Role of mesenchymal stem cells in neurogenesis and nervous system repair

Neurochemistry International 59 (2011) 347–356

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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Review

Role of mesenchymal stem cells in neurogenesis and nervous system repair Daniel J. Maltman a,b, Steven A. Hardy a,b, Stefan A. Przyborski a,b,* a b

School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK Reinnervate Limited, NETPark Incubator, Thomas Wright Way, Sedgefield, Co Durham TS21 3FD, UK

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 March 2011 Received in revised form 27 May 2011 Accepted 9 June 2011 Available online 21 June 2011

Bone marrow-derived mesenchymal stem cells (MSCs) are attractive candidates for use in regenerative medicine since they are easily accessible and can be readily expanded in vivo, and possess unique immunogenic properties. Moreover, these multipotent cells display intriguing environmental adaptability and secretory capacity. The ability of MSCs to migrate and engraft in a range of tissues has received significant attention. Evidence indicating that MSC transplantation results in functional improvement in animal models of neurological disorders has highlighted exciting potential for their use in neurological cell-based therapies. The manner in which MSCs elicit positive effects in the damaged nervous system remains unclear. Cell fusion and/or ‘transdifferentiation’ phenomena, by which MSCs have been proposed to adopt neural cell phenotypes, occur at very low frequency and are unlikely to fully account for observed neurological improvement. Alternatively, MSC-mediated neural recovery may result from the release of soluble molecules, with MSC-derived growth factors and extracellular matrix components influencing the activity of endogenous neural cells. This review discusses the potential of MSCs as candidates for use in therapies to treat neurological disorders and the molecular and cellular mechanisms by which they are understood to act. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Mesenchymal stem cell Neurogenesis Nervous system repair Mechanisms Paracrine Factors Review

1. Introduction Mesenchymal stem cells (MSCs) are stromal cells found in a wide range of adult tissues including the bone marrow. These cells are the origin of several mesodermal cell lineages, characterised by an ability to differentiate into derivatives including bone, fat, and cartilage. MSCs were originally identified as a fibroblastic cell population within the bone marrow, which is distinct from the haematopoietic lineage (Friedenstein et al., 1976). They can be readily isolated from a range of sources including bone marrow, adipose tissue, peripheral blood, umbilical cord blood, amniotic fluid, tendon and ligaments, chorionic villi of the placenta, synovial membranes, olfactory mucosa, deciduous teeth and foetal liver, lung and spleen (Delorme et al., 2010; Igura et al., 2004; in’t Anker et al., 2003; Kuznetsov et al., 2001; Miura et al., 2003; Rosada et al., 2003; Salingcarnboriboon et al., 2003; Seo et al., 2004; Tome et al., 2009; Tsai et al., 2004; Vandenabeele et al., 2003). In fact it now seems that MSCs are to be found in most post-natal organs and tissues, although individual populations may display subtle

* Corresponding author at: School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK. Tel.: +44 0191 334 1201; fax: +44 0191 334 3988. E-mail address: [email protected] (S.A. Przyborski). 0197-0186/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.06.008

differences related to their specific source tissue (da Silva Meirelles et al., 2006). The isolation of MSCs from vascular tissues (da Silva Meirelles et al., 2006), supports the theory that MSCs originate from perivascular cells (Farrington-Rock et al., 2004; Shi and Gronthos, 2003), a viewpoint that might account for the widespread distribution of MSCs in the body. The bone marrow remains the most commonly used source for MSC isolation, and even though MSCs represent only a tiny fraction (around 0.01%) of the total marrow population (Pittenger et al., 1999), their adherent nature facilitates rapid expansion and enrichment from heterogeneous starting cultures. Thus, the most common method of MSC isolation involves aspiration of bone marrow directly onto tissue culture plasticware, where MSCs will adhere to the culture surface, while contaminating haematopoietic cells remain in suspension. MSCs appear spherical during cell division, after which they increase in size and spread out, acquiring stromal, fibroblastic morphology. As cell division continues, MSCs form colonies, and eventually monolayer cultures as these colonies merge. Confirmation of MSC populations involves the analysis of celltype specific markers. At present, no single definitive MSC marker exists and it is therefore common practice to monitor a panel of molecules to build highly descriptive expression profiles. For example, flow cytometry is often used to confirm MSC phenotype by examining the expression of MSC-positive cell surface markers

