversial, mainly because of the non-univocal data regarding the role sustained by professional phagocytes under CNS inflammatory conditions. *In vivo* studies have in fact produced opposite evidences that might underline once more the bimodal action of some immune regulators [209]. NPC transplantation promote the infiltration of CD11b+ myeloid cells into the brain of MCAo mice, thus suggesting that myeloid cell activation might be required for transplanted NPCs to exert part of their neuroprotective action [189]. Indeed, MCAo mice in which CD11b+ microglia have been selectively ablated showed exacerbation of the ischemia-dependent brain injury [113]. However, several studies have showed a significant reduction of microglia/macrophages in the brain of mice, with either ischemic or haemorrhagic stroke, together with improved neuronal survival and locomotor functions after NPC trans‐ plantation [22, 140]. Also in this case, NPCs have been engineered in order to increase their immune modulatory capacity. Recently, mouse NPCs were transduced with IL-10, which has been proved to efficiently suppress EAE symptoms and promote survival of neurons and oligodendrocytes [210-212]. Mouse NPCs, transduced with a lentiviral vector encoding IL-10, showed enhanced ability to induce remyelination, neuronal repair and immune suppression

*In vitro*, NPCs are shown to increase the apoptosis of PLP139-151-specific Th1 pro-inflammatory (but not Th2 anti-inflammatory) cells through the engagement of death receptors, including FasL,TNF-relatedapoptosis-inducingligand(TRAIL), andAPO3L,onthe surfaceofNPCs [16]. Mouse and rat NPCs also inhibit T cell activation and proliferation in response to T cell recep‐

regulatory T cells [136]. Interestingly, i.v. injection of NPCs also protects against

immune cells [185].

cells (e.g. astrocytes and neurons) and CNS-infiltrating blood-borne CD45+

sis of blood-borne CNS-infiltrating encephalitogenic T cells [185]

after systemic injection in EAE mice compared to control NPCs [213].

Human NPCs have been proved to suppress the proliferation and alter the cytokine secretion profiles of activated T cells on both xenogeneic antigen-specific T cells derived from EAE induced non-human primates (common marmosets), and allogeneic mitogen-activated T cells. Co-culture of human NPCs with T cells, revealed their immune modulatory capacity through both direct cell-to-cell contacts as well as via the release of soluble mediators into the culture medium [15]. Notably, in contrast to the mouse counterpart, human NPCs have a limited cytotoxicity against T cells *in vitro*, given that FasL is only barely detectable on their surface. However, human NPCs exposed to cytokines express high levels of TNF-α, resulting in a higher cytotoxic potential against monocytes and macrophages [217]. In line with this, immortalized human NPCs were also proved, trough direct *in vitro* experiments, to reduce T cell activation and proliferation. Conditioned media collected from human NSCs (HB1.F3 line) *in vitro*, directly suppress the proliferation of activated human T cells through both induction of apoptosis and cell cycle arrest. Nonetheless, human NPC-released mediators alter the cytokine production pattern of T lymphocytes, increasing the expression of IL-4, IL-10, TNFα, and IFN-γ and decreasing IL-2, thus affecting the overall activation [200].

#### **4.4. NPC interactions with the dysfunctional non-CNS microenvironment**

InparalleltotheobservedimmunemodulationandneuroprotectionintotheCNS,other studies have shown that systemically injected cells may act also outside the injured CNS. Different studies, in fact, have reported the capacity of NPCs to target and synergize with immune cells at the level of secondary lymphoid organs (e.g. draining lymph nodes) and the spleen, result‐ ing in the attenuation of the inflammatory response following EAE, stroke and SCI.

It was initially showed *in vitro* that NPCs strongly inhibited the ability of EAE-derived lympho‐ cytes to produce pro-inflammatory Th1 cytokines in response to MOG35-55 stimulation. In addition, specificallyactivatedTcells isolatedfromEAEmice treatedwithNPCs,weredeficient in their ability to adoptively transfer EAE (to a naïve host), thus suggesting a long-lasting inhibitionof encephalitogenicityofTcells [20].Furtherdatahavebeenprovidedabout a specific and almost exclusive targeting of the peripheral immune system in SJL mice with PLP-in‐ duced EAE, in which NPCs had been injected subcutaneously (s.c.) at 3 and 10 dpi [21]. This alternative administration protocol showed a significant clinical improvement in EAE mice despiteinjectedcellswereneverbeenconsistentlyfoundintotheinflamedCNS.Sub-cutaneous‐

ly injected, s.c.-injected cells were mainly found accumulating and persisting (up to 2 months) at the level of the perivascular areas of the draining lymph nodes, where they interacted with resident cells. Similarly to what observed in the CNS parenchyma, NPCs accumulated as focal clusters around blood vessels of the hilum and medullary/paracortical areas. Here they established close interactions with endothelial cells and cell-to-cell contacts with CD11c+ DCs, F4/80+ professional phagocytes and MHC class II+ immune cells [21]. Further, *ex vivo* analyses of lymph nodes isolated from NPC-treated EAE mice, showed hampered activation and matura‐ tion of myeloid DCs. This was associated, according to both *in vivo* and *in vitro* analyses, to the release of BMP-4 and TNF-α or TLR agonists. The BMP-dependent effect is highly specific for immune regulatory NPCs and, in turn, leads to the restraint of encephalitogenic T cell expan‐ sion at sites of antigen presentation. In addition to BMP-4, transplanted NPCs modulated the local increase of major stem cell fate determinants, including BMP-7, the extracellular matrix protein tenascin C, Shh and Noggin. The pattern of NPC accumulation, the secretion of extracellularmatrixproteinsandstemcellregulators,andthelackofexpressionofneurallineage antigens (e.g. PSA-NCAM, class III β–tubulin, NeuN, NG-2 and GFAP) once more suggest the establishment of perivascular *atypical ectopic niche-like* areas in the peripheral lymph nodes, similarly to what already seen in the CNS [15]. Supported by these experiments, a successive step forward was undertaken to test NPCs therapeutic capacity in a non-human primate model of EAE. Systemically injected foetal human NPCs into MOG74-96-immunized common marmo‐ sets delivered at either the clinical onset of the disease or at subclinical occurrence of MRI detectablebrainlesions,werefoundnotonlyatthelevelofperivascularinflammatoryCNSareas but also in secondary lymphoid organs. In parallel to these observations, human NPCs inter‐ fere *invitro*withanumberofkeyfunctions, suchas thedifferentiationofmyeloidprecursor cells (MPCs) into immature DC (iDC) and the maturation of iDC to functional mature DCs. A significant impairment of the differentiation of CD14+ MPCs into CD1a+ iDCs has been report‐ ed when MPCs were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 in presence of NPCs [15]. In the same study, NPCs influenced the up regulation of the co-stimulatory molecules CD80, CD86 and MHC-II on LPS-treated DCs, thus impairing their capacity to induce a proliferative allogeneic response in mixed leukocyte reaction *in vitro*. Clinically wise,the i.v. NPC injection resulted more efficacious than the i.t. NPC treatment.This might be related to either the higher number of surviving cells or to an additional peripheral effect. Systemic NPCs were, in fact, subjected to selective capturing into cervical lymph nodes where they persisted up to 3 months while establishing close contacts with blood-borne inflammatory cells [15].

reduce the initial cerebral oedema and inflammatory cell infiltration caused by stroke [22]. NPC accumulation into the spleen, in this case, modulated brain inflammation by reducing the level of major inflammatory mediators in stroke, such as TNF-α, IL-6 and nuclear factor-

It has been recently shown that mouse NPCs (from fully mismatched C57BL/6 mice) cotransplanted with pancreatic islet under the kidney capsule of Balb/c diabetic mice prevents acute islet allograft rejection. This effect was related to a significant reduction of CD4+

inducing active tolerance. These data suggest that the peripheral immune-modulation exerted by NPCs could alleviate the immune reaction leading to organ rejection. Unfortunately this condition appeared strictly associated with the development of NPC-derived tumours mainly

Whether most of the immune regulatory effects of systemically injected NPCs act mainly into the CNS or in the periphery is still under debate. Peripheral lymphoid organs have been demonstrated to play an important role in the regulation of the immune responses to myelin antigens in EAE and a very sophisticated modulation of T-cell self-reactivity is known to take place [219-221]. A very recent study proposes a molecular mechanism sustaining NPCs immune modulation capacity in EAE. The preventive (0 dpi) or therapeutic (10 dpi) i.v. administration of NPCs resulted in their accumulation in lymph nodes and spleen, with rare cells observed into the CNS and without any evidence of myelin repair. Nevertheless, treated mice showed partial clinical recovery. Remarkably, the authors achieved the same results even transplanting NPC conditioned-medium or minimally irradiated NPCs (unable to differenti‐ ate but capable of secreting cytokines and neurotrophins), evidence sustaining a true periph‐ eral function of NPCs. In particular, the observed clinical amelioration seem to be related to the selective inhibition of encephalitogenic Th17 cell differentiation through secreted factors. LIF has been identified as the key factor responsible for the observed inhibition of Th17 cell differentiation and the authors elucidated the signalling pathway behind this novel mecha‐ nism of action, where LIF antagonizes IL-6-induced Th17 cell differentiation through ERKdependent inhibition of STAT3 phosphorylation [23]. Further studies will be needed to establish the absolute relevance of these pre-clinical data in EAE, where peripheral lymphoid organs play an important role in the regulation of the immune responses to myelin antigens, and their potential for future applications in MS. All the preclinical data describing NPC

/CD25+

/FoxP3+

**Observed Effect(s)**

Cell

differentiatio

**Proposed Mechanism(s)**

None [10]

**Refs**

T cells

309

regulatory T cells in the spleen,

Systemic stem cell therapies and brain diseases

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

kappa B (NF-κB), and consequently improved neurologic outcome.

sustained by insulin secretion from the co-transplanted islets [218].

therapeutic effect upon systemic administration are summarized in Table 1.

*animal*

1.5-2x104 i.c.v. or i.t.

*Route Time*

*Experimental Autoimmune Encephalomyelitis (EAE)*

Disease peak

**Disease Model Species Transplant Features**

Acute EAE Rat Rat

*Cell type Cell no./*

neurospheres

and with a concomitant enrichment of CD4+

Similarly to what observed in EAE models, i.v. administered human NPCs, in a rat model of ICH, revealed a peripheral therapeutic function in attenuating the inflammatory response to the insult [22]. In line with previous studies, cells were rarely observed into the injured brain while the majority of NPCs were found distributed within the systemic organs. In particular, few NPCs were observed in mesenteric lymph nodes while large numbers were detected in the spleen, especially in the marginal zone area, which is typically enriched in macrophages. Once again NPCs were found in close contact with immune cells and some of them were establishing cell-to-cell contact interactions with CD11b+ spleen macrophages. This result was probably due to the existence of a link between brain and spleen inflammation, called "brainspleen inflammatory coupling". Remarkably, splenectomy prior to ICH has been shown to reduce the initial cerebral oedema and inflammatory cell infiltration caused by stroke [22]. NPC accumulation into the spleen, in this case, modulated brain inflammation by reducing the level of major inflammatory mediators in stroke, such as TNF-α, IL-6 and nuclear factorkappa B (NF-κB), and consequently improved neurologic outcome.

ly injected, s.c.-injected cells were mainly found accumulating and persisting (up to 2 months) at the level of the perivascular areas of the draining lymph nodes, where they interacted with resident cells. Similarly to what observed in the CNS parenchyma, NPCs accumulated as focal clusters around blood vessels of the hilum and medullary/paracortical areas. Here they established close interactions with endothelial cells and cell-to-cell contacts with CD11c+

lymph nodes isolated from NPC-treated EAE mice, showed hampered activation and matura‐ tion of myeloid DCs. This was associated, according to both *in vivo* and *in vitro* analyses, to the release of BMP-4 and TNF-α or TLR agonists. The BMP-dependent effect is highly specific for immune regulatory NPCs and, in turn, leads to the restraint of encephalitogenic T cell expan‐ sion at sites of antigen presentation. In addition to BMP-4, transplanted NPCs modulated the local increase of major stem cell fate determinants, including BMP-7, the extracellular matrix protein tenascin C, Shh and Noggin. The pattern of NPC accumulation, the secretion of extracellularmatrixproteinsandstemcellregulators,andthelackofexpressionofneurallineage antigens (e.g. PSA-NCAM, class III β–tubulin, NeuN, NG-2 and GFAP) once more suggest the establishment of perivascular *atypical ectopic niche-like* areas in the peripheral lymph nodes, similarly to what already seen in the CNS [15]. Supported by these experiments, a successive step forward was undertaken to test NPCs therapeutic capacity in a non-human primate model of EAE. Systemically injected foetal human NPCs into MOG74-96-immunized common marmo‐ sets delivered at either the clinical onset of the disease or at subclinical occurrence of MRI detectablebrainlesions,werefoundnotonlyatthelevelofperivascularinflammatoryCNSareas but also in secondary lymphoid organs. In parallel to these observations, human NPCs inter‐ fere *invitro*withanumberofkeyfunctions, suchas thedifferentiationofmyeloidprecursor cells (MPCs) into immature DC (iDC) and the maturation of iDC to functional mature DCs. A

ed when MPCs were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 in presence of NPCs [15]. In the same study, NPCs influenced the up regulation of the co-stimulatory molecules CD80, CD86 and MHC-II on LPS-treated DCs, thus impairing their capacity to induce a proliferative allogeneic response in mixed leukocyte reaction *in vitro*. Clinically wise,the i.v. NPC injection resulted more efficacious than the i.t. NPC treatment.This might be related to either the higher number of surviving cells or to an additional peripheral effect. Systemic NPCs were, in fact, subjected to selective capturing into cervical lymph nodes where they persisted up to 3 months while establishing close contacts with blood-borne

Similarly to what observed in EAE models, i.v. administered human NPCs, in a rat model of ICH, revealed a peripheral therapeutic function in attenuating the inflammatory response to the insult [22]. In line with previous studies, cells were rarely observed into the injured brain while the majority of NPCs were found distributed within the systemic organs. In particular, few NPCs were observed in mesenteric lymph nodes while large numbers were detected in the spleen, especially in the marginal zone area, which is typically enriched in macrophages. Once again NPCs were found in close contact with immune cells and some of them were establishing cell-to-cell contact interactions with CD11b+ spleen macrophages. This result was probably due to the existence of a link between brain and spleen inflammation, called "brainspleen inflammatory coupling". Remarkably, splenectomy prior to ICH has been shown to

professional phagocytes and MHC class II+

significant impairment of the differentiation of CD14+

inflammatory cells [15].

F4/80+

308 Neural Stem Cells - New Perspectives

DCs,

immune cells [21]. Further, *ex vivo* analyses of

MPCs into CD1a+

iDCs has been report‐

It has been recently shown that mouse NPCs (from fully mismatched C57BL/6 mice) cotransplanted with pancreatic islet under the kidney capsule of Balb/c diabetic mice prevents acute islet allograft rejection. This effect was related to a significant reduction of CD4+ T cells and with a concomitant enrichment of CD4+ /CD25+ /FoxP3+ regulatory T cells in the spleen, inducing active tolerance. These data suggest that the peripheral immune-modulation exerted by NPCs could alleviate the immune reaction leading to organ rejection. Unfortunately this condition appeared strictly associated with the development of NPC-derived tumours mainly sustained by insulin secretion from the co-transplanted islets [218].

Whether most of the immune regulatory effects of systemically injected NPCs act mainly into the CNS or in the periphery is still under debate. Peripheral lymphoid organs have been demonstrated to play an important role in the regulation of the immune responses to myelin antigens in EAE and a very sophisticated modulation of T-cell self-reactivity is known to take place [219-221]. A very recent study proposes a molecular mechanism sustaining NPCs immune modulation capacity in EAE. The preventive (0 dpi) or therapeutic (10 dpi) i.v. administration of NPCs resulted in their accumulation in lymph nodes and spleen, with rare cells observed into the CNS and without any evidence of myelin repair. Nevertheless, treated mice showed partial clinical recovery. Remarkably, the authors achieved the same results even transplanting NPC conditioned-medium or minimally irradiated NPCs (unable to differenti‐ ate but capable of secreting cytokines and neurotrophins), evidence sustaining a true periph‐ eral function of NPCs. In particular, the observed clinical amelioration seem to be related to the selective inhibition of encephalitogenic Th17 cell differentiation through secreted factors. LIF has been identified as the key factor responsible for the observed inhibition of Th17 cell differentiation and the authors elucidated the signalling pathway behind this novel mecha‐ nism of action, where LIF antagonizes IL-6-induced Th17 cell differentiation through ERKdependent inhibition of STAT3 phosphorylation [23]. Further studies will be needed to establish the absolute relevance of these pre-clinical data in EAE, where peripheral lymphoid organs play an important role in the regulation of the immune responses to myelin antigens, and their potential for future applications in MS. All the preclinical data describing NPC therapeutic effect upon systemic administration are summarized in Table 1.



