The therapeutic contribution of nanomedicine to treat neurodegenerative diseases via neural stem cell differentiation

Biomaterials 123 (2017) 77e91

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Review

The therapeutic contribution of nanomedicine to treat neurodegenerative diseases via neural stem cell differentiation
ronique Pre
at a, Anne des Rieux a, b, * Dario Carradori a, c, Joel Eyer c, Patrick Saulnier c, Ve a Universit
e Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Avenue E. Mounier 73, 1200 Brussels, Belgium b Universit
e Catholique de Louvain, Institute of Materials and Condensed Matter, 1348 Louvain-la-Neuve, Belgium c Universit
e d’Angers, Unit
e Micro et Nanom
edecines Biomim
etiques, MINT, Institut de Biologie en Sant
e PBH-IRIS, 49033 Angers, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2016 Received in revised form 22 December 2016 Accepted 27 January 2017 Available online 27 January 2017

The discovery of adult neurogenesis drastically changed the therapeutic approaches of central nervous system regenerative medicine. The stimulation of this physiologic process can increase memory and motor performances in patients affected by neurodegenerative diseases. Neural stem cells contribute to the neurogenesis process through their differentiation into specialized neuronal cells. In this review, we describe the most important methods developed to restore neurological functions via neural stem cell differentiation. In particular, we focused on the role of nanomedicine. The application of nanostructured scaffolds, nanoparticulate drug delivery systems, and nanotechnology-based real-time imaging has significantly improved the safety and the efficacy of neural stem cell-based treatments. This review provides a comprehensive background on the contribution of nanomedicine to the modulation of neurogenesis via neural stem cell differentiation. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Nanomedicine Nanoparticles Nanotechnology Neural stem cell differentiation Neurodegenerative disease Neurogenesis

1. Introduction The dogma of a static brain was destroyed when Smart and Leblond showed for the first time that glial cells are dividing throughout the mouse brain parenchyma [1]. A few years later, Altman and Das reported the migration of postnatally born neuroblasts from the subventricular zone to the olfactory bulb, providing the first strong evidence of neurogenesis in the adult brain [2]. Important discoveries were made in the following decades, such as the presence of adult-born neurons in the dentate gyrus of rats [3] and in the vocal control nucleus of birds [4], but the perception of neurogenesis has drastically changed only since the 1990s. One of the most important discoveries was the observation that the proliferation of progenitor cells, and the subsequent number of newborn neurons, was dynamic. Several factors such as hormonal stress [5], age [6], and alcohol [7] could modulate this

Abbreviations: CNS, central nervous system; NSC, neural stem cells; SVZ, subventricular zone; SGZ, subgranular zone.
Catholique de Louvain, Louvain Drug * Corresponding author. Universite Research Institute, Advanced Drug Delivery and Biomaterials, Avenue E. Mounier 73, 1200 Brussels, Belgium. E-mail address: [email protected] (A. des Rieux). http://dx.doi.org/10.1016/j.biomaterials.2017.01.032 0142-9612/© 2017 Elsevier Ltd. All rights reserved.

process. The improvement of immunohistological techniques represented another step forward in the description of neurogenesis by providing more sensitive analyses [8,9]. Moreover, the ability to isolate, cultivate, and differentiate neuronal precursor cells in vitro provided crucial data on the cellular and biomolecular mechanisms involved in adult neurogenesis [10e12]. In humans, the evaluation of neurogenesis was initially performed by the quantification of the number of cells expressing neuroblast markers such as doublecortin (DCX) or polysialylatedneural cell adhesion molecule (PSA-NCAM) in postmortem brains [13]. These markers were found in significant amounts in two regions of the brain: the subgranular zone (SGZ) and the subventricular zone (SVZ) (Fig. 1). These markers are highly expressed during the fetal and perinatal phases, then expression dramatically decreases during the first postnatal months, and finally slowly declines throughout life [14]. Human adult neurogenesis was recently confirmed by Spalding and colleagues [15]. They measured the annual neuron turnover (1.75%) and concluded that neurons were generated during adulthood at similar rates in humans and mice. The discovery of adult neurogenesis also showed the limits of this physiologic process. Indeed, neurogenesis is restricted to small areas of the brain (the SGZ and SVZ), called niches, and its impact

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on the adult organism is very limited. The identification of neural stem cells (NSC) and their role in adult neurogenesis motivated researchers to explore the regenerative potential of these cells. The design of therapeutics able to modulate the differentiation of NSC, and consequently the rate of neurogenesis, represents a promising strategy in the treatment of many neurodegenerative diseases. In this review we underline the significant advancements achieved from the conception to the clinical application of therapies targeting NSC differentiation. We review the therapeutic potential of NSC and analyze how their differentiation could contribute to the treatment of neurodegenerative disorders. Although much progress has been made, issues are still associated with the therapeutic use of NSC. In this review, we will underline how nanomedicine can contribute to the improvement of NSC-based therapy. 2. Neural stem cells 2.1. Definition The current definition of NSC, related to their peculiar biological properties, was based on retrospective in vitro studies [10,16,17]. “We define a neural stem cell as a stem cell derived from any part of the nervous system and which primarily makes cells expressing neural markers (those of astrocytes, oligodendrocytes and neurons) in in vitro culture” [18]. While there was evidence of adult neurogenesis, the cells involved were not characterized until the end of the 1990s. At that time, NSC were identified and their role in neurogenesis was understood and described. Long term expansion and differentiation into neural lineages of specific cells isolated from the brain hinted the existence of adult NSC [10,12]. NSC are characterized by the ability to self-renew and to differentiate into neurons, astrocytes, and oligodendrocytes [19,20]. It has long been postulated that adult neurogenesis

originated from these tri-potent NSC, which are mostly restricted to the SVZ and the SGZ (Fig. 2A). Unlike other somatic stem cells, the information regarding the localization and the properties of NSC precursors is very limited. The embryonic origins of NSC are not well understood. Adult SGZ-NSC could originate from the ventral hippocampus during the late fetal stage [21], while adult SVZ-NSC are regionally specified at an early embryonic stage [22,23]. 2.2. Biological functions The role of adult somatic stem cells is normally related to the modulation of homeostasis in the tissues. When adult NSC were discovered, it was initially assumed that their function was exclusively to provide a regenerative source of new neurons and glial cells in pathological conditions. Instead, evidence suggested that the primary function of adult NSC was to confer additional plasticity to the brain. Direct and indirect mechanisms were described to regulate such plasticity [24] (Fig. 2B). Intrinsic transcriptional programs directed to gene expression or external signals triggering an intracellular cascade greatly impact the behavior of NSC. Consequently, the identification of the origin of the niche signals is challenging (e.g., the role of calcium levels on the activity of postnatal developing regions [25e27]). Although NSC are multipotent in vitro, recent genetic fate-mapping and clonal lineage-tracing of NSC have highlighted the lack of similarities between NSC differentiation in vitro and in vivo [28]. The niche environment seems to limit adult NSC differentiation. In the adult SGZ, NSC can generate dentate gyrus granular cells while in the adult SVZ, NSC produce neuroblasts, which migrate to the olfactory bulb where they differentiate into interneurons [29]. Moreover, NSC localization in the niches would determine the type of cells derived from NSC. In the SVZ, ventral NSC mostly develop into calbindin-expressing cells, whereas dorsal NSC develop into thyrosine-hydroxylaseexpressing cells [30]. These two markers are associated with two different cell types: long-axon and dopaminergic neurons,