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such as CD29, CD44, CD54, CD55, CD73, CD90, CD105, CD106, CD117 and CD166, and negative surface markers, usually antigens of haematopoietic cells, such as CD11b, CD14, CD31, CD33, CD34 and CD45 (Pittenger et al., 1999; Shyu et al., 2006). In addition to cell surface antigens, intracellular proteins including fibronectin, vimentin and a-smooth muscle actin (a-SMA) are also positive markers for MSCs (Pittenger et al., 1999). As previously noted, much attention is currently focused on the utilisation of cell surface marker expression as a means of producing more homogenous MSC cultures by fluorescence-activated cell sorting (FACS) (Alsalameh et al., 2004; Dennis et al., 2002). Demonstration of function remains the most convincing proof of cellular identity, and in the case of MSCs this includes proving the ability to differentiate into cells possessing phenotypes of, for example, bone, fat, and cartilaginous tissues. Such differentiation can be induced in vitro by the addition of specific inductive factors to the culture medium. For example, osteogenic differentiation, MSCs can be treated with a cocktail of dexamethasone, ascorbic acid 2-phosphate and b-glycerophosphate, while adipogenesis can be achieved by cyclic treatment with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine (IBMX), followed by treatment with insulin. MSCs are regarded as exciting candidates to provide reparative/regenerative action against a variety of disease types (Pittenger et al., 1999). They are traditionally categorised as ‘multipotent’, since they possess the potential to differentiate into multiple, closely related cell lineages specific to their germ layer of origin (i.e. bone, fat and cartilage). It stands to reason that MSCs have attracted attention as potential candidates for the treatment of diseases affecting mesodermal tissues. However, in recent years it has become clear that MSCs could be used for neurological treatments, based on convincing evidence that their administration results in functional recovery in various animal models of neural perturbation. What remains unclear is the nature of the major mechanism(s) responsible. Three main theories have attempted to explain MSC-mediated neurogenesis/neural repair. These are, ‘trans’-differentiation, cell fusion, and paracrine activity through the release of soluble factors. While there is evidence for all three phenomena, there is debate over the contribution that each of these models can realistically make to neural recovery. 2. Possible roles for MSCs in the treatment of neurological deficits For several reasons, MSCs are emerging as particularly strong candidates for cellular therapies (Fig. 1). First, they can be isolated from a wide range of autologous sources (Gronthos et al., 2001; Igura et al., 2004; in’t Anker et al., 2003; Kuznetsov et al., 2001; Miura et al., 2003; Rosada et al., 2003; Salingcarnboriboon et al., 2003; Seo et al., 2004; Tsai et al., 2004; Vandenabeele et al., 2003), some of which are readily accessible (particularly bone marrow and adipose tissue), using robust, well-established techniques (da Silva Meirelles et al., 2006; Gronthos et al., 2001; Pittenger et al., 1999). Second, their high proliferative potential allows for rapid MSC expansion ex vivo, while maintaining multipotentiality. Finally, MSCs are potentially suitable for use in allogeneic as well as autologous transplantation (Aggarwal and Pittenger, 2005), because they express intermediate levels of MHC Class I antigens and negligible levels of MHC Class II antigens, as do differentiated MSC derivatives (Le Blanc et al., 2003). This means that immune responses commonly associated with allogeneic transplantation may be avoidable, thereby minimising the need for rigorous immune suppression following treatment. It has also been reported that MSCs do not express co-stimulatory molecules (Majumdar et al., 2003). Moreover, following allogeneic trans-

Paent or Donor

MSC

In vitro expansion

In vitro differenaon

Genec manipulaon

Extracon of soluble factors

Paent Fig. 1. Possible routes to MSC-based cell therapy. A number of key properties make MSCs strong candidates for future use in regenerative medicine. They can be readily isolated and rapidly expanded ex vivo, and their low immunogenicity may allow for xenographic as well as autologous transplantation. Because MSCs adapt to different microenvironments it may be possible to administer them either without prior stimulation, or following pre-differentiation or preconditioning in specific preparation for their intended use. The natural homing and secretory properties of MSCs may allow them to be exploited as vehicles for the delivery of specific therapeutic proteins following genetic manipulation. Finally, MSC-derived factors could form the basis of cell-free therapeutic cocktails.

