Although NPCs have been successfully transplanted in several preclinical models of neurological disorders, the remarkable functional recovery obtained upon NPC transplantation has generally showed little correlation with the absolute numbers of terminally differentiated neural cells originated by transplanted NPCs (see for review Martino and Pluchino 2006). As such, transplanted NPCs very scarcely differentiate into tyrosine hydroxylase (TH)-immunoreactive neurons, when transplanted in rodents with experimental Parkinson’s disease (PD) (Ben-Hur et al. 2004; Ourednik et al. 2002; Rafuse et al. 2005; Richardson et al. 2005) or Huntington’s disease (HD) (McBride et al. 2004; Ryu et al. 2004), despite significant behavioural improvement. Similarly, mice with spinal cord injury (SCI) (Fujiwara et al. 2004; Hofstetter et al. 2005), acute stroke (Chu et al. 2004), or intracerebral haemorrhage (Jeong et al. 2003; Lee et al. 2008) show clinical recovery regardless of transplanted NPCs acquiring preferential astroglial fate in vivo. Other evidence shows that NPCs injected into models of SCI do not differentiate at all into terminally differentiated neuronal cells (Cummings et al. 2005; Fujiwara et al. 2004; Lu et al. 2003; Teng et al. 2002). In EAE, very low differentiation of NPCs into myelin-forming oligodendrocytes is accompanied by neurophysiological evidence of axonal protection and remyelination (Ben-Hur et al. 2003b; Pluchino et al. 2003). In the very same context, much way over 20 % of transplanted cells reaching inflammatory demyelinated areas do not express differentiation markers (Pluchino et al. 2003). It is yet to be shown that transplanted cells can remyelinate efficiently in clinically relevant models of MS. This may depend both on grafting precursor cells that are already programmed to the oligodendroglial lineage (Ben-Hur et al. 1998; Hu et al. 2009) and the use of appropriate models of disease that allow remyelination to occur.

This scarce terminal differentiation and propensity to maintain an undifferentiated phenotype within the host tissue has suggested that transplanted NPCs might be therapeutic efficacious via a number of bystander mechanism(s) alternative to cell replacement.

The poor repair capacity of the adult CNS results from a number of failing repair programmes, including the apparent inability of endogenous progenitors and stem cells to properly respond to disease states, to replace damaged cells and from lack of regenerative capacity of injured axons (Franklin 2002; Franklin and Ffrench-Constant 2008; Martino 2004; Pluchino et al. 2008).

NPC therapy is emerging as a mode of treatment that can enhance the host brain’s ability to repair itself in both aspects (Einstein and Ben-Hur 2008; Pluchino and Martino 2008). Recent studies have focused also on several bystander tissue-protective and -trophic properties of transplanted NPCs. NPCs seeded on a synthetic biodegradable scaffold and grafted into the cord of hemi-sectioned rats induced significant clinical recovery while reducing the necrosis of the surrounding parenchyma and preventing extensive secondary cell loss, inflammation, and glial scar formation (Teng et al. 2002). Moreover, the NPC graft induced a permissive environment for axonal regeneration (Teng et al. 2002). Substantial endogenous reconstitution of the brain structural connectivity has been found following injection of NPCs in biodegradable scaffolds into regions of extensive brain degeneration caused by hypoxia (Park et al. 2002a, b) or following i.c. transplantation of NPCs after ischemia/reperfusion injury in mice (Capone et al. 2007). Transplanted NPCs have rescued endogenous dopaminergic neurons of the mesostriatal system in a PD model in rodents (Ourednik et al. 2002), while prevention of death of motor neurons has been observed when NPCs were transplanted in models of amyotrophic lateral sclerosis (ALS) (Ferrer-Alcon et al. 2007; Kerr et al. 2003; Suzuki et al. 2007). NPC-driven bystander tissue protection has generally led to a reduction of glial scar formation and increase of survival and/or functions of endogenous glial and neuronal progenitors surviving to the pathological insult.

