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

(1)

Review

The therapeutic contribution of nanomedicine to treat

neurodegenerative diseases via neural stem cell differentiation

Dario Carradori

^a^,^c

, Joel Eyer

^c

, Patrick Saulnier

^c

, V eronique Pr eat

^a

, Anne des Rieux

^a^,^b^,^*

aUniversite Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Avenue E. Mounier 73, 1200 Brussels, Belgium

bUniversite Catholique de Louvain, Institute of Materials and Condensed Matter, 1348 Louvain-la-Neuve, Belgium

cUniversite d’Angers, Unite Micro et Nanomedecines Biomimetiques, MINT, Institut de Biologie en Sante PBH-IRIS, 49033 Angers, France

a r t i c l e i n f o

Article history:

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

Nanomedicine Nanoparticles Nanotechnology

Neural stem cell differentiation Neurodegenerative disease Neurogenesis

a b s t r a c t

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 functionsvianeural 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 neu- rogenesisvianeural stem cell differentiation.

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 theﬁrst 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

process. The improvement of immunohistological techniques rep- resented 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 cellsin vitro provided crucial data on the cellular and biomolecular mechanisms involved in adult neurogenesis[10e12].

In humans, the evaluation of neurogenesis was initially per- formed by the quantification of the number of cells expressing neuroblast markers such as doublecortin (DCX) or polysialylated- neural 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 Abbreviations:CNS, central nervous system; NSC, neural stem cells; SVZ, sub-

ventricular zone; SGZ, subgranular zone.

*Corresponding author. Universite Catholique de Louvain, Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Avenue E. Mounier 73, 1200 Brussels, Belgium.

E-mail address:anne.desrieux@uclouvain.be(A. des Rieux).

Contents lists available atScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o m a t e r i a l s

(2)

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 retrospectivein vitrostudies[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 differentiationin vitroandin 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-hydroxylase- expressing cells[30]. These two markers are associated with two different cell types: long-axon and dopaminergic neurons,

A

B C

D

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 thisﬁgure legend, the reader is referred to the web version of this article.)

(3)

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 nomen- clature 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 non- differentiated 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 IIIbtubulin (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 polysialylated- neural 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 neural- speciﬁc 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 non- neural 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 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) andfinally 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 penetrateviaspecific 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 Ca^þþchannels, 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 thisfigure legend, the reader is referred to the web version of this article.)

(4)

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 NSCin vitroafter 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 differentiationin vivoorin situhas not yet been established. Brain repairviaNSC 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 differentiationin 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 basicﬁbroblast 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

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 Table 1

Markers for adult neurogenesis.

Type of cells Markers Reference

Neural stem cells/Progenitors Nestin [49]

SRY-related HMG-box gene (Sox2) [62]

Musashi-1 (Msi1) [50]

Paired box gene 6 (Pax6) [63]

Prominin (CD133) [64]

Glialﬁbrillary acid protein (GFAP) [59]

NG2-glia positive cells Chondroitin sulfate proteoglycan neuron/glia antigen 2 (NG2) [51]

Neural lineage (early) IIIbtubulin (TUJ1) [54]

Doublecortin (DCX, C-18) [55]

Polysialylated-neural cell adhesion molecule (PSA-NCAM) [56]

Neurogenetic Differentiation (NeuroD) [56]

Neural lineage (mature) Neuronal nuclear epitope (NeuN) [58]

Microtubule-associated protein 2 (MAP2) [57]

Neuron-speciﬁc enolase (NSE) [57]

Calbindin [65]

Thyrosine-hydroxylase (TH) [66]

Calretinin [65]

Neuroﬁlaments (NF) [65]

Astrocytic lineage Glialﬁbrillary acid protein (GFAP) [59]

Calcium binding proteins (S100/S100b) [60]

Glutammate-aspartate transporter (EAAT1) [67]

Glial lineage Galactocerebroside (GalC) [68]

2⁰,3⁰-cyclic-nucleotide 3⁰-phosphodiesterase (CNP) [61]

Myelin basic protein (MBP) [61]

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

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 cultivatedin vitrofor in vivotransplantation 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 exclusivelyin situ. (For interpretation of the references to colour in thisﬁgure legend, the reader is referred to the web version of this article.)

