neuro-immune interactions of neural stem cell transplants: from

Experimental Neurology xxx (2013) xxx–xxx

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Experimental Neurology

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Review

Neuro-immune interactions of neural stem cell transplants: From animal diseasemodels to human trials

Elena Giusto 1, Matteo Donegà 1, Chiara Cossetti, Stefano Pluchino ⁎Dept of Clinical Neurosciences, John van Geest Centre for Brain Repair, Wellcome Trust/Medical Research Council, Stem Cell Institute, National Institute for Health Research (NIHR),Biomedical Research Centre, University of Cambridge, CB2 0PY, UK

⁎ Corresponding author. Fax: +44 1223 331174.E-mail address: [email protected] (S. Pluchino).URL: http://www.pluchinolab.org (S. Pluchino).

1 These authors contributed equally to this work.

0014-4886/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.expneurol.2013.03.009

Please cite this article as: Giusto, E., et al., Ntrials, Exp. Neurol. (2013), http://dx.doi.org

a b s t r a c t
a r t i c l e i n f o
Article history:Received 14 September 2012Revised 5 March 2013Accepted 8 March 2013Available online xxxx

Keywords:Neural stem cellsStem cell therapyImmune modulationNeuro-immune interactionsClinical trials

Stem cell technology is a promising branch of regenerative medicine that is aimed at developing newapproaches for the treatment of severely debilitating human diseases, including those affecting the centralnervous system (CNS).Despite the increasing understanding of themechanisms governing their biology, the application of stemcell ther-apeutics remains challenging. The initial idea that stem cell transplantswork in vivo via the replacement of endog-enous cells lost or damaged owing to disease has been challenged by accumulating evidence of their therapeuticplasticity. This new concept covers the remarkable immune regulatory and tissue trophic effects that transplantedstem cells exert at the level of the neuralmicroenvironment to promote tissue healing via combination of immunemodulatory and tissue protective actions, while retaining predominantly undifferentiated features.Among a number of promising candidate stem cell sources, neural stem/precursor cells (NPCs) are under exten-sive investigation with regard to their therapeutic plasticity after transplantation. The significant impact in vivoof experimental NPC therapies in animalmodels of inflammatory CNS diseases has raised great expectations thatthese stem cells, or themanipulation of themechanisms behind their therapeutic impact, could soon be translat-ed to human studies.This review aims to provide an update on the most recent evidence of therapeutically-relevant neuro-immune interactions following NPC transplants in animal models of multiple sclerosis, cerebral stroke andtraumas of the spinal cord, and consideration of the forthcoming challenges related to the early translationof some of these exciting experimental outcomes into clinical medicines.

© 2013 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Spinal cord injuries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Towards clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Introduction

The discovery of adult neurogenesis and the development of proto-cols that allow in vitro growth and significantly large scale-up of stem

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euro-immune interactions of/10.1016/j.expneurol.2013.0

and precursor cells of the brain (Reynolds and Weiss, 1992) have fos-tered the development of innovative therapies aimed at stem cell trans-plantation for acute and chronic disorders of the nervous system(Cossetti et al., 2012).Motivated by the expectation of achievingCNS re-pair and/or regeneration via functional neural cell replacement, thesestudies have demonstrated a potential benefit of neural stem/precursorcell (NPC)-based experimental treatments in animal models of severalneurological diseases (Martino et al., 2011). However, mounting evi-dence suggests that the effects orchestrated by transplanted NPCs are

neural stem cell transplants: From animal disease models to human3.009

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2 E. Giusto et al. / Experimental Neurology xxx (2013) xxx–xxx

not only associatedwith the generation of newneurons or glial cells butalso that the pathological setting in which these cells are transplantedcritically determines the outcome (Cossetti et al., 2012). Cell replace-ment is therefore only one of the multiple ways in which transplantedNPCs promote tissue repair, and amuchmore complex therapeutic sce-nario should be foreseen. The concept of stem cell therapeutic plasticity(Martino and Pluchino, 2006) (or functional multipotency) (Teng et al.,2011) has therefore emerged, as it describes the multiple way(s)grafted NPCs whichmediate systemic homeostasis, e.g. by the secretionof tissue trophic factors, as well as interaction with tissue-resident vs.-infiltrating immune cells, at the level of the inflammatory tissue con-text in which they are either transplanted or to which they migrateafter transplantation.

The newest picture is therefore that stem cell therapies, contraryto single-molecule-based pharmaceutical interventions, hold the po-tential to deliver a complex series of information to a multitude of tar-gets in the diseased microenvironment (Cossetti et al., 2012). Whileno final mechanisms (or direct evidence) of stem cell-to-host im-mune system interaction is yet available, a number of studies arenow focussing on the cellular signalling that exists between graftedstem cells and endogenous target cells, with the aim of clarifying itsphysiological or circumstantial nature, and elucidating its molecularsignature and therapeutic potential.

Here we will review the most recent evidence of immune modula-tion following syngeneic NPC transplants in animal models of multi-ple sclerosis, spinal cord injury and stroke, and discuss the nextchallenges related to the translation of some of these exciting exper-imental outcomes into clinical medicines.

Multiple sclerosis

Multiple sclerosis (MS) is a complex, highly debilitatingCNS autoim-mune disease that constitutes the most common cause of neurologicaldisability in young adults (Compston and Coles, 2002). The main path-ological hallmark of MS is the presence of highly heterogeneous, chron-ic inflammatory and demyelinating perivascular lesions within the CNS(Compston and Coles, 2002; Dyment and Ebers, 2002; Flugel et al.,2001; Lucchinetti et al., 2000; Noseworthy et al., 2000; Wingerchuket al., 2001). Most of the demyelinated regions undergo partialremyelination and show structural repair and recovery of function(Barkhof et al., 2003; Chang et al., 2002; Compston, 1996, 1997;Prineas et al., 1993; Raine and Wu, 1993). However, remyelination inMS is typically patchy and incomplete, and ultimately fails (Blakemoreet al., 2002; Franklin, 2002; Franklin and Ffrench-Constant, 2008). Thefailure of remyelination in MS has multiple causes:

(i) Inadequate provision of OPCs (recruitment failure) (Chari et al.,2003) or a failure of recruited OPCs to differentiate intoremyelinating oligodendrocytes (differentiation failure) (Jepsonet al., 2012; Syed et al., 2011);

(ii) Ageing of the perilesional microenvironment where recruitedOPCs show impaired differentiation into oligodendrocytes(Ruckh et al., 2012);

(iii) Inhospitable environment generated by pro-inflammatoryTh1/Th17 cells and cytokines (Steinman, 2007); and

(iv) Anatomical barriers around chronic lesions impeding the re-cruitment of OPCs (Franklin, 2002).

Furthermore, recurring inflammation may have profound conse-quences on the health of anatomically intact axons, resulting in pro-gressive and irreversible damage/dysfunction that accounts for thedegenerative nature of MS (Franklin and Ffrench-Constant, 2008;Patrikios et al., 2006). The sequential involvement of most of theabove processes underlies the clinical course of MS, which ischaracterised by recurrent episodes of relapses that eventually leavetemporary or persistent deficits, to finally deteriorate into a second-ary chronic progressive phase (Compston and Coles, 2002).

Please cite this article as: Giusto, E., et al., Neuro-immune interactions oftrials, Exp. Neurol. (2013), http://dx.doi.org/10.1016/j.expneurol.2013.0

The issue of (stem) cell therapies for MS has therefore gained incomplexity as its success relies on the capacity of transplanted(stem) cells to target the specific sites of disease, integrate into thehost tissue and eventually differentiate into neural functional cells(neurons and glia), while surviving in the chronically inflamed CNSenvironment. This adds crucial concerns of identification of cell source,its constitutive vs. reactive immunogenicity, window of opportunity,route of cell delivery, as well as ways in which to help the integrationand long-term survival of grafted cells in the ‘inhospitable’ inflammato-ry CNS environment. All these are critical aspects of stem cell therapiesthat must be critically considered when envisaging therapeutic celltransplants for MS (Martino et al., 2011).