A

C

D B

Fig. 1. Neural stem cell niches in the brain. Localization of the subgranular (SGZ) and subventricular (SVZ) zones in an adult human brain. A, lateral section of the brain. B, frontal section of the brain. C, subventricular zone, highlighted in red. D, subgranular zone, highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Adult NSC localization and regulation in the SVZ and SGZ niches. A) Cellular organization in the SVZ and the SGZ. In the SVZ, ependymal cells (or type E cells, blue) form the walls of the ventricle, NSC (or type B1 cells, green) are the primary neuronal precursors able to give rise to intermediate progenitor cells (or type C cells, brown) which in turn become neuroblasts (type A cells, red). In the SGZ, NSC (or type 1 cells, green) generate intermediate progenitor cells (brown) which in turn become neuroblasts (red) and finally develop into dentate granule cells (light blue). In the SVZ and the SGZ, astrocytes (gray) contribute to the niche architecture and sustain cell growth. B) Interconnections between extrinsic and intrinsic NSC regulation pathways. Extrinsic signals can bind to NSC plasma membrane receptors or penetrate via specific channels and trigger intracellular cascades inducing modifications in gene expression (blue arrows). The secretions of the choroid plexus such as insulin-like growth factor 1 (IGF1) [38,39] or ions such as calcium [26] are examples of extrinsic signals that can modulate NSC differentiation by their interaction with IGF1 receptor and Caþþ channels, respectively. Intrinsic regulatory processes can also direct gene expression in NSC by affecting intrinsic transcriptional programs (red arrows). Sonic hedgehog is the major activating ligand to initiate Hedgehog signaling in the brain and has been shown to play an important role in NSC proliferation and differentiation [40]. Both the extrinsic and intrinsic pathways are interconnected. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

respectively. In the SGZ, the adult NSC population reacts differently to environmental stimuli depending on their lineage [31]. Learning and memory processes are strictly related to adult neurogenesis in the SVZ [32e34], while SGZ-NSC-derived granule cells of the dentate gyrus have been implicated in long-term spatial memory and pattern separation [35,36]. Transplanted NSC are also able to release immunomodulatory and neurotrophic factors (bystander effect) such as nerve growth factor, brain-derived growth factor, and leukemia inhibiting factor [37]. 2.3. Therapeutic significance The therapeutic relevance of NSC was investigated after several studies clearly demonstrated that the inhibition of neurogenesis decreased neurological functions [41,42], while its stimulation resulted in behavioural performance recovery, e.g., learning and memory tasks [43,44]. Consequently, adult neurogenesis modulation could have a positive impact on the treatment of neurodegenerative diseases, which are mostly characterized by neurological function deterioration, such as Alzheimer’s (memory impairment) and Parkinson’s (motor impairment) diseases. Although the mechanism of NSC is regulated by many physiological stimuli, NSC are considered to be key determinants in neurogenesis. Therefore, the control of NSC differentiation has emerged as a promising approach to manipulate neural cells for therapeutic purposes. 3. Neural stem cell differentiation 3.1. Neurogenesis Adult neurogenesis was described both in the SGZ and in the SVZ of the brain. It consists of several developmental stages (proliferation, differentiation, maturation and integration) that are characterized by distinct cell phenotypes. SGZ and SVZ do not have the same precursors; consequently, there is not a unique nomenclature to identify cells involved in the adult neurogenesis [31,45,46]. Several markers are used to detect the different cell

phenotypes involved in the neurogenic process (Table 1) [47,48]. (i) Stem-like/precursor cells. Nestin was the first marker described to identify stem-like/precursor cells and is the most widely used [49]. Nestin is a specific class of intermediate filament proteins that are expressed in nondifferentiated cells. Another marker commonly used is the neural RNA-binding protein Musashi 1 (Msi1), which has been identified in proliferating neural/glial precursors [50]. Recently, the chondroitin sulfate proteoglycan neuron/glia antigen 2 (NG2)-glia positive cells have been identified. They represent the major proliferative cell population in the healthy adult brain outside the neurogenic niches [51]. Since NG2-glia positive cells can give rise to different neuronal cell types, including neurons, and maintain their undifferentiated population, they are considered to be very close to neural stem cells [52] and thus an interesting target to treat neurodegenerative diseases [53]. (ii) Immature neurons. Class III b tubulin (Tuj1) [54], a protein expressed in post-mitotic neuron cytoskeleton, doublecortin (DCX) [55], which encodes a microtubule-associated protein present in migrating neuroblasts, and the polysialylatedneural cell adhesion molecule (PSA-NCAM), which is a product of post-translational modification of NCAM, are the most accepted markers for early neurons [56]. Another widely used marker at this stage is NeuroD, the transcription factor of bHLH [56]. (iii) Mature neurons. The most widely used markers to identify mature neurons are the microtubule-associated protein 2 (MAP-2), the neuron-specific enolase (NSE) and the neuralspecific nuclear protein (NeuN) [57,58]. Some non-neural cells can also be positive for the markers mentioned above. To avoid ambiguous interpretation of the results, it is recommended to perform multiple staining including nonneural markers such as glial fibrillary acidic protein (GFAP) [59] or calcium binding proteins (S100 and S100b) [60] for astrocytes, and 20 ,30 -cyclic-nucleotide 30 -phosphodiesterase (CNP) or myelin

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D. Carradori et al. / Biomaterials 123 (2017) 77e91 Table 1 Markers for adult neurogenesis. Type of cells