plantation, MSCs have been shown to evade immune recognition and remain readily detectable in recipients at extended time points (Aggarwal and Pittenger, 2005). Transplantation of MSCs has also been associated with reduced incidence of graft-versushost-disease (GVHD), and there are even reports of MSC transplantation being used in the treatment of GVHD (Le Blanc et al., 2004). The major clinical implications of these findings are that MSCs could potentially be transplanted between unrelated individuals. MSCs hold clear promise as a source of cell-based therapies for a wide variety of illnesses, including those affecting the heart, bone, kidneys and skin, and a number of clinical studies have already been performed (Salem and Thiemermann, 2010). For the purposes of this review, we will focus on the potential role of MSCs as therapeutic agents in neurology. 3. Post-transplantation migration and engraftment of MSCs in the nervous system An important first step when considering a cell type for use in regenerative medicine is to investigate the ability of that cell to migrate, engraft and survive at sites of injury. The stem cell niche plays a crucial role in the maintenance and function of stem cells, providing a delicate balance between cell– cell interactions, extracellular matrix contact, oxygen tension, pH and exposure to growth factors, etc. (Fuchs et al., 2004). A number of studies have investigated the ability of MSCs to tolerate the essentially alien microenvironment presented by nervous tissues. Specifically, the survival and engraftment of transplanted MSCs has been assessed. It has been demonstrated that upon transplantation into the brain, MSCs can survive and undergo subsequent engraftment and migration.

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Azizi et al. (1998) examined the transplantation of both rat and human MSCs directly into the rat corpus striatum, which is a component of the basal ganglia and functions primarily in the coordination of movement. In positive controls, rat astrocytes were injected into the corpus striatum, since astrocytes had previously been shown to undergo post-transplantation engraftment and migration throughout the CNS, in a manner analogous to neural stem cells (Andersson et al., 1993; Zhou and Lund, 1992). Two weeks post-transplantation, engraftment success rates of 20% and 30% were observed for human and rat MSCs, respectively, indicating that xeno-transplantation as well as autologous transplantation of MSCs is possible due to their unique immunological properties. Furthermore, MSCs were found in several successive layers of the brain up to 72 days post-transplantation, and this was mirrored in the pattern of migration of astrocytes (Azizi et al., 1998) which were already known to engraft and migrate in a manner similar to neural stem cells following transplantation into the brain (Andersson et al., 1993). There are many other reports of similar observations in alternative systems including mouse and higher order primates (Ankeny et al., 2004; Deng et al., 2006a,b; Isakova et al., 2006; Kopen et al., 1999; Phinney et al., 2006). Such research demonstrates the ability of MSCs to engraft and migrate, which reinforces the promise surrounding MSCs as potential candidates in cellular therapies of neural disorders. Beyond analysis of engraftment and migration, administration of MSCs has resulted in various degrees of functional recovery in models of Huntington’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, stroke, traumatic brain injury and also damage to the spinal cord and peripheral nerves (Chen et al., 2003; Chopp et al., 2000; Cuevas et al., 2002; Dezawa et al., 2004; Glavaski-Joksimovic et al., 2009; Kamada et al., 2011; Lescaudron et al., 2003; Li et al., 2001a,b, 2005; Mahmood et al., 2001; Mandalfino et al., 2000; Mazzini et al., 2008; Osaka et al., 2010). MSC treatment has also been reported to improve function/behaviour in psychiatric disorders such as depression (Tfilin et al., 2010). Restoration of functional activity can be assessed in many ways ranging from simple behavioural tests to more complex cell function assays. The following sections discuss specific examples in the context of the working hypotheses put forward to explain the mechanisms by which MSC mediate neural recovery. 4. Mechanisms of MSC-mediated neural functional recovery Advancement in our understanding of how MSCs exert neurogenic effects will facilitate their development as therapeutic agents. To date, the primary mechanism(s) by which MSCs promote neural function remains uncertain. Opinion is divided between three main models: transdifferentiation, cell fusion, and the secretion of trophic factors. In the first, MSCs are proposed to