**Disease Model Species Transplant Features**

Chronic EAE Mouse Mouse MSC-

Marmoset

Relapsing EAE Mouse Mouse NPCs

MCAo (10' or

90')

Mouse CCR5-

Chronic EAE Passive EAE

Chronic EAE Common

*Cell type Cell no./*

derived NPCs

transduced mouse BMderived NPCs

Relapsing EAE Mouse Mouse NPCs 1x106 i.v. Disease

Relapsing EAE Mouse Mouse NPCs 0.5x106 s.c. 3 and 10

and Olig2 transduced NPCs

MCAo Rat Rat NPCs 1x105 i.t. 2 dpi Cell

MCAo (180') Rat Rat NPCs 1x105 i.t. 2 dpi Tissue

Rat Human NPCs 5x106 i.v. 1 dpi Cell

*animal*

3.5x104-6. 1x106

Human NPCs 2-6x106 i.t. or i.v. Disease

*Route Time*

1.5x106 i.v. 22 dpi

i.t. 21, 28 and 35 dpi

(peak)

onset

onset or first relapse

dpi, or 10 dpi only

onset or first relapse

1.5x105 i.c.v. Disease

*Stroke*

**Observed Effect(s)**

Tissue trophism

Immune regulation

Immune regulation (central)

Immune regulation (central)

Immune regulation (peripheral)

Immune regulation (central) and Tissue trophism

differentiatio n (neuronal)

differentiatio n (neuronal, glial)

trophism

**Proposed Mechanism(s)**

Systemic stem cell therapies and brain diseases

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

None [258]

None [259]

Suppression of encephalitogen ic T cells, imparment of dendritic cell maturation

Induction of T cell apoptosis

BMP-4 dependent hindrance of dendritic cell maturation

None [260]

None [204]

None [12, 187]

[190]

Increased angiogenesis [15]

[16]

[21]

**Refs**

311


**Disease Model Species Transplant Features**

Acute EAE Rat Rat

310 Neural Stem Cells - New Perspectives

Chronic EAE Mouse Mouse

Chronic EAE Mouse Human ES

Chronic EAE Mouse IL-10-

Chronic EAE Mouse Mouse and

Chronic EAE Passive EAE

*Cell type Cell no./*

neurospheres

neurospheres

cell-derived NPCs

transduced mouse NPCs

human ES cell-derived NPCs

Chronic EAE Mouse Mouse NPCs 1x106 i.v. or i.t. 22 dpi (Low) cell

Mouse Mouse NPCs 1x106 i.v. 8 dpi Immune

1.5x106 i.v. or

i.c.v.

*animal*

*Route Time*

2x104 i.c.v. 0 dpi Immune

2.5x103 i.c.v. 6 dpi Immune

5x105 i.c.v. 7 dpi Immune

2x106 i.v. 0 or 10 dpi Immune

10, 22 or 30 dpi

**Observed Effect(s)**

n (neuronal and glial and tissue trophism

regulation (central)

differentiatio n and tissue trophism

regulation (local)

regulation (peripheral)

regulation (local)

Immune regulation (local, peripheral) and cell differentiatio

n

regulation (peripheral)

**Proposed Mechanism(s)**

None [136]

[11]

[139]

[20]

[142]

[213]

[23]

Inhibition of reactive gliosis

Reduction of CNS

inflammatory infiltrates, increase of regulatory T cells

Suppression of encephalitogen ic T cells

Suppression of encephalitogen ic T cells

Induction of T cell apoptosis, promotion of myelin debris clearance

LIF-mediated inhibition of Th17 cell differentiation **Refs**


**Disease Model Species Transplant Features**

Contusion (T8) Mouse Mouse NPCs 1x106

Contusion (T12) Mouse Mouse NPCs

and MOG35-55 immunization

**5. Pros and cons of NPC systemic administration**

s.c.: subcutaneous; TAT-Hsp70: TAT-heat shock protein.

Compression (T8)

models.

*Cell type Cell no./*

*animal*

or1x105

Mouse Human NPCs 1x107 i.v. 7 dpi Cell

*Route Time*

*Spinal Cord Injury (SCI)*

5x105 i.c.v. 7 dpi Immune

BBB: blood-brain barrier; BM: bone marrow; BMP-4: bone morphogenetic protein 4; CCAo: common carotid artery occlusion; CCR5: C-C chemokine receptor type 5; dpi: days post immunization/injury; ES cells: embryonic stem cells; GDNF: glial-derived neurotrophic factor; HIF-1α: hypoxia-inducible factor 1α; i.ca.: intracarotid; ICH: intracerebral haemorrhage; i.c.v.: intracerebroventricular; i.p.c.: intraparenchymal (perilesional); i.t.: intrathecal; i.v.: intravenous; LIF: leukemia inhibitory factor; MCAo: middle cerebral artery occlusion; MSC: mesenchymal stem cells; ROS: reactive oxygen species;

**Table 1.** Neuro-immune interaction following systemic neural stem cell transplantation in experimental disease

In parallel to the investigation concerning the principal mechanism(s) sustaining NPC therapeutic efficacy, other questions, such as (i) the ideal administration route, (ii) the amount of cells to be transplanted and (iii) the optimal time point for cell delivery need to be answered. Among the different possible routes of cell administration, intravenous cell delivery represents one of the most attractive because of its technical simplicity and clinical practicability. However, i.v. and i.t. administrations result in lower numbers of cells infiltrating the CNS, compared to local stereotaxic-driven intracerebral injections, a reason why local injections of cells are commonly preferred in clinical trials (see next section) despite the higher invasiveness of the procedure. Even though initially investigated for multifocal disorders (e.g. MS), in order

i.v. or i.p.c.

**Observed Effect(s)**

n (glial and neuronal)

trophism

differentiatio n (neuronal, glial)

regulation (local) and Tissue trophism

Acute Tissue

**Proposed Mechanism(s)**

Systemic stem cell therapies and brain diseases

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

neuronal circuitries

Reduction of apoptosis and modulation of TNF-α expression

None [167]

T-cell mediated activation of microglia with a protective phenotype

**Refs**

313

[13]

[226]


BBB: blood-brain barrier; BM: bone marrow; BMP-4: bone morphogenetic protein 4; CCAo: common carotid artery occlusion; CCR5: C-C chemokine receptor type 5; dpi: days post immunization/injury; ES cells: embryonic stem cells; GDNF: glial-derived neurotrophic factor; HIF-1α: hypoxia-inducible factor 1α; i.ca.: intracarotid; ICH: intracerebral haemorrhage; i.c.v.: intracerebroventricular; i.p.c.: intraparenchymal (perilesional); i.t.: intrathecal; i.v.: intravenous; LIF: leukemia inhibitory factor; MCAo: middle cerebral artery occlusion; MSC: mesenchymal stem cells; ROS: reactive oxygen species; s.c.: subcutaneous; TAT-Hsp70: TAT-heat shock protein.

**Table 1.** Neuro-immune interaction following systemic neural stem cell transplantation in experimental disease models.

#### **5. Pros and cons of NPC systemic administration**

**Disease Model Species Transplant Features**

Mouse Mouse 17.2 NSCs

> transduced rat NPCs

> transduced rat NPCs

transduced mouse NPCs

ICH Rat Human NPCs 5x106 i.v. 1 dpi Tissue

CCAo + global hypoxiaischemia

MCAo and CCAo

MCAo (120') Rat GDNF-

312 Neural Stem Cells - New Perspectives

MCAo (90') Rat HIF-1α-

MCAo (45') Mouse TAT-Hsp70-

*Cell type Cell no./*

MCAo (180' Rat Human NPCs 1x106 i.v. 2 dpi Tissue

MCAo (45') Mouse Mouse NPCs 1x106 i.v. 3 dpi Immune

Rat Rat NPCs 1.5x105 i.t. 7 dpi Immune

1x106 or 5x105

*animal*

*Route Time*

3x105 i.ca. 2 dpi Tissue

5x105 i.c.v. 3 dpi Tissue

1x106 i.c.v. 1 dpi Tissue

i.v. or i.p.c.

**Observed Effect(s)**

trophism

trophism

trophism, Cell

differentiatio n (neuronal)

regulation (local) and Tissue trophism

regulation and tissue trophism

trophism

trophism, reduction of ROS formation and BBB leakage

trophism, Cell

differentiatio

Acute Tissue

**Proposed Mechanism(s)**

None [261]

None [198]

Reduction of microglial activation and neuronal death

Neuroprotectio n mediated by NGF and modulation of class I MHC expression

Promotion of angiogenesis

Neuroprotectio

Neuroprotectio

n and integration in endogenous

n and enhanced neurogenesis [18]

[140]

[141]

[227]

[232]

[137]

Increased angiogenesis **Refs**

In parallel to the investigation concerning the principal mechanism(s) sustaining NPC therapeutic efficacy, other questions, such as (i) the ideal administration route, (ii) the amount of cells to be transplanted and (iii) the optimal time point for cell delivery need to be answered. Among the different possible routes of cell administration, intravenous cell delivery represents one of the most attractive because of its technical simplicity and clinical practicability. However, i.v. and i.t. administrations result in lower numbers of cells infiltrating the CNS, compared to local stereotaxic-driven intracerebral injections, a reason why local injections of cells are commonly preferred in clinical trials (see next section) despite the higher invasiveness of the procedure. Even though initially investigated for multifocal disorders (e.g. MS), in order to deliver exogenous cells to all the disseminated inflammatory foci, all the previous experi‐ mental data suggest that intravenous or intrathecal administration routes could be desirable even for focal damages, such as those occurring in stroke and spinal cord injury [222]. In experimental animal studies, i.p.c [223-225], i.v. [137, 185] i.a. [222], i.t. [204, 226] and i.c.v. [142, 227] protocols have been tested so far. However, only few comparative studies have been conducted, testing pros and cons of the different administration routes. These studies (mainly in animal models of stroke) evidenced the obvious capacity of intraparenchymal injection to deliver higher numbers of cells *in situ*, compared to i.c.v. and i.v. [228]. By contrast, systemic injections are thought to lead to a wider distribution of cells around the focal lesioned area. This aspect is extremely important if we consider that human stem cells (and in particular hNPCs) are still a limited resource [229]. Intravenously injected NPCs are firstly delivered to peripheral organs, such as lungs, liver, spleen and kidney [16, 230]. This whole-body distri‐ bution of exogenous systemic injected NPCs significantly reduces cell homing to the injured brain [222]. To avoid this problem, at least partially, intra-arterial administration could be a valid alternative (possibly coupled with pre-interventional imaging-based planning) to selectively cover an injured volume supplied by several target vessels. Intracarotid injection has already been proved to be functional for delivering stem cells in models of stroke, TBI and SCI, resulting in higher numbers of extravasating cells (20%) compared to i.v. injections [18]. Nevertheless, although the number of cells infiltrating the CNS has been sometimes described as fundamental, or at least proportional to their therapeutic effect [231], others have shown that very low numbers of cells [140] can result into similar outcomes (in term of functional recovery) compared to higher numbers of locally injected cells. This effect may be explained by the fact that cell replacement is unlikely the only mechanism sustaining stem-cell thera‐ peutic potential. Higher starting numbers of cells, in fact, increase the therapeutic potential of intracerebral administered cells, but did not affected the efficacy of the i.v. injected cells. This again suggests that the number of cells is much more important for focal than systemic injections [232].

are subjected to highly inflammatory conditions causing cell death [205, 236]. On the contrary, the sub acute phase (few days after insult, in rodents) of the injury seems to be characterized by better conditions for stem cell survival and a permissive microenvironment for tissue repair/ healing [237]. Although higher inflammation generally correlates with higher number of cells infiltrating the CNS, it has been shown that greater numbers of cells accumulated into the spinal cord after i.v. injection at 7 dpi compared to 3 and 10 dpi [167]. However, the optimal time window for cell transplantation is still elusive and depends mainly on the type of pathology and aim of the treatment. While neuroprotection should be addressed in the early stage of the inflammatory disease, just after the initial insult, cell replacement and neurore‐ generation should be targeted in a later stage, when the lesion has stabilized. Indeed, admin‐ istration route, number of cells and time window and seem intimately related and it is not so difficult to envisage a future in which a combination of early i.v. and late i.p.c administration of different stem cell sources will be enrolled for the treatment of so far incurable CNS

Systemic stem cell therapies and brain diseases

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315

In the last two decades the clinical potential of stem cells in the field of regenerative/restorative medicine has been often matter of debate, mainly because of its inconsistent outcome. As an example, the first attempt to treat a CNS disorder by means of stem cell transplantation took place in the '80s: autologous adrenal medulla cells were intracerebrally transplanted into the striatum of PD patients to provide a local source of catecholamine. The study was proved safe although with minimal beneficial effect. Further, the first intrastriatal grafts of human foetal ventral mesencephalic (neuronal preparations) tissue have provided proof-of-concept that cell therapy can work in patients affected by PD [238]. However, subsequent randomized, doubleblind, placebo-controlled trials brought to much more sceptical conclusions because of patients showing functional decline (post transplantation) due to dyskinesias (graft-induced involun‐

Prospectively, many factors can be contended to (partially) justify these patchy results. First, it is now clear that different cell types are needed for different diseases. If on one side PD and amyotrophic lateral sclerosis (ALS) patients will require cells with dopaminergic and motor neuron properties respectively, on the other side, cell replacement in AD patients is much more complicated by the necessity to replace a large variety of cell populations lost in different brain areas. Second, even though initially expected and long-term envisaged, neuronal replacement and circuitry integration of transplanted NPCs have been poorly proved. Third, it as to be considered that pre-clinical animal studies only represent models of human conditions, and, as such, they offer an exceptionally homogeneous platform, where the genetic background, age, and environment are all alike. Clearly, this is not the case with patients. Further, even if multiple models have been established to investigate different aspects of a given disease, none of them can faithfully emulate the human pathology in its complexity [241, 242]. This is particularly challenging considering the rate of progression and lack of validated surrogate disease markers typical of many neurodegenerative disorders. While these aspects are most

tary movements), originated by excessive graft function [239, 240].

disorders.

**6. Clinical trials**

Importantly, when evaluating the optimal protocol, we should consider the procedure itself, so that the risk should not outweigh the benefits of the treatment. From this point of view, i.e. cell injection might be accompanied by increased mortality during cell delivery, probably due to further ischemia or thrombosis [233, 234]. By contrast, cell transplantation trough the vertebral artery, into patients affected by SCI, showed no adverse effects [235].

Another important unsolved issue for experimental stem cell therapies is the ideal time point of transplantation. As described, the inflammatory activation of the CNS, characterizing MS, stroke, SCI, epilepsy, AD, PD, HD is necessary for the homing of systemically injected cells. Because of the rapid and dynamic changes occurring into the CNS during these inflammatory conditions, the time of transplantation should be evaluated carefully. In fact, cell death, excitotossicity, reactive oxygen species accumulation, inflammatory cell infiltrations and glial scar formation, cause a rapid evolution in the damaged tissue, while creating an hostile microenvironment for the engraftment of exogenous cells. This is important irrespectively to the route of administration. For example, the acute focal transplantation of cells into the ischemic brain or the injured spinal cord reduces the therapeutic efficacy of the cells, which are subjected to highly inflammatory conditions causing cell death [205, 236]. On the contrary, the sub acute phase (few days after insult, in rodents) of the injury seems to be characterized by better conditions for stem cell survival and a permissive microenvironment for tissue repair/ healing [237]. Although higher inflammation generally correlates with higher number of cells infiltrating the CNS, it has been shown that greater numbers of cells accumulated into the spinal cord after i.v. injection at 7 dpi compared to 3 and 10 dpi [167]. However, the optimal time window for cell transplantation is still elusive and depends mainly on the type of pathology and aim of the treatment. While neuroprotection should be addressed in the early stage of the inflammatory disease, just after the initial insult, cell replacement and neurore‐ generation should be targeted in a later stage, when the lesion has stabilized. Indeed, admin‐ istration route, number of cells and time window and seem intimately related and it is not so difficult to envisage a future in which a combination of early i.v. and late i.p.c administration of different stem cell sources will be enrolled for the treatment of so far incurable CNS disorders.