Markers

Reference

Neural stem cells/Progenitors

Nestin SRY-related HMG-box gene (Sox2) Musashi-1 (Msi1) Paired box gene 6 (Pax6) Prominin (CD133) Glial fibrillary acid protein (GFAP) Chondroitin sulfate proteoglycan neuron/glia antigen 2 (NG2) III b tubulin (TUJ1) Doublecortin (DCX, C-18) Polysialylated-neural cell adhesion molecule (PSA-NCAM) Neurogenetic Differentiation (NeuroD) Neuronal nuclear epitope (NeuN) Microtubule-associated protein 2 (MAP2) Neuron-specific enolase (NSE) Calbindin Thyrosine-hydroxylase (TH) Calretinin Neurofilaments (NF) Glial fibrillary acid protein (GFAP) Calcium binding proteins (S100/S100b) Glutammate-aspartate transporter (EAAT1) Galactocerebroside (GalC) 20 ,30 -cyclic-nucleotide 30 -phosphodiesterase (CNP) Myelin basic protein (MBP)

[49] [62] [50] [63] [64] [59] [51] [54] [55] [56] [56] [58] [57] [57] [65] [66] [65] [65] [59] [60] [67] [68] [61] [61]

NG2-glia positive cells Neural lineage (early)

Neural lineage (mature)

Astrocytic lineage

Glial lineage

Some of the markers listed in the table (e.g., GFAP) are not exclusively expressed by a single cell type.

basic protein (MBP) for oligodendrocytes [61]. 3.2. Endogenous and exogenous NSC In the last 20 years, several protocols have been developed to cultivate and differentiate NSC in vitro after the isolation of NSC from niches (isolated NSC) or after derivation from pluripotent restored adult somatic cells (induced pluripotent stem cell-derived NSC, iPSC-derived NSC) [69]. However, the development of standard procedures to induce NSC differentiation in vivo or in situ has not yet been established. Brain repair via NSC differentiation can be achieved by following different strategies, which essentially depend on whether NSC are exogenous or endogenous (Fig. 3). iPSC-derived and isolated NSC are considered exogenous NSC when expanded/treated in vitro and followed by an in vivo transplantation. Niche-localized NSC are considered endogenous NSC and their differentiation is stimulated in situ. According to their origin, different approaches have been developed to induce NSC differentiation in vitro/in vivo/in situ (Table 2). 3.3. In vitro stimulation of NSC differentiation iPSC-derived and isolated NSC can preserve their multipotent profile in the presence of repressor-type bHLH genes [70,71], hypoxia [72], serum-free media [73], and media enriched with growth factors. Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) are the most used growth factors to maintain NSC in an undifferentiated state [74]. Exposure to specific compounds and conditions can induce NSC differentiation. Numerous mechanisms controlling the behavior of NSC were elucidated by deciphering the role of intrinsic and extrinsic signals in NSC circuits. Consequently, some of the strategies to induce NSC differentiation were based on the targeted modulation of these signals. Experimental evidence has highlighted the importance of the regulatory feedback loop between micro RNA (miR) and transcription factors, which can differentially influence NSC behavior [75]. miR upregulation via miR-9 transfection [76] or miR-195 downregulation via MBD1-expressing

Fig. 3. NSC differentiation for brain repair. Strategies developed to achieve brain repair with the use of exogenous or endogenous NSC (in green). A) Exogenous NSC can be obtained by the induction of other somatic cells (e.g., fibroblasts) or by isolation from neuronal niches (e.g., the SVZ and SGZ). Exogenous NSC are cultivated in vitro for in vivo transplantation and their differentiation can be designed to occur during cell cultivation or after cell transplantation. B) Endogenous NSC are localized to the neuronal niches and their differentiation can occur exclusively in situ. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

lentivirus [77] can increase embryonic mouse NSC differentiation into neurons and astrocytes. The signals from cell-cell contact were also identified as modulators of NSC differentiation: co-cultures of NSC and protoplasmic astrocytes or amniotic cells promotes embryonic rat NSC differentiation into neurons [78]. The alteration of epigenetic marks is an important NSC lineage modulator as well. The overexpression of activator-type bHLH genes such as Mash1, neurogenin2, and NeuroD promotes neuronal-specific gene expression while it inhibits glia-specific gene expression [70]. Deciphering cell circuits and their regulatory signal network provides crucial information for NSC differentiation strategies [79]. In addition to endogenous modulators, pharmacological agents were found to impact NSC differentiation (Table 2). Incubations with 4-aminothiazoles (e.g., neuropathiazol) or oleanoic acid led to

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Table 2 NSC-differentiation in vitro, in vivo and in situ. Molecule/Condition

Type of cells/area

Study design

Outcomes

Reference Year

neuropathiazol

in vitro

þ neurons

[80] 2006

ELFEFs NSC co-culture with astrocytes or amniotic cells miR-9 transfection

primary neural progenitor cells isolated from adult rat hippocampus NSC isolated from the brain cortex of nb mice NSC isolated from embryonic rats

in vitro in vitro

þ neurons þ neurons

[89] 2008 [78] 2012

NSC isolated from embryonic mice

in vitro

[76] 2013

MBD1-expressing lentivirus

NSC isolated from embryonic mice

in vitro

BDNF

NSC isolated from the forebrain cortex of nb mice

in vitro

oleanolic acid valproic acid

NSC isolated from the embryonic mouse striatum NSC isolated from embryonic rat forebrains

in vitro in vitro

þ þ þ þ þ þ þ þ

all-trans-retinoic acid

NSC isolated from embryonic rat forebrains

in vitro

þ neurons

1,25-Dihydroxyvitamin D3 opioid peptides NFL

NSC isolated from adult mouse brain NSC isolated from embryonic rat striatum NSC isolated from nb rat SVZ

in vitro in vitro in vitro

NSC transplantation

human NSC

in vivo

growth factor-overexpressing NSC transplantation morphine D-amphetamine ELFEFs retroviral Zfp488 perfluorooctane sulfonate

NSC isolated fromtransgenic nb mouse hippocampus

in vivo

adult mouse hippocampus adult mouse hippocampus adult mouse hippocampus adult mouse corpus callosum cortical tissues of neonatal mice

in in in in in

situ situ situ situ situ

PCDHIIx shRNA/siRNA simvastatin ketamine nicotine long noncoding RNA Pnky oxygen supply modulation

lateral ventricle of mice injured areas of adult rat brain nb rat SVZ adult rat hippocampus embryonic and postnatal mouse brain developing cerebral cortex of mice

in in in in in in

situ situ situ situ situ situ

þ oligodendrocytes þ neurons þ neurons þ oligodendrocytes prevention of further cognitive deterioration AD deficit recovery, synaptic density increment þ astrocytes þ neurons þ neurons motor function restoration þ neurons þ oligodendrocytes þ neurons enhancement of neurological functions þ neurons þ neurons þ neurons þ radial glia

neurons astrocytes neurons astrocytes neurons oligodendrocytes neurons neurons

[77] 2013 [90] 2013 [81] 2015 [84] 2015; [82] 2008 [84] 2015; [83] 1998 [87] 2015 [88] 2015 [91] 2016 [99] 2015 [100] 2016 [86] [87] [97] [95] [94]

2015 2013 2010 2011 2013

[101] 2014 [93] 2015 [102] 2015 [103] 2015 [104] 2015 [96] 2016

ELFEFs, extremely low-frequency electromagnetic fields; Exo, exogenous; endo, endogenous; nb, new-born; SVZ, subventricular zone.