(5)

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 cellsin vitro[84]. The impact of addictive drugs on NSC differentiation and neurogenesis was also demonstrated [85].

Morphine promotes astrocyte differentiation [86] while D- amphetamine[87]and opioid peptides [88]increase neuron differentiationin vitroandin situ,in the hippocampus of adult mice.

NSC differentiation can also be stimulated by extremely low- frequency electromagnetic fields (ELFEFs) which affect several biological parameters such as the intracellular calcium level. ELFEFs upregulate the Ca^2þchannels and increase the Ca^2þinflux 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 andþ11.9% 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 resultsin vitrodid not fully translatedin vivo/in situ(orvice versa).

One solution is to directly apply therapiesin vivo, at the desired site of action, to target endogenous NSC. Indeed, strategies aiming atin situdifferentiation 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, probablyvia PPARgnuclear 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 Table 2

NSC-differentiationin vitro,in vivoandin situ.

Molecule/Condition Type of cells/area Study

design

Outcomes Reference

Year neuropathiazol primary neural progenitor cells isolated from adult rat

hippocampus

in vitro þneurons [80]2006

ELFEFs NSC isolated from the brain cortex of nb mice in vitro þneurons [89]2008

NSC co-culture with astrocytes or amniotic cells

NSC isolated from embryonic rats in vitro þneurons [78]2012

miR-9 transfection NSC isolated from embryonic mice in vitro þneurons

þastrocytes

[76]2013 MBD1-expressing lentivirus NSC isolated from embryonic mice in vitro þneurons

þastrocytes

[77]2013

BDNF NSC isolated from the forebrain cortex of nb mice in vitro þneurons

þoligodendrocytes

[90]2013

oleanolic acid NSC isolated from the embryonic mouse striatum in vitro þneurons [81]2015

valproic acid NSC isolated from embryonic rat forebrains in vitro þneurons [84]2015;[82]

2008

all-trans-retinoic acid NSC isolated from embryonic rat forebrains in vitro þneurons [84]2015;[83]

1998

1,25-Dihydroxyvitamin D3 NSC isolated from adult mouse brain in vitro þoligodendrocytes [87]2015

opioid peptides NSC isolated from embryonic rat striatum in vitro þneurons [88]2015

NFL NSC isolated from nb rat SVZ in vitro þneurons

þoligodendrocytes

[91]2016

NSC transplantation human NSC in vivo prevention of further cognitive

deterioration

[99]2015 growth factor-overexpressing NSC

transplantation

NSC isolated fromtransgenic nb mouse hippocampus in vivo AD deﬁcit recovery, synaptic density increment

[100]2016

morphine adult mouse hippocampus in situ þastrocytes [86]2015

D-amphetamine adult mouse hippocampus in situ þneurons [87]2013

ELFEFs adult mouse hippocampus in situ þneurons [97]2010

retroviral Zfp488 adult mouse corpus callosum in situ motor function restoration [95]2011

perﬂuorooctane sulfonate cortical tissues of neonatal mice in situ þneurons

þoligodendrocytes

[94]2013

PCDHIIx shRNA/siRNA lateral ventricle of mice in situ þneurons [101]2014

simvastatin injured areas of adult rat brain in situ enhancement of neurological functions[93]2015

ketamine nb rat SVZ in situ þneurons [102]2015

nicotine adult rat hippocampus in situ þneurons [103]2015

long noncoding RNA Pnky embryonic and postnatal mouse brain in situ þneurons [104]2015

oxygen supply modulation developing cerebral cortex of mice in situ þradial glia [96]2016

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

(6)

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 perturba- tion 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 vivoapplication 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 thein situstimulation 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 modelsviaNSC 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 atin vivoNSC 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 sug- gest 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 performedin vivoto explain this result, but the preservation of NSC multipotency was demonstrated in vitroafter 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 onco- lytic 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 5- fluorocytosine (5-FU) intra-cerebrally. The objective was to facili- tate 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 hydro- chloride, 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 (see3.6 Challenges). Focusing on strategies based onin situstimulation of endogenous NSC differentiation could provide promising alterna- tives that might be easier to translate into therapy for the treatment Fig. 4.Major challenges associated with NSC-based therapies for CNS repair.