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differentiate into neural cell types capable of directly replacing neural tissue that has been damaged as a result of disease. In the second model, MSCs are thought to undergo spontaneous fusion events with host neural cells, and in doing so acquire the phenotypic properties of those neural cells. In the third mechanism, MSCs exert neurotrophic effects by releasing a complement of molecules that promote endogenous repair mechanisms. Such molecules may include neurotrophic growth factors and cytokines as well as extracellular matrix proteins and associated matrix regulatory components. Together it is envisaged that these factors combine to stimulate resident neural cells, thereby activating neurogenesis or neuroprotective mechanisms (Table 1). 4.1. Transdifferentiation of MSCs into neural cell types In recent years the notion that multipotent stem cells are restricted in their potency to the formation of cell types of only germ layer has been challenged. Numerous reports have claimed that different stem cells are capable of crossing the germ layer boundary to form cell types of alternative layers – a process termed ‘transdifferentiation’. A series of studies have proposed that MSCs too possess greater differentiation potential than previously thought, including the ability to form both endodermal and ectodermal tissue (Krause et al., 2001; Petersen et al., 1999; Sanchez-Ramos et al., 2000; Woodbury et al., 2000, 2002). 4.1.1. MSC transdifferentiation into neural cell types in vitro Studies demonstrating putative MSC transdifferentiation towards neuronal and/or glial fates in vitro employ widely varying culture conditions. Broadly, these protocols involve chemical induction (media supplementation with cocktails of small molecules or chemicals) (Deng et al., 2001; Hellmann et al., 2006; Hung et al., 2002; Ikeda et al., 2005; Jori et al., 2005; Kohyama et al., 2001; Levy et al., 2003; Munoz-Elias et al., 2004; Rismanchi et al., 2003; Woodbury et al., 2000, 2002), biological treatment or gene transfection (of MSCs with biomolecules known to mediate neurogenesis) (Barzilay et al., 2008; Dezawa et al., 2004; Hermann et al., 2004; Kamada et al., 2005; Kohyama et al., 2001; Krampera et al., 2007; Tatard et al., 2007; Tohill et al., 2004; Trzaska et al., 2007, 2009), or co-culture of MSCs with neural cell types (in which MSCs are induced to adopt neural phenotypes as a result of direct cell–cell interactions and/or the interplay of soluble factors) (Choong et al., 2007; Jiang et al., 2002, 2003; SanchezRamos et al., 2000; Wislet-Gendebien et al., 2003, 2005). Two significant studies that demonstrated the concept of MSC in vitro transdifferentiation are those of Woodbury and SanchezRamos (Sanchez-Ramos et al., 2000; Woodbury et al., 2000, 2002). However, recent research has raised doubt over the authenticity of neural derivatives that result from specific in vitro MSC differentiation protocols. One of the major concerns surrounds the

Table 1 Example growth factors/cytokines expressed by MSCs. Growth factor

Full name

Reference

BDNF bFGF CNTF FGF-2 GDNF HGF IGF IL6 NGF NT-3 VEGF

Brain-derived neurotrophic factor Basic fibroblast growth factor Ciliary neurotrophic factor Fibroblast growth factor 2 Glial cell line-derived neurotrophic factor Hepatocyte growth factor Insulin-like growth factor Interleukin 6 Nerve growth factor Neurotrophin 3 Vascular endothelial growth factor

Chen et al. (2002, 2005), Kan et al. (2010), and Nicaise et al. (2011) Chen et al. (2002, 2005) Chen et al. (2002, 2005) Nicaise et al. (2011) Chen et al. (2002, 2005) and Nicaise et al. (2011) Chen et al. (2002, 2005) Nicaise et al. (2011) Djouad et al. (2007) Nicaise et al. (2011) Chen et al. (2002, 2005) Chen et al. (2002, 2005) and Djouad et al. (2007)

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remarkably short timescales of differentiation, since neural differentiation is a highly complex process, which in vivo requires the spatial and temporal regulation of a wide array of genes. The possibility for MSCs to undergo the complex molecular events associated with neural differentiation within a matter of hours to form mature, functional neural derivatives, seems highly questionable and improbable. For instance, the differentiation of MSCs into ‘gold standard’ mesenchymal derivatives, such as bone or fat, occurs over a timescale of several days and weeks, and the earliest phenotypic indications of differentiation appear only after 5 days post-induction of differentiation. Several groups have attempted to explain the response of MSCs cultured under these so called neural transdifferentiation conditions. Evidence suggests that apparent transdifferentiation events are in fact artificial responses to cell culture growth conditions rather than the result of true neural differentiation pathways. Most notably, the acquirement of neural morphology has been attributed to cell–stress response(s) (Bertani et al., 2005; Lu et al., 2004; Neuhuber et al., 2004; Woodbury et al., 2000). For example, the formation of neural-like structures did not involve genuine neurite outgrowth which is a highly dynamic process involving microtubule re-modelling and the extension of neurites from a central cell soma, a principle event during neuronal differentiation. Instead, the MSC membrane and cytoplasm retracted towards the centre of the cell, forming spherical cell bodies tight around the nucleus, reminiscent of a neural soma. This resulted in the putative neurites which were in fact cytoplasmic extensions remaining adhered to growth surfaces during membrane retraction. This was subsequently found to be the consequence of cytoskeletal collapse and could be mimicked by treatment MSCs with actin cytoskeleton-disrupting pharmacological agents, such as cytochalasin B and D (Bertani et al., 2005; Croft and Przyborski, 2006). Primary fibroblasts, HEK293 cells (a human embryonic kidney cell line) and PC-12 cells (a human pheochromocytoma cell line) have all been shown to undergo the same morphological changes in response to chemical induction (Bertani et al., 2005; Croft and Przyborski, 2006; Lu et al., 2004; Neuhuber et al., 2004). Furthermore, MSCs acquire putative neuronal morphologies in the presence of protein synthesis inhibitors (Tondreau et al., 2004), and it is highly unlikely that any form of differentiation could take place under such conditions. In addition, microarray data shows that chemical induction of transdifferentiation in MSCs does not significantly affect neural marker transcript levels (Lu et al., 2004). And finally, MSC expression of neural markers during transdifferentiation protocols cannot not in itself be taken as bona fide neural differentiation since MSCs are known to spontaneously express neural proteins, even under standard culture conditions (Deng et al., 2006a,b; Lamoury et al., 2006; Woodbury et al., 2000).