#### **6. Clinical trials**

to deliver exogenous cells to all the disseminated inflammatory foci, all the previous experi‐ mental data suggest that intravenous or intrathecal administration routes could be desirable even for focal damages, such as those occurring in stroke and spinal cord injury [222]. In experimental animal studies, i.p.c [223-225], i.v. [137, 185] i.a. [222], i.t. [204, 226] and i.c.v. [142, 227] protocols have been tested so far. However, only few comparative studies have been conducted, testing pros and cons of the different administration routes. These studies (mainly in animal models of stroke) evidenced the obvious capacity of intraparenchymal injection to deliver higher numbers of cells *in situ*, compared to i.c.v. and i.v. [228]. By contrast, systemic injections are thought to lead to a wider distribution of cells around the focal lesioned area. This aspect is extremely important if we consider that human stem cells (and in particular hNPCs) are still a limited resource [229]. Intravenously injected NPCs are firstly delivered to peripheral organs, such as lungs, liver, spleen and kidney [16, 230]. This whole-body distri‐ bution of exogenous systemic injected NPCs significantly reduces cell homing to the injured brain [222]. To avoid this problem, at least partially, intra-arterial administration could be a valid alternative (possibly coupled with pre-interventional imaging-based planning) to selectively cover an injured volume supplied by several target vessels. Intracarotid injection has already been proved to be functional for delivering stem cells in models of stroke, TBI and SCI, resulting in higher numbers of extravasating cells (20%) compared to i.v. injections [18]. Nevertheless, although the number of cells infiltrating the CNS has been sometimes described as fundamental, or at least proportional to their therapeutic effect [231], others have shown that very low numbers of cells [140] can result into similar outcomes (in term of functional recovery) compared to higher numbers of locally injected cells. This effect may be explained by the fact that cell replacement is unlikely the only mechanism sustaining stem-cell thera‐ peutic potential. Higher starting numbers of cells, in fact, increase the therapeutic potential of intracerebral administered cells, but did not affected the efficacy of the i.v. injected cells. This again suggests that the number of cells is much more important for focal than systemic

Importantly, when evaluating the optimal protocol, we should consider the procedure itself, so that the risk should not outweigh the benefits of the treatment. From this point of view, i.e. cell injection might be accompanied by increased mortality during cell delivery, probably due to further ischemia or thrombosis [233, 234]. By contrast, cell transplantation trough the

Another important unsolved issue for experimental stem cell therapies is the ideal time point of transplantation. As described, the inflammatory activation of the CNS, characterizing MS, stroke, SCI, epilepsy, AD, PD, HD is necessary for the homing of systemically injected cells. Because of the rapid and dynamic changes occurring into the CNS during these inflammatory conditions, the time of transplantation should be evaluated carefully. In fact, cell death, excitotossicity, reactive oxygen species accumulation, inflammatory cell infiltrations and glial scar formation, cause a rapid evolution in the damaged tissue, while creating an hostile microenvironment for the engraftment of exogenous cells. This is important irrespectively to the route of administration. For example, the acute focal transplantation of cells into the ischemic brain or the injured spinal cord reduces the therapeutic efficacy of the cells, which

vertebral artery, into patients affected by SCI, showed no adverse effects [235].

injections [232].

314 Neural Stem Cells - New Perspectives

In the last two decades the clinical potential of stem cells in the field of regenerative/restorative medicine has been often matter of debate, mainly because of its inconsistent outcome. As an example, the first attempt to treat a CNS disorder by means of stem cell transplantation took place in the '80s: autologous adrenal medulla cells were intracerebrally transplanted into the striatum of PD patients to provide a local source of catecholamine. The study was proved safe although with minimal beneficial effect. Further, the first intrastriatal grafts of human foetal ventral mesencephalic (neuronal preparations) tissue have provided proof-of-concept that cell therapy can work in patients affected by PD [238]. However, subsequent randomized, doubleblind, placebo-controlled trials brought to much more sceptical conclusions because of patients showing functional decline (post transplantation) due to dyskinesias (graft-induced involun‐ tary movements), originated by excessive graft function [239, 240].

Prospectively, many factors can be contended to (partially) justify these patchy results. First, it is now clear that different cell types are needed for different diseases. If on one side PD and amyotrophic lateral sclerosis (ALS) patients will require cells with dopaminergic and motor neuron properties respectively, on the other side, cell replacement in AD patients is much more complicated by the necessity to replace a large variety of cell populations lost in different brain areas. Second, even though initially expected and long-term envisaged, neuronal replacement and circuitry integration of transplanted NPCs have been poorly proved. Third, it as to be considered that pre-clinical animal studies only represent models of human conditions, and, as such, they offer an exceptionally homogeneous platform, where the genetic background, age, and environment are all alike. Clearly, this is not the case with patients. Further, even if multiple models have been established to investigate different aspects of a given disease, none of them can faithfully emulate the human pathology in its complexity [241, 242]. This is particularly challenging considering the rate of progression and lack of validated surrogate disease markers typical of many neurodegenerative disorders. While these aspects are most likely destined to remain unsolved pitfalls, others (including the amount of cells to be transplanted, the manipulation protocols used, the time of transplantation, the route of cell delivery and the statistics adopted to analyse the data) need to be ameliorated through the establishment of common guidelines. In particular, the International Society for Stem Cell Research (ISSCR) composed with a group of international experts (scientists, surgeons, ethicists and patient advocates) "The ISSCR Guidelines for the Clinical Translation of Stem Cells" [243] to trace a roadmap guiding the application of experimental stem cell therapeutics in patients. Importantly, when translating into clinical trials, the choice of the "ideal patient" imposes major scientific and ethical constraints. Indeed, if on one side the treatment of the most chronic/severe patients who were not able to respond to previous treatment lowers the blame for a possible ineffectiveness of a therapy, on the other side the scenario offered by such a compromised tissue may hinder the potential effect of the treatment.

**Sponsor and place**

ReNeuro n, Ltd. at Glasgow Southern General Hospital, Glasgow (UK)

Neuralst em, Inc. at Emory Universit y, Atlanta (USA)

Azienda Ospedali era Santa Maria, Terni (Italy)

StemCell s, Inc. at Universit y of Californi a, San Francisco (USA)

Pelizaeus Merzbach er disease (PMD)

Amyotro phic Lateral Sclerosis (ALS)

Stable Ischemic Stroke (PISCES)

**Disease Trial phas e**

**Patien ts (no)**

**Age at enrolm ent (y)**

I 12 60-85 24 CTX0E30

I 18 > 18 48 NSI-566R

ALS I 18 20-75 36 Foetal,

I 4 0.5-5 12 HuCNS-

**Follo w up (mont hs)**

*Cell type Cell no./*

3 (Foetal, Brainderived, c-myc immortal ized, Allogene ic, single donor)

SC (Foetal, Spinal cordderived, Allogene ic, single donor)

Brainderived, Allogene ic, single donor

SC®

2.5x105/ injection

3x108 Multiple

*patient*

2-20x106 Single

0.5-1x106 Multiple

injection s, Intraspin al

Multiple injection s, Single dose, Intraspin al

injection s, Single dose, Intracere bral

> 6 months

NA Y (9

months pt)

≥ 1.5 years Y (≥ 4 months pt)

AnR Eva

NA AR Angelo

Feldman , MD, PhD

Vescovi, PhD

AnR Stephen Huhn, MD

injection, Four Ascendin g doses, Intracere bral (putame n)

**Transplant Features Statu**

*Route Time after disease/ injury*

> 0.5-5 years

**s**

Systemic stem cell therapies and brain diseases

NA AR Keith

Muir, MD

*Immune suppress ion*

**Principal Investig ator**

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

**Trial Identifier**

NCT0115 1124

NCT0134 8451

NCT0164 0067

NCT0100 5004

NA

[250]

[248, 249]

NA

**Outc ome and Note s**

317

The primary importance of patient's care dramatically impacts also on the choice of the best route of cell delivery. Indeed, if one side the intravenous injection allows for a less invasive procedure, on the other, the number of cells delivered to the site of interest is lower compared to local injections. Further, the intracerebral transplantation has been widely accepted, by both clinicians and patients, after years of clinical applications and technical improvements. These, together with the relatively limited availability of human NPCs explains why most of the clinical trials started so far have nevertheless favoured the adoption of more invasive proce‐ dures, such as intraparenchymal/intracerebroventricular ones (see Table 2). However, as discussed, the correlation between the number of cells entering the CNS and their efficacy still need to be confirmed.



likely destined to remain unsolved pitfalls, others (including the amount of cells to be transplanted, the manipulation protocols used, the time of transplantation, the route of cell delivery and the statistics adopted to analyse the data) need to be ameliorated through the establishment of common guidelines. In particular, the International Society for Stem Cell Research (ISSCR) composed with a group of international experts (scientists, surgeons, ethicists and patient advocates) "The ISSCR Guidelines for the Clinical Translation of Stem Cells" [243] to trace a roadmap guiding the application of experimental stem cell therapeutics in patients. Importantly, when translating into clinical trials, the choice of the "ideal patient" imposes major scientific and ethical constraints. Indeed, if on one side the treatment of the most chronic/severe patients who were not able to respond to previous treatment lowers the blame for a possible ineffectiveness of a therapy, on the other side the scenario offered by such

The primary importance of patient's care dramatically impacts also on the choice of the best route of cell delivery. Indeed, if one side the intravenous injection allows for a less invasive procedure, on the other, the number of cells delivered to the site of interest is lower compared to local injections. Further, the intracerebral transplantation has been widely accepted, by both clinicians and patients, after years of clinical applications and technical improvements. These, together with the relatively limited availability of human NPCs explains why most of the clinical trials started so far have nevertheless favoured the adoption of more invasive proce‐ dures, such as intraparenchymal/intracerebroventricular ones (see Table 2). However, as discussed, the correlation between the number of cells entering the CNS and their efficacy still

**Transplant Features Statu**

*Route Time after disease/ injury*

> ≥ 3 months

**s**

AR Armin Curt, MD

*Immune suppress ion*

Y (9 months pt)

**Principal Investig ator**

**Trial Identifier**

NCT0132 1333

NA

**Outc ome and Note s**

a compromised tissue may hinder the potential effect of the treatment.

need to be confirmed.

316 Neural Stem Cells - New Perspectives

**Disease Trial phas e**

**Patien ts (no)**

**Age at enrolm ent (y)**

I/II 12 18-60 12 HuCNS-

**Follo w up (mont hs)**

*Cell type Cell no./*

SC® (Foetal, Brainderived, Allogene ic, single donor)

*patient*

2x107 Multiple

injection s, Single dose, Intramed ullar

**Sponsor and place**

StemCell s, Inc. at Universit y Hospital Balgrist-Uniklinik Zurich, (Switzerl and)

Thoracic spinal cord injuries (SCI)


pt: post-transplant; NA: information not available. AR: Active Recruiting; AnR: Active not Recruiting; C: Completed.

Nevertheless, translational research did not (and should not) stop. As a matter of fact, the huge amount of data collected so far led to the development of numerous early stage clinical trials. There are currently 1750 studies employing the use of stem cells in interventional clinical trials, among which 280 are testing NPCs. Within the total number of clinical studies, 277 worldwide open trials involve SCs and patients affected by CNS disorders (Figure 2), such as MS, stroke, SCI, epilepsy, ALS, PD, AD, HD, neuronal ceroid lipofuscinose (NCL), Pelizaeus–Merzbacher disease (PMD), age-related macular degeneration (AMD) [246]. Several phase I and II clinical studies with NPCs (9 in highly debilitating CNS disorders, Table 2) have now been started with the primary aim to verify the safety (mainly in terms of toxicity) and feasibility - rather than the efficacy - of the treatment. Further, clinical studies are often accompanied by nonofficial secondary end-points usually concerning the potential impact in the clinical outcomes. These explorative trials have certainly a key role in stem cell medicine development, as both phase II dose-escalation studies and the inclusion of non-fatal diseases with larger population

**Figure 2. Worldwide open experimental clinical trials involving the use of stem cells for CNS disorders.** The map shows the distribution of open stem cell-based clinical trials enrolling patients affected by CNS pathologies and treat‐ ed with different source of SCs. Figure has been generate by clinicaltrials.gov, using the keywords *stem cells* AND *CNS diseases*; the inclusion criteria were only open studies; while the exclusion criteria where studies with unknown status. Stem cells in the figure include: neural stem/precursor cells, mesenchymal stem cells from the bone marrow and the

Systemic stem cell therapies and brain diseases

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

319

In May 2006 at Oregon Health and Science University (OHSU, Portland, OR, USA) the first human study involving the transplantation of allogeneic NPCs was started on a fatal rare neurometabolic syndrome, such as the NCL Batten's disease. In this open-label dose-escalating phase I trial, a total of 6 subjects with infantile and late-infantile NCL were transplanted in a single-stage procedure. StemCell, Inc. proprietary, single donor allogeneic free-floating

bases will definitely be facilitated once human safety will be established.

adipose tissue, hematopoietic stem cells, embryonic stem cells.

**Table 2.** Active clinical trials with neural stem/precursor cells.

Other challenging problems that need to be faced when approaching the clinic, are related to safety, product potency, and manufacturing quality of the cell source. Indeed, principles of good tissue practice (GTP) and good manufacturing practice (GMP) are mandatory require‐ ments, especially when dealing with cells of human origin [244].

Last, but not least, some major issues related to the long-term safety of the cellular product need to be solved. It is important to stress how, differently from the classical drug-therapy, a cell-based treatment cannot be discontinued, since once the cells are within the patient they cannot be removed. Therefore, long-term pre-clinical data need to be collected before trans‐ lating from the bench to the bed-side to avoid the occurrence of dramatic outcomes, such as the one involving a young patient suffering of Ataxia Telangiectasia who developed a donorderived brain tumour following neural stem cell transplantation [245].

**Sponsor and place**

StemCell s, Inc. at Oregon Health and Science Universit y, Portland (USA)

StemCell s, Inc. Retina Foundati on of the Southwe st, Dallas (USA)

Agerelated Macular Degenera tion (AMD)

Neuronal Ceroid Lipofusci nosis (NCL)

**Disease Trial phas e**

318 Neural Stem Cells - New Perspectives

**Patien ts (no)**

**Age at enrolm ent (y)**

I 6 1.5-12 13 HuCNS-

I/II 16 > 50 12 HuCNS-

**Table 2.** Active clinical trials with neural stem/precursor cells.

ments, especially when dealing with cells of human origin [244].

derived brain tumour following neural stem cell transplantation [245].

**Follo w up (mont hs)**

*Cell type Cell no./*

SC®

SC®

*patient*

0.5-1x109 Multiple

0.2-1x106 Single

pt: post-transplant; NA: information not available. AR: Active Recruiting; AnR: Active not Recruiting; C: Completed.

Other challenging problems that need to be faced when approaching the clinic, are related to safety, product potency, and manufacturing quality of the cell source. Indeed, principles of good tissue practice (GTP) and good manufacturing practice (GMP) are mandatory require‐

Last, but not least, some major issues related to the long-term safety of the cellular product need to be solved. It is important to stress how, differently from the classical drug-therapy, a cell-based treatment cannot be discontinued, since once the cells are within the patient they cannot be removed. Therefore, long-term pre-clinical data need to be collected before trans‐ lating from the bench to the bed-side to avoid the occurrence of dramatic outcomes, such as the one involving a young patient suffering of Ataxia Telangiectasia who developed a donor-

injection, Single dose, Subretin al

injection s, Single dose, Intracere bral

**Transplant Features Statu**

*Route Time after disease/ injury*

**s**

C Robert Steiner, MD

AR David Birch, PhD

*Immune suppress ion*

months pt)

NA Y (12

NA Y (3

months pt)

**Principal Investig ator**

**Trial Identifier**

NCT0033 7636

NCT0163 2527

NA

**Outc ome and Note s**

[247]

**Figure 2. Worldwide open experimental clinical trials involving the use of stem cells for CNS disorders.** The map shows the distribution of open stem cell-based clinical trials enrolling patients affected by CNS pathologies and treat‐ ed with different source of SCs. Figure has been generate by clinicaltrials.gov, using the keywords *stem cells* AND *CNS diseases*; the inclusion criteria were only open studies; while the exclusion criteria where studies with unknown status. Stem cells in the figure include: neural stem/precursor cells, mesenchymal stem cells from the bone marrow and the adipose tissue, hematopoietic stem cells, embryonic stem cells.

Nevertheless, translational research did not (and should not) stop. As a matter of fact, the huge amount of data collected so far led to the development of numerous early stage clinical trials. There are currently 1750 studies employing the use of stem cells in interventional clinical trials, among which 280 are testing NPCs. Within the total number of clinical studies, 277 worldwide open trials involve SCs and patients affected by CNS disorders (Figure 2), such as MS, stroke, SCI, epilepsy, ALS, PD, AD, HD, neuronal ceroid lipofuscinose (NCL), Pelizaeus–Merzbacher disease (PMD), age-related macular degeneration (AMD) [246]. Several phase I and II clinical studies with NPCs (9 in highly debilitating CNS disorders, Table 2) have now been started with the primary aim to verify the safety (mainly in terms of toxicity) and feasibility - rather than the efficacy - of the treatment. Further, clinical studies are often accompanied by nonofficial secondary end-points usually concerning the potential impact in the clinical outcomes. These explorative trials have certainly a key role in stem cell medicine development, as both phase II dose-escalation studies and the inclusion of non-fatal diseases with larger population bases will definitely be facilitated once human safety will be established.

In May 2006 at Oregon Health and Science University (OHSU, Portland, OR, USA) the first human study involving the transplantation of allogeneic NPCs was started on a fatal rare neurometabolic syndrome, such as the NCL Batten's disease. In this open-label dose-escalating phase I trial, a total of 6 subjects with infantile and late-infantile NCL were transplanted in a single-stage procedure. StemCell, Inc. proprietary, single donor allogeneic free-floating cultured, foetal-derived brain human NPCs (HuCNS- SC®) were directly administered to the cerebral hemispheres and lateral ventricles. Immune suppression was administered for 12 months after transplantation. This study has now been completed with one out of 6 patients died for disease progression, 11 months after treatment. The cell transplantation and combi‐ nation with prolonged immune suppression were both well tolerated [247].