NPC trophic effects relate in part to increased in vivo bioavailability of major neurotrophins (e.g. nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], ciliary neurotrophic factor [CNTF], glial-derived neurotrophic factor [GDNF]) (Chu et al. 2004; Einstein et al. 2006; Lu et al. 2003; Pluchino et al. 2003; Teng et al. 2002 and others). The multiple roles of neurotrophins as mediators in cell cycle regulation, cell survival, and differentiation during development and adulthood make them potential candidates for the regulation of endogenous NPC proliferation and differentiation following brain injury and for modulation of host environment into more permissive for regeneration. For example, neurotrophins secreted by transplanted NPCs help promote corticospinal axon growth after transplantation onto an organotypic co-culture system, containing dissected brain cortex and spinal cord (Kamei et al. 2007). Moreover, several neurotrophins that may be released by NPCs were shown to inhibit EAE. Insulin-like growth factor (IGF)-1 and glial growth factor (GGF)-2 are neurotrophic factors that promote survival and proliferation in the oligodendrocyte lineage (Barres et al. 1992; Canoll et al. 1996, 1999; Mason et al. 2000). Treatment with these factors was beneficial clinically and pathologically in animals with EAE (Akassoglou et al. 1998; Cannella et al. 1998; Yao et al. 1996). Interestingly, their effect was mediated not only by enhancing oligodendrocyte survival (Butzkueven et al. 2002; Linker et al. 2002) but also by decreasing neuroinflammation (Cannella et al. 1998; Flugel et al. 2001; Ruffini et al. 2001; Villoslada et al. 2000).

The broad (tissue) trophic support by transplanted stem cells has been studied extensively in models of spinal cord injury and attributed to multiple mechanisms. These include production of neurotrophic growth factors that enhance axonal regeneration (Lu et al. 2003); induction of matrix metalloproteinases that degrade the extracellular matrix and cell surface molecules that impede axonal regeneration, thus enabling axons to extend through the glial scar (Zhang et al. 2007); induction of angiogenesis in the lesioned tissue, which provides trophic support and enables tissue repair (Rauch et al. 2009); providing proper realignment and guidance to enable axonal regeneration along long fibre tracts (Pfeifer et al. 2004); and increasing remyelination in the lesion by both endogenous and graft-derived myelin-forming cells to enhance action potential conduction and limit secondary axonal degeneration (Keirstead et al. 2005). In addition, NPC transplantation has been shown to facilitate endogenous myelin repair following chronic cuprizone-induced demyelination, by induction of resident OPC proliferation and differentiation (Einstein et al. 2009).

Recent work has also indicated that transplanted stem cells, including NPCs, can enhance endogenous neurogenesis in certain physiological and pathological conditions (Hattiangady et al. 2007; Munoz et al. 2005). Mice exposed prenatally to opioids display impaired learning associated with reduced neurogenesis, and transplantation of NPCs improves learning functions, as well as host brain-derived neurogenesis in the DG of the hippocampus (Ben-Shaanan et al. 2008). A similar neurotrophic effect was also reported in physiological ageing. While neurogenesis in the DG declines severely by middle age, transplantation of NPCs stimulates the endogenous NPCs in the SGZ to produce new dentate granule cells (Hattiangady et al. 2007).

Thus, transplanted NPCs may enhance the adult CNS capacity to repair itself by restoring the ability of endogenous progenitors and stem cells to both respond properly to disease state and replace damaged CNS cells and the ability of severed axons to regenerate. Yet, a current and comprehensive illustration of whether these and other ­(tissue)trophic properties of NPCs are also relevant to EAE and MS is partially lacking.

Important progress towards the application of stem cell therapies in MS has been made recently with the observation that NPC transplantation attenuates the clinical course of acute (Einstein et al. 2003), chronic (Einstein et al. 2006; Pluchino et al. 2003) and relapsing (Pluchino et al. 2005; Pluchino et al. 2009) EAE in rodents and primates (Pluchino et al. 2009a). While dealing with the expected regenerative potential of stem cell transplantation as direct cell-replacement therapy, recent work has highlighted peculiar additional immune modulatory mechanisms by which transplanted NPCs exhibit therapeutic effects in preclinical models of MS. The first indication of novel (anti-inflammatory?) effect of NPCs was obtained when neurospheres were transplanted i.c. in acute spinal cord homogenate (SCH)-induced EAE Lewis rats (Einstein et al. 2003). These EAE rats show acute, reversible paralytic disease that is the result of disseminated CNS inflammation without demyelination or axonal injury. NPC transplantation in SCH-induced EAE Lewis rats attenuated the inflammatory brain process and clinical severity of disease (Einstein et al. 2003).