migration. Neural-like marker expression profiles following transplantation into the nervous system, has contributed to the speculation surrounding MSCs as potential alternatives to neural stem/progenitor cells (NSPCs) for cellular therapies against neurological and neurodegenerative disorders (Azizi et al., 1998). Following administration into the lateral ventricle of neonatal mice, MSCs have been found to proliferate and migrate throughout the forebrain and cerebellum, in a manner reflective of NSPCs (Kopen et al., 1999). These transplanted cells strongly expressed the astrocytic marker, glial fibrillary acidic protein (GFAP), and to a lesser degree a neuronal neurofilament marker. Since the cells were administered in an undifferentiated state, it was proposed that expression of neural markers was induced by exposure to the brain microenvironment. This may have significant clinical implications such as potential for MSCs to be administered to patients without any prior stimulation in vitro. In a separate study it was reported that MSCs transdifferentiated in vivo to produce dopaminergic neurons following transplantation into a mouse model of Parkinson’s disease (Kopen et al., 1999). This was supported by the expression of tyrosine hydroxylase, an enzyme marker of dopaminergic neurons. Tyrosine hydroxylase expression would be required by dopaminergic cells since this enzyme catalyses the conversion of tyrosine to dihydroxyphenylalanine, or DOPA, a precursor of dopamine. More recently, tyrosine hydroxylase expression has been associated with chemically induced differentiation of human umbilical cordderived MSCs (Tio et al., 2010). Such experiments may hold promise for the treatment of neurological diseases such as Parkinson’s, the main causative mechanism of which is loss of dopamine neurons in the substantia nigra. However, more work is required to validate true dopaminergic functionality. In a subsequent study, the same group reported that MSCs expressed two mature neuronal markers, NeuN and MAP2, as well as the mature astrocytic marker, GFAP following transplantation (Li et al., 2001a,b). Even if neural marker-expressing MSCs were found to possess genuine neural functionality, it seems doubtful that the low measured rates of transdifferentiation could fully account for the neurological improvements reported in models of neurological and neurodegenerative disorders. The number of MSCs which express markers of neural differentiation, is very low, as percentage of the total number of MSCs that successfully engraft following transplantation (Deng et al., 2006a,b). For instance, only 1% express neuronal markers and only 5% of these express glial markers. There are several other studies reporting the transdifferentiation of MSCs into neural cell types in vivo (Li et al., 2002; Lu et al., 2006; Mezey and Chandross, 2000; Munoz-Elias et al., 2004). However, the mechanism(s) by which this occurs remain to be elucidated.

4.1.2. MSC transdifferentiation into neural cell types in vivo The growth of cells in culture is highly artificial, and places cells under considerable stress, forcing the adoption of unnatural morphologies. This has been highlighted in the previous section, with the two-dimensional culture environment contributing to artifactual morphology during the implementation of MSC transdifferentiation protocols. The two-dimensional environment undoubtedly influences cellular behaviour and function. Therefore, in order to gain a more accurate understanding of MSC potential for transdifferentiation into neural cell types, the phenomenon must also be investigated in more representative culture systems or in vivo. Earlier we described that MSCs are able to survive when transplanted into the unfamiliar environment of the nervous system, where they can undergo successful engraftment and