In June 2012, the Azienda Ospedaliera Santa Maria (Terni, Italy) enrolled the very first of total 18 ALS patients to treat with intraspinally implanted allogeneic free-floating cultured, foetal-

Systemic stem cell therapies and brain diseases

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321

Importantly, there are not yet clinical trials with NPCs in MS. However, a consensus paper has recently been produced by a group of experts to define the uniform guidelines on the devel‐ opment of haematopoietic and non-haematopoietic stem cell therapies for MS [9]. All the

While in this paragraph we offer an overview of the current clinical trials involving solely human NPCs, it has to be said that, in the light of the neuroprotective/immunomodulatory (rather than cell replacement) properties attributed to stem cells, the therapeutic plasticity of cells of non-neural origin are being tested as well. Among these, MSCs are emerging as a good potential candidate, mainly because of their great accessibility and remarkable proliferation. Also, growing evidence suggests that other than giving origin to multiple derivatives of the mesodermal lineage (from which they derive), under particular conditions MSCs seem able to *transdifferentiate* into neuro-ectodermal cells *in vitro* [251-254]. However, this ability to convert from one lineage to another is still highly questionable and opened to different interpretations. Several studies have also proved the ability of MSCs to survive, migrate and eventually bring about functional recovery when transplanted into the CNS of different experimental models of neurological diseases (for a review see [255]). However, the mechanisms yielding to such

Since 2006, the advent of induced pluripotent stem cells (iPSCs, [24]) technology has brought new excitement in medical research and clinical therapy, since these cells provide a valuable alternative without being constrained by ethical issues and immunological incompatibility [256]. Although still under debate about their long-term safety, the methods for iPSC genera‐ tion, reprogramming and differentiation efficiency, iPSCs represent a break-trough for both study of disease mechanisms and investigation of potential new treatments (for a perspective

The potential impact of this technological platform has been further boosted by the scientific stream emerged from iPSC technology that is the "direct reprogramming" from one somatic lineage to another. In fact, the direct conversion of fibroblasts to functional neurons (iN cells) or iNSCs [25, 26], for example, represents one of the most exciting, ultimate technologies for future application in CNS pathologies. Thanks to these next generation techniques, it will be possible to derive virtually unlimited numbers of specific neural/neuronal population bypassing the pluripotent stage, thus likely eliminating the potential presence of unwanted undifferentiated cells. However, many issues, such as the purity of the cell preparation, the use of virus-based technologies and the proper *in vivo* integration and differentiation still need to be better addressed. Importantly, the availability of such a high number of cells will release the intravenous protocol from one of its major limit, thus casting new light on its clinical

current clinical trials envolving NPCs for CNS disorders are described in Table 2.

derived brain NPCs. (clinicaltrials.gov identifier no. NCT01640067).

rescue are unlikely ascribable merely to cell replacement.

analysis, see [257]).

potentiality.

In September of 2009, NeuralStem, Inc. sponsored a phase I trial in ALS at the Emory University School of Medicine (Atlanta, GA, USA), using proprietary single donor allogeneic, adherent cultured, foetal-derived spinal NPCs (NSI-566RSC). NSI-566 cells were surgically implanted on a total of 12 patients via multiple injections directly into the thoracic spinal cord (either unilateral or bilateral). The clinical assessments demonstrated no evidence of acceleration of disease progression with the planned 18 months post-transplantation follow up [248, 249]. StemCell, Inc. is also sponsoring other two phase I trials with HuCNS-SC® in X chromosome linked connatal leukodistrophy PMD (in which oligodendrocytes cannot myelinate axons) and AMD. With the PMD trial at the University of California, San Francisco (UCSF, San Francisco, CA, USA), HuCNS-SC® were directly delivered through multiple injections into the brain of a total of 4 male patients (clinicaltrials.gov identifier no. NCT01005004). Data regarding this clinical trial has been recently published [250]. The transplantation procedure, the immuno‐ suppression and the cells were well tolerated by all the 4 patients. No adverse effects related to the implant were detected. MRI investigation before and after the transplantation of cells, revealed, after 9 months, a consistent donor cell-derived myelination *in situ*, in three of the patients. However, these data are just published and under intense scientific discussion. With the AMD trial at the Retina Foundation of the Southwest (Dallas, TX, USA), HuCNS-SC® are being delivered directly into the subretinal space of one eye in a single transplant procedure in a total 16 patients. The estimated completion date of this study is March 2014 (clinicaltri‐ als.gov identifier no. NCT01632527).

In June 2012, the Glasgow Southern General Hospital (Glasgow, Scotland) enrolled the first patient (of 12 total) of the dose-escalating Pilot Investigation of Stem Cells in Stroke (PISCES) phase I trial to be transplanted in a single-stage procedure with direct cerebral (intraparen‐ chymal) delivery of Reneuron, Ltd. proprietary single donor allogeneic adherent cultured, cmyc immortalized foetal-derived brain human NPCs (CTX0E03) (clinicaltrials.gov identifier no. NCT01151124).

In March 2011, the University Hospital Balgrist (Zurich, Switzerland) enrolled the first patient (of 12 total) with chronic thoracic (T2–T11) SCI (3 to 12 months after complete and incomplete cord injuries) to be transplanted with HuCNS-SC® in a further StemCell, Inc. sponsored phase I/II clinical trial estimated to be concluded in March 2016. A single dose (20x106 cells) of HuCNS-SC® has been directly implanted through multiple injections into the thoracic spinal cord, and immune suppression administered for 9 months after transplantation (clinicaltri‐ als.gov identifier no. NCT01321333). In November 2012 started the consequent long-term follow up of the 12 patients subjected to HuCNS-SC® transplantation that will last until March 2018 (clinicaltrials.gov identifier no. NCT01725880).

In June 2012, the Azienda Ospedaliera Santa Maria (Terni, Italy) enrolled the very first of total 18 ALS patients to treat with intraspinally implanted allogeneic free-floating cultured, foetalderived brain NPCs. (clinicaltrials.gov identifier no. NCT01640067).

cultured, foetal-derived brain human NPCs (HuCNS- SC®) were directly administered to the cerebral hemispheres and lateral ventricles. Immune suppression was administered for 12 months after transplantation. This study has now been completed with one out of 6 patients died for disease progression, 11 months after treatment. The cell transplantation and combi‐

In September of 2009, NeuralStem, Inc. sponsored a phase I trial in ALS at the Emory University School of Medicine (Atlanta, GA, USA), using proprietary single donor allogeneic, adherent cultured, foetal-derived spinal NPCs (NSI-566RSC). NSI-566 cells were surgically implanted on a total of 12 patients via multiple injections directly into the thoracic spinal cord (either unilateral or bilateral). The clinical assessments demonstrated no evidence of acceleration of disease progression with the planned 18 months post-transplantation follow up [248, 249]. StemCell, Inc. is also sponsoring other two phase I trials with HuCNS-SC® in X chromosome linked connatal leukodistrophy PMD (in which oligodendrocytes cannot myelinate axons) and AMD. With the PMD trial at the University of California, San Francisco (UCSF, San Francisco, CA, USA), HuCNS-SC® were directly delivered through multiple injections into the brain of a total of 4 male patients (clinicaltrials.gov identifier no. NCT01005004). Data regarding this clinical trial has been recently published [250]. The transplantation procedure, the immuno‐ suppression and the cells were well tolerated by all the 4 patients. No adverse effects related to the implant were detected. MRI investigation before and after the transplantation of cells, revealed, after 9 months, a consistent donor cell-derived myelination *in situ*, in three of the patients. However, these data are just published and under intense scientific discussion. With the AMD trial at the Retina Foundation of the Southwest (Dallas, TX, USA), HuCNS-SC® are being delivered directly into the subretinal space of one eye in a single transplant procedure in a total 16 patients. The estimated completion date of this study is March 2014 (clinicaltri‐

In June 2012, the Glasgow Southern General Hospital (Glasgow, Scotland) enrolled the first patient (of 12 total) of the dose-escalating Pilot Investigation of Stem Cells in Stroke (PISCES) phase I trial to be transplanted in a single-stage procedure with direct cerebral (intraparen‐ chymal) delivery of Reneuron, Ltd. proprietary single donor allogeneic adherent cultured, cmyc immortalized foetal-derived brain human NPCs (CTX0E03) (clinicaltrials.gov identifier

In March 2011, the University Hospital Balgrist (Zurich, Switzerland) enrolled the first patient (of 12 total) with chronic thoracic (T2–T11) SCI (3 to 12 months after complete and incomplete cord injuries) to be transplanted with HuCNS-SC® in a further StemCell, Inc. sponsored phase

HuCNS-SC® has been directly implanted through multiple injections into the thoracic spinal cord, and immune suppression administered for 9 months after transplantation (clinicaltri‐ als.gov identifier no. NCT01321333). In November 2012 started the consequent long-term follow up of the 12 patients subjected to HuCNS-SC® transplantation that will last until March

cells) of

I/II clinical trial estimated to be concluded in March 2016. A single dose (20x106

nation with prolonged immune suppression were both well tolerated [247].

als.gov identifier no. NCT01632527).

320 Neural Stem Cells - New Perspectives

2018 (clinicaltrials.gov identifier no. NCT01725880).

no. NCT01151124).

Importantly, there are not yet clinical trials with NPCs in MS. However, a consensus paper has recently been produced by a group of experts to define the uniform guidelines on the devel‐ opment of haematopoietic and non-haematopoietic stem cell therapies for MS [9]. All the current clinical trials envolving NPCs for CNS disorders are described in Table 2.

While in this paragraph we offer an overview of the current clinical trials involving solely human NPCs, it has to be said that, in the light of the neuroprotective/immunomodulatory (rather than cell replacement) properties attributed to stem cells, the therapeutic plasticity of cells of non-neural origin are being tested as well. Among these, MSCs are emerging as a good potential candidate, mainly because of their great accessibility and remarkable proliferation. Also, growing evidence suggests that other than giving origin to multiple derivatives of the mesodermal lineage (from which they derive), under particular conditions MSCs seem able to *transdifferentiate* into neuro-ectodermal cells *in vitro* [251-254]. However, this ability to convert from one lineage to another is still highly questionable and opened to different interpretations. Several studies have also proved the ability of MSCs to survive, migrate and eventually bring about functional recovery when transplanted into the CNS of different experimental models of neurological diseases (for a review see [255]). However, the mechanisms yielding to such rescue are unlikely ascribable merely to cell replacement.

Since 2006, the advent of induced pluripotent stem cells (iPSCs, [24]) technology has brought new excitement in medical research and clinical therapy, since these cells provide a valuable alternative without being constrained by ethical issues and immunological incompatibility [256]. Although still under debate about their long-term safety, the methods for iPSC genera‐ tion, reprogramming and differentiation efficiency, iPSCs represent a break-trough for both study of disease mechanisms and investigation of potential new treatments (for a perspective analysis, see [257]).

The potential impact of this technological platform has been further boosted by the scientific stream emerged from iPSC technology that is the "direct reprogramming" from one somatic lineage to another. In fact, the direct conversion of fibroblasts to functional neurons (iN cells) or iNSCs [25, 26], for example, represents one of the most exciting, ultimate technologies for future application in CNS pathologies. Thanks to these next generation techniques, it will be possible to derive virtually unlimited numbers of specific neural/neuronal population bypassing the pluripotent stage, thus likely eliminating the potential presence of unwanted undifferentiated cells. However, many issues, such as the purity of the cell preparation, the use of virus-based technologies and the proper *in vivo* integration and differentiation still need to be better addressed. Importantly, the availability of such a high number of cells will release the intravenous protocol from one of its major limit, thus casting new light on its clinical potentiality.

GABA: Gamma-aminobutyric acid

GFAP: Glial-fibrillary acidic protein

GMP: Good manufacturing practice

hNPC: Human neural stem/precursor cell

HVc: hyperstriatum ventrale, pars caudalis

ICAM: Intercellular adhesion molecule

ICH: Intracerebral hemorrhage

IGF: Insulin-like growth factor

IML: Inner molecular layer

iN cells: Induced neuronal cells

iNSC: Induced neural stem cell IPC: Intermediate progenitor cell iPS: Induced pluripotent stem cell

iNOS: inducible nitric oxide synthase

LFA: Leukocyte-function associated antigen

GDNF: Glial-derived neurotrophic factor

GF-CSF: granulocyte macrophage colony stimulating factor

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GCL: Granule cell layer

GTP: Good tissue practice HD: Huntington's disease

Hsp70: Heat shock protein 70

i.c.v.: Intracerebroventricular

i.p.c.: Intraparenchyma

Ig: Immunoglobulin

IL: Interleukin

INF: Interferon

i.a.: Intraartery

i.t.: Intrathecal i.v.: Intravenous

HuCNS-SC: Human CNS stem cell

#### **Abbreviations**

AD: Alzheimer's disease ALS: Amyotrophic lateral sclerosis APC: Antigen presenting cell ASCL1: Achaete-scute homolog 1 BBB: Blood brain barrier BCSFB: Blood-cerebrospinal fluid barrier BDNF: Brain-derived neurotrophic factor BLMB: Blood-leptomeningeal barrier BMP: Bone morphogenetic protein BMSC: Bone marrow-derived stem cell CCAo: Common carotid artery occlusion CCL: Chemokine (C-C motif) ligand CCR: C-C chemokine receptor CNS: Central nervous system CNTF: Ciliary neurotrophic factor CSF: Cerebrospinal fluid CXCR: C-X-C chemokine receptor DCs: dendritic cells DCX: Doublecortin DG: Dentate gyrus DGC: Dentate granule cell Dlx: Distal-less homeobox d.p.t.: days post transplantation EAE: Experimental autoimmune encephalomyelitis EC: Endothelial cell ES cells: Embryonic stem cells FACS: Fluorescence-activated cell sorting FGF: Fibroblast growth factor

GABA: Gamma-aminobutyric acid GCL: Granule cell layer GDNF: Glial-derived neurotrophic factor GFAP: Glial-fibrillary acidic protein GF-CSF: granulocyte macrophage colony stimulating factor GMP: Good manufacturing practice GTP: Good tissue practice HD: Huntington's disease hNPC: Human neural stem/precursor cell Hsp70: Heat shock protein 70 HuCNS-SC: Human CNS stem cell HVc: hyperstriatum ventrale, pars caudalis i.a.: Intraartery i.c.v.: Intracerebroventricular i.p.c.: Intraparenchyma i.t.: Intrathecal i.v.: Intravenous ICAM: Intercellular adhesion molecule ICH: Intracerebral hemorrhage Ig: Immunoglobulin IGF: Insulin-like growth factor IL: Interleukin IML: Inner molecular layer iN cells: Induced neuronal cells INF: Interferon iNOS: inducible nitric oxide synthase iNSC: Induced neural stem cell IPC: Intermediate progenitor cell iPS: Induced pluripotent stem cell LFA: Leukocyte-function associated antigen

**Abbreviations**

AD: Alzheimer's disease

322 Neural Stem Cells - New Perspectives

BBB: Blood brain barrier

ALS: Amyotrophic lateral sclerosis

ASCL1: Achaete-scute homolog 1

BCSFB: Blood-cerebrospinal fluid barrier BDNF: Brain-derived neurotrophic factor

BLMB: Blood-leptomeningeal barrier

BMSC: Bone marrow-derived stem cell CCAo: Common carotid artery occlusion

CCL: Chemokine (C-C motif) ligand

CCR: C-C chemokine receptor CNS: Central nervous system

CSF: Cerebrospinal fluid

DGC: Dentate granule cell Dlx: Distal-less homeobox

d.p.t.: days post transplantation

ES cells: Embryonic stem cells

FGF: Fibroblast growth factor

FACS: Fluorescence-activated cell sorting

EAE: Experimental autoimmune encephalomyelitis

DCs: dendritic cells DCX: Doublecortin DG: Dentate gyrus

EC: Endothelial cell

CNTF: Ciliary neurotrophic factor

CXCR: C-X-C chemokine receptor

BMP: Bone morphogenetic protein

APC: Antigen presenting cell

LIF: Leukemia inhibitory factor LPS: Lipopolysaccharide MAdCAM: Mucosal addressin cell adhesion molecule MCAo: Middle cerebral artery occlusion MCP: Monocyte chemoattractant protein MHC: Major histocompatibility complex MMS: Medial migratory stream MOG: Myelin oligodendrocyte glycoprotein MPC: Myeloid precursor cell MRI: Magnetic resonance imaging MS: Multiple sclerosis MSC: Mesenchymal stem cell NCL: Neuronal ceroid lipofuscinose NeuN: Neuronal nuclei NF: Neurofilament NF-kB: Nuclear factor-kB NGF: Nerve growth factor NO: Nitric oxide NPC: Neural stem/precursor cell NSC: Neural stem cell OB: Olfactory bulb OPC: Oligodendrocyte progenitor cells PD: Parkinson's disease PGE2: prostaglandine 2 PLP: Proteolipid protein PSA-NCAM: Polysialylated neural cell adhesion molecule PSGL: P-selectin glycoprotein ligand pt: Post-transplantation RA: Radial astrocyte RMS: Rostral migratory stream

s.c.: Subcutaneous

SC: Stem cell

SCF: Stem cell factor

SCI: Spinal cord injury

SE: Status epilepticus

SGZ: Subgranular zone

Shh: Sonic hedgehog

TCR: T cell receptor

TJ: Tight junction

SDF: Stromal cell-derived factor

TGF: Transforming growth factor

TLE: Temporal lobe epilepsy

TNF: Tumor necrosis factor

V-SVZ: Ventricular-subventricular zone

VCAM: Vascular cell adhesion molecule

VEGF: Vascular endothelial growth factor

Matteo Donegà1,2, Elena Giusto1,2, Chiara Cossetti1

\*Address all correspondence to: spp24@cam.ac.uk

MRC Stem Cell Institute, University of Cambridge, UK

2 NIHR Biomedical Research Centre, University of Cambridge, UK

and Stefano Pluchino1\*

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325

1 Dept of Clinical Neurosciences, John van Geest Centre for Brain Repair, Wellcome Trust-

TLR: Toll-like receptor

VLA: Very late antigen

**Author details**

SIDS: Stroke-induced immune depression syndrome

SCF: Stem cell factor SCI: Spinal cord injury SC: Stem cell SDF: Stromal cell-derived factor SE: Status epilepticus SGZ: Subgranular zone Shh: Sonic hedgehog SIDS: Stroke-induced immune depression syndrome TCR: T cell receptor TGF: Transforming growth factor TJ: Tight junction TLE: Temporal lobe epilepsy TLR: Toll-like receptor TNF: Tumor necrosis factor V-SVZ: Ventricular-subventricular zone VCAM: Vascular cell adhesion molecule VEGF: Vascular endothelial growth factor VLA: Very late antigen