TuJ1 (a neural marker) expression in 90% of positive cells in treated primary neural progenitor cells isolated from adult rat hippocampus and NSC isolated from the embryonic striatum of mice respectively [80,81]. Valproic acid [82] and all-trans-retinoic acid [83] stimulate NSC differentiation by the inhibition of histone deacetylase and the transcriptional activation of NeuroD, respectively. Initially, the differentiation efficiency was unsatisfactory but the recent combination of valproic acid and all-trans-retinoic acids significantly increased the percentage of MAP-2 (neural marker) positive cells in vitro [84]. The impact of addictive drugs on NSC differentiation and neurogenesis was also demonstrated [85]. Morphine promotes astrocyte differentiation [86] while Damphetamine [87] and opioid peptides [88] increase neuron differentiation in vitro and in situ, in the hippocampus of adult mice. NSC differentiation can also be stimulated by extremely lowfrequency electromagnetic fields (ELFEFs) which affect several biological parameters such as the intracellular calcium level. ELFEFs upregulate the Ca2þ channels and increase the Ca2þ influx which induce the signaling cascade associated with the promoter of specific bHLH that control NSC differentiation [26,27]. NSC isolated from the brain cortex of new-born mice and exposed to ELFEFs showed more neuronal marker positive cells (þ11.8% of MAP-2 and þ11.9% of beta III tubulin) compared to the controls [89]. 3.4. In vivo/in situ stimulation of NSC differentiation Recent evidence suggests that a combination of extracellular signals and niche environmental conditions, hardly reproducible

in vitro, affect NSC behavior in the organism [92]. Consequently, most of the strategies which provided promising results in vitro did not fully translated in vivo/in situ (or vice versa). One solution is to directly apply therapies in vivo, at the desired site of action, to target endogenous NSC. Indeed, strategies aiming at in situ differentiation of endogenous NSC would bypass issues related to exogenous NSC transplantation, such as challenging supply of exogenous NSC (ethical concerns), immune response against allogeneic transplantation and post-grafting cell viability (Fig. 4). Consequently, the administration of active compounds is the most common approach to enhance neurological function in neurodegenerative disease animal models (Table 2). Simvastatin increased the percentage of MAP-2 (neural marker) and GFAP (astrocytic marker) positive cells in a rat traumatic brain injury model [93]. Its administration enhanced neurological functions such as sensory functions, motor functions, beam balance performance, and reflexes. Perfluorooctane sulfonate induced neural and oligodendrocytic NSC differentiation in healthy mice, probably via PPARg nuclear receptor activation, a pathway also involved in the retinoid-induced cascade for NSC differentiation [94]. The corpus callosum in mice exhibiting cuprizone-induced demyelination showed a significant increase in Olig2 (oligodendrocytic marker) positive cells after treatment with retroviral Zfp488 [95]. The overexpression of Zfp488 protein (activator of oligodendrocyte differentiation) induced a significant motor function restoration in demyelination-injured mice. Another approach to induce NSC differentiation would be the oxygen supply modulation at the neurogenic niches. It was recently

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Fig. 4. Major challenges associated with NSC-based therapies for CNS repair.

showed by Lange et al. that NSC differentiation coincided with recovery from hypoxia in the developing cerebral cortex by ingrowth of blood vessels [96]. They demonstrated that selective perturbation of brain angiogenesis in embryos increased NSC expansion by preventing the relief from hypoxia while exposure to increased oxygen levels stimulated NSC differentiation. The in vivo application of ELFEFs promoted proliferation and differentiation of hippocampal NSC in C57bl/6 mice [97]. NSC differentiated into neurons, which were functionally integrated in the dentate gyrus network 30 days after the ELFEF treatment. Spatial learning and memory were enhanced, highlighting the important therapeutic implications of ELFEFs for the treatment of neurodegenerative diseases. Moreover, it was demonstrated that ELFEFs increased the survival of hippocampal newborn cells [98]. Altogether, there is concurrent information demonstrating that the in situ stimulation of NSC differentiation is a valid approach to enhance neurological functions during neurodegenerative diseases. The therapeutic effect achieved in neurologically compromised animal models (e.g., restoration of cognitive functions), together with the stimulation of the neurogenic process and neuroprotection in healthy animal models via NSC differentiation, provide precious insight for the clinical translation of NSC-based therapeutic strategy. 3.5. Clinical trials Depending on the results obtained during pre-clinical studies, NSC differentiation-based therapies have been recently translated into the clinic [105]. Thirty-seven NSC-based clinical trials are currently on going, involving patients affected by gliomas, ischemic stroke (IS), amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI) and Parkinson’s disease (PD) [106]. Surprisingly, most of these therapies aimed at in vivo NSC differentiation of exogenous grafted NSC (Table 3), while most of the pre-clinical studies focused on differentiation of endogenous NSC (Table 2). In the clinical trial NCT02117635, CTX cells (a human neural stem cell line) were injected by stereotaxy in the striatum of IS patients (site of lesion) (Phase I). The treatment promoted a partial recovery of neurologic

functions [107] but no anatomical modifications. This would suggest that NSC do not directly differentiate into neurons but rather act as cellular mediators by secreting paracrine factors. The aim of the clinical trial NCT01640067 (Phase I) was to assess the safety of NSC and their efficacy. The transplantation of foetal NSC in the spinal cord of ALS patients stopped the progression of the disease for up to 18 months and did not cause side effect [108]. No mechanistic study was performed in vivo to explain this result, but the preservation of NSC multipotency was demonstrated in vitro after the recovery of remaining NSC in the syringe used for the injection, and culture of transplanted NSC. Transplantation of genetically modified NSC has also been used for the treatment of gliomas and is being evaluated in clinical trials [109]. The strategies consisted by using genetically modified NSC as vehicles to target tumor cells without harming healthy brain tissue. Very promising pre-clinical studies showed that NSC-based oncolytic virus delivery [110] and iPSC-derived NSC engineered with therapeutic/diagnostic transgenes [111] were able to suppress tumor growth and to significantly extend the survival of glioblastoma-bearing mice. In another study (NCT01172964), E. Coli cytosine deaminase-expressing NSC were co-injected with 5fluorocytosine (5-FU) intra-cerebrally. The objective was to facilitate the conversion of 5-FU into its active form (fluorouracil) directly in the tumor. Another approach was to perform an intracranial injection at the tumor site of carboxylesterase-expressing NSC to increase glioblastoma cell sensitivity to irinotecan hydrochloride, an anti-cancer drug (Camptosar) (NCT02192359). Unfortunately, more detailed information regarding the results and efficiency of these clinical trials are not available. It seems that none of the described clinical treatments caused severe adverse events. To the extent of our knowledge, no NSC-based therapy aiming at treating neurodegenerative diseases reached phase III yet. The limited clinical translation could be due to the fact that the current dominant approach is based on exogenous NSC transplantation, which is often associated with important issues (see 3.6 Challenges). Focusing on strategies based on in situ stimulation of endogenous NSC differentiation could provide promising alternatives that might be easier to translate into therapy for the treatment

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Table 3 NSC-based clinical trials. Targeted disease

Type of cells

Approach

Identification

ALS SCI SCI IS PD ALS gliomas gliomas

exo NSC exo NSC exo NSC exo NSC exo NSC endo NSC gen. mod. NSC gen. mod. NSC

transplantation transplantation transplantation transplantation transplantation in situ stimulation transplantation transplantation

NCT01640067 NCT02326662 NCT01772810 NCT02117635 NCT02452723 NCT00397423 NCT01172964 NCT02192359

Phase I (concluded in 2015) I/II (ongoing 2016) I (ongoing 2016) II (recruiting 2016) I (recruiting 2016) II (completed 2007) I (completed 2015) I (recruiting 2016)

ALS, amyotrophic lateral sclerosis; SCI, spinal cord injury; IS, ischemic stroke; PD, Parkinson’s disease; exo, exogenous; endo, endogenous; gen. mod., genetically modified. Resource: https://clinicaltrials.gov.

of neurodegenerative diseases. 3.6. Challenges Despite important and encouraging progress, the intrinsic complexity of the CNS still precludes the potential of many NSCbased therapeutic approaches. The structural fragility of the CNS limits invasive approaches whereas the stage, the area, and the type of the pathology strongly influence the impact and the effect of the treatments [112,113]. One limiting factor is the lack of correlation between in vitro and in vivo NSC behavior. The ability of NSC to differentiate into specialized lineages depends on their microenvironment. Understanding the chemical and physical signals as well as the cell-cell interactions represents the most important challenge to dynamically modulate NSC differentiation in vivo [92]. Another challenge is the control of the biological activity and behavior of these cells following their transplantation or stimulation. In many clinical trials involving NSC, little is known about the mechanisms, the location, and the extent of the modulation of neurogenesis. Additional problems are related to NSC in vitro cultivation, such as strict chemically defined conditions (xeno-free) and the risk of adaptive genetic changes [114]. Moreover, in vivo transplantation is often associated with cell survival, graft rejection and cell source issues, which complicate the design of exogenous NSC-based therapies [115]. The current inability of medical science and fundamental research to provide information and solutions to these problems represents a significant risk for the patients, and thus impairs the clinical translation of NSC research. At the same time, under the pressure of the public and the media, the population has overestimated expectations about the ability of NSC transplantation to cure neurodegenerative diseases. Pressure on governments and regulatory authorities has already exposed patients to severe risks by the implementation of clinical trials with incomplete scientific knowledge, e.g., in 2013 with the “Caso Stamina” [116]. Cattaneo and Bonfanti highlighted the importance of a constructive dialogue between science and society, which should bypass the media [117]. The major challenges of NSC-based therapies are reviewed in Fig. 4. 4. Nanomedicine to modulate NSC differentiation Nanotechnology is the development and the application of materials and systems that possess peculiar physicochemical properties such as dimensions in the range between 1 and 100 nm. Nanomedicine is the medical application of nanotechnology [118]. Its main objective is the improvement of conventional therapies by providing new skills and/or overcoming the limitations associated with conventional pharmaceutical forms. In the last decades, nanomedicine-based approaches showed promising preclinical results by improving the efficiency of several therapies such as anticancer treatments (e.g., doxorubicin-loaded liposomes showed

reduced risk of acute and cumulative cardiotoxicity compared to the free molecule [119]) [120]. The toxicity of nanomedicines is a crucial topic, especially to obtain approval by the regulatory authorities (e.g., Food and Drug Administration or European Medicine Agency). No standard protocols to evaluate the general safety of these therapeutics has been proposed, making the comparison between different studies challenging [121]. However, it has been clearly established that size, shape and composition of nanomedicines play an important role on the safety of human health by directly impacting their biological reactivity and accumulation/ clearance in the body [122]. The reduction of the size and the increase of the surface area can induce an inflammatory response and genotoxicity for a same mass dose of nanomedicine [123]. Indeed, one of the critical point is the potential activation of the immune system. It could nullify the expected therapeutic effect of nanomedicines (e.g., by macrophage sequestration) or induce acute immunotoxicity (e.g., anaphylactic and hypersensitivity reactions) [124]. Nevertheless, strategies can be used to limit the negative impact of nanoparticles on the immune system by modifying their size, by using less-immunogenic materials and by modifying their surface. For instance, the PEGylation of nanoparticle surface is widely used to reduce opsonisation and thus to “hide” nanoparticles from the immune system recognition [125]. To the extent of our knowledge, no mention of toxicity has been related in the studies reported in this review (Table 4). 4.1. Therapeutic contribution The modulation of NSC differentiation by conventional medicine exhibits important limitations, and nanomedicines have emerged to overcome some limitations (Fig. 5) [126]. The limitations include: (i) Poor correlations between in vitro and in vivo NSC behavior. Nanostructured scaffolds are promising candidates to mimic the in vivo extracellular conditions; the selection of appropriate nanoscale material and architecture can ensure the differentiation of post-transplanted NSC. Brain injuries can induce cell migration from the neurogenic regions to the affected areas of the brain but the percentage of replaced cells is very low. After a stroke for instance, SVZ-derived cells can migrate into the injured striatum and differentiate into the damaged cell phenotype (striatal medium-sized spiny neurons, DARP-32 positive) but only 0.2% of injured neurons are functionally replaced [127,128]. It has been documented that the lack of signals, present during the development, and the expression of inhibitory molecules, during neurodegenerative diseases, hinders axon regeneration and projection [129]. Moreover, the distance between the neurogenic niches and the injured areas of the brain (e.g., the striatum of PD patients) is a limiting factor in the recovery process mediated by adult neurogenesis. Consequently, the effectiveness of NSC differentiationbased strategies could be compromised by the difficulty to support NSC-differentiated cell migration and integration within the

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Table 4 Nanomedicine-based approaches for the modulation of NSC differentiation. Strategy

System

Study design

Outcomes

Reference

nanostructured scaffold

DNA-peptide nanotubes carbon nanotubes graphene nanofibers PLA/gelatin nanofibers rolled graphene oxide foams and electric stimuli patterned porous silicon photonic crystals self-assembling peptide -PCLePLGA nanofibers

in in in in in in in

[173] [145] [174] [146] [175] [176] [147]

self-assembling peptide nanofibers salmon fibrin fibers

in vivo implantation in vivo implantation

NSC differentiation in neurons actuation of NSC differentiation NSC differentiation in oligodendrocytes iPSC differentiation in neural-like cells NSC proliferation and differentiation NSC differentiation NSC proliferation and differentiation in neurons and oligodendrocytes robust survival and neurite outgrowth NSC proliferation and differentiation, vessel growth NSC recognition in co-culture increased neurofilamnet expression

[168] 2011 [161] 2016

increased number of MAP-2 positive cells

[153] 2016

increased number of MAP-2 positive cells

[153] 2016 [163] 2016 [151] 2016; [150] 2015; [149] 2012 [169] 2016 [162] 2016

[165] 2015

nanoparticulate drug delivery system

nanotechnology-based real-time imaging

mixed

vitro vivo vitro vitro vitro vitro vivo implantation

nilo1-titanium dioxide nanoparticles in vitro neurogenin2-loaded biodegradable in vitro nanoparticles polymeric nanoparticle-based nanogel loaded in in vitro retinoic acid polymeric nanoparticle-based bloc micelle in vitro system loaded in retinoic acid DNA microcircle magnetic nanoparticles in vitro retinoic-acid loaded polymeric nanoparticles in vivo (intracranial)

2014 2014 2014 2016 2016 2016 2016

[177] 2016 [178] 2016

NFL-lipid nanocapsules miR-124-nanoparticles

in vivo (intracranial) in vivo (intracerebral)

curcumin-loaded nanoparticles

in vivo (intraperitoneal and intracranial) in vivo

sustained gene expression NSC differentiation, neuroprotection, AD deficit recovery targeting of SVZ-NSC neurogenic niche modulation, PD deficit recovery NSC differentiation in neurons, AD deficit recovery NSC differentiation imaging

in vivo

NSC differentiation imaging

[164] 2016

in vivo

Tracking of NSC behavior after neural transplantation In vivo T1 magnetic resonance imaging of transplanted NSC In vivo magnetic resonance imaging of iPSC-derived NSC NSC differentiation monitoring

[166] 2016

NSC differentiation in neurons NSC differentiation in neurons attenuation of neuronal loss and rescues memory deficiencies

[172] 2013 [114] 2015 [115] 2016

bicistronic vector TUPIS functionalized nanovehicle deoxythymidine oligonucleotides Gd(III)/Cy3 functionalized gold nanoparticles USPIO/MIRB nanoparticles DNAegadoliniumegold nanoparticles

in vivo

poly-L-lysine-gFe2O3 coated nanoparticles

in vivo

3D graphene oxide-encapsulated gold nanoparticles nanotopographical siRNA delivery gold nanoparticle-decorated scaffold retinoic-acid- and siRNA-loaded SPION nanoparticles

in vitro in vitro in vitro in vivo

[152] 2013

[179] 2016 [167] 2016 [180] 2013

PLA, polylactic acid; PCL, polycaprolactone; PLGA, poly(lactic-co-glycolic acid); USPIO, ultrasmall superparamagnetic iron oxide; MIRB, Molday ION Rhodamine B; SPION, Superparamagnetic iron oxide nanoparticles.

Fig. 5. Nanomedicine-based strategies for NSC differentiation. A) NSC differentiation induced by cell incorporation in a polymeric scaffold mimicking the natural environment of NSC. B) A nanoparticulate drug delivery system able to improve the efficacy of the drug and/or enrich the system with supplementary properties such as NSC-targeting features. C) A nanotechnology-based real-time imaging method to trace post-transplanted NSC. The combination of some or all of these approaches is also possible.

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existent cellular network. Some methods have been developed to guide the migration of transplanted neurons and support their connection using drug-loaded hydrogels (e.g. semaphoring Cloaded hydrogel [130]) or co-culture of neural and non-neural cells (e.g., neurotrophin-3 gene-modified Schwann cells and TrkC genemodified NSC in gelatin sponge scaffolds [131]). On the one hand, nanostructured scaffold might increase the viability of replaced cells by providing a more favourable microenvironment and boost neurogenesis in non-neurogenetic regions [86], such as the striatum of PD patients. On the other hand, nanostructured scaffold could be associated to active drugs (e.g. chemotrophic proteins [132]) which can guide and maintain the migration and integration of NSC-differentiated cells via sustained drug release. (ii) Unfavorable physicochemical profile of the drugs. The galenic formulation and the therapeutic effect of the molecules can be limited either by their low solubility or high environmental sensitivity (e.g., to the pH, the temperature, and enzymatic proteolysis). Indeed, some of the molecules used to stimulate NSC differentiation listed in Table 2 can be poorly soluble in water (vitamins) or highly sensitive (proteins). The nanoscale size reduction of low soluble drugs (e.g., simvastatin [133]) improves the dissolution rate of the molecules in aqueous media, facilitating their administration. The nanoencapsulation protects growth factors from the environment [134] and increases their levels in the CNS, preserving their activity [135]. (iii) Poor drug bioavailability. Another critical factor is the capacity of the molecule to reach the therapeutic dose in the required timeframe. The control of drug release contributes to bioavailability. The release of molecules can be controlled by different nanoparticulate systems (e.g., nanospheres or nanocapsules), the structure of systems (e.g., monolayer or multilayers), and the composition of systems (e.g., chitosan or hyaluronic acid-based polymers) [136]. The association of these drugs with nanoparticulate systems led to the increased half-life and bioavailability of drugs compared to the free forms of medications (e.g., for retinoic acid [137]). Furthermore, the nanoscale size can enhance the cellular uptake of the drug [138]. (iv) Limited knowledge about the mechanisms of NSC differentiation. Nanotechnology-based realtime imaging can involve noninvasive tools, which allow for the monitoring of NSC differentiation dynamics after in vivo transplantation. The real-time traceability of NSC offers spatial and temporal information of the processes involved in differentiation, as well as the interactions between exogenous NSC and endogenous cells. (v) Transplantation-associated issues. The incidence of tumors is one of the most important concerns in NSC transplantation. Tumor development has been rarely reported in the majority of the described stem cell-transplantation-based clinical trials [139] but it is not unheard of. One article reports the formation of tumors in the spinal cord and brain of a 14 years old boy with ataxia telangiectasia following NSC injection [140]. The reason proposed by the authors is that the donor-derived cells might have been able to establish tumors because patients with this kind of affection often have an impaired immune system. This example of a donor-derived brain tumor developing after fetal neural cell transplantation is worrying and suggests that further work should be done to assess the safety of this therapy. However, exogenous NSC-based therapeutic approaches still show many limitations [114,115]. The development of systems able to specifically stimulate endogenous NSC differentiation is a promising solution to overcome the transplantation-associated issues [141]. Until now, the presence of the blood-brain barrier (BBB) and the lack of NSCtargeting compounds limited the development of conventional in situ NSC differentiation strategies. The surface functionalization of nanoparticles [142] with NSC-targeting molecules, such as NFL [91], and BBB-crossing compounds, such as OX26 [143] and lipoproteins [144], could allow selective drug delivery systems to reach

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endogenous NSC and induce their differentiation. 4.2. Nanostructured scaffolds Nanostructured scaffolds have been developed for in vivo transplantation of NSC. The first carbon-nanotube structured PLGA matrix made by Landers et al. induced the differentiation of iPSCderived NSC into neuronal cells after electric stimuli in vitro [145]. This nanostructured scaffold is a promising candidate to improve cell survival and functional integration in patients with neurodegenerative diseases who are receiving NSC transplantation (e.g., PD). Recently, Hoveizi et al. produced PLA/gelatin nanofibers seeded with iPSC-derived NSC to investigate the influence of the nanostructured scaffold on NSC differentiation [146]. The authors demonstrated that iPSC-derived NSC were able to attach, proliferate, and differentiate on the PLA/gelatin fibers and that the system was a potential cell carrier for transplantation. In another work, Raspa et al. used self-assembling peptides (Ac-FAQ) in association with poly(ε-caprolactone)poly(D,L-lactide-co-glycolide) (PCLePLGA) to produce electrospun fibers [147]. The nanofibrous systems were highly biocompatible in vivo when implanted in rats and they promoted NSC differentiation in vitro after NSC were seeded onto flat electrospun covered coverslips. The survival of the transplanted cells, their ability to differentiate and to integrate within the existent cellular network are some of the most important challenges associated to NSC transplantation-based therapies. Nanostructured scaffolds have demonstrated to be a potential tool to overcome these limits by increasing cellular viability/proliferation and by ensuring NSC differentiation/integration. The reported studies showed that nanostructured scaffold can reproduce the in vivo microenvironmental conditions either of the niches or of the area receiving the transplant. Thus, they can positively contribute to the development of NSC transplantation-based strategies and enhance the therapeutic benefits of these approaches. 4.3. Nanoparticulate drug delivery systems Bernardino and Ferreira were the first to produce retinoic acidloaded nanoparticles [148,149]. Pro-neurogenic gene expression was increased after the intracranial injection of nanoparticles into the mouse SVZ due to the activation of nuclear retinoic acid receptors. Recently, a neuroprotective effect and an enhanced vascular regulation induced by their formulation was reported in PD [150] and IS [151] mouse models, respectively. Curcumin-loaded nanoparticles modulate NSC differentiation and are associated with the recovery of functional deficits in an AD rat model [152]. The administration of these nanoparticles via intraperitoneal injection increased the expression of genes involved in neuronal differentiation (neurogenin, neuroD1, etc.) and reversed learning and memory impairments probably via the activation of the Wnt/bcatenin pathway. Papadimitriou et al. developed two different types of polymeric nanoparticles, crosslinked to form a nanogel or self-assembled to form a block micelle system, which were loaded with retinoic acid and tested in vitro on NSC of the SVZ of mice [153]. They demonstrated that both the nanogel and the block micelle system reached the cytoplasm and ensured a higher bioavailability of the retinoic acid, which increased the NSC differentiation in MAP-2 (neural marker) positive cells. Fe3O4 magnetic nanoparticles in association with ELFEFs enhanced neuronal [154] and osteogenic [155] differentiation of bone marrowderived mesenchymal stem cells. Since ELFEFs already showed the efficacy of inducing neural differentiation of NSC, its association to magnetic nanoparticle would have the potential to enhance the impact on NSC differentiation. Genome editing in iPSC via

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nanoparticle-based drug delivery systems has been developed as a promising reprogramming strategy for personalized medicine [156,157]. Direct delivery of mRNA or microRNA into human iPSC have provided human models for specific disease phenotypes, including neurodegenerative diseases, which are useful to design the most appropriate therapy by understanding their mechanisms and pathogenesis [158]. Moreover, genome editing of iPSC-derived NSC using nanomedicines would supply an unlimited source of any human cell type, avoiding the cross-species issues of animalderived models, and most of the ethical concerns related to stem cells (e.g., the utilization of human embryos) [159]. Direct delivery of nucleic acids to the CNS increased neuron regeneration and/or slow the progression of neurological impairments. Although transfection methods mediated by viral vectors are efficiently applied to induce NSC differentiation (as shown in part 3 [76] [77] [95]), from a clinical point of view, non-viral vectors are preferred [160]. Nanomedicine provides non-viral vector, such as nanoparticlebased systems, which are suitable for cell reprogramming. Li et al. induced mature neuron differentiation via a biodegradable nanoparticle-mediated transfection method [161]. They delivered neurogenin 2 (bHLH transcription factor) to transplanted human fetal tissue-derived NSC in the lesion site of a rat brain and generated a significantly larger number of neurofilament (neural marker) positive cells. Saravia et al. reported for the first time the ability of a nanoparticle-based formulation to deliver miR-124 and to modulate the endogenous neurogenic niche in PD animal model [162]. They demonstrated not only neurogenesis at the SVZolfactory bulb axis but also the migration and maturation of new neurons into the lesioned striatum and the enhancement of the motor functions in PD-like mice. Fernandez and Chari recently achieved the highest transfection level (54%) reported so far on NSC [163]. They demonstrated that the association between DNA microcircles, which are small DNA vectors without a bacterial backbone, and magnetic nanoparticles resulted in a sustained gene expression for 4 weeks. These results are really promising and bide well for a clinical translation of their system. Nanoparticles are able to increase the cellular uptake of drugs with unfavorable physicochemical profile and to preserve the biological activity of fragile molecules. All these studies reported that NSC differentiation was more impacted by nanoparticle-associated drugs than by their free form. This effect is probably due to an increased bioavailability of the active molecules. Consequently, nanoparticulate drug delivery systems had higher therapeutic effects than the drug in solution when administered in neurodegenerative disease animal models. Moreover, nanoparticles have been recently used as non-viral vectors for cell reprogramming. This strategy offers several advantages over viral-based carriers such as lower immunogenicity and ability to carry multidrug cargos. The NSC-differentiated neuronal cells generated by this methodology were shown to perform similarly to NSC-differentiate neuronal cells generated by other technics. 4.4. Nanotechnology-based real-time imaging Recently, gold nanoparticles with deoxythymidine oligonucleotides Gd(III) and Cy3 have been shown to be useful tools for MRI imaging of transplanted NSC [164]. A majority of transplanted NSC (71%) was detectable in the brain over 2 weeks posttransplantation. In another work, the differentiation peak time (12 days post-transplantation) and the migration/apoptosis phases of the transplanted NSC were identified [165]. The authors developed a polymeric nanovehicle that induced NSC differentiation with retinoic acid and was detectable in real-time imaging due to a bicistronic vector TUPIS [165]. Umashankar et al. proposed a live-

imaging method to monitor superparamagnetic iron oxide/Molday ION Rhodamine B (USPIO/MIRB)-labelled NSC after transplantation [166]. NSC were incubated with USPIO/MIRB nanoparticles and then identified by dual magnetic resonance and optical imaging. Although USPIO/MIRB may have advantageous labelling and detection features for NSC tracking, the immunoresponse produced in vivo needs further examinations before their
et al. demonstrated that poly-Lutilization in the clinic. Jir
akova lysine-gFe2O3 coated nanoparticles are a potential tool for the detection and monitoring of transplanted iPSC-derived NSC [167]. Contrarily to cobalt zinc ferrite coated nanoparticles, poly-L-lysinegFe2O3 coated nanoparticles did not affect cell proliferation and differentiation. By making NSC detectable by magnetic resonance without affecting the cellular behavior, they provide a suitable noninvasive tool for cell tracking in NSC-based therapies. The duration, the localization and the extend of NSC proliferation/differentiation are important parameters to consider when designing a NSC-based therapy. Real-time imaging can provide this information by the support of nanotechnology. The technics described could allow to select the most effective posology of the bioactive molecule (e.g., when it induces the highest percentage of NSC-differentiated neurons) or to identify the most appropriate area of transplantation (e.g., the zone where transplanted-NSC survive longer). Consequently, nanotechnology-based real-time imaging tools are useful to investigate the fate of transplanted-NSC in vivo/in situ or to improve the efficacy of NSC-based therapies to treat neurodegenerative diseases. 4.5. Nanomedicine for in situ NSC differentiation In situ NSC differentiation is considered one of the most promising strategies for the treatment of neurodegenerative diseases (Fig. 6). Nevertheless, no work based on this approach has yet reached the clinical phase. The lack of NSC-targeting molecules primarily promotes the development of non-selective systems. Only a few nanoparticulate systems have been designed to target endogenous NSC and to deliver active molecules directly to the neurogenic niches. Titanium dioxide nanoparticles coupled to Nilo1 interacted with NSC in vitro [168] but no further information is available on the in vivo efficacies of such a system. More recently, Carradori et al. developed NFL-functionalized lipid nanocapsules that selectively target SVZ-NSC in vitro and, after intracranial injection, in vivo [169]. The versatility of the lipid nanocapsules [170] together with the NSC-targeting property make this system a promising tool for therapeutic applications. For several nanoparticulate drug delivery systems, the in vivo proof-of-principle is lacking but significant results have been achieved in vitro [171,172]. The development of systems able to target endogenous NSC represents a promising strategy to overcome transplantationassociated issues. The risk of death or rejection of transplanted NSC would be totally excluded. Also, the procedural limitations derived from in vitro manipulation of NSC before their transplantation (e.g., cultivation in restricted conditions or genetic modifications, see 3.5 Challenges) would be avoided. Moreover, endogenous NSC targeting would increase the efficacy of the treatments by enhancing the drug bioavailability and, consequently, limiting its counter effects. Considering that people affected by neurodegenerative diseases are often physically debilitated, the development of less invasive strategies, such as in situ NSC differentiation via targeting nanomedicines, could be a successful approach. 5. Conclusion The discovery of adult neurogenesis has had a significant impact

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Fig. 6. In situ NSC differentiation. A) The nanoparticulate drug delivery system (e.g., NFL-LNC [169] loaded in bioactive molecules) can selectively target NSC (e.g., SVZ-NSC) and induce their in situ differentiation (e.g., via retinoic acid stimulation). B) The nanoparticulate drug delivery system selectively reaches SVZ-NSC and delivers the active molecule. C) The active molecule induces NSC differentiation and affected areas are restored by cellular repair/replacement.

on CNS regenerative medicine. The regulation of neurogenesis has gained therapeutic importance, and the role played by NSC in the modulation of neurogenesis was quickly apparent, even if the biological mechanisms behind the modulation are not well understood. NSC differentiation, and consequently neurogenesis, are reported to have therapeutic effects in the treatment of several neurodegenerative diseases. Many methods have been developed to induce NSC differentiation in vitro/in vivo/in situ and some of them have been included in clinical trials. Despite the rapid clinical translation, several issues are still challenging with this therapeutic approach. The intrinsic complexity of the CNS, the lack of in vitroin vivo correlation, transplantation-associated issues, and the biological activity control remain difficult obstacles to overcome. In our opinion, nanomedicine represents one of the most promising strategies to overcome such obstacles. Nanotechnology offers many tools to improve the efficacy of the conventional NSC differentiation-based treatments, as well as the comprehension of the biological mechanisms behind NSC differentiation. The design of NSC-targeting systems could be one of the most promising and safe strategies in CNS regenerative medicine. Acknowledgments We greatly acknowledge our financial supports. Dario Carradori is supported by NanoFar “European Doctorate in Nanomedicine” EMJD program funded by EACEA. This work is also supported by AFM (Association Française contre les Myopathies), by ARC (Asso
gion des Paysciation de Recherche sur le Cancer), by CIMATH (Re de-la-Loire), and by MATWIN (Maturation & Accelerating Translation With Industry). This work is supported by grants from the
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