4.2. Spontaneous cell fusion of MSCs with host neural cells As putative neural transdifferentiation of MSCs in vivo appears to occur at very low frequency, it is highly likely that other mechanisms contribute to the functional recovery observed in models of neurological disease. Spontaneous fusion between transplanted MSCs and host neural cells has been suggested as an alternative model. A number of independent studies have examined spontaneous stem cell fusion events. Two examples (Terada et al., 2002; Ying et al., 2002) proposed that stem cells could demonstrate alternative phenotypes as a result of spontaneous fusion with other cells, with resultant hybrid cells adopting the phenotypic traits of the recipient cell type. It is known that bone marrow cells can spontaneously fuse with other cell types in co-culture systems, and in so doing, acquire

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phenotypic traits of these cells. It appears as though the acquirement of recipient cell properties also extends to plasticity (Terada et al., 2002; Ying et al., 2002). Demonstration of cell fusion events does not disprove the theory of transdifferentiation, and while it has been shown that MSCs can spontaneously fuse with neural cell types (Crain et al., 2005), it seems that MSCs can transdifferentiate into neural cell types by independent mechanisms (Terada et al., 2002). Although cell fusion represents a possible mechanism of MSCmediated neural functional recovery, the evidence indicates that the phenomenon takes place at extremely low frequencies. For example a fusion rate of 2–11 clones per million cells has been observed (Terada et al., 2002). The low frequency of both transdifferentiation and spontaneous cell fusion suggests that the contribution of these processes to MSC-mediated neurogenesis/neural repair is likely to be minor. In general, the extreme rarity of MSC transdifferentiation and spontaneous cell fusion events strongly suggests that other mechanisms play an important role in MSC-mediated neural recovery. 4.3. MSCs as trophic mediators of neural differentiation There is significant evidence to suggest that stem cells play important secretory/uptake roles in the regulation of trophic factors and cytokines in their niche microenvironments in vivo. Trophic roles for stem cells have been described for both embryonic and adult stem cell populations, including haematopoietic and neural stem cells (Guo et al., 2006; Lu et al., 2003; Terada et al., 2002). With regard to MSCs, there are several studies demonstrating trophic function in vivo, and perhaps the best characterised is the role they perform in the regulation of haematopoiesis (Cabanes et al., 2007). The third major mechanism receiving attention as an explanation for MSC promotion of neurological recovery relates to trophic influence on neighbouring cells. It is thought that cocktails of factors capable of ameliorating neural damage are secreted by MSCs into neural niche microenvironments. Such paracrine effects may include direct neurotrophic/neuroprotective activity on resident NSPCs (Chen et al., 2007; Haynesworth et al., 1996), as well as neurotrophic (NTF) delivery and physical guidance mechanisms directed by extracellular matrix (ECM) molecules. In some cases the actual presence of MSCs at sites of neural damage may be important. A recent study using a model of periventricular white matter injury reported functional improvements following MSC injection. It was concluded that while MSC-secreted factors may have contributed to reduced hypomyelination, the presence of the MSCs themselves was also beneficial (Chen et al., 2010). In the following sections MSC-secreted molecules are discussed according to their categorisation as either neurotrophic or nonneurotrophic factors. 4.3.1. Neurotrophic factors secreted by MSCs MSCs secrete a wide array of neurotrophins, growth factors, cytokines and other soluble factors (Crigler et al., 2006). Studies in vitro and in vivo demonstrate that such factors can promote cell proliferation, survival and differentiation. Several of the soluble factors secreted by MSCs are known to exert neurogenic effects on NSPCs, including the proteins nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and ciliary neurotrophic factor (CNTF) (Chen et al., 2002, 2005). The known secretome of MSCs also consists of a wide variety of interleukin (IL) molecules, including IL-6, -7, -8, -11, -12, -14 and 15, in keeping with their