The route of cell administration has always been a major con-straint for stem cell transplantation in CNS diseases and appeared tobe very much dependent on the presence of focal vs. multifocal le-sions to target. With MS being a multifocal disease, it is unrealisticto propose lesion-targeted injection of cells (Pluchino and Martino,2008). This is also complicated by the fact that it is generally difficultto determine which of the multiple lesions identified bymagnetic res-onance imaging (MRI) would underscore clinical significance, andwhether they would eventually be amenable to effective (therapeuti-cally relevant) remyelination upon (stem) cell therapy (Chen et al.,2007).

Following a number of successful proof-of-concept in vivoremyelination studies with focally-transplanted glial progenitor celltypes, including oligodendrocytes (Blakemore and Crang, 1988), OPCs(Groves et al., 1993; Windrem et al., 2004, 2008) and Schwann cells(Blakemore, 1977; Zujovic et al., 2012), NPCs were the very first candi-date stem cells for systemic cell treatment of experimental autoimmuneencephalomyelitis (EAE), as an animal model of MS [reviewed inPluchino et al. (2004) and Goldman et al. (2012)]. During the lastdecade, rodents and non-human primates with acute, relapsing andchronic EAE (Martino and Pluchino, 2006) have been treated withNPCs injected intracerebroventricularly (icv), intrathecally (it) or intra-venously (iv). These studies have shown that systemically injectedNPCs enter the CNS where they survive for months in perivascular in-flammatory areas while retaining mostly undifferentiated features,and remarkably reduce the clinical and pathological burden of the dis-ease (Martino and Pluchino, 2006). Interestingly, the majority of thesereports have substantially failed to show convincing differentiationand integration of transplanted NPCs in vivo, but rather contributed tothe provocative idea that NPC transplants would work throughmecha-nisms other than direct cell differentiation that imply the interactionbetween NPCs and immune cells (Cossetti et al., 2012; Martino andPluchino, 2007).

The first evidence that NPCs possess immune-like features camefrom the observation that systemically injected NPCs use functionalleukocyte-specific cell adhesion molecules (such as CD44 and very lateantigen [VLA]-4) and inflammatory chemokine receptors (e.g. CCR2,CCR5 and CXCR4) to interact with activated ependymal and endo-thelial cells and ultimately enter the brain (Ben-Hur et al., 2003;Einstein et al., 2003; Pluchino et al., 2003). Once into the CNS, NPCsare found around inflamed blood vessels, in close contact with endoge-nous neural cells (e.g. astrocytes and neurons) and CNS-infiltratingblood-borne CD45+ immune cells, while creating niche-like areas thatare ultrastructurally and molecularly reminiscent of the prototypicalstem cell niches from which NPCs were derived (Pluchino et al.,2005). NPC transplants are also associated with significantly reducedglial scar formation (Pluchino et al., 2003) and local inflammatory re-sponse (Ben-Hur et al., 2003; Einstein et al., 2006; Pluchino et al.,2005, 2009a, 2009b), which in turn lead to the increased survival andrecruitment of endogenous neural cells (e.g. oligodendroglial progeni-tor cells) participating in the brain's intrinsic reparative responseupon myelin damage (Einstein et al., 2009; Pluchino et al., 2005). Theunderlying molecular mechanisms by which transplanted NPCs conferthis broad tissue protection were first indirectly linked to the increased



3E. Giusto et al. / Experimental Neurology xxx (2013) xxx–xxx

in vivo bioavailability of major neurotrophins (Chu et al., 2004a, 2004b;Einstein et al., 2006; Lu et al., 2003; Pluchino et al., 2003; Teng et al.,2002) and to the modulation of the host environment into one that ismore permissive for regeneration. Several neurotrophins that arefound increased after NPC transplants have also been shown to inhibitEAE, and neurotrophins like insulin-like growth factor (IGF)-1 andglial growth factor (GGF)-2 promote the survival and proliferation of ol-igodendrocyte lineage cells (Barres et al., 1992; Canoll et al., 1996, 1999;Mason et al., 2000).

However, the observation that (icv)-injected NPCs primarily atten-uate brain inflammation, in correlation with a reduction of CD3+ Tcells and an increase in CD25+ and CD25+/CD62L+ regulatory Tcells (Einstein et al., 2003), suggested a completely novel mechanismof action that deserved further investigation. The iv injection of NPCsalso protects against chronic neural tissue loss as well as disease-related disability in EAE, via induction of the apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells (Pluchino et al.,2005). In vitro, NPCs increase the apoptosis of antigen-specific Th1pro-inflammatory (but not Th2 anti-inflammatory) cells selectivelythrough the engagement of death receptors, including FasL, TRAILand APO3L, on the surface of NPCs (Pluchino et al., 2005). Mouse andrat NPCs also inhibit T cell activation and proliferation in response toT cell receptor (TCR)-mediated stimuli (Einstein et al., 2003;Fainstein et al., 2008). NPC-T lymphocyte co-cultures suggest thatpart of the anti-proliferative effect of NPCs might depend on the inhi-bition of IL-2 and IL-6 signalling on T lymphocytes (Fainstein et al.,2008). Knight et al. show that NPCs have a selective pro-apoptoticeffect on Th17 cells in vitro via a FasL-dependent mechanism, identify-ing the axis Fas-Birc3 as an additional survival pathway for NPCs(Knight et al., 2010). NPCs also suppress T cell proliferation by thereactive production of nitric oxide (NO) and prostaglandin E2 (PGE2).Interleukin (IL)-10-transduced NPCs show enhanced ability to induceremyelination, neuronal repair and immune suppression after systemicNPC injection in EAEmice (Yang et al., 2009). HumanNPCs suppress theproliferation of non-human primate activated T cells through both di-rect cell-to-cell contacts and via the release of soluble mediators intothe culture supernatant (Kim et al., 2009; Pluchino et al., 2009a).

Other studies have shown that systemically injected NPCs alsoinhibit EAE by a peripheral immune modulation in lymph nodes(Einstein et al., 2007; Pluchino et al., 2009b). Einstein et al. first showedthat EAE-derived lymph node cells were strongly inhibited by NPCs inthe production of pro-inflammatory cytokines in response toMOG35-55. Furthermore, primed T cells from mice treated with NPCswere also deficient in their ability to adoptively transfer EAE, thus dem-onstrating a long-lasting inhibition of the encephalitogenicity of the Tcells that are transferred to a naïve host, rather than an effect specificto the in vivo environment (Einstein et al., 2007).

We have reported a specific and almost exclusive targeting of theperipheral immune system in SJL mice with PLP-induced EAE inwhich NPCs were injected subcutaneously (sc) at 3 and 10 dayspost-immunisation (dpi) (Pluchino et al., 2009b). After sc injectionin EAE, NPCs consistently accumulate and persist in draining lymphnodes, where they increase the availability of major stem cell regula-tors (including bone morphogenetic protein [BMP]-4) and interactwith lymphoid dendritic cells (DCs) that are hindered in their matura-tion to professional antigen-presenting cells (APCs). In vitro, NPCsspecifically impair the maturation of immature DCs (iDCs) via aBMP-4-dependent mechanism (Pluchino et al., 2009b). A recentstudy has shown that the preventive intravenous administration ofNPCs ameliorates EAE by selectively inhibiting the differentiationof encephalitogenic T helper 17 (Th17) through secreted factors.Recently, Cao et al. have identified leukaemia inhibitory factor (LIF)as the first key factor responsible for the observed inhibition ofTh17 cell differentiation by transplanted NPCs, and elucidated thesignalling pathway behind this novel mechanism of action, whereLIF antagonises interleukin (IL)-6-induced Th17 cell differentiation


through the ERK-dependent inhibition of STAT3 phosphorylation(Cao et al., 2011).

Whether most of the immune regulatory effects of systemicallyinjected NPCs in EAE act directly in the CNS or in the periphery isstill an unanswered question and further studies are needed to estab-lish the absolute relevance of these pre-clinical data in EAE – whereperipheral lymphoid organs play an important role in the regulationof the immune responses to self myelin antigens – and indeed thepossibility that both sites of action may become interrelated andpathophysiologically relevant to the future applications of NPCs inMS (Kokaia et al., 2012; Martino et al., 2011). Evidence of theneuro-immune interactions observed following NPC transplantationin EAE is shown in Table 1 and summarized in Fig. 1.