(7)

of neurodegenerative diseases.

3.6. Challenges

Despite important and encouraging progress, the intrinsic complexity of the CNS still precludes the potential of many NSC- based 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 betweenin vitroandin 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 differentiationin 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 Sta- mina”[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 inFig. 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 thein vivoextracellular 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 differentiation- based strategies could be compromised by the difﬁculty to support NSC-differentiated cell migration and integration within the Table 3

NSC-based clinical trials.

Targeted disease Type of cells Approach Identiﬁcation Phase

ALS exo NSC transplantation NCT01640067 I (concluded in 2015)

SCI exo NSC transplantation NCT02326662 I/II (ongoing 2016)

SCI exo NSC transplantation NCT01772810 I (ongoing 2016)

IS exo NSC transplantation NCT02117635 II (recruiting 2016)

PD exo NSC transplantation NCT02452723 I (recruiting 2016)

ALS endo NSC in situstimulation NCT00397423 II (completed 2007)

gliomas gen. mod. NSC transplantation NCT01172964 I (completed 2015)

gliomas gen. mod. NSC transplantation NCT02192359 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.

(8)

Table 4

Nanomedicine-based approaches for the modulation of NSC differentiation.

Strategy System Study design Outcomes Reference

nanostructured scaffold DNA-peptide nanotubes in vitro NSC differentiation in neurons [173]2014

carbon nanotubes in vivo actuation of NSC differentiation [145]2014

graphene nanoﬁbers in vitro NSC differentiation in oligodendrocytes [174]2014

PLA/gelatin nanoﬁbers in vitro iPSC differentiation in neural-like cells [146]2016

rolled graphene oxide foams and electric stimuliin vitro NSC proliferation and differentiation [175]2016

patterned porous silicon photonic crystals in vitro NSC differentiation [176]2016

self-assembling peptide -PCLePLGA nanoﬁbers in vivoimplantation NSC proliferation and differentiation in neurons and oligodendrocytes

[147]2016 self-assembling peptide nanofibers in vivoimplantation robust survival and neurite outgrowth [177]2016 salmonfibrinfibers in vivoimplantation NSC proliferation and differentiation, vessel

growth

[178]2016 nanoparticulate drug

delivery system

nilo1-titanium dioxide nanoparticles in vitro NSC recognition in co-culture [168]2011 neurogenin2-loaded biodegradable

nanoparticles

in vitro increased neuroﬁlamnet expression [161]2016

polymeric nanoparticle-based nanogel loaded in retinoic acid

in vitro increased number of MAP-2 positive cells [153]2016 polymeric nanoparticle-based bloc micelle

system loaded in retinoic acid

in vitro increased number of MAP-2 positive cells [153]2016 DNA microcircle magnetic nanoparticles in vitro sustained gene expression [163]2016 retinoic-acid loaded polymeric nanoparticles in vivo(intracranial) NSC differentiation, neuroprotection, AD

deﬁcit recovery

[151]2016;[150]

2015;[149]2012

NFL-lipid nanocapsules in vivo(intracranial) targeting of SVZ-NSC [169]2016

miR-124-nanoparticles in vivo(intracerebral) neurogenic niche modulation, PD deﬁcit recovery

[162]2016 curcumin-loaded nanoparticles in vivo(intraperitoneal

and intracranial)

NSC differentiation in neurons, AD deﬁcit recovery

[152]2013 nanotechnology-based

real-time imaging

bicistronic vector TUPIS functionalized nanovehicle

in vivo NSC differentiation imaging [165]2015

deoxythymidine oligonucleotides Gd(III)/Cy3 functionalized gold nanoparticles

in vivo NSC differentiation imaging [164]2016

USPIO/MIRB nanoparticles in vivo Tracking of NSC behavior after neural

transplantation

[166]2016 DNAegadoliniumegold nanoparticles in vivo In vivo T1 magnetic resonance imaging of

transplanted NSC

[179]2016 poly-L-lysine-gFe2O3 coated nanoparticles in vivo In vivo magnetic resonance imaging of

iPSC-derived NSC

[167]2016 mixed 3D graphene oxide-encapsulated gold

nanoparticles

in vitro NSC differentiation monitoring [180]2013

nanotopographical siRNA delivery in vitro NSC differentiation in neurons [172]2013

gold nanoparticle-decorated scaffold in vitro NSC differentiation in neurons [114]2015 retinoic-acid- and siRNA-loaded SPION

nanoparticles

in vivo attenuation of neuronal loss and rescues memory deﬁciencies

[115]2016

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.