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housekeeping role as mediators of haematopoiesis (Djouad et al., 2007). Besides long-established roles for IL molecules in haematopoiesis, it is increasingly evident that these molecules have important functions during neural development. The possibly of cross-talk between haematopoietic and neural differentiation pathways supports a role for MSCs as trophic mediators in both capacities (Chen et al., 2007; Majumdar et al., 1998). Proteomic analysis of the MSC secretome would suggest that a combination of soluble factors and cytokines, rather than any single factor is likely to be responsible for neurological recovery following MSC transplantation (Mehler et al., 1993). The beneficial effects of MSC-derived factors in mediating neurological recovery are highly documented in the literature. The effects of these factors can be broadly classified as angiogenic (the formation of new blood vessels), neurogenic (the formation of new neural tissue), neuroprotective (the protection of neural tissue from degeneration and apoptosis), synaptogenic (the formation of synapses and synaptic contacts), and inhibition of scarring (prevention of scarring that would prevent reconstruction of neural circuitry following injury or damage) (Chopp and Li, 2002; Fiore et al., 2002; Hagg, 2005; Hsu et al., 2007; Palmer et al., 1999; Schanzer et al., 2004; Schinkothe et al., 2008) (Fig. 2). Here we will focus on a selection of key studies which demonstrate that MSCs act as trophic mediators of neurological recovery. Chen and colleague demonstrated that the major mechanism behind MSC-mediated repair of neural tissue in an in vitro model of stroke was a result of cytokine and soluble factor secretion by MSCs (Chen and Chopp, 2006). Crucially, it was argued that the MSC secretome was significantly influenced by the microenvironment in which MSCs were cultured, indicating that environmental cues modify the secretory profile of MSCs. In an artificial neural setting, MSCs respond to brain extracts from an amyotrophic lateral sclerosis (ALS) model by up-regulating the expression of NGF and BDNF, while down-regulating FGF-2, insulin-like growth factor and GDNF (Nicaise et al., 2011). A further point of interest was the finding that the MSC secretion profile varied depending on the time point at which the brain tissue was extracted following middle cerebral artery occlusion. This illustrates that the timing of MSCbased treatments could be critical if they are to be applied successfully in the clinic. Finally, in a more recent study it was reported that MSC-induced proliferation and differentiation of

[(Fig._2)TD$IG]

Fig. 2. MSC interaction with the neural microenvironment. MSCs interact with their surrounding microenvironment. In neural tissues, MSCs adapt their secretory profile (blue arrows) and express neural antigens. The repertoire of MSC-derived molecules likely to contribute to neurogenesis includes extracellular matrix (ECM) proteins and growth factors. MSC interaction with host neural progenitors may promote neurogenesis (red arrows) through a variety of mechanisms including structural guidance and direct neurotrophic signalling.

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resident NSCs in the subventricular zone was associated with increased BDNF levels (Kan et al., 2010). These data suggest that secretion of neurotrophic factors and cytokines may also play a role in MSC-mediated neural repair in vivo, and that increased MSC secretion of neurotrophic factors occurs in response to neural damage. MCS have also been ‘pre-conditioned’ prior to transplantation into a spinal damage model by co-culture with Schwann cells (Xu et al., 2010). This work found that transplantation of cocultured MSCs resulted in greater measured recovery than with MSCs not co-cultured. Outside the CNS, rat sciatic nerve injury has been used as a model to ascertain whether MSC transplantation could treat peripheral nerve damage (Chen et al., 2007). Functional recovery was assessed using the sciatic function index (SFI), measured on a scale of 0 to 100 (0 being indicative of normal nerve function, 100 being indicative of complete sciatic nerve dysfunction). SFI was determined periodically for up to 10 weeks post-transplantation, and significantly higher values were reported in animals subjected to MSC transplantation compared to controls, from the fifth week after treatment onwards. Increases in the levels of NGF, BDNF, GDNF and CNTF, and the ECM proteins, collagen I, collagen IV, fibronectin and laminin, were detected in MSCs cultured in ischemic brain supernatants (Chen et al., 2002). These data support the notion that transplanted MSCs release trophic factors and cytokines to mediate functional recovery following neurological damage in the central and peripheral nervous system. There are however multiple possible ways in which secreted bioactive molecules may play a role. For example, to investigate whether MSC-derived factors act directly on NSPCs, MSCs have been administered into the dentate gyrus of the hippocampus, an area known to be rich in NSPCs (Munoz et al., 2005). A substantial increase in the proliferation and migration of host neural stem cells was noted, at the expense of MSC proliferation and migration. The increase in proliferation and migration of resident neural progenitor cells was attributed to increased levels of NGF, VEGF, CNTF and bFGF in the hippocampus following MSC transplantation, strongly suggesting that MSCs may have been at least partly responsible for their secretion. This study also claimed that MSC-derived soluble factors not only influence NSPCs to undergo proliferation and differentiation, but also activate resident astrocytes. Therefore it is possible that activated astrocytes secreted the increased levels of NTFs observed, thereby themselves directly influencing neurogenesis. Astrocytic contribution to neural recovery concomitant with the direct effects of MSC-derived factors is supported by reports that astrocytes indeed play a trophic role in neurogenesis (Munoz et al., 2005; Rudge et al., 1992). Using a rat model of Huntington’s Disease, increased levels of BDNF, collagen 1 and fibronectin were detected in the striatum following MSC transplantation. None of the transplanted MSCs displayed evidence of neural transdifferentiation (Rossignol et al., 2011). 4.3.2. Non-neurotrophic factors secreted by MSCs Some of the studies into NTF secretion by MSCs outlined above also hint that non-neurotrophic factors may have neurogenic roles, particularly ECM proteins. MSCs are essentially a population of stromal cells and they secrete and deposit a wide range of extracellular matrix (ECM) proteins, including collagen, laminin and fibronectin (Nakayama et al., 2003). It is therefore reasonable to assume that MSCs will contribute to the ECM composition of the neural niche upon engraftment in neural tissues. Such contribution would most likely alter the native composition of the NSPC microenvironment which in turn is likely to influence the behaviour of resident cells. The influence of the ECM on neural cell behaviour is liable to include both physical and trophic effects. Evidence from in vitro studies suggests that the ECM plays important structural roles during neurogenesis, such as providing