Spinal cord injuries

Spinal cord injuries (SCIs) are devastating and debilitating condi-tions affecting all regions of the world – predominantly in youngadults – which are associated with severe physical, psychological,social and economic burdens on patients and their families[reviewed in Ho et al. (2007) and van den Berg et al. (2010)]. An im-portant premise for the development of effective treatments for SCIsis the precise understanding of the main pathophysiological eventsfollowing the acute injury and how these interact in the develop-ment of established anatomical and functional deficits (Rowlandet al., 2008).

The most common mechanisms of SCI developed into two exclu-sive broad chronological phases that are sustained by the primaryand secondary mechanisms of injury. Primary injuries include shear-ing, laceration, and acute stretching vs. acceleration–decelerationevents, which very rarely lead to complete transection or disruptionof the anatomical continuity of the spinal cord (Rowland et al.,2008). The severity of neurological injury, the level of the injury andthe presence of a zone of partial cord preservation are accepted pre-dictors of recovery and survival after SCI. Indeed, the presence ofspared axons crossing the injury site holds great therapeutic potentialas a substrate of a number of emerging therapeutic strategies (Wilsonet al., 2012).

A number of processes are triggered by primary injuries and lead tomuch more prolonged secondary injury phases that start within a fewhours and remain active up to weeks after SCIs; thus, progressively ex-acerbating the consequences of the mechanical injury to the cord. Im-mediate, acute, intermediate and chronic secondary injury phaseshave been identified, characterised and staged (Rowland et al., 2008).Some of these phases include the early traumatic severing of theaxons, the death of neurons and glia and the instantaneous loss of func-tion at and below the injury level (spinal shock) (Ditunno et al., 2004;Kakulas, 2004). The spinal cord undergoes diffuse swelling and thecentral grey matter shows signs of petechial haemorrhage (Tator andKoyanagi, 1997). Cell necrosis and the activation of local astrocytesand microglia are also observed immediately after the injury(Watanabe et al., 1999). Subsequent secondary events include free rad-ical production, ionic (e.g. Ca++) deregulation (Tymianski et al., 1993)and glutamate excitotoxicity (Li and Stys, 2000) (immediate — hours);changes in the blood–brain barrier (BBB) permeability (Noble andWrathall, 1989), activation of a multifaceted inflammatory responsethat involves soluble inflammatory mediators and a variety of cellssuch as astrocytes, microglia, T cells, neutrophils and cord-infiltratingmonocytes (Donnelly and Popovich, 2008; Fleming et al., 2006), oligo-dendrocyte apoptotic cell death and demyelination (Crowe et al.,1997) (acute — days); astroglial proliferation and formation of a glioticscar (Silver and Miller, 2004) that interferes with axonal sprouting(intermediate — weeks to months); and retrograde Wallerian axonaldegeneration (Coleman and Perry, 2002), dynamicmaturation of the le-sion up to the development of cysts and/or syrinxes, and delayed



Table 1Neuro-immune interaction following neural stem cell transplants in animal disease models.

Disease model Species Transplant features Observed effect(s) Proposed mechanism(s) References

Cell type Cell no./animal Route Time

Experimental Autoimmune Encephalomyelitis (EAE)Acute EAE Rat Rat neurospheres 1.5–2 × 104 icv or it Disease

peakCell differentiation (neuronal and glial)and tissue trophism

None (Ben-Hur et al., 2003)

Acute EAE Rat Rat neurospheres 2 × 104 icv 0 dpi Immune regulation (central) None (Einstein et al., 2003)Chronic EAE Mouse Mouse NPCs 1 × 106 iv or it 22 dpi (Low) cell differentiation and

tissue trophismInhibition of reactive gliosis (Pluchino et al., 2003)

Chronic EAE Mouse Mouse neurospheres 2.5 × 103 icv 6 dpi Immune regulation (local) Reduction of CNS inflammatory infil-trates, increase in regulatory T cells

(Einstein et al., 2006)

Chronic EAEPassive EAE

Mouse Mouseneurospheres

1 × 106 iv 8 dpi Immune regulation (peripheral) Suppression of encephalitogenic T cells (Einstein et al., 2007)

Chronic EAE Mouse Human ES cell-derived NPCs

5 × 105 icv 7 dpi Immune regulation (local) Suppression of encephalitogenic T cells (Aharonowiz et al., 2008)

Chronic EAE Mouse IL-10-transducedmouse NPCs

1.5 × 106 Iv or icv 10, 22 or30 dpi

Immune regulation (local, peripheral)and cell differentiation

Induction of T cell apoptosis,promotion of myelin debris clearance

(Yang et al., 2009)

Chronic EAE Mouse Mouse and humanES cell-derived NPCs

2 × 106 iv 0 or 10 dpi Immune regulation (peripheral) LIF-mediated inhibition of Th17 celldifferentiation

(Cao et al., 2011)

Chronic EAE Mouse Mouse MSC-derived NPCs 3.5 × 104–6.1 × 106

it 21, 28 and35 dpi

Tissue trophism None (Harris et al., 2012)

Chronic EAEPassive EAE

Mouse CCR5-transducedmouse BM-derived NPCs

1.5 × 106 iv 22 dpi (peak) Immune regulation None (Yang et al., 2012)

Chronic EAE CommonMarmoset

Human NPCs 2–6 × 106 it or iv Disease onset Immune regulation (central) Suppression of encephalitogenic T cells,impairment of dendritic cell maturation

(Pluchino et al., 2009a)

Relapsing EAE Mouse Mouse NPCs 1 × 106 iv Disease onsetor first relapse

Immune regulation (central) Induction of T cell apoptosis (Pluchino et al., 2005)

Relapsing EAE Mouse Mouse NPCs 0.5 × 106 sc 3 and 10 dpi,or 10 dpi only

Immune regulation (peripheral) BMP-4-dependent hindrance ofdendritic cell maturation

(Pluchino et al., 2009b)

Relapsing EAE Mouse Mouse NPCs and olig2-transduced NPCs

1.5 × 105 icv Disease onsetor first relapse

Immune regulation (central) and tissuetrophism

None (Sher et al., 2012)

Spinal Cord Injuries (SCI)Compression (T7) Rat Mouse NPCs 3–4 × 105 ipc 14 or 56 dpi Cell differentiation (oligodendroglial) Remyelination (Karimi-Abdolrezaee et al., 2006)Compression (T7) Rat Mouse NPCs 4 × 105 ipc 49 dpi Cell differentiation (oligodendroglial) Remyelination (Karimi-Abdolrezaee et al., 2010)Compression (T8–T9) Rat Human NPCs 2.5 × 105 ipc 7 dpi Cell differentiation (neuronal and glial) None (Mothe et al., 2011)Contusion (C5) Common

marmosetHuman NPCs 1 × 106 il 9 dpi Cell differentiation (neuronal and glial) Integration in endogenous neuronal

circuitries(Iwanami et al., 2005)

Contusion (T8) Rat Rat NPCs 3–5 × 105 il or ipc 10 dpi Cell differentiation (glial) None (Cao et al., 2001; Karimi-Abdolrezaee et al., 2006)

Contusion (T8) Mouse Mouse NPCs 1 × 106 or 1 × 105 Iv or ipc Acute Tissue trophism Reduction of apoptosis (Bottai et al., 2008)Contusion (T8–T9) Rat Rat NPCs 1 × 105 ipc 7 dpi Cell differentiation (neuronal and glial) None (Hofstetter et al., 2005)Contusion (T9) Mouse Human NPCs 7.5 × 104 ipc 9 dpi Cell differentiation (neuronal and glial) Integration into endogenous neuronal

circuitry(Cummings et al., 2005)

Contusion (T9–T10) Rat Rat G-NPCs 5 × 105 il Acute Axonal (re)growth Reduction of astrogliosis and promotionof axon sprouting

(Hill et al., 2004)

Contusion (T9–T10) Mouse Mouse NPCs + VPAand HDAC inhibitor

5 × 105 il 7 dpi Cell differentiation (neuronal and glial) Integration into endogenous neuronalcircuitries

(Abematsu et al., 2010)