(9)

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 C- loaded hydrogel[130]) or co-culture of neural and non-neural cells (e.g., neurotrophin-3 gene-modified Schwann cells and TrkC gene- modified 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 cellsviasustained drug release. (ii) Unfavor- able 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 inTable 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 nano- encapsulation 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 real- time 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 forma- tion 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 NSC- targeting compounds limited the development of conventionalin situNSC 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

endogenous NSC and induce their differentiation.

4.2. Nanostructured scaffolds

Nanostructured scaffolds have been developed for in vivo transplantation of NSC. Theﬁrst carbon-nanotube structured PLGA matrix made by Landers et al. induced the differentiation of iPSC- derived NSC into neuronal cells after electric stimuliin 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, prolifer- ate, and differentiate on the PLA/gelatinfibers 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 electrospunfibers[147]. The nanofibrous systems were highly biocompatiblein vivowhen implanted in rats and they promoted NSC differentiation in vitro after NSC were seeded ontoflat 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 thein vivomicroenvironmental conditions either of the niches or of the area receiving the trans- plant. 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 thefirst to produce retinoic acid- loaded 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 nanoparticlesviaintraperitoneal 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/b- catenin 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 testedin vitroon 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. Fe3O4mag- netic nanoparticles in association with ELFEFs enhanced neuronal [154] and osteogenic [155] differentiation of bone marrow- derived 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

(10)

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 animal- derived 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 nanoparticle- based 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 thefirst 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 SVZ- olfactory 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 car- gos. 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 post- transplantation. 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/Mol- day 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 immunores- ponse producedin vivoneeds further examinations before their utilization in the clinic. Jirakova et al. demonstrated that poly-L- 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-lysine- gFe2O3coated 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 situor to improve the efﬁcacy of NSC-based therapies to treat neurodegenerative diseases.

4.5. Nanomedicine for in situ NSC differentiation

In situNSC 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 thein vivoefﬁcacies of such a system. More recently, Carradori et al. developed NFL-functionalized lipid nanocapsules that selectively target SVZ-NSCin vitroand, 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, thein vivoproof-of-principle is lacking but signiﬁcant results have been achievedin vitro[171,172].

The development of systems able to target endogenous NSC represents a promising strategy to overcome transplantation- associated 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 debil- itated, the development of less invasive strategies, such asin situ NSC differentiationvia targeting nanomedicines, could be a suc- cessful approach.

5. Conclusion

The discovery of adult neurogenesis has had a signiﬁcant impact

(11)

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 invitro/in vivo/in situand 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 invitro- in vivocorrelation, 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 ourﬁnancial 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- ciation de Recherche sur le Cancer), by CIMATH (Region des Pays- de-la-Loire), and by MATWIN (Maturation&Accelerating Trans- lation With Industry). This work is supported by grants from the Universite Catholique de Louvain (Fonds Speciaux de Recherche, F.S.R.). A. des Rieux is a F.R.S.-FNRS Research Associate.

References

[1] I. Smart, C.P. Leblond, Evidence for division and transformations of neuroglia cells in the mouse brain, as derived from radioautography after injection of thymidine-H3, J. Comp. Neurol. 116 (1961) 349e367.

[2] J. Altman, G.D. Das, Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats, J. Comp. Neurol. 124 (1965) 319e335.

[3] M.S. Kaplan, J.W. Hinds, Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs, Science 197 (1977) 1092e1094.

[4] S.A. Goldman, F.P. Nottebohm, Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain, Proc.

Natl. Acad. Sci. U. S. A. 80 (1983) 2390e2394.

[5] E. Gould, H.A. Cameron, D.C. Daniels, C.S. Woolley, B.S. McEwen, Adrenal hormones suppress cell division in the adult rat dentate gyrus, J. Neurosci. 12 (1992) 3642e3650.