support and guidance for developmental events, including neurite outgrowth (Aizman et al., 2009; Li et al., 2009). The large number of different ECM proteins creates the potential for enormous variation in ECM composition from one tissue to the next, and such variation is likely to be important in supporting tissuespecific signalling pathways. In the nervous system it is already established that different collagen isoforms impart wide-ranging functionality with regard to specific neural processes (Fox, 2008). In what may be considered an indirect trophic role, the ECM can facilitate cell-to-cell signalling by allowing trophic molecules to be concentrated and delivered to cells in an appropriate manner. For example, specific structures within the ECM have been proposed to promote the action of growth factors in the NSPC niche (Kerever et al., 2007). Furthermore, as perceived roles for the ECM extend beyond the provision of structural support, it has emerged that certain ECM proteins themselves possess biological activity, and may therefore play direct trophic roles in the neural niche. Socalled non-collagenous domains of some collagen types exhibit specific activities upon proteolytic release from their parent proteins (Ortega and Werb, 2002). An example is the antiangiogenic protein endostatin which is derived from collagen XVIII (O’Reilly et al., 1997). Serial analysis of gene expression (SAGE) of the MSC transcriptome has confirmed that expression of a variety of ECM and adhesion molecules that may contribute to MSC-mediated recovery of neural injury in vivo (Tremain et al., 2001). MSCderived molecules with roles in neural development include netrin-4 and reticulon-4 (previously reported involvement in axonal guidance), ninjurin-1/2 and astrotactin (previously reported involvement in neural cell adhesion), and prosaposin and pleiotrophin (previously reported neurite-inducing properties). In the case of prosaposin, it was recently found that the secretion of this protein by MSCs mediates neuroprotection in vitro (Li et al., 2010). It is increasingly likely therefore that MSC-mediated amelioration of neural damage involves not only neurotrophic factors acting directly on resident NSPCs but also a complement of ECM proteins which exert functional platforms from which these factors can operate, as well as providing chemotactic neurite guidance. For example, Aizman and co-workers showed that ECM derived from MSCs influences the growth of neural cells in vitro (Aizman et al., 2009), while Li and colleagues have shown that in co-culture assays, MSC-derived ECM proteins contributed to neurite outgrowth and guidance (Li et al., 2009). 4.3.3. In vitro systems for the study of MSC-derived neurotrophic activity Co-culture and conditioned media (CM) systems allow the direct study of the trophic influence of MSCs on other cell types. In relation to MSC-mediated neurogenesis, co-culture of MSCs has been performed with a variety of neural cell types, either for the sole purpose of in vitro analysis, or as a precursor to transplantation. Co-culture experiments using bone marrowderived MSCs and embryonic NSCs have demonstrated that MSCs stimulated to express neural antigens secrete positive signals which direct neuronal differentiation of the NSCs (Croft and Przyborski, 2009). Conditioned media refers to culture medium in which cells have been grown. Consequently it contains a mixture of cell-derived molecules, including those which have been actively secreted. The study of MSC-CM represents an attractive basis for improved understanding of MSC-derived neurotrophic activity and also the identification of candidate trophic factors. A study using rat MSC-conditioned media showed that MSCderived factors induced neural stem cells to acquire a predominately oligodendrocytic phenotype, at the expense of an astrocytic

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cell fate (Rivera et al., 2006). The MSC-CM was introduced to adult rat neural stem cells for a period of 7 days, followed by analysis of neural-specific markers. A significant increase in expression of