Contusion (T9–T10) Mouse Human iPS cell-derivedNPCs

1 × 106 il 7 dpi Cell differentiation (neuronal and glial)and tissue trophism

Integration into endogenous neuronalcircuitries

(Fujimoto et al., 2012)

Contusion (T10) Mouse Mouse NPCs 5 × 105 il 9 dpi Cell differentiation (oligodendroglial) Remyelination (Yasuda et al., 2011)Contusion (T10) Mouse Human iPS cell-derived

NPCs5 × 105 ipc 9 dpi Cell differentiation (neuronal and glial),

tissue trophismIntegration into endogenous neuronalcircuitries, remyelination andneuroprotection

(Nori et al., 2011)

Contusion (T12) Mouse Mouse NPCs 5 × 105 it 7 dpi Immune regulation (local) andtissue trophism

T cell mediated activation of microgliawith a protective phenotype

(Ziv et al., 2006)

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Contusion (T12) Mouse Mouse NPCs 0.75–1.5 × 105 ipc 7 and 21 dpi Immune regulation (local) Reduction of the M1-like macrophagepopulation

(Cusimano et al., 2012)

Hemisection (C1) Rat Rat NPCs 5 × 105 il Acute Cell differentiation (neuronal) Integration into endogenous neuronalcircuitries

(Bonner et al., 2011)

Hemisection (C3) Rat NT-3-transduced ratNPCs

1 × 105 il Acute Tissue trophism Neuroprotection (Lu et al., 2003)

Hemisection (C3) Rat Rat NPCs 4.8–5.4 × 105 il Acute Cell differentiation (astro andoligodendroglial)

None (Vroemen et al., 2003)

Hemisection (C4) Rat Rat G-NPCs 1 × 105 il or ipc 7–10 dpi Cell differentiation (astro andoligodendroglial)

None (Han et al., 2004)

Hemisection (C4) Rat Rat NPCs 4 × 105 il Acute Cell differentiation (neuronal and glial) None (Lepore and Fischer, 2005)Transection (T10) Rat Mouse NPCs + gelfoam 3 × 105 il Acute Tissue trophism Neuroprotection (Gu et al., 2012)Hemisection (T9–T10) Rat Mouse NPCs + PLGA

scaffold5 × 105 il Acute Tissue trophism Reduction of astrogliosis (Teng et al., 2002)

StrokeMCAo Rat Rat NPCs 1 × 105 it 2 dpi Cell differentiation (neuronal) None (Zhang et al., 2003)MCAo (10′ or 90′) Rat Human NPCs 5 × 106 iv 1 dpi Cell differentiation (neuronal, glial) None (Chu et al., 2003, 2004b)MCAo (30′ or 60′) Rat Human NPCs 3 × 105 ipc 7 or 14 dpi Cell differentiation (neuronal and glial) None (Darsalia et al., 2007;

Kelly et al., 2004)MCAo (ED-1 or 120′) Rat Mouse ES cell-derived

NPCs1 × 105 ipc 7 dpi Cell differentiation (neuronal and glial) None (Buhnemann et al., 2006;

Wei et al., 2005)MCAo (70′) Rat Human NPCs 8 × 105 ipc 21–28 dpi Tissue trophism None (Pollock et al., 2006)MCAo (180′) Rat Human NPCs 1 × 106 iv 2 dpi Tissue trophism None (Jiang et al., 2006)MCAo (NA) Cynomolgus

monkeyHuman neurospheres 2–4 × 105 ipc 7 dpi Cell differentiation (neuronal) None (Roitberg et al., 2006)

MCAo (NA) Mouse Mouse ES cellderived NPCs

5 × 104 ipc 7 dbi Tissue trophism None (Pignataro et al., 2007)

MCAo (90′) Rat Rat adult and EScell-derived NPCs

1 × 105 ipc Acute Cell differentiation, tissue trophism None (Takahashi et al., 2008)

MCAo (20′) Rat Human ES cell-derivedNPCs

5 × 104 ipc 1 dpi Tissue trophism Reduction of endogenous apoptosis (Zhang et al., 2009)

MCAo (60′) Rat Human NPCs 4.5 × 103–4.5 × 105

ipc 28 dpi Tissue trophism Restoration of endogenous neurogenesis (Stroemer et al., 2009)

MCAo (120′) Rat GDNF-transduced ratNPCs

5 × 105 icv 3 dpi Tissue trophism, cell differentiation(neuronal)

None (Chen et al., 2009)

MCAo (45′) Mouse Mouse NPCs 1 × 106 iv 3 dpi Immune regulation (local) and tissuetrophism

Reduction of microglial activation andneuronal death

(Bacigaluppi et al., 2009)

MCAo (90′) Rat HIF-1 α-transduced ratNPCs

1 × 106 icv 1 dpi Tissue trophism Promotion of angiogenesis (Wu et al., 2010)

MCAo (permanent) Rat Human ES cell-derivedNPCs

1.2 × 105 ipc 14 dpi Tissue trophism Enhancement of endogenousneurogenesis

(Jin et al., 2011)


5 × 104 ipc 1 dpi Tissue trophism Enhancement of endogenousneurogenesis

(Daadi et al., 2010)

MCAo (permanent) Rat Human ES cell-derivedNPCs + Matrigel

1.2 × 105 ipc 21 dpi Tissue trophism, cell differentiation(neuronal)

Integration into endogenous neuronalcircuitries

(Jin et al., 2010)


5 × 104 ipc 1 dpi Tissue trophism Enhancement of endogenousneurogenesis and angiogenesis

(Zhang et al., 2011)

MCAo (permanent) Rat Human NPCs 1 × 105 ipc 7 dpi Tissue trophism Neuroprotection (Andres et al., 2011b)CCAo (10′ + 10′) Mongolian

gerbilsHuman NPCs 5 × 105 ipc 4 dpi Tissue trophism and cell differentiation

(neuronal)Integration into endogenous neuronalcircuitries and neuroprotection

(Ishibashi et al., 2004)

ICH Rat Human NPCs 5 × 106 iv 1 dpi Tissue trophism, cell differentiation(glial and neuronal)

Neuroprotection and integration intoendogenous neuronal circuitries

(Jeong et al., 2003)

ICH Mouse VEGF-transduced humanNPCs

2 × 105 ipc 7 dpi Tissue trophism Promotion of angiogenesis (Lee et al., 2007)

Abbreviations: BM: bone marrow; BMP-4: bone morphogenetic protein 4; CCAo: common carotid artery occlusion; dpi: days post immunisation/injury; dbi: days before immunisation/injury; ED-1: endothelin-1; ES cells: embryonic stemcells; GDNF: glial-derived neurotrophic factor; G-NPCs: glial-restricted NPCs; HDAC: histone deacetylase inhibitor; HIF-1 α: hypoxia-inducible factor 1 α; ICH: intracerebral haemorrhage; icv: intracerebroventricular; il: intralesional; ipc:intraparenchymal (perilesional); iPS cells: induced pluripotent stem cells; it: intrathecal; iv: intravenous; LIF: leukaemia inhibitory factor; MCAo: middle cerebral artery occlusion; MSC: mesenchymal stem cells; NT-3: neurotrophin 3; sc:subcutaneous; VEGF: vascular endothelial growth factor; VPA: valproic acid.

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Fig. 1. Overview diagram of the novel evidence of stem cell mediated effects – including neuro-immune interactions – that have inspired the present pipeline of neural stem celltherapeutic product development in multiple sclerosis, spinal cord injuries and brain stroke. In neurospheres, magenta is for Vimentin and green is for Phospho-Histone; in differ-entiated NPCs, red is for glial fibrillary acidic protein (GFAP) and green is for microtubule-associated protein 2 (MAP-2). Nuclei are stained with 4′,6-diamidino-2-phenylindole(DAPI). The magnetic resonance image (MRI) is a representative diffusion weighted image (DWI) coronal scan from the brain of a mouse 24 h after transient (45′) middle cerebralartery occlusion (MCAo).


neuronal dysfunction and neuropathic pain (Stoodley, 2000) (chronic—several months).