Subsequent studies examined the effect of NPC transplantation, upon either i.c. or i.v. cell injection, in the myelin oligodendrocyte glycoprotein (MOG)35-55-induced EAE in C57BL/6 mice. In this model, there is an acute paralytic disease due to a T cell-mediated autoimmune process that causes severe axonal injury and demyelination. Subsequently, the mice remain with fixed neurologic sequel, the severity of which is correlated with the extent of axonal loss (Wujek et al. 2002). NPC transplantation in MOG35-55-induced EAE mice attenuated the inflammatory process, rescued the endogenous pool of oligodendrocyte progenitor cells, reduced acute and chronic axonal injury and demyelination, and improved the overall clinical and neurophysiological performance of the mice (Einstein et al. 2006; Pluchino et al. 2003). Recent evidence has now suggested that this latter phenomenon may be dependent on the capacity of transplanted NPCs to engage multiple mechanisms of action within specific microenvironments in vivo (Martino and Pluchino 2006). Among a wide range of potential therapeutic actions, and in addition to the cell replacement and (tissue) trophic capacities (Pluchino et al. 2003), remarkable immune modulatory capacities are described for transplanted NPCs within specific CNS (Einstein et al. 2003, 2006; Pluchino et al. 2003, 2005; Swanborg 2001) and non-CNS areas (Einstein et al. 2007). As such, considerable proof that NPC-mediated bystander immune regulation may take place both in the CNS, at the level of the atypical perivascular niches (Pluchino et al. 2005), as well as in secondary lymphoid organs, such as the lymph nodes (Einstein et al. 2007) or the spleen (Lee et al. 2008), has recently been provided. However, the exact mechanisms by which transplanted NPCs attenuate CNS vs. peripheral inflammation are not yet clear.

One school of thought has first suggested an immune suppressive effect, by which NPCs induce apoptosis of Th1—but not Th2—cells selectively, via the inflammation-driven up-regulation of membrane expression of functional death receptor ligands (e.g. FasL, TRAIL, Apo3L) on NPCs (Pluchino et al. 2005). Alternatively, it has been suggested that NPCs inhibit T cell activation and proliferation by a non-specific, bystander immune suppressive action (Einstein et al. 2007). This notion emerged from co-culture experiments that showed a striking inhibition of the activation and proliferation of EAE-derived, as well as naive T cells by NPCs, following stimulation by various stimuli (Einstein et al. 2003, 2007). The suppressive effect of NPCs on T cells was accompanied by a significant suppression of pro-inflammatory cytokines, such as interleukin (IL)-2, TNFα, and IFNγ (Einstein et al. 2007). Moreover, NPCs inhibited multiple inflammatory signals, as exemplified by attenuation of T cell receptor-IL2- and IL6-mediated immune cell activation and/or proliferation (Fainstein et al. 2008). The relevance of such NPC/T cell interaction was first suggested when NPCs i.v. injected prior to EAE disease onset (e.g. at 8 days after the immunization) were transiently found in peripheral lymphoid organs, where they interacted with T cells to reduce their encephalitogenicity (Einstein et al. 2007). In this protocol i.v. NPC injection at an early time point, transplanted cells did not cross the blood–brain barrier, and their entire effect was mediated by peripheral immune suppression, resulting in reduced immune cell infiltration into the CNS and consequently milder CNS damage.

Recent studies have started addressing the role of individual molecular candidates in regulating this novel immune modulatory (or regulatory) capacity of transplanted NPCs in EAE. As such, when injected s.c. in a setup of passive cell vaccination in EAE mice, NPCs have shown remarkable capacity to target and synergize with immune cells in secondary lymphoid organs (but not in the CNS), where they stably (e.g. for more than 2 months after cell injection) modify the perivascular microenvironment. Within this context, surviving NPCs have hindered the activation of myeloid dendritic cells (DC) via a bone morphogenetic protein (BMP)-4-dependent mechanism, which was completely reverted by the BMP antagonist Noggin (Pluchino et al. 2009b; Pluchino et al. 2009).

Indeed, such a broad immune regulatory capacity has more recently been shown also for human somatic (Pluchino et al. 2009a) as well as human ES-cell-derived NPCs (Aharonowiz et al. 2008) after transplantation in non-human primates and mice with EAE, respectively. Again, the therapeutic effects of both these latter NPC sources was not related to graft or host-driven remyelination but was rather mediated by an immune regulatory mechanism that protected the CNS from immune-mediated injury.

Taken together, the results of experimental studies that we describe here have started to reveal the mechanisms by which neural stem cells act as immune regulators and in so doing create environments within the CNS that favour protection and repair. Such mechanistic insights are essential if the full therapeutic potential of CNS stem cells is to be achieved.