#### **Author details**

s.c.: Subcutaneous

LIF: Leukemia inhibitory factor

MMS: Medial migratory stream

MPC: Myeloid precursor cell

MSC: Mesenchymal stem cell

MS: Multiple sclerosis

NeuN: Neuronal nuclei

NF-kB: Nuclear factor-kB NGF: Nerve growth factor

NPC: Neural stem/precursor cell

OPC: Oligodendrocyte progenitor cells

PSGL: P-selectin glycoprotein ligand

RMS: Rostral migratory stream

PSA-NCAM: Polysialylated neural cell adhesion molecule

NF: Neurofilament

NO: Nitric oxide

NSC: Neural stem cell

PD: Parkinson's disease PGE2: prostaglandine 2 PLP: Proteolipid protein

pt: Post-transplantation

RA: Radial astrocyte

OB: Olfactory bulb

MRI: Magnetic resonance imaging

NCL: Neuronal ceroid lipofuscinose

MCAo: Middle cerebral artery occlusion MCP: Monocyte chemoattractant protein MHC: Major histocompatibility complex

MOG: Myelin oligodendrocyte glycoprotein

MAdCAM: Mucosal addressin cell adhesion molecule

LPS: Lipopolysaccharide

324 Neural Stem Cells - New Perspectives

Matteo Donegà1,2, Elena Giusto1,2, Chiara Cossetti1 and Stefano Pluchino1\*

\*Address all correspondence to: spp24@cam.ac.uk

1 Dept of Clinical Neurosciences, John van Geest Centre for Brain Repair, Wellcome Trust-MRC Stem Cell Institute, University of Cambridge, UK

2 NIHR Biomedical Research Centre, University of Cambridge, UK

#### **References**

[1] Altman J, Das GD. Autoradiographic and histological evidence of postnatal hippo‐ campal neurogenesis in rats. The Journal of comparative neurology. 1965 Jun;124(3): 319-35.

[11] Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Na‐

Systemic stem cell therapies and brain diseases

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

327

[12] Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transi‐

[13] Bottai D, Madaschi L, Di Giulio AM, Gorio A. Viability-dependent promoting action of adult neural precursors in spinal cord injury. Molecular medicine (Cambridge,

[14] Lee ST, Chu K, Park JE, Lee K, Kang L, Kim SU, et al. Intravenous administration of human neural stem cells induces functional recovery in Huntington's disease rat

[15] Pluchino S, Gritti A, Blezer E, Amadio S, Brambilla E, Borsellino G, et al. Human neural stem cells ameliorate autoimmune encephalomyelitis in non-human primates.

[16] Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L, Salani G, et al. Neurospherederived multipotent precursors promote neuroprotection by an immunomodulatory

[17] van der Meulen AA, Biber K, Lukovac S, Balasubramaniyan V, den Dunnen WF, Boddeke HW, et al. The role of CXC chemokine ligand (CXCL)12-CXC chemokine re‐ ceptor (CXCR)4 signalling in the migration of neural stem cells towards a brain tu‐

[18] Guzman R, De Los Angeles A, Cheshier S, Choi R, Hoang S, Liauw J, et al. Intracaro‐ tid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.

[19] Martino G, Pluchino S. The therapeutic potential of neural stem cells. Nature re‐

[20] Einstein O, Fainstein N, Vaknin I, Mizrachi-Kol R, Reihartz E, Grigoriadis N, et al. Neural precursors attenuate autoimmune encephalomyelitis by peripheral immuno‐

[21] Pluchino S, Zanotti L, Brambilla E, Rovere-Querini P, Capobianco A, Alfaro-Cervello C, et al. Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function. PloS one.

[22] Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, et al. Anti-inflammatory mech‐ anism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain.

mour. Neuropathology and applied neurobiology. 2009 Dec;35(6):579-91.

Stroke; a journal of cerebral circulation. 2008 Apr;39(4):1300-6.

suppression. Annals of neurology. 2007 Mar;61(3):209-18.

ent forebrain ischemia. Neuroscience letters. 2003 Jun 5;343(2):129-33.

ture. 2003 Apr 17;422(6933):688-94.

Mass. 2008 Sep-Oct;14(9-10):634-44.

model. Neuroscience research. 2005 Jul;52(3):243-9.

Annals of neurology. 2009 Sep;66(3):343-54.

views. 2006 May;7(5):395-406.

2009;4(6):e5959.

2008 Mar;131(Pt 3):616-29.

mechanism. Nature. 2005 Jul 14;436(7048):266-71.


[11] Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Na‐ ture. 2003 Apr 17;422(6933):688-94.

**References**

319-35.

326 Neural Stem Cells - New Perspectives

13211-6.

6(5):247-55.

Jan;41(1):73-80.

1966 Mar;126(3):337-89.

ment. Brain. 2008 Oct;131(Pt 10):2564-78.

cells. Physiological reviews. 2011 Oct;91(4):1281-304.

sue research. 2012 Jul;349(1):321-9.

[1] Altman J, Das GD. Autoradiographic and histological evidence of postnatal hippo‐ campal neurogenesis in rats. The Journal of comparative neurology. 1965 Jun;124(3):

[2] Altman J, Das GD. Autoradiographic and histological studies of postnatal neurogen‐ esis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats, with special reference to post‐ natal neurogenesis in some brain regions. The Journal of comparative neurology.

[3] Goldman SA, Nottebohm F. Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain. Proceedings of the National

[4] Picard-Riera N, Decker L, Delarasse C, Goude K, Nait-Oumesmar B, Liblau R, et al. Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice. Proceedings of the National Academy of Sciences of the United States of America. 2002 Oct 1;99(20):

[5] Pluchino S, Martino G. The therapeutic plasticity of neural stem/precursor cells in multiple sclerosis. Journal of the neurological sciences. 2008 Feb 15;265(1-2):105-10. [6] Pluchino S, Muzio L, Imitola J, Deleidi M, Alfaro-Cervello C, Salani G, et al. Persis‐ tent inflammation alters the function of the endogenous brain stem cell compart‐

[7] Cossetti C, Alfaro-Cervello C, Donega M, Tyzack G, Pluchino S. New perspectives of tissue remodelling with neural stem and progenitor cell-based therapies. Cell and tis‐

[8] Martino G, Pluchino S, Bonfanti L, Schwartz M. Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem

[9] Martino G, Franklin RJ, Van Evercooren AB, Kerr DA. Stem cell transplantation in multiple sclerosis: current status and future prospects. Nat Rev Neurol. 2010 May;

[10] Ben-Hur T, Einstein O, Mizrachi-Kol R, Ben-Menachem O, Reinhartz E, Karussis D, et al. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia. 2003

Academy of Sciences of the United States of America. 1983 Apr;80(8):2390-4.


[23] Cao W, Yang Y, Wang Z, Liu A, Fang L, Wu F, et al. Leukemia inhibitory factor in‐ hibits T helper 17 cell differentiation and confers treatment effects of neural progeni‐ tor cell therapy in autoimmune disease. Immunity. 2011 Aug 26;35(2):273-84.

[37] Fuentealba LC, Obernier K, Alvarez-Buylla A. Adult neural stem cells bridge their

Systemic stem cell therapies and brain diseases

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

329

[38] Alvarez-Buylla A, Garcia-Verdugo JM. Neurogenesis in adult subventricular zone. J

[39] Merkle FT, Mirzadeh Z, Alvarez-Buylla A. Mosaic organization of neural stem cells

[40] Doetsch F. The glial identity of neural stem cells. Nature neuroscience. 2003 Nov;

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

[42] Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in

[43] Sawamoto K, Wichterle H, Gonzalez-Perez O, Cholfin JA, Yamada M, Spassky N, et al. New neurons follow the flow of cerebrospinal fluid in the adult brain. Science

[44] Kokovay E, Wang Y, Kusek G, Wurster R, Lederman P, Lowry N, et al. VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression. Cell stem cell. 2012 Aug 3;11(2):220-30.

[45] Lehtinen MK, Zappaterra MW, Chen X, Yang YJ, Hill AD, Lun M, et al. The cerebro‐ spinal fluid provides a proliferative niche for neural progenitor cells. Neuron. 2011

[46] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activa‐

[47] Chapouton P, Jagasia R, Bally-Cuif L. Adult neurogenesis in non-mammalian verte‐

[48] Fernando RN, Eleuteri B, Abdelhady S, Nussenzweig A, Andang M, Ernfors P. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Pro‐ ceedings of the National Academy of Sciences of the United States of America. 2011

[49] Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, et al. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature. 2011 Sep

[50] Mosher KI, Andres RH, Fukuhara T, Bieri G, Hasegawa-Moriyama M, He Y, et al. Neural progenitor cells regulate microglia functions and activity. Nature neuro‐

neurogenic regions of the adult brain. Cell stem cell. 2008 Sep 11;3(3):265-78.

in the adult brain. Science (New York, NY. 2007 Jul 20;317(5836):381-4.

niche. Cell stem cell. 2012 Jun 14;10(6):698-708.

cells. Annual review of neuroscience. 2009;32:149-84.

(New York, NY. 2006 Feb 3;311(5761):629-32.

tion mechanism. Cell. 2009 Apr 17;137(2):216-33.

brates. Bioessays. 2007 Aug;29(8):745-57.

Neurosci. 2002 Feb 1;22(3):629-34.

6(11):1127-34.

Mar 10;69(5):893-905.

Apr 5;108(14):5837-42.

science. 2012 Nov;15(11):1485-7.

1;477(7362):90-4.


[37] Fuentealba LC, Obernier K, Alvarez-Buylla A. Adult neural stem cells bridge their niche. Cell stem cell. 2012 Jun 14;10(6):698-708.

[23] Cao W, Yang Y, Wang Z, Liu A, Fang L, Wu F, et al. Leukemia inhibitory factor in‐ hibits T helper 17 cell differentiation and confers treatment effects of neural progeni‐

[24] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐ ic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76.

[25] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M. Direct con‐ version of fibroblasts to functional neurons by defined factors. Nature. 2010 Feb

[26] Thier M, Worsdorfer P, Lakes YB, Gorris R, Herms S, Opitz T, et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell stem cell. 2012 Apr

[27] Altman J. Autoradiographic and histological studies of postnatal neurogenesis. 3. Dating the time of production and onset of differentiation of cerebellar microneurons

[28] Ming GL, Song H. Adult neurogenesis in the mammalian central nervous system.

[29] Gage FH. Mammalian neural stem cells. Science (New York, NY. 2000 Feb

[30] Lois C, Alvarez-Buylla A. Proliferating subventricular zone cells in the adult mam‐ malian forebrain can differentiate into neurons and glia. Proceedings of the National

Academy of Sciences of the United States of America. 1993 Mar 1;90(5):2074-7.

[31] Kaplan MS, Bell DH. Mitotic neuroblasts in the 9-day-old and 11-month-old rodent

[32] Cameron HA, Woolley CS, McEwen BS, Gould E. Differentiation of newly born neu‐ rons and glia in the dentate gyrus of the adult rat. Neuroscience. 1993 Sep;56(2):

[33] Goldman SA, Chen Z. Perivascular instruction of cell genesis and fate in the adult

[34] Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their

[35] Tavazoie M, Van der Veken L, Silva-Vargas V, Louissaint M, Colonna L, Zaidi B, et al. A specialized vascular niche for adult neural stem cells. Cell stem cell. 2008 Sep

[36] Shen Q, Wang Y, Kokovay E, Lin G, Chuang SM, Goderie SK, et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell

in rats. The Journal of comparative neurology. 1969 Jul;136(3):269-93.

Annual review of neuroscience. 2005;28:223-50.

hippocampus. J Neurosci. 1984 Jun;4(6):1429-41.

brain. Nature neuroscience. 2011 Nov;14(11):1382-9.

niche. Cell. 2004 Mar 19;116(6):769-78.

stem cell. 2008 Sep 11;3(3):289-300.

25;463(7284):1035-41.

25;287(5457):1433-8.

337-44.

11;3(3):279-88.

6;10(4):473-9.

328 Neural Stem Cells - New Perspectives

tor cell therapy in autoimmune disease. Immunity. 2011 Aug 26;35(2):273-84.


[51] Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso C, et al. Hu‐ man neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Sci‐ ence (New York, NY. 2007 Mar 2;315(5816):1243-9.

[63] Ehm O, Goritz C, Covic M, Schaffner I, Schwarz TJ, Karaca E, et al. RBPJkappa-de‐ pendent signaling is essential for long-term maintenance of neural stem cells in the

Systemic stem cell therapies and brain diseases

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

331

[64] Lavado A, Lagutin OV, Chow LM, Baker SJ, Oliver G. Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

[65] Song J, Zhong C, Bonaguidi MA, Sun GJ, Hsu D, Gu Y, et al. Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision. Nature. 2012

[66] Dayer AG, Ford AA, Cleaver KM, Yassaee M, Cameron HA. Short-term and longterm survival of new neurons in the rat dentate gyrus. The Journal of comparative

[67] Schwartz M, Shechter R. Protective autoimmunity functions by intracranial immuno‐ surveillance to support the mind: The missing link between health and disease. Mo‐

[68] Ziv Y, Ron N, Butovsky O, Landa G, Sudai E, Greenberg N, et al. Immune cells con‐ tribute to the maintenance of neurogenesis and spatial learning abilities in adult‐

[69] Gould E, Tanapat P. Lesion-induced proliferation of neuronal progenitors in the den‐

[70] Yang L, Benardo LS, Valsamis H, Ling DS. Acute injury to superficial cortex leads to a decrease in synaptic inhibition and increase in excitation in neocortical layer V pyr‐

[71] Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal

[72] Huehnchen P, Prozorovski T, Klaissle P, Lesemann A, Ingwersen J, Wolf SA, et al. Modulation of adult hippocampal neurogenesis during myelin-directed autoimmune

[73] Roosendaal SD, Hulst HE, Vrenken H, Feenstra HE, Castelijns JA, Pouwels PJ, et al. Structural and functional hippocampal changes in multiple sclerosis patients with in‐

[74] Rasmussen S, Imitola J, Ayuso-Sacido A, Wang Y, Starossom SC, Kivisakk P, et al. Reversible neural stem cell niche dysfunction in a model of multiple sclerosis. Annals

[75] Tepavcevic V, Lazarini F, Alfaro-Cervello C, Kerninon C, Yoshikawa K, Garcia-Ver‐ dugo JM, et al. Inflammation-induced subventricular zone dysfunction leads to olfac‐

adult hippocampus. J Neurosci. 2010 Oct 13;30(41):13794-807.

PLoS biology. 2010;8(8).

Sep 6;489(7414):150-4.

neurology. 2003 Jun 9;460(4):563-72.

lecular psychiatry. 2010 Apr;15(4):342-54.

hood. Nature neuroscience. 2006 Feb;9(2):268-75.

neuroinflammation. Glia. 2011 Jan;59(1):132-42.

of neurology. 2011 May;69(5):878-91.

tate gyrus of the adult rat. Neuroscience. 1997 Sep;80(2):427-36.

amidal cells. Journal of neurophysiology. 2007 Jan;97(1):178-87.

tact memory function. Radiology. 2010 May;255(2):595-604.

neurogenesis. Science (New York, NY. 2003 Dec 5;302(5651):1760-5.