Despite the important advances in the understanding of SCI path-ophysiology, to date, virtually all therapies that have shown promiseat the preclinical stage of study have failed to translate into clinicallyeffective treatments. For this reason, the potential of NPC transplanta-tion to drive spinal cord repair has been widely investigated, as NPCsmay provide an effective treatment by either directly replacing thosecells lost owing to the injury (e.g. oligodendrocytes), influencing/modifying the environment in a way that supports axonal regenera-tion, providing neuroprotection, or re-setting the inflammatory re-sponse to a mode that heals the damaged tissue (Sahni and Kessler,2010).

Adult mammalian NPCs from different sources have beentransplanted into a wide range of SCI models with significant clinicalimprovement. Most of the studies have delivered NPCs focally toincrease their viability at the injury site. While evidence exists insupport of an astroglial differentiation default mode shown bytransplanted embryonic NPCs in both the developing and the adultcord (Lepore et al., 2006), some pioneering work suggests that NPCssurvive, migrate and generate functional remyelinating oligodendro-cytes, which promote functional recovery when transplanted sub-acutely (namely 2 weeks) – but not chronically (namely 8 weeks) –

after SCI (Karimi-Abdolrezaee et al., 2006).However, other approaches have more convincingly established

that the transplantation of somatic NPCs in experimental SCI yields agenerally low degree of survival – as the injury creates a highly toxicenvironment – and differentiation (Bottai et al., 2008; Cao et al.,2002), most of which was biassed towards a glial fate (Cao et al.,2001; Pfeifer et al., 2004; Vroemen et al., 2003), thus challenging the ini-tial expectation of achieving predominant neuronal/oligodendroglialcell replacement (Cao et al., 2001; Vroemen et al., 2003) in vivo.

There is also a growing belief that the severity (and type) of theinjury, as well as the time after injury at which cells are transplanted,are twomajor key factors influencing the capability of grafted NPCs toaffect the healing of the damaged spinal cord tissue. As such, rat spi-nal cord-derived somatic NPCs failed to induce any detectable


functional recovery when transplanted hyperacutely at the level ofinjury in a severe (35-g) clip-induced SCI model (Parr et al., 2007),but were indeed significantly efficacious when transplanted as earlyas 9 days after injury in a milder (27-g) model of SCI (Parr et al.,2008). Furthermore, the homogeneity vs. heterogeneity of the neuralstem/progenitor cell preparation is a key point. As such, prominentneuronal differentiation is reliably achieved with primary foetal tis-sue and when glially and neuronally restricted progenitors are com-bined, but not when only neuronally restricted progenitors aregrafted to the injured spinal cord [reviewed in (Fischer, 2000)].

Strategies to overcome the observed poor survival and differenti-ation potential of transplanted NPCs have included combinationwith valproic acid (Abematsu et al., 2010) or neurotrophic growthfactors (Bonner et al., 2010, 2011), which promoted neuronal differ-entiation and established functional synapses between host axonsand graft neurons at the injury site.

The use of engineered NPCs transduced with transcription factorsor survival genes (Hwang et al., 2009; Lee et al., 2009), as well as theco-transplantation with ‘scaffold’ cells such as mesenchymal/stromalstem cells or olfactory ensheathing cells (Oh et al., 2010; Wang et al.,2010), has increased the survival, migration and differentiation oftransplanted NPCs and the final functional recovery observed.

Furthermore, the combined transplantation of adult NPCs withminocycline treatment and growth factor delivery promoted cell sur-vival and remyelination of spared axons into a rat model of compres-sive SCI (Karimi-Abdolrezaee et al., 2006). Transplanted NPCs weremainly incorporated into the white matter of the dorsal or lateral col-umn and substantially differentiated along the oligodendroglial lineage.Exogenous (NPC-derived) myelin basic protein (MBP)-positive cellswere found in close association with endogenous neuronal processes,finally correlating with the recovery of locomotor function (Karimi-Abdolrezaee et al., 2006). Interestingly, the net functional significanceof exogenous remyelination in promoting behavioural recovery hasbeen further confirmed by the recent observation that mice receivingNPC-derived oligodendrocytes isolated from myelin-deficient micefailed to acquire locomotor functionality after SCI (Yasuda et al.,2011). The concomitant use of chondroitinase ABC (ChABC) to reduce




the presence of chondroitin sulphate proteoglycans (CSPGs) togetherwith NPC transplantation in a chronic model of compressive SCI greatlyincreased the long-term survival, migration and integration of graftedcells (Karimi-Abdolrezaee et al., 2010).

In the majority of cases, the transplantation of NPCs has resultedin the significant recovery of functions that were highly specific tothe treatment applied, as they were completely abolished in humanNPC-transplanted SCI mice treated with diphtheria toxin (DT; ashuman cells are about 100,000 times more sensitive to DT thanmouse cells) (Cummings et al., 2005). Interestingly, when morelineage-restricted neural precursors have been transplanted, a muchhigher rate of neuronal differentiation has been achieved, presumablybecause these latter cells are less sensitive than NPCs to inhibitorysignals coming from the environment (Han et al., 2002; Yan et al.,2007).

Based on some of these assumptions, several strategies have beendeveloped to promote regeneration and functional recovery by deliv-ering biomaterial scaffolds engineered with cells and/or bioactivemolecules (e.g. hydrogels, sponges, single- and multi-channelledguidance tubes, and nanofibre scaffolds (Bamber et al., 2001; Chenet al., 2010; Hurtado et al., 2006; Johnson et al., 2010; Taylor et al.,2006; Tobias et al., 2003; Wang et al., 2011; Xiong et al., 2012; Zenget al., 2011). Interestingly, the implantation of a scaffold–neuralstem cell unit into an adult rat hemisection model of SCI led to overalltissue preservation with limited atrophy and scar formation that fi-nally resulted in significant functional improvement up to 1 yearpost-engraftment (Teng et al., 2002).

Alternative routes of administration (e.g. systemic) have also beeninvestigated to avoid the damage to the spared cord tissue at the timeof focal cell injection, as well as other procedure-related complica-tions, with the final aim of improving the chances of translation intoclinical practise. After injection into the tail vein of nude SCI mice,human NPCs were able to reach the injury site (Takeuchi et al.,2007), leading to significantly better behavioural recovery comparedwith SCI mice transplanted intraspinally with NPCs (Bottai et al.,2008).

Importantly, the shaping of new intraspinal circuits should betightly controlled to avoid serious side effects such as aberrant hostfibre sprouting associated with allodynia-like hypersensitivity(Hofstetter et al., 2005; Macias et al., 2006). These latter effects oftransplanted NPCs may derive from their ability to release biologicalmolecules able to protect damaged cells and/or promote endogenousCNS repair/regeneration. Indeed, in vitro (Hawryluk et al., 2012;Kamei et al., 2007) and in vivo (Lu et al., 2003) studies have shownhow NPCs can release a milieu of biological factors able to attract in-jured axons and promote the growth of host spinal axons after injury.

As such, strong parallel evidence suggests that undifferentiatedNPCs can also influence the injury environment at the spinal cordthrough the establishment of functional interactions with endogenousneural and immune cells. On the one hand, the transplantation ofmouse NPCs both provided a cellular substrate and secreted severalneurotrophic factors (e.g. nerve growth factor [NGF], brain-derivedneurotrophic growth factor [BDNF] and GDNF), leading to substantialhost axonal re-growth (Lu et al., 2003). Also, adult NPCs themselveswere shown to induce the up-regulation of neurotrophic factors andchemokines such as BDNF, NGF and LIF, and inflammatory moleculessuch as TNF-α (Bottai et al., 2008). In line with this, NPCs, engineeredto express a human NT-3 capable of binding to both trkB and trkC,showed increased survival and enhanced myelin formation, althoughthey led only to a modest improvement in locomotor function, whentransplanted into a chronic SCI model (Kusano et al., 2010). Further-more, when combined with a self-antigen (e.g. myelin)-specific T cellvaccination that is expected to promote recovery from CNS insults pro-vided that their activity (in terms of onset, intensity and duration) iswell controlled (Butovsky et al., 2001, 2006), NPCs transplanted inmice with SCI migrated to the injury site and synergistically promoted


functional recovery via modulation of the nature and intensity ofthe local T cell and microglial response, expression of BDNF andNoggin, and appearance of newly formed neurons from endogenousprecursor-cell pools (Ziv et al., 2006). This immune regulatory effectwas also present when NPCs were transplanted locally (at the lesionborders) into an extremely severe (e.g. 200 kdynes)model of contusionSCI. Sub-acutely (but not chronically) transplanted NPCs remainedundifferentiated and altered the inflammatory infiltrate of the in-jured spinal cord by reducing the proportion of ‘classically-activated’(M1) inflammatory macrophages, and increasing that of regulatoryT cells, in turn promoting the healing of the injured cord (Cusimanoet al., 2012).