[6] H.G. Kuhn, H. Dickinson-Anson, F.H. Gage, Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation, J. Neurosci. 16 (1996) 2027e2033.

[7] K. Nixon, F.T. Crews, Binge ethanol exposure decreases neurogenesis in adult rat hippocampus, J. Neurochem. 83 (2002) 1087e1093.

[8] G. Kempermann, H.G. Kuhn, F.H. Gage, More hippocampal neurons in adult mice living in an enriched environment, Nature 386 (1997) 493e495.

[9] H.G. Kuhn, J. Winkler, G. Kempermann, L.J. Thal, F.H. Gage, Epidermal growth factor andﬁbroblast growth factor-2 have different effects on neural progenitors in the adult rat brain, J. Neurosci. 17 (1997) 5820e5829.

[10] B.A. Reynolds, S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science 255 (1992) 1707.

[11] T.J. Kilpatrick, P.F. Bartlett, Cloning and growth of multipotential neural precursors: requirements for proliferation and differentiation, Neuron 10 (1993) 255e265.

[12] T.D. Palmer, J. Takahashi, F.H. Gage, The adult rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci. 8 (1997) 389e404.

[13] C. Wang, F. Liu, Y.Y. Liu, C.H. Zhao, Y. You, L. Wang, J. Zhang, B. Wei, T. Ma, Q. Zhang, Y. Zhang, R. Chen, H. Song, Z. Yang, Identiﬁcation and character- ization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain, Cell Res. 21 (2011) 1534e1550.

[14] G. Christian, J. Frisen, Neural stem cells and neurogenesis in the adult, Cell Stem Cell 10 (2012) 657e659.

[15] K.L. Spalding, O. Bergmann, K. Alkass, S. Bernard, M. Salehpour, H.B. Huttner, E. Bostr€om, I. Westerlund, C. Vial, B.A. Buchholz, G. Possnert, D.C. Mash, H. Druid, J. Frisen, Dynamics of hippocampal neurogenesis in adult humans, Cell 153 (2013) 1219e1227.

[16] T.D. Palmer, E.A. Markakis, A.R. Willhoite, F. Safar, F.H. Gage, Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS, J. Neurosci. 19 (1999) 8487e8497.

[17] V.G. Kukekova, E.D. Laywella, O. Suslova, K. Daviesc, B. Schefﬂera, L.B. Thomasa, T.F. O’Briene, M. Kusakabef, D.A. Steindlera, Multipotent stem/

progenitor cells with similar properties arise from two neurogenic regions of adult human brain, Exp. Neurol. 156 (1999) 333e344.

[18] W. Murrell, E. Palmero, J. Bianco, B. Stangeland, M. Joel, L. Paulson, B. Thiede, Z. Grieg, I. Ramsnes, H.K. Skjellegrind, S. Nygård, P. Brandal, C. Sandberg, E. Vik-Mo, S. Palmero, I.A. Langmoen, Expansion of multipotent stem cells from the adult human brain, PLoS One 8 (2013) e71334.

[19] A.J. Wagers, I.L. Weissman, Plasticity of adult stem cells, Cell 116 (2004) 639e648.

[20] H.E. Shenghui, D. Nakada, S.J. Morrison, Mechanisms of stem cell self- renewal, Annu. Rev. Cell. Dev. Bi 25 (2009) 377e406.

[21] G. Li, L. Fang, G. Fernandez, S.J. Pleasure, The ventral hippocampus is the embryonic origin for adult neural stem cells in the dentate gyrus, Neuron 78 (2013) 658e672.

[22] L.C. Fuentealba, S.B. Rompani, J.I. Parraguez, K. Obernier, R. Romero, C.L. Cepko, A. Alvarez-Buylla, Embryonic origin of postnatal neural stem cells, Cell 161 (2015) 1644e1655.

[23] S. Furutachi, H. Miya, T. Watanabe, H. Kawai, N. Yamasaki, Y. Harada, I. Imayoshi, M. Nelson, K.I. Nakayama, Y. Hirabayashi, Y. Gotoh, Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells, Nat. Neurosci. 18 (2015) 657e665.

Fig. 6.In situNSC 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 theirin situdifferentiation (e.g.,viaretinoic 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.