[63] Ehm O, Goritz C, Covic M, Schaffner I, Schwarz TJ, Karaca E, et al. RBPJkappa-de‐ pendent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus. J Neurosci. 2010 Oct 13;30(41):13794-807.

[51] Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso C, et al. Hu‐ man neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Sci‐

[52] Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, et al. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks

[53] Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, et al. Neurogenesis in the adult human hippocampus. Nature medicine. 1998 Nov;

[54] Quinones-Hinojosa A, Sanai N, Soriano-Navarro M, Gonzalez-Perez O, Mirzadeh Z, Gil-Perotin S, et al. Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells. The Journal of comparative neurol‐

[55] Ming GL, Song H. Adult neurogenesis in the mammalian brain: significant answers

[56] Yang Z, Ming GL, Song H. Postnatal neurogenesis in the human forebrain: from two

[57] Guerrero-Cazares H, Gonzalez-Perez O, Soriano-Navarro M, Zamora-Berridi G, Gar‐ cia-Verdugo JM, Quinones-Hinojosa A. Cytoarchitecture of the lateral ganglionic eminence and rostral extension of the lateral ventricle in the human fetal brain. The

[58] Sanai N, Nguyen T, Ihrie RA, Mirzadeh Z, Tsai HH, Wong M, et al. Corridors of mi‐ grating neurons in the human brain and their decline during infancy. Nature. 2011

[59] Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A. Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci. 2001 Sep 15;21(18):

[60] Bonaguidi MA, Song J, Ming GL, Song H. A unifying hypothesis on mammalian neu‐ ral stem cell properties in the adult hippocampus. Current opinion in neurobiology.

[61] Seri B, Garcia-Verdugo JM, Collado-Morente L, McEwen BS, Alvarez-Buylla A. Cell types, lineage, and architecture of the germinal zone in the adult dentate gyrus. The

[62] Lugert S, Vogt M, Tchorz JS, Muller M, Giachino C, Taylor V. Homeostatic neurogen‐ esis in the adult hippocampus does not involve amplification of Ascl1(high) inter‐

and significant questions. Neuron. 2011 May 26;70(4):687-702.

Journal of comparative neurology. 2011 Apr 15;519(6):1165-80.

Journal of comparative neurology. 2004 Oct 25;478(4):359-78.

mediate progenitors. Nature communications. 2012;3:670.

migratory streams to dribbles. Cell stem cell. 2011 Nov 4;9(5):385-6.

ence (New York, NY. 2007 Mar 2;315(5816):1243-9.

chain migration. Nature. 2004 Feb 19;427(6976):740-4.

4(11):1313-7.

330 Neural Stem Cells - New Perspectives

ogy. 2006 Jan 20;494(3):415-34.

Oct 20;478(7369):382-6.

2012 Oct;22(5):754-61.

7153-60.


tory deficits in a targeted mouse model of multiple sclerosis. The Journal of clinical investigation. 2011 Dec;121(12):4722-34.

[87] Guerra-Crespo M, Gleason D, Sistos A, Toosky T, Solaroglu I, Zhang JH, et al. Trans‐ forming growth factor-alpha induces neurogenesis and behavioral improvement in a

Systemic stem cell therapies and brain diseases

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

333

[88] Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced neuropro‐ tection, neurogenesis, and angiogenesis after focal cerebral ischemia. The Journal of

[89] Jin K, Mao XO, Sun Y, Xie L, Greenberg DA. Stem cell factor stimulates neurogenesis in vitro and in vivo. The Journal of clinical investigation. 2002 Aug;110(3):311-9.

[90] Shingo T, Sorokan ST, Shimazaki T, Weiss S. Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem

[91] Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, et al. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neu‐

[92] Xu R, Wu C, Tao Y, Yi J, Yang Y, Zhang X, et al. Nestin-positive cells in the spinal cord: a potential source of neural stem cells. Int J Dev Neurosci. 2008 Nov;26(7):

[93] Ke Y, Chi L, Xu R, Luo C, Gozal D, Liu R. Early response of endogenous adult neural progenitor cells to acute spinal cord injury in mice. Stem cells (Dayton, Ohio). 2006

[94] Vaquero J, Ramiro MJ, Oya S, Cabezudo JM. Ependymal reaction after experimental

[95] Beattie MS, Bresnahan JC, Komon J, Tovar CA, Van Meter M, Anderson DK, et al. Endogenous repair after spinal cord contusion injuries in the rat. Experimental neu‐

[96] Felix MS, Popa N, Djelloul M, Boucraut J, Gauthier P, Bauer S, et al. Alteration of forebrain neurogenesis after cervical spinal cord injury in the adult rat. Frontiers in

[97] Parent JM, Valentin VV, Lowenstein DH. Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway. J Neurosci.

[98] Jessberger S, Romer B, Babu H, Kempermann G. Seizures induce proliferation and dispersion of doublecortin-positive hippocampal progenitor cells. Experimental neu‐

[99] Overstreet-Wadiche LS, Bromberg DA, Bensen AL, Westbrook GL. Seizures acceler‐ ate functional integration of adult-generated granule cells. J Neurosci. 2006 Apr

spinal cord injury. Acta neurochirurgica. 1981;55(3-4):295-302.

chronic stroke model. Neuroscience. 2009 May 5;160(2):470-83.

clinical investigation. 2003 Jun;111(12):1843-51.

cells. J Neurosci. 2001 Dec 15;21(24):9733-43.

roaxis. J Neurosci. 1996 Dec 1;16(23):7599-609.

813-20.

Apr;24(4):1011-9.

rology. 1997 Dec;148(2):453-63.

neuroscience. 2012;6:45.

2002 Apr 15;22(8):3174-88.

12;26(15):4095-103.

rology. 2005 Dec;196(2):342-51.


[87] Guerra-Crespo M, Gleason D, Sistos A, Toosky T, Solaroglu I, Zhang JH, et al. Trans‐ forming growth factor-alpha induces neurogenesis and behavioral improvement in a chronic stroke model. Neuroscience. 2009 May 5;160(2):470-83.

tory deficits in a targeted mouse model of multiple sclerosis. The Journal of clinical

[76] Ransohoff RM. Animal models of multiple sclerosis: the good, the bad and the bot‐

[77] Nait-Oumesmar B, Picard-Riera N, Kerninon C, Decker L, Seilhean D, Hoglinger GU, et al. Activation of the subventricular zone in multiple sclerosis: evidence for early glial progenitors. Proceedings of the National Academy of Sciences of the United

[78] Zivadinov R, Zorzon M, Monti Bragadin L, Pagliaro G, Cazzato G. Olfactory loss in multiple sclerosis. Journal of the neurological sciences. 1999 Oct 15;168(2):127-30. [79] Zhang R, Zhang Z, Zhang C, Zhang L, Robin A, Wang Y, et al. Stroke transiently in‐ creases subventricular zone cell division from asymmetric to symmetric and increas‐ es neuronal differentiation in the adult rat. J Neurosci. 2004 Jun 23;24(25):5810-5. [80] Jin K, Wang X, Xie L, Mao XO, Zhu W, Wang Y, et al. Evidence for stroke-induced neurogenesis in the human brain. Proceedings of the National Academy of Sciences

[81] Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nature medicine. 2002 Sep;

[82] Yamashita T, Ninomiya M, Hernandez Acosta P, Garcia-Verdugo JM, Sunabori T, Sa‐ kaguchi M, et al. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J Neurosci. 2006 Jun 14;26(24):

[83] Thored P, Wood J, Arvidsson A, Cammenga J, Kokaia Z, Lindvall O. Long-term neu‐ roblast migration along blood vessels in an area with transient angiogenesis and in‐ creased vascularization after stroke. Stroke; a journal of cerebral circulation. 2007

[84] Thored P, Arvidsson A, Cacci E, Ahlenius H, Kallur T, Darsalia V, et al. Persistent production of neurons from adult brain stem cells during recovery after stroke. Stem

[85] Nadareishvili Z, Hallenbeck J. Neuronal regeneration after stroke. The New England

[86] Kang SS, Keasey MP, Arnold SA, Reid R, Geralds J, Hagg T. Endogenous CNTF me‐ diates stroke-induced adult CNS neurogenesis in mice. Neurobiology of disease.

of the United States of America. 2006 Aug 29;103(35):13198-202.

investigation. 2011 Dec;121(12):4722-34.

tom line. Nature neuroscience. 2012 Aug;15(8):1074-7.

States of America. 2007 Mar 13;104(11):4694-9.

8(9):963-70.

332 Neural Stem Cells - New Perspectives

6627-36.

Nov;38(11):3032-9.

2012 Aug 31;49C:68-78.

cells (Dayton, Ohio). 2006 Mar;24(3):739-47.

journal of medicine. 2003 Jun 5;348(23):2355-6.


[100] Mathern GW, Leiphart JL, De Vera A, Adelson PD, Seki T, Neder L, et al. Seizures decrease postnatal neurogenesis and granule cell development in the human fascia dentata. Epilepsia. 2002;43 Suppl 5:68-73.

[113] Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci.

Systemic stem cell therapies and brain diseases

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

335

[114] Smith JA, Das A, Ray SK, Banik NL. Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain research bulletin. 2012 Jan

[115] Shechter R, London A, Varol C, Raposo C, Cusimano M, Yovel G, et al. Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in re‐

covery from spinal cord injury in mice. PLoS medicine. 2009 Jul;6(7):e1000113.

[116] Engelhardt B, Ransohoff RM. The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends in immunology. 2005 Sep;

[117] Kivisakk P, Imitola J, Rasmussen S, Elyaman W, Zhu B, Ransohoff RM, et al. Localiz‐ ing central nervous system immune surveillance: meningeal antigen-presenting cells activate T cells during experimental autoimmune encephalomyelitis. Annals of neu‐

[118] Weller RO, Kida S, Zhang ET. Pathways of fluid drainage from the brain--morpho‐ logical aspects and immunological significance in rat and man. Brain pathology (Zur‐

[119] Carare RO, Bernardes-Silva M, Newman TA, Page AM, Nicoll JA, Perry VH, et al. Solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroim‐

munology. Neuropathology and applied neurobiology. 2008 Apr;34(2):131-44.

[120] Moalem G, Leibowitz-Amit R, Yoles E, Mor F, Cohen IR, Schwartz M. Autoimmune T cells protect neurons from secondary degeneration after central nervous system ax‐

[121] Yoles E, Hauben E, Palgi O, Agranov E, Gothilf A, Cohen A, et al. Protective autoim‐ munity is a physiological response to CNS trauma. J Neurosci. 2001 Jun 1;21(11):

[122] Kipnis J, Mizrahi T, Yoles E, Ben-Nun A, Schwartz M. Myelin specific Th1 cells are necessary for post-traumatic protective autoimmunity. Journal of neuroimmunology.

[123] Schwartz M, Hauben E. T cell-based therapeutic vaccination for spinal cord injury.

[124] Hauben E, Butovsky O, Nevo U, Yoles E, Moalem G, Agranov E, et al. Passive or ac‐ tive immunization with myelin basic protein promotes recovery from spinal cord

2007 Mar 7;27(10):2596-605.

rology. 2009 Apr;65(4):457-69.

ich, Switzerland). 1992 Oct;2(4):277-84.

otomy. Nature medicine. 1999 Jan;5(1):49-55.

Progress in brain research. 2002;137:401-6.

contusion. J Neurosci. 2000 Sep 1;20(17):6421-30.

4;87(1):10-20.

26(9):485-95.

3740-8.

2002 Sep;130(1-2):78-85.


[113] Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci. 2007 Mar 7;27(10):2596-605.

[100] Mathern GW, Leiphart JL, De Vera A, Adelson PD, Seki T, Neder L, et al. Seizures decrease postnatal neurogenesis and granule cell development in the human fascia

[101] Haughey NJ, Liu D, Nath A, Borchard AC, Mattson MP. Disruption of neurogenesis in the subventricular zone of adult mice, and in human cortical neuronal precursor cells in culture, by amyloid beta-peptide: implications for the pathogenesis of Alz‐

[102] Yu Y, He J, Zhang Y, Luo H, Zhu S, Yang Y, et al. Increased hippocampal neurogene‐ sis in the progressive stage of Alzheimer's disease phenotype in an APP/PS1 double

[103] Rodriguez JJ, Jones VC, Verkhratsky A. Impaired cell proliferation in the subventric‐ ular zone in an Alzheimer's disease model. Neuroreport. 2009 Jul 1;20(10):907-12.

[104] Ermini FV, Grathwohl S, Radde R, Yamaguchi M, Staufenbiel M, Palmer TD, et al. Neurogenesis and alterations of neural stem cells in mouse models of cerebral amy‐

[105] Zhang C, McNeil E, Dressler L, Siman R. Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of fam‐

[106] Demars M, Hu YS, Gadadhar A, Lazarov O. Impaired neurogenesis is an early event in the etiology of familial Alzheimer's disease in transgenic mice. Journal of neuro‐

[107] Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, et al. Increased hippocam‐ pal neurogenesis in Alzheimer's disease. Proceedings of the National Academy of

[108] Parent JM, Vexler ZS, Gong C, Derugin N, Ferriero DM. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Annals of neurology. 2002 Dec;

[109] Bonfanti L. From hydra regeneration to human brain structural plasticity: a long trip

[110] Schwartz M, Shechter R. Systemic inflammatory cells fight off neurodegenerative

[111] Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science (New York, NY. 2005 May

[112] Hanisch UK, Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nature neuroscience. 2007 Nov;10(11):1387-94.

through narrowing roads. TheScientificWorldJournal. 2011;11:1270-99.

ilial Alzheimer's disease. Experimental neurology. 2007 Mar;204(1):77-87.

heimer's disease. Neuromolecular medicine. 2002;1(2):125-35.

transgenic mouse model. Hippocampus. 2009 Dec;19(12):1247-53.

loidosis. The American journal of pathology. 2008 Jun;172(6):1520-8.

Sciences of the United States of America. 2004 Jan 6;101(1):343-7.

dentata. Epilepsia. 2002;43 Suppl 5:68-73.

334 Neural Stem Cells - New Perspectives

science research. 2010 Aug 1;88(10):2103-17.

disease. Nat Rev Neurol. 2010 Jul;6(7):405-10.

52(6):802-13.

27;308(5726):1314-8.


[125] Hauben E, Ibarra A, Mizrahi T, Barouch R, Agranov E, Schwartz M. Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens. Proceedings of the National Academy of Sciences of the United States of America. 2001 Dec 18;98(26):15173-8.

experimental allergic encephalomyelitis. Molecular and cellular neurosciences. 2003

Systemic stem cell therapies and brain diseases

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

337

[137] Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell trans‐ plantation promotes functional recovery in rats with experimental intracerebral hem‐

[138] Chu K, Kim M, Jung KH, Jeon D, Lee ST, Kim J, et al. Human neural stem cell trans‐ plantation reduces spontaneous recurrent seizures following pilocarpine-induced

[139] Einstein O, Grigoriadis N, Mizrachi-Kol R, Reinhartz E, Polyzoidou E, Lavon I, et al. Transplanted neural precursor cells reduce brain inflammation to attenuate chronic experimental autoimmune encephalomyelitis. Experimental neurology. 2006 Apr;

[140] Bacigaluppi M, Pluchino S, Peruzzotti-Jametti L, Kilic E, Kilic U, Salani G, et al. De‐ layed post-ischaemic neuroprotection following systemic neural stem cell transplan‐

[141] Sun C, Zhang H, Li J, Huang H, Cheng H, Wang Y, et al. Modulation of the major histocompatibility complex by neural stem cell-derived neurotrophic factors used for regenerative therapy in a rat model of stroke. Journal of translational medicine.

[142] Aharonowiz M, Einstein O, Fainstein N, Lassmann H, Reubinoff B, Ben-Hur T. Neu‐ roprotective effect of transplanted human embryonic stem cell-derived neural pre‐

[143] Zlokovic BV. The blood-brain barrier in health and chronic neurodegenerative disor‐

[144] Wolburg H, Lippoldt A. Tight junctions of the blood-brain barrier: development, composition and regulation. Vascular pharmacology. 2002 Jun;38(6):323-37.

[145] Begley DJ. ABC transporters and the blood-brain barrier. Current pharmaceutical de‐

[146] Agrawal S, Anderson P, Durbeej M, van Rooijen N, Ivars F, Opdenakker G, et al. Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis. The Jour‐

[147] Engelhardt B, Ransohoff RM. Capture, crawl, cross: the T cell code to breach the

[148] Nag S, Venugopalan R, Stewart DJ. Increased caveolin-1 expression precedes de‐ creased expression of occludin and claudin-5 during blood-brain barrier breakdown.

nal of experimental medicine. 2006 Apr 17;203(4):1007-19.

blood-brain barriers. Trends in immunology. 2012 Aug 24.

Acta neuropathologica. 2007 Nov;114(5):459-69.

cursors in an animal model of multiple sclerosis. PloS one. 2008;3(9):e3145.

tation involves multiple mechanisms. Brain. 2009 Aug;132(Pt 8):2239-51.

orrhage. Stroke; a journal of cerebral circulation. 2003 Sep;34(9):2258-63.

status epilepticus in adult rats. Brain Res. 2004 Oct 15;1023(2):213-21.