Thus, NPC transplantation in SCIs contributes to anatomical andbehavioural recovery in SCI models through cell replacement andintegration of grafted cells into local circuits, as well as tissue trophiceffects. Recent evidence has identified an additional novel protectivemechanism that grafted undifferentiated NPCs exert on innate im-mune responses to remodel the perilesional inflammatory environ-ment towards a tissue-healing mode (Kokaia et al., 2012; Martinoet al., 2011).

Evidence of the main outcomes following syngeneic NPC trans-plantation in experimental SCIs is shown in Table 1 and summarizedin Fig. 1.

Stroke

Clinical recovery after stroke remains very poor despite advances intherapy, and stem cell treatment is considered a promising alternative(Lindvall and Kokaia, 2011). Transplantation of NPCs with different de-livery strategies, intraparenchymal (ipc) or icv injection, as well as sys-temic administration, has been shown to improve clinical signs inexperimental stroke models (Liu et al., 2009; Pluchino et al., 2010).

Irrespective of the route of administration, transplanted NPCsmigrate towards the infarct site in models of transientmiddle cerebralartery occlusion (MCAo) and intracerebral haemorrhage (ICH) (Chuet al., 2003, 2004b; Darsalia et al., 2007; Jeong et al., 2003; Kellyet al., 2004; Modo et al., 2002; Roitberg et al., 2006; Zhang et al.,2003), where gradients of pro-inflammatory cytokines andchemokines are released (Bacigaluppi et al., 2008), and CCL2/CCR2and CXCL12/CXCR4 play a crucial role in transendothelial recruitmentand intraparenchymal migration, respectively (Andres et al., 2011a;Darsalia et al., 2007; Imitola et al., 2004). Once they reach the ischaemicboundary zone (IBZ), grafted NPCs interact with the inflammatory envi-ronment, as suggested by the increase in the gene expression levels ofVEGF, CXCL12/SDF1-α and TGF-β in the NPC-transplanted mousebrain with middle cerebral artery (MCA) occlusion 4 h after the insult(Capone et al., 2007). Similarly, the up-regulation of the cell adhesionmolecule VCAM-1 onto the surface of endothelial cells facilitates thetargeting and the subsequent extravasation of CD49d expressing NPCsto the site of injury (Guzman et al., 2008).

Intracerebrally transplanted NPCs survive up to 1 month, migratetowards the peri-infarct area and partially differentiate into matureneurons, finally resulting in a reduced lesion volume and functionalrecovery. Their beneficial effects were first attributed mainly to a com-bined action between trophic support and the ability of newly generat-ed neurons to establish new synapses (Ishibashi et al., 2004). NPC-derived neurons have been described to produce neurotransmitters,form dendrites and show electrophysiological properties characteristicof integrated functional neurons (Buhnemann et al., 2006; Englund etal., 2002; Park et al., 2002). In most of the cases, it has been observedthat transplanted NPCs remained confined to the penumbra (Jin et al.,2011), leading to a decrease in white matter atrophy and a reductionin endogenous apoptotic cells (Shen et al., 2010). Remarkably, NPCsgrafted close to the lesion edges showed less prominent survival, mostlikely because of a gradient of factors and molecules released from thecore of the injury that might have influenced NPC migration and




maturation (Darsalia et al., 2007). Histopathological and MRI analysesshowed that NPCs may also have profound effects on the white matterreorganisation within the IBZ (Jiang et al., 2006). More importantly, thenumber of cells surviving the transplantation does not strictly correlatewith the observed clinical restoration. Indeed, a very low percentage ofsurviving cells (0.28%) could result in a robust behavioural recovery(Bacigaluppi et al., 2009).

On the other hand, NPCs injected systemically into MCAo micemostly maintain an undifferentiated phenotype, while accumulatingat the boundaries of the lesioned area (Bacigaluppi et al., 2009; Sunet al., 2010). Therefore, besides the (limited) cell replacement, NPCsare believed to also exert tissue trophic and immune modulatoryeffects (Bacigaluppi et al., 2008) in stroke models [reviewed by(Kokaia et al., 2012; Martino et al., 2011)]. In line with this, thesub-acute (delayed) NPC injection afterMCAo has been shown to signif-icantly down-regulate multiple RNA species involved in inflammation,including IFN-γ, TNF-α, IL-1β, IL-6 and leptin receptor (Bacigaluppi etal., 2009). NPCs may exert an immune modulatory action, while in anundifferentiated state, causing a profound down-regulation of inflam-matory lymphoid (T cells) and myeloid cells (macrophages) withininflamed brain areas. While the inhibition of the T cell responses byNPCs is quite an established concept (Ben-Hur, 2008), the effects onmicroglia/macrophages at the ischaemic injury site remain controver-sial, as professional phagocytes can exert both protective anddeleteriouseffects after brain injuries, including stroke (Iadecola and Anrather,2011). In addition to having a beneficial effect on axonal sprouting(Daadi et al., 2010), NPC transplantation promotes the infiltration ofCD11b+ myeloid cells in the brain of MCAo mice (Capone et al., 2007;Daadi et al., 2010), thus suggesting that some myeloid cell activationmight be required for transplanted NPCs to exert part of theirneuroprotective action (Capone et al., 2007). Mice with MCAo, selec-tively ablated of CD11b-positive microglia or mineralocorticoid recep-tor (MR)-expressing macrophages, show exacerbation or reductionof the ischaemia-dependent brain injury, respectively (Frieler et al.,2011; Lalancette-Hebert et al., 2007). However, other studies show asignificant reduction in microglia/macrophages in the brain of micewith either ischaemic or haemorrhagic stroke afterNPC transplantation,with improved neuronal survival and locomotor functions (Bacigaluppiet al., 2009; Lee et al., 2008). Interestingly, when injected systemicallyinto mice with collagenase-induced intracerebral haemorrhage (ICH),only very few transplanted NPCs migrated into the brain, with themajority of them accumulating predominantly at the level of the spleen.In ICH mice, only the hyperacute (e.g. 2-h) NPC injection resulted indecreased brain oedema, inflammatory infiltration and neurological de-terioration. Consistently, splenectomyprior to ICH induction eliminatedthe positive effect on oedema and the inflammation of transplantedNPCs (Lee et al., 2008).

Thus, preclinical research in animal models of stroke shows remark-able behavioural and pathological recovery through a number ofbystander mechanisms that grafted NPCs employ to neutralize freeradicals, inflammatory cytokines, excitotoxins, lipases peroxidasesand other toxic metabolites released following an ischaemic event(Bacigaluppi et al., 2009; Ourednik et al., 2002). Once again, NPC trans-plants exert different therapeutic effects (e.g. cell replacement,neurotrophic support, central vs. peripheral immunomodulation, etc.)in response to the (inflammatory) signature of the tissue in whichthey are transplanted, or migrate to after systemic cell injection(Kokaia et al., 2012; Martino et al., 2011). Evidence of the main out-comes following syngeneic NPC transplantation in experimental strokeis shown in Table 1 and summarized in Fig. 1.

Towards clinical trials

Based on the encouraging results collected pre-clinically duringthe last 5–7 years (Table 1), phase I clinical trials have started to beconducted, both in fatal and non-fatal incurable neurological diseases


where the risk/benefit ratio is in theory favourable (Aboody et al.,2011). Besides the unquestionable care regarding the characterisationand manufacture of the medicinal product (Rayment and Williams,2010), one of the other important hurdles in the design of clinicalstudy for (stem) cell therapy trials is defining end-points, as thesewill be the measure of the trial's failure or success. This is particularlychallenging given the inflammatory and degenerative nature of someof the target neurological disorders under consideration and the com-plexity posed by the rate of progression and lack of validated surro-gate disease markers. The overall goal of these phase I NPC humanstudies is therefore to determine whether the transplantation ofNPCs is feasible and safe – before checking for efficacy – with the pri-mary aim of determining the maximum tolerated dose and potentialdose-limiting toxicities. Secondary end-points are usually correlativestudies (e.g. imaging the biodistribution of transplanted cells,assessing their immunogenicity, tracking their survival and fate invivo) that would expand the knowledge gained from conductingthese pilot studies. Long-term follow-up for the assessment of latetoxicity is also important, particularly in patients with non-fatal con-ditions, who might live for many years after transplant.

Following the experimental evidence that human NPCs engraft ro-bustly, migrate extensively and produce sufficient levels of palmitoylprotein thioesterase-1 (PPT1) to alter the behaviour and neuropa-thology of immunodeficient Ppt1−/− mice, as a model of infantileneuronal ceroid lipofuscinosis (NCL) (Tamaki et al., 2009), the firstopen-label dose-escalating phase I human study involving the trans-plantation of allogeneic NPCs was started in May 2006 at the OregonHealth and Science University (OHSU, Portland, OR, USA), investigat-ing infantile and late-infantile NCL Batten disease. The rationale wasthat early intervention with stem cell transplants would supply anexogenous source of the missing enzyme sufficient for the uptakeand cross-correction of host cells. A total of 6 subjects underwenttransplantation in a single-stage procedure with direct delivery ofStemCell, Inc. proprietary single-donor allogeneic free-floating cul-tured, foetally derived brain human NPCs (HuCNS-SC®) to the cere-bral hemispheres and lateral ventricles. Immune suppression wasadministered for 12 months after transplantation. This study hasnow been completed and only 1 out of 6 patients has been reportedto die from disease progression 11 months after treatment. The celltransplantation and combination with prolonged immune suppres-sion were both well tolerated (Steiner et al., 2010).

Neural stem/precursor cell transplants also have the potential toreplace lost or injured cell types, including neurons, astrocytes and oli-godendrocytes (Lepore and Maragakis, 2007). While NPC-derived neu-ronsmay reconstruct circuits or create novel connections; graft-derivedglial cells may be able to replace dysfunctional endogenous dead cells,thus restoring some level of normal (synaptic) communication andpreventing further excitotoxic motor neuron cell death, as describedfor neurodegenerative diseases such as amyotrophic lateral sclerosis(ALS) and leukodystrophies (Allaman et al., 2011; Lobsiger andCleveland, 2007).

In September 2009, NeuralStem, Inc. initiated a phase I trial inamyotrophic lateral sclerosis (ALS) at the Emory University School ofMedicine (Atlanta, GA, USA), using proprietary single-donor allogeneicadherent cultured, foetally derived spinal NPCs (NSI-566RSC). NSI-566cells were surgically implanted in a total of 12 patients via multiple in-jections directly into the thoracic spinal cord (either unilateral or bilat-eral). Combined immune suppression with early methylprednisoloneand prednisone, and late maintenance tacrolimus and mycophenolatemofetil was administered until the end of the study. The clinical assess-ments demonstrated no evidence of acceleration of disease progressionwith the planned 18 month post-transplantation follow-up (Glass et al.,2012; Riley et al., 2012). In June 2012, the Azienda Ospedaliera SantaMaria (Terni, Italy) enrolled the first of a total of 18 ALS patients(subdivided into three independent groups, according to the disabilityat enrolment) and treated themwith intraspinally implanted allogeneic,



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

Sponsor and place Disease Trialphase

Patients(no)

Age atenrolment(y)

Follow-up(months)

Transplant features Status Principalinvestigator

Trialidentifier

Outcomeand notes

Cell type Cell no./patient

Route Time afterdisease/injury

Immunesuppression

StemCells, Inc. atUniversity HospitalBalgrist-UniklinikZurich, (Switzerland)

Thoracic spinalcord injuries(SCI)

I/II 12 18–60 12 HuCNS-SC®(foetal, brain-derived, allogeneic,single donor)

2 × 107 Multiple injections,single dose,intramedullar

≥3 months Y (9 months pt) AR Armin Curt,MD

NCT01321333 NA

ReNeuron, Ltd. atGlasgow SouthernGeneral Hospital,Glasgow (UK)

Stableischaemicstroke (PISCES)

I 12 60–85 24 CTX0E303 (foetal,brain-derived,c-myc immortalised,allogeneic, singledonor)

2–20 × 106 Single injection,four ascendingdoses,intracerebral(putamen)

0.5–5 years NA AR Keith Muir,MD

NCT01151124 NA

Neuralstem, Inc. atEmory University,Atlanta (USA)

Amyotrophiclateral sclerosis(ALS)

I 18 >18 48 NSI-566RSC (foetal,spinal cord-derived,allogeneic, singledonor)

0.5–1 × 106 Multipleinjections,intraspinal

≥1.5 years Y (≥4 months pt) AnR Eva Feldman,MD, PhD

NCT01348451 (Glass et al.,2012; Rileyet al., 2012)

Azienda OspedalieraSanta Maria, Terni(Italy)

ALS I 18 20–75 36 Foetal, brain-derived, allogeneic,single donor

NA Multiple injections,single dose,intraspinal

>6 months NA AR Angelo Vescovi,PhD

NCT01640067 NA

StemCells, Inc. atUniversity ofCalifornia, SanFrancisco (USA)

Pelizaeus–Merzbacherdisease (PMD)

I 4 0.5–5 12 HuCNS-SC® 3 × 108 Multipleinjections,single dose,intracerebral

NA Y (9 months pt) AnR Stephen Huhn,MD

NCT01005004 (Gupta et al.,2012)

StemCells, Inc. atOregon Health andScience University,Portland (USA)

Neuronal ceroidlipofuscinosis(NCL)

I 6 1.5–12 13 HuCNS-SC® 0.5–1 × 109 Multipleinjections,single dose,intracerebral

NA Y (12 months pt) C Robert Steiner,MD

NCT00337636 (Steiner et al.,2010)

StemCells, Inc. RetinaFoundation of theSouthwest, Dallas(USA)

Age-relatedmaculardegeneration(AMD)

I/II 16 >50 12 HuCNS-SC® 0.2–1 × 106 Single injection,single dose,subretinal

NA Y (3 months pt) AR David Birch,PhD

NCT01632527 NA

Abbreviations: pt: post-transplant; NA: information not available. AR: actively recruiting; AnR: actively not recruiting; C: completed.

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ctgov:NCT01321333

ctgov:NCT01151124

ctgov:NCT01348451

ctgov:NCT01640067

ctgov:NCT01005004

ctgov:NCT00337636

ctgov:NCT01632527



free-floating, cultured, foetally derived brain NPCs. This study has todate completed the enrolment of the fourth and final patient of treat-ment group A [main inclusion criteria: inability to walk and forcedvital capacity (FVC) b 60%], who underwent transplantation in Novem-ber 2012 [source: http://www.neurothon.com/trial.htm]. The patientsin this study will be followed for 36 months post-transplantation(clinicaltrials.gov identifier no. NCT01640067).

StemCell, Inc. has also sponsored another two phase I trials withHuCNS-SC® in the X chromosome-linked co-natal leukodystrophyPelizaeus–Merzbacher disease (PMD) and age-related macular degen-eration (AMD). With the PMD trial at the University of California, SanFrancisco (UCSF, San Francisco, CA, USA; clinicaltrials.gov identifier no.NCT01005004), HuCNS-SC® was directly delivered through multipleinjections into the brain of a total of 4 male patients, and immune sup-pressionwas administered for 9 months after transplantation. Multipleassessments were conducted to evaluate safety and to detect evidenceof myelin formation after HuCNS-SC® transplantation, as suggested bythe transplantation of xenogeneic human NPCs into the brains of neo-natal or juvenile immunodeficient shiverer × RAG2−/− (Shi-id) mice, amodel of PMD (Uchida et al., 2012). After a 1-year follow-up no clinicalor radiological adverse effects were directly attributed to the donorcells. Furthermore, serial neurological evaluations, developmental as-sessments, as well as MRI and MR spectroscopy, including high-angular resolution diffusion tensor imaging (DTI), suggest durable cellengraftment and donor-derived myelin (starting at 9 months post-transplant) in the white matter of at least 3 of the 4 transplantedpatients (Gupta et al., 2012).

The direct endogenous oligodendrocyte cell replacement operatedby human NPC transplants in early chronic SCI in immunodeficientNonObese Diabetic/severe combined immunodeficiency (NOD-scid)mice (Salazar et al., 2010) inspired a further StemCell, Inc.-sponsoredphase I/II clinical trial started in March 2011 at the University Hospi-tal Balgrist (Zurich, Switzerland). A single dose (20 × 106 cells) ofHuCNS-SC® was directly implanted through multiple injections intothe thoracic spinal cord of the first patient (of a total of 12) withchronic thoracic (T2–T11) SCI, and immune suppression administeredfor 9 months after transplantation. This trial is enrolling patients 3–12 months after complete and incomplete cord injuries. The estimatedcompletion date of this study is March 2016 (clinicaltrials.gov identifierno. NCT01321333).

A rescue effect on endogenous retinal ganglion cells (RGCs) (McGillet al., 2012) is the idea behind the StemCell, Inc.-sponsored AMD trial atthe Retina Foundation of the Southwest (Dallas, TX, USA), whereHuCNS-SC® is being delivered directly into the subretinal space of oneeye in a single transplant procedure in a total of 16 patients. Immunesuppression is not administered. The patient enrolment started in June2012, and the estimated completion date isMarch2014 (clinicaltrials.govidentifier no. NCT01632527).

In June 2012, the Glasgow Southern General Hospital (Scotland)enrolled the first patient (of a total of 12) into the dose-escalatingPilot Investigation of Stem Cells in Stroke (PISCES) phase I trial tobe transplanted in a single-stage procedure with direct cerebral(intraparenchymal) delivery of Reneuron, Ltd. proprietary single-donor allogeneic adherent cultured, c-myc immortalised foetallyderived brain human NPCs (CTX0E03). Immune suppression is not ad-ministered (clinicaltrials.gov identifier no. NCT01151124). Two recentstudies in rats have provided some evidence in support of the multipleways in which the CTX NPCs promote repair in the stroke-damagedbrain, both through the up-regulation of (neo)angiogenesis (Hickset al., in press), and through the recruitment of pro-neurogenicCD11b+ macrophages/microglial cells in the ischaemic brain (Hassaniet al., 2012).

Finally, there are not as yet any clinical trials using NPCs in MS, de-spite the solid pre-clinical data with somatic NPC transplants in EAE(Payne et al., 2011). However, there is general sense that they willstart soon; and that the approach to MS with non-hematopoietic


(neuroprotective) stem cell strategies will significantly benefit fromthe collection and interpretation of the data from those phase I trialsthat are now being done on diseases that are only apparently notstrictly linked to MS (Martino et al., 2010). A consensus paper hasbeen produced by a group of experts to define the uniform guidelineson the development of hematopoietic and non-hematopoietic stemcell therapies for MS. The main topics of this document include theidentification of the ideal route of stem cell injection, the adequatedosage and type of cells, the appropriate protocols of in vitro expan-sion, the disease level of patients and the statistical power of trials(Martino et al., 2010). Furthermore, new expectations are arisingfrom the use of induced pluripotent stem (iPS) cell-derived NPCs,which would eventually be available as autologous (e.g., if derivedfrom the somatic cells of the same patient) cell sources.

These explorative trials play a key role in stem cell medicine forinflammatory CNS disorders, as they hold a great potential that goesfar beyond the straightforward interpretation of disease-specificoutcomes.

Both phase II dose-escalation studies and the inclusion of non-fatal diseases with larger population bases will finally be facilitatedonce human safety is established. The focus of phase II studies willalso include certain clinical outcomes (e.g., transition to a differentgrade of disability) that can be measured and result in a benefitfor the patient. The active clinical trials with NPCs are shown inTable 2.

Conclusions and future directions

Stem cell-based therapies hold great promise in regenerative neu-roscience (Park et al., 2010). The huge advances made over the lastfew years have enormously deepened our knowledge about the biol-ogy of stem cells, leading to global reconsideration of their therapeu-tic potential and mechanisms of action, as well as their intrinsiclimitations (Martino et al., 2011). Importantly, the use of animalmodels that closely mimic different aspects of human pathologieshas also contributed to increasing our perception of the technicaland biological challenges that still need to be faced before translatinginto clinical practise.

Neural stem/precursor cells not only give rise to neural cells forfunctional replacement, but also exert immune modulatory and tissuetrophic effects, both in vitro and in vivo, after transplantation(Martino et al., 2011). As such, stem cells can be thought of as dynamicmicrosystems able to engage in active communications with surround-ing neighbours and adapt accordingly. We also know that transplantedcells present the peculiar ability to sense the environment, specificallyhoming to the sites of damage (Martino et al., 2011). This is of extremeimportance when considering multifocal diseases, where the in situ in-jection of stem cells is far from being achievable.

These new stem cell therapies may indeed help to explain the evi-dence that somatic stem cell sources of diverse embryonic origin, evenwith low capabilities of neural differentiation, efficiently promote CNSrepair (Martino and Pluchino, 2006; Uccelli et al., 2008). Accordingly,stem cell therapeutic plasticity (or functionalmultipotency) should beviewed as the intrinsic capacity of stem cells to adapt their fate andfunction(s) to specific environmental needs occurring as a result ofdifferent pathological conditions (Martino and Pluchino, 2006; Tenget al., 2011).

Nevertheless, before introducing stem cell treatment wholesale intoclinics, it is crucial to confront several inevitable issues. First, the biolog-ical and functional differences occurring between the rodent and thehuman host, which may in part explain the numerous failures encoun-tered so far, where exciting pre-clinical data did not necessarily trans-late into promising clinical outcomes. Thus, an accurate choice of themodel and the transition to non-human primates is still of vital impor-tance. Further, the establishment of common guidance to preciselydefine the treatment strategies, the maximum tolerated cell dose, the


http://www.neurothon.com/trial.htm



standard conditions for scale-up and manipulation (prior to injection),the window of opportunity to treat and the most appropriate routefor cell delivery are all high priority tasks to guarantee the uniformityof measures (Martino et al., 2010).

The next big challenge will be to understand the dynamics and thelayers of host-to-graft-to-host interactions and to develop reliablesurrogate markers that may enable us to assess some of the key out-come measures of stem cell therapies, including (but not only) stemcell survival, distribution and differentiation/integration. We also en-visage that the in-depth understanding of stem cell-mediated im-mune modulatory actions has a real chance of resulting in (orcontributing to) the development of more efficacious therapies forneurological disorders.

Unfortunately though, many of these efforts risk being corruptedby so-called stem cell tourism. Patients are attracted by false promisesmade by dubious scientists all over the world. In most cases, what isbeing sold as amiracle is in fact a fallacy that may be seriously harmfulto patients' safety. An example of this is a young patient suffering fromataxia telangiectasia who developed a donor-derived brain tumourfollowing neural stem cell transplantation (Amariglio et al., 2009).

Such experiences should once more encourage scientists, clini-cians and regulators to work together to ensure that the huge effortsmade so far and the credibility of the scientific community are not ob-fuscated by the development of deregulated cell-based therapies.

Acknowledgments

The authors are grateful to Gianvito Martino for the vision and con-tinuous inspiration, Jayden A. Smith for critically reviewing the article,Paula Francis for proof edits and Letterio Politi for the diffusionweightedimage shown in Fig. 1. This work has received support from theNationalMultiple Sclerosis Society (NMSS, partial grant RG-4001-A1), the ItalianMultiple Sclerosis Association (AISM, grant 2010/R/31), the ItalianMinistry of Health (GR08-7), Wings for Life, Banca Agricola Popolare diRagusa (BAPR), the European Research Council (ERC) under the ERC-2010-StG grant agreement no. 260511-SEM_SEM and the EuropeanCommunity (EC) 7th Framework Program (FP7/2007-2013) undergrant agreement no. 280772-iONE.

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