Dec;24(4):1074-82.

198(2):275-84.

2010;8:77.

ders. Neuron. 2008 Jan 24;57(2):178-201.

sign. 2004;10(12):1295-312.


experimental allergic encephalomyelitis. Molecular and cellular neurosciences. 2003 Dec;24(4):1074-82.

[137] Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. Human neural stem cell trans‐ plantation promotes functional recovery in rats with experimental intracerebral hem‐ orrhage. Stroke; a journal of cerebral circulation. 2003 Sep;34(9):2258-63.

[125] Hauben E, Ibarra A, Mizrahi T, Barouch R, Agranov E, Schwartz M. Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: comparison with other myelin antigens. Proceedings of the National Academy of Sciences of the United States of America.

[126] Hohlfeld R, Kerschensteiner M, Stadelmann C, Lassmann H, Wekerle H. The neuro‐ protective effect of inflammation: implications for the therapy of multiple sclerosis.

[127] Eugster HP, Frei K, Kopf M, Lassmann H, Fontana A. IL-6-deficient mice resist mye‐ lin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. European

[128] Lacroix S, Chang L, Rose-John S, Tuszynski MH. Delivery of hyper-interleukin-6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth. The Journal of comparative neurology. 2002 Dec 16;454(3):213-28.

[129] Klusman I, Schwab ME. Effects of pro-inflammatory cytokines in experimental spinal

[130] Whiteley W, Jackson C, Lewis S, Lowe G, Rumley A, Sandercock P, et al. Inflamma‐ tory markers and poor outcome after stroke: a prospective cohort study and system‐

[131] Okada S, Nakamura M, Mikami Y, Shimazaki T, Mihara M, Ohsugi Y, et al. Blockade of interleukin-6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury. Journal of neuroscience research. 2004

[132] Serada S, Fujimoto M, Mihara M, Koike N, Ohsugi Y, Nomura S, et al. IL-6 blockade inhibits the induction of myelin antigen-specific Th17 cells and Th1 cells in experi‐ mental autoimmune encephalomyelitis. Proceedings of the National Academy of Sci‐

[133] Gertz K, Kronenberg G, Kalin RE, Baldinger T, Werner C, Balkaya M, et al. Essential role of interleukin-6 in post-stroke angiogenesis. Brain. 2012 Jun;135(Pt 6):1964-80.

[134] Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science (New York, NY. 1992 Mar

[135] Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proceedings of the National Academy of Sciences of the United States of

[136] Einstein O, Karussis D, Grigoriadis N, Mizrachi-Kol R, Reinhartz E, Abramsky O, et al. Intraventricular transplantation of neural precursor cell spheres attenuates acute

atic review of interleukin-6. PLoS medicine. 2009 Sep;6(9):e1000145.

ences of the United States of America. 2008 Jul 1;105(26):9041-6.

Journal of neuroimmunology. 2000 Jul 24;107(2):161-6.

journal of immunology. 1998 Jul;28(7):2178-87.

cord injury. Brain Res. 1997 Jul 11;762(1-2):173-84.

2001 Dec 18;98(26):15173-8.

336 Neural Stem Cells - New Perspectives

Apr 15;76(2):265-76.

27;255(5052):1707-10.

America. 2000 Nov 7;97(23):12846-51.


[149] Readnower RD, Chavko M, Adeeb S, Conroy MD, Pauly JR, McCarron RM, et al. In‐ crease in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury. Journal of neuroscience re‐ search. 2010 Dec;88(16):3530-9.

[161] Mahad D, Callahan MK, Williams KA, Ubogu EE, Kivisakk P, Tucky B, et al. Modu‐ lating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis

Systemic stem cell therapies and brain diseases

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

339

[162] Quandt J, Dorovini-Zis K. The beta chemokines CCL4 and CCL5 enhance adhesion of specific CD4+ T cell subsets to human brain endothelial cells. Journal of neuropa‐

[163] Alt C, Laschinger M, Engelhardt B. Functional expression of the lymphoid chemo‐ kines CCL19 (ELC) and CCL 21 (SLC) at the blood-brain barrier suggests their in‐ volvement in G-protein-dependent lymphocyte recruitment into the central nervous system during experimental autoimmune encephalomyelitis. European journal of

[164] Nourshargh S, Hordijk PL, Sixt M. Breaching multiple barriers: leukocyte motility through venular walls and the interstitium. Nat Rev Mol Cell Biol. 2010 May;11(5):

[165] Carman CV. Mechanisms for transcellular diapedesis: probing and pathfinding by 'invadosome-like protrusions'. Journal of cell science. 2009 Sep 1;122(Pt 17):3025-35.

[166] Lindvall O, Kokaia Z. Stem cell research in stroke: how far from the clinic? Stroke; a

[167] Takeuchi H, Natsume A, Wakabayashi T, Aoshima C, Shimato S, Ito M, et al. Intrave‐ nously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1- and HGF-dependent manner. Neuroscience letters. 2007 Oct

[168] Sandner B, Prang P, Rivera FJ, Aigner L, Blesch A, Weidner N. Neural stem cells for

[169] Shetty AK. Progress in cell grafting therapy for temporal lobe epilepsy. Neurothera‐

[170] Ahmed AU, Lesniak MS. Glioblastoma multiforme: can neural stem cells deliver the therapeutic payload and fulfill the clinical promise? Expert review of neurotherapeu‐

[171] Carbajal KS, Schaumburg C, Strieter R, Kane J, Lane TE. Migration of engrafted neu‐ ral stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis. Proceedings of the National Academy of Sciences of the United

[172] Andres RH, Choi R, Pendharkar AV, Gaeta X, Wang N, Nathan JK, et al. The CCR2/ CCL2 interaction mediates the transendothelial recruitment of intravascularly deliv‐ ered neural stem cells to the ischemic brain. Stroke; a journal of cerebral circulation.

spinal cord repair. Cell and tissue research. 2012 Jul;349(1):349-62.

pathogenesis. Brain. 2006 Jan;129(Pt 1):212-23.

immunology. 2002 Aug;32(8):2133-44.

366-78.

16;426(2):69-74.

peutics. 2011 Oct;8(4):721-35.

tics. 2011 Jun;11(6):775-7.

2012 Oct;42(10):2923-31.

States of America. 2010 Jun 15;107(24):11068-73.

thology and experimental neurology. 2004 Apr;63(4):350-62.

journal of cerebral circulation. 2011 Aug;42(8):2369-75.


[161] Mahad D, Callahan MK, Williams KA, Ubogu EE, Kivisakk P, Tucky B, et al. Modu‐ lating CCR2 and CCL2 at the blood-brain barrier: relevance for multiple sclerosis pathogenesis. Brain. 2006 Jan;129(Pt 1):212-23.

[149] Readnower RD, Chavko M, Adeeb S, Conroy MD, Pauly JR, McCarron RM, et al. In‐ crease in blood-brain barrier permeability, oxidative stress, and activated microglia in a rat model of blast-induced traumatic brain injury. Journal of neuroscience re‐

[150] Jiao H, Wang Z, Liu Y, Wang P, Xue Y. Specific role of tight junction proteins clau‐ din-5, occludin, and ZO-1 of the blood-brain barrier in a focal cerebral ischemic in‐

[151] van Assema DM, Lubberink M, Bauer M, van der Flier WM, Schuit RC, Windhorst AD, et al. Blood-brain barrier P-glycoprotein function in Alzheimer's disease. Brain.

[152] Errede M, Girolamo F, Ferrara G, Strippoli M, Morando S, Boldrin V, et al. Blood-Brain Barrier Alterations in the Cerebral Cortex in Experimental Autoimmune Ence‐ phalomyelitis. Journal of neuropathology and experimental neurology. 2012 Oct;

[153] Hickey WF. Migration of hematogenous cells through the blood-brain barrier and the initiation of CNS inflammation. Brain pathology (Zurich, Switzerland). 1991 Jan;1(2):

[154] Engelhardt B, Wolburg-Buchholz K, Wolburg H. Involvement of the choroid plexus in central nervous system inflammation. Microscopy research and technique. 2001

[155] Archambault AS, Sim J, Gimenez MA, Russell JH. Defining antigen-dependent stages of T cell migration from the blood to the central nervous system parenchyma. Euro‐

[156] Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science (New York,

[157] Steiner O, Coisne C, Cecchelli R, Boscacci R, Deutsch U, Engelhardt B, et al. Differen‐ tial roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell ar‐ rest, polarization, and directed crawling on blood-brain barrier endothelium. J

[158] Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N. Preven‐ tion of experimental autoimmune encephalomyelitis by antibodies against alpha 4

[159] Coisne C, Mao W, Engelhardt B. Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model

[160] Man S, Tucky B, Cotleur A, Drazba J, Takeshita Y, Ransohoff RM. CXCL12-induced monocyte-endothelial interactions promote lymphocyte transmigration across an in vitro blood-brain barrier. Science translational medicine. 2012 Feb 1;4(119):119ra14.

pean journal of immunology. 2005 Apr;35(4):1076-85.

beta 1 integrin. Nature. 1992 Mar 5;356(6364):63-6.

of multiple sclerosis. J Immunol. 2009 May 15;182(10):5909-13.

search. 2010 Dec;88(16):3530-9.

2012 Jan;135(Pt 1):181-9.

71(10):840-54.

338 Neural Stem Cells - New Perspectives

Jan 1;52(1):112-29.

NY. 1996 Apr 5;272(5258):60-6.

Immunol. 2010 Oct 15;185(8):4846-55.

97-105.

sult. J Mol Neurosci. 2011 Jun;44(2):130-9.


[173] Peng H, Huang Y, Rose J, Erichsen D, Herek S, Fujii N, et al. Stromal cell-derived fac‐ tor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells. Journal of neuroscience research. 2004 Apr 1;76(1):35-50.

[185] Pluchino S, Zanotti L, Deleidi M, Martino G. Neural stem cells and their use as thera‐ peutic tool in neurological disorders. Brain research. 2005 Apr;48(2):211-9.

Systemic stem cell therapies and brain diseases

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

341

[186] Einstein O, Friedman-Levi Y, Grigoriadis N, Ben-Hur T. Transplanted neural precur‐ sors enhance host brain-derived myelin regeneration. J Neurosci. 2009 Dec 16;29(50):

[187] Chu K, Kim M, Park KI, Jeong SW, Park HK, Jung KH, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ische‐

[188] Martino G, Pluchino S. Neural stem cells: guardians of the brain. Nature cell biology.

[189] Capone C, Frigerio S, Fumagalli S, Gelati M, Principato MC, Storini C, et al. Neuro‐ sphere-derived cells exert a neuroprotective action by changing the ischemic micro‐

[190] Jiang Q, Zhang ZG, Ding GL, Zhang L, Ewing JR, Wang L, et al. Investigation of neu‐ ral progenitor cell induced angiogenesis after embolic stroke in rat using MRI. Neu‐

[191] Lu P, Jones LL, Snyder EY, Tuszynski MH. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord in‐

[192] Lee HJ, Kim KS, Park IH, Kim SU. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke

[193] Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D, et al. Functional re‐ covery following traumatic spinal cord injury mediated by a unique polymer scaf‐ fold seeded with neural stem cells. Proceedings of the National Academy of Sciences

[194] Kim SU, de Vellis J. Stem cell-based cell therapy in neurological diseases: a review.

[195] Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science (New York, NY.

[196] Zhang WR, Sato K, Iwai M, Nagano I, Manabe Y, Abe K. Therapeutic time window of adenovirus-mediated GDNF gene transfer after transient middle cerebral artery

[197] Kobayashi T, Ahlenius H, Thored P, Kobayashi R, Kokaia Z, Lindvall O. Intracere‐ bral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogen‐

15694-702.

2007 Sep;9(9):1031-4.

mia. Brain Res. 2004 Aug 6;1016(2):145-53.

environment. PloS one. 2007;2(4):e373.

roImage. 2005 Nov 15;28(3):698-707.

model. PloS one. 2007;2(1):e156.

1993 May 21;260(5111):1130-2.

jury. Experimental neurology. 2003 Jun;181(2):115-29.

of the United States of America. 2002 Mar 5;99(5):3024-9.

occlusion in rat. Brain Res. 2002 Aug 23;947(1):140-5.

Journal of neuroscience research. 2009 Aug 1;87(10):2183-200.


[185] Pluchino S, Zanotti L, Deleidi M, Martino G. Neural stem cells and their use as thera‐ peutic tool in neurological disorders. Brain research. 2005 Apr;48(2):211-9.

[173] Peng H, Huang Y, Rose J, Erichsen D, Herek S, Fujii N, et al. Stromal cell-derived fac‐ tor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells.

[174] DeGrendele HC, Estess P, Siegelman MH. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science (New York, NY. 1997 Oct

[175] Rampon C, Weiss N, Deboux C, Chaverot N, Miller F, Buchet D, et al. Molecular mechanism of systemic delivery of neural precursor cells to the brain: assembly of brain endothelial apical cups and control of transmigration by CD44. Stem cells

[176] Carman CV, Springer TA. A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them. The Journal of cell

[177] Mueller A, Mahmoud NG, Strange PG. Diverse signalling by different chemokines through the chemokine receptor CCR5. Biochemical pharmacology. 2006 Sep

[178] Weiss N, Deboux C, Chaverot N, Miller F, Baron-Van Evercooren A, Couraud PO, et al. IL8 and CXCL13 are potent chemokines for the recruitment of human neural pre‐ cursor cells across brain endothelial cells. Journal of neuroimmunology. 2010 Jun;

[179] Jaderstad J, Jaderstad LM, Li J, Chintawar S, Salto C, Pandolfo M, et al. Communica‐ tion via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host. Proceedings of the National Academy of Sci‐

[180] Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signal‐

[181] Gerdes HH, Bukoreshtliev NV, Barroso JF. Tunneling nanotubes: a new route for the exchange of components between animal cells. FEBS letters. 2007 May 22;581(11):

[182] Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune re‐

[183] Vultur A, Cao J, Arulanandam R, Turkson J, Jove R, Greer P, et al. Cell-to-cell adhe‐ sion modulates Stat3 activity in normal and breast carcinoma cells. Oncogene. 2004

[184] Ben-Hur T, Ben-Menachem O, Furer V, Einstein O, Mizrachi-Kol R, Grigoriadis N. Effects of proinflammatory cytokines on the growth, fate, and motility of multipoten‐ tial neural precursor cells. Molecular and cellular neurosciences. 2003 Nov;24(3):

ences of the United States of America. Mar 16;107(11):5184-9.

sponses. Nat Rev Immunol. 2009 Aug;9(8):581-93.

ing and therapy. Circulation research. 2008 Nov 21;103(11):1204-19.

Journal of neuroscience research. 2004 Apr 1;76(1):35-50.

24;278(5338):672-5.

340 Neural Stem Cells - New Perspectives

14;72(6):739-48.

223(1-2):131-4.

2194-201.

623-31.

Apr 8;23(15):2600-16.

(Dayton, Ohio). 2008 Jul;26(7):1673-82.

biology. 2004 Oct 25;167(2):377-88.


esis after stroke in adult rats. Stroke; a journal of cerebral circulation. 2006 Sep;37(9): 2361-7.

[209] Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation.

Systemic stem cell therapies and brain diseases

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

343

[210] Molina-Holgado F, Grencis R, Rothwell NJ. Actions of exogenous and endogenous IL-10 on glial responses to bacterial LPS/cytokines. Glia. 2001 Feb;33(2):97-106. [211] Croxford JL, Feldmann M, Chernajovsky Y, Baker D. Different therapeutic outcomes in experimental allergic encephalomyelitis dependent upon the mode of delivery of IL-10: a comparison of the effects of protein, adenoviral or retroviral IL-10 delivery

[212] Boyd ZS, Kriatchko A, Yang J, Agarwal N, Wax MB, Patil RV. Interleukin-10 receptor signaling through STAT-3 regulates the apoptosis of retinal ganglion cells in re‐ sponse to stress. Investigative ophthalmology & visual science. 2003 Dec;44(12):

[213] Yang J, Jiang Z, Fitzgerald DC, Ma C, Yu S, Li H, et al. Adult neural stem cells ex‐ pressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis. The Journal of clinical investigation. 2009 Dec;119(12):

[214] Fainstein N, Vaknin I, Einstein O, Zisman P, Ben Sasson SZ, Baniyash M, et al. Neu‐ ral precursor cells inhibit multiple inflammatory signals. Molecular and cellular neu‐

[215] Knight JC, Scharf EL, Mao-Draayer Y. Fas activation increases neural progenitor cell

[216] Wang L, Shi J, van Ginkel FW, Lan L, Niemeyer G, Martin DR, et al. Neural stem/ progenitor cells modulate immune responses by suppressing T lymphocytes with ni‐ tric oxide and prostaglandin E2. Experimental neurology. 2009 Mar;216(1):177-83.

[217] Ricci-Vitiani L, Casalbore P, Petrucci G, Lauretti L, Montano N, Larocca LM, et al. In‐ fluence of local environment on the differentiation of neural stem cells engrafted on‐

[218] Melzi R, Antonioli B, Mercalli A, Battaglia M, Valle A, Pluchino S, et al. Co-graft of allogeneic immune regulatory neural stem cells (NPC) and pancreatic islets mediates tolerance, while inducing NPC-derived tumors in mice. PloS one. 2010;5(4):e10357.

[219] Flugel A, Berkowicz T, Ritter T, Labeur M, Jenne DE, Li Z, et al. Migratory activity and functional changes of green fluorescent effector cells before and during experi‐

[220] de Vos AF, van Meurs M, Brok HP, Boven LA, Hintzen RQ, van der Valk P, et al. Transfer of central nervous system autoantigens and presentation in secondary lym‐

mental autoimmune encephalomyelitis. Immunity. 2001 May;14(5):547-60.

phoid organs. J Immunol. 2002 Nov 15;169(10):5415-23.

to the injured spinal cord. Neurological research. 2006 Jul;28(5):488-92.

survival. Journal of neuroscience research. 2010 Mar;88(4):746-57.

into the central nervous system. J Immunol. 2001 Mar 15;166(6):4124-30.

Nature medicine. 2011 Jul;17(7):796-808.

5206-11.

3678-91.

rosciences. 2008 Nov;39(3):335-41.


[209] Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nature medicine. 2011 Jul;17(7):796-808.

esis after stroke in adult rats. Stroke; a journal of cerebral circulation. 2006 Sep;37(9):

[198] Chen B, Gao XQ, Yang CX, Tan SK, Sun ZL, Yan NH, et al. Neuroprotective effect of grafting GDNF gene-modified neural stem cells on cerebral ischemia in rats. Brain

[199] Nishimura Y, Natsume A, Ito M, Hara M, Motomura K, Fukuyama R, et al. Interfer‐ on-beta delivery via human neural stem cell abates glial scar formation in spinal cord

[200] Kim HM, Hwang DH, Lee JE, Kim SU, Kim BG. Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue spar‐

[201] Lee HJ, Kim KS, Kim EJ, Choi HB, Lee KH, Park IH, et al. Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intra‐ cerebral hemorrhage stroke model. Stem cells (Dayton, Ohio). 2007 May;25(5):

[202] Lee HJ, Lim IJ, Lee MC, Kim SU. Human neural stem cells genetically modified to overexpress brain-derived neurotrophic factor promote functional recovery and neu‐ roprotection in a mouse stroke model. Journal of neuroscience research. 2010 Nov

[203] Modo M, Stroemer RP, Tang E, Patel S, Hodges H. Effects of implantation site of stem cell grafts on behavioral recovery from stroke damage. Stroke; a journal of cere‐

[204] Zhang ZG, Jiang Q, Zhang R, Zhang L, Wang L, Zhang L, et al. Magnetic resonance imaging and neurosphere therapy of stroke in rat. Annals of neurology. 2003 Feb;

[205] Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proceedings of the National Academy of Sciences of the United States of America.

[206] Roitberg BZ, Mangubat E, Chen EY, Sugaya K, Thulborn KR, Kordower JH, et al. Survival and early differentiation of human neural stem cells transplanted in a non‐ human primate model of stroke. Journal of neurosurgery. 2006 Jul;105(1):96-102.

[207] Darsalia V, Kallur T, Kokaia Z. Survival, migration and neuronal differentiation of human fetal striatal and cortical neural stem cells grafted in stroke-damaged rat stria‐

[208] Ben-Hur T. Immunomodulation by neural stem cells. Journal of the neurological sci‐

tum. The European journal of neuroscience. 2007 Aug;26(3):605-14.

2361-7.

342 Neural Stem Cells - New Perspectives

1204-12.

15;88(15):3282-94.

53(2):259-63.

Res. 2009 Aug 11;1284:1-11.

injury. Cell transplantation. 2012 Oct 12.

bral circulation. 2002 Sep;33(9):2270-8.

2004 Aug 10;101(32):11839-44.

ences. 2008 Feb 15;265(1-2):102-4.

ing after spinal cord injury. PloS one. 2009;4(3):e4987.


[221] Mohindru M, Kang B, Kim BS. Functional maturation of proteolipid pro‐ tein(139-151)-specific Th1 cells in the central nervous system in experimental autoim‐ mune encephalomyelitis. Journal of neuroimmunology. 2004 Oct;155(1-2):127-35.

tential against ischemic stroke after intrastriatal and systemic transplantation. Stem

Systemic stem cell therapies and brain diseases

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

345

[233] Walczak P, Zhang J, Gilad AA, Kedziorek DA, Ruiz-Cabello J, Young RG, et al. Dualmodality monitoring of targeted intraarterial delivery of mesenchymal stem cells af‐ ter transient ischemia. Stroke; a journal of cerebral circulation. 2008 May;39(5):

[234] Li L, Jiang Q, Ding G, Zhang L, Zhang ZG, Li Q, et al. Effects of administration route on migration and distribution of neural progenitor cells transplanted into rats with focal cerebral ischemia, an MRI study. J Cereb Blood Flow Metab. Mar;30(3):653-62.

[235] Sykova E, Homola A, Mazanec R, Lachmann H, Konradova SL, Kobylka P, et al. Au‐ tologous bone marrow transplantation in patients with subacute and chronic spinal

[236] Parr AM, Kulbatski I, Tator CH. Transplantation of adult rat spinal cord stem/ progenitor cells for spinal cord injury. Journal of neurotrauma. 2007 May;24(5):

[237] Cusimano M, Biziato D, Brambilla E, Donega M, Alfaro-Cervello C, Snider S, et al. Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary

[238] Brundin P, Strecker RE, Londos E, Bjorklund A. Dopamine neurons grafted unilater‐ ally to the nucleus accumbens affect drug-induced circling and locomotion. Experi‐

[239] Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W, Kao R, et al. Transplanta‐ tion of embryonic dopamine neurons for severe Parkinson's disease. The New Eng‐

[240] Olanow CW, Goetz CG, Kordower JH, Stoessl AJ, Sossi V, Brin MF, et al. A doubleblind controlled trial of bilateral fetal nigral transplantation in Parkinson's disease.

[241] Lindvall O. Why is it taking so long to develop clinically competitive stem cell thera‐

[242] Daley GQ. The promise and perils of stem cell therapeutics. Cell stem cell. 2012 Jun

[243] Hyun I, Lindvall O, Ahrlund-Richter L, Cattaneo E, Cavazzana-Calvo M, Cossu G, et al. New ISSCR guidelines underscore major principles for responsible translational

[244] Aboody K, Capela A, Niazi N, Stern JH, Temple S. Translating stem cell studies to the clinic for CNS repair: current state of the art and the need for a Rosetta stone.

pies for CNS disorders? Cell stem cell. 2012 Jun 14;10(6):660-2.

stem cell research. Cell stem cell. 2008 Dec 4;3(6):607-9.

tissue damage in the injured spinal cord. Brain. 2012 Feb;135(Pt 2):447-60.

mental brain research Experimentelle Hirnforschung. 1987;69(1):183-94.

cells (Dayton, Ohio). Jun;30(6):1297-310.

cord injury. Cell transplantation. 2006;15(8-9):675-87.

land journal of medicine. 2001 Mar 8;344(10):710-9.

Annals of neurology. 2003 Sep;54(3):403-14.

Neuron. 2011 May 26;70(4):597-613.

1569-74.

835-45.

14;10(6):740-9.


tential against ischemic stroke after intrastriatal and systemic transplantation. Stem cells (Dayton, Ohio). Jun;30(6):1297-310.

[233] Walczak P, Zhang J, Gilad AA, Kedziorek DA, Ruiz-Cabello J, Young RG, et al. Dualmodality monitoring of targeted intraarterial delivery of mesenchymal stem cells af‐ ter transient ischemia. Stroke; a journal of cerebral circulation. 2008 May;39(5): 1569-74.

[221] Mohindru M, Kang B, Kim BS. Functional maturation of proteolipid pro‐ tein(139-151)-specific Th1 cells in the central nervous system in experimental autoim‐ mune encephalomyelitis. Journal of neuroimmunology. 2004 Oct;155(1-2):127-35. [222] Guzman R, Choi R, Gera A, De Los Angeles A, Andres RH, Steinberg GK. Intravas‐ cular cell replacement therapy for stroke. Neurosurgical focus. 2008;24(3-4):E15. [223] Takahashi K, Yasuhara T, Shingo T, Muraoka K, Kameda M, Takeuchi A, et al. Em‐ bryonic neural stem cells transplanted in middle cerebral artery occlusion model of rats demonstrated potent therapeutic effects, compared to adult neural stem cells.

[224] Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Schut D, Fehlings MG. Synergistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and growth factors promote functional repair and plasticity of the chronically injured spi‐

[225] Zhang P, Li J, Liu Y, Chen X, Lu H, Kang Q, et al. Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri-in‐ farct angiogenesis after focal cerebral ischemia. Neuropathology. 2011 Aug;31(4):

[226] Ziv Y, Avidan H, Pluchino S, Martino G, Schwartz M. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spi‐ nal cord injury. Proceedings of the National Academy of Sciences of the United

[227] Wu W, Chen X, Hu C, Li J, Yu Z, Cai W. Transplantation of neural stem cells express‐ ing hypoxia-inducible factor-1alpha (HIF-1alpha) improves behavioral recovery in a

[228] Jin K, Sun Y, Xie L, Mao XO, Childs J, Peel A, et al. Comparison of ischemia-directed migration of neural precursor cells after intrastriatal, intraventricular, or intravenous

[229] Lundberg J, Le Blanc K, Soderman M, Andersson T, Holmin S. Endovascular trans‐ plantation of stem cells to the injured rat CNS. Neuroradiology. 2009 Oct;51(10):

[230] Chu K, Park KI, Lee ST, Jung KH, Ko SY, Kang L, et al. Combined treatment of vas‐ cular endothelial growth factor and human neural stem cells in experimental focal

[231] Vendrame M, Cassady J, Newcomb J, Butler T, Pennypacker KR, Zigova T, et al. In‐ fusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke; a journal of cerebral

[232] Doeppner TR, Ewert TA, Tonges L, Herz J, Zechariah A, ElAli A, et al. Transduction of neural precursor cells with TAT-heat shock protein 70 chaperone: therapeutic po‐

cerebral ischemia. Neuroscience research. 2005 Dec;53(4):384-90.

circulation. 2004 Oct;35(10):2390-5.

transplantation in the rat. Neurobiology of disease. 2005 Mar;18(2):366-74.

Brain Res. 2008 Oct 9;1234:172-82.

384-91.

344 Neural Stem Cells - New Perspectives

661-7.

nal cord. J Neurosci. 2010 Feb 3;30(5):1657-76.

States of America. 2006 Aug 29;103(35):13174-9.

rat stroke model. J Clin Neurosci. 2010 Jan;17(1):92-5.


[245] Amariglio N, Hirshberg A, Scheithauer BW, Cohen Y, Loewenthal R, Trakhtenbrot L, et al. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS medicine. 2009 Feb 17;6(2):e1000029.

[257] Yamanaka S. Induced pluripotent stem cells: past, present, and future. Cell stem cell.

Systemic stem cell therapies and brain diseases

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

347

[258] Harris VK, Yan QJ, Vyshkina T, Sahabi S, Liu X, Sadiq SA. Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis. Journal of the neurological sciences.

[259] Yang J, Yan Y, Ma CG, Kang T, Zhang N, Gran B, et al. Accelerated and enhanced effect of CCR5-transduced bone marrow neural stem cells on autoimmune encepha‐

[260] Sher F, Amor S, Gerritsen W, Baker D, Jackson SL, Boddeke E, et al. Intraventricular‐ ly injected Olig2-NSCs attenuate established relapsing-remitting EAE in mice. Cell

[261] Jiang Q, Zhang ZG, Ding GL, Silver B, Zhang L, Meng H, et al. MRI detects white matter reorganization after neural progenitor cell treatment of stroke. NeuroImage.

lomyelitis. Acta neuropathologica. 2012 Oct;124(4):491-503.

2012 Jun 14;10(6):678-84.

2012 Feb 15;313(1-2):167-77.

transplantation. 2012 Mar 28.

2006 Sep;32(3):1080-9.


[257] Yamanaka S. Induced pluripotent stem cells: past, present, and future. Cell stem cell. 2012 Jun 14;10(6):678-84.

[245] Amariglio N, Hirshberg A, Scheithauer BW, Cohen Y, Loewenthal R, Trakhtenbrot L, et al. Donor-derived brain tumor following neural stem cell transplantation in an

[246] Feng Z, Gao F. Stem cell challenges in the treatment of neurodegenerative disease.

[247] Steiner R, Huhn, S., Koch, T., Al-Uzri, A., Guillaime, D., Sutcliffe, T., Vogel, H., and Selden, N. CNS transplantation of purified human neural stem cells in infantile and late-infantile neuronal ceroid lipofuscinoses: Summary of the Phase I trial. Mol Genet

[248] Glass JD, Boulis NM, Johe K, Rutkove SB, Federici T, Polak M, et al. Lumbar intraspi‐ nal injection of neural stem cells in patients with amyotrophic lateral sclerosis: results of a phase I trial in 12 patients. Stem cells (Dayton, Ohio). 2012 Jun;30(6):1144-51. [249] Riley J, Federici T, Polak M, Kelly C, Glass J, Raore B, et al. Intraspinal stem cell transplantation in amyotrophic lateral sclerosis: a phase I safety trial, technical note, and lumbar safety outcomes. Neurosurgery. 2012 Aug;71(2):405-16; discussion 16.

[250] Gupta N, Henry RG, Strober J, Kang SM, Lim DA, Bucci M, et al. Neural stem cell engraftment and myelination in the human brain. Science translational medicine.

[251] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multili‐ neage potential of adult human mesenchymal stem cells. Science (New York, NY.

[252] Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science (New York, NY. 2000 Dec 1;290(5497):

[253] Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. Journal of neuroscience research. 2000 Aug

[254] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Experimental

[255] Joyce N, Annett G, Wirthlin L, Olson S, Bauer G, Nolta JA. Mesenchymal stem cells for the treatment of neurodegenerative disease. Regenerative medicine. 2010 Nov;

[256] Gao A, Peng Y, Deng Y, Qing H. Potential therapeutic applications of differentiated induced pluripotent stem cells (iPSCs) in the treatment of neurodegenerative diseas‐

ataxia telangiectasia patient. PLoS medicine. 2009 Feb 17;6(2):e1000029.

CNS neuroscience & therapeutics. 2012 Feb;18(2):142-8.

Metab. 2010;99(S35).

346 Neural Stem Cells - New Perspectives

2012 Oct 10;4(155):155ra37.

1999 Apr 2;284(5411):143-7.

neurology. 2000 Aug;164(2):247-56.

es. Neuroscience. 2012 Oct 13.

1775-9.

15;61(4):364-70.

5(6):933-46.


**Chapter 12**

**Cell Adhesion Molecules in Neural Stem Cell**

Additional information is available at the end of the chapter

Shan Bian

**1. Introduction**

http://dx.doi.org/10.5772/55136

2004; Merkle and Alvarez-Buylla, 2006).

**and Stem Cell-Based Therapy for Neural Disorders**

Neural stem/progenitor cells (NS/PCs), found in both the developing and the adult mamma‐ lian central nervous system (CNS), are a heterogeneous population of multipotent cells with the potential to self-renew by symmetric cell division or to differentiate into neurons, astrocytes and oligodendrocytes through asymmetric cell division (Gage, 2000; Alvarez-Buylla et al., 2001; Temple, 2001; Götz and Huttner, 2005). NS/PCs have been found in almost all regions of the developing mammalian CNS, including the basal forebrain, cerebral cortex, ganglionic eminence, hippocampus, cerebellum, neural crest and spinal cord (Temple, 2001). Throughout development, NS/PCs give rise to neurons and glial cell populations of the CNS. In the adult CNS, NS/PCs are mainly found in the subventricular zone (SVZ) and subgranular layer (SGL) of hippocampal dentate gyrus (DG) (Göritz and Frisén, 2012). The ependymal cells lining the central canal of spinal cord of the adult mouse could be another potential source of adult NS/ PCs (Meletis et al., 2008). Because neurogenesis and gliogenesis occur during different stages of mammalian brain development, it was long assumed that neurons and glial cells in the CNS were generated from distinct precursor populations, known as early-embryonic, late-embry‐ onic, and adult NS/PCs. However, abundant evidence has since now demonstrated that embryonic and adult NS/PCs are likely lineage-related. Neuroepithelial cells behaving as NS/ PCs during very early developmental stages of the mammalian CNS give rise to radial glial cells around embryonic day 12 (E12). As the progeny of neuroepithelial cells, radial glial cells act as NS/PCs in the fetal and perinatal brain, and develop into astrocyte-like stem cells in the adult brains. Astrocyte-like adult stem cells function as stem cells to generate new nerve cells in the adult mammalian CNS. (Doetsch et al., 1999; Alvarez-Buylla et al., 2001; Merkle et al.,

> © 2013 Bian; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

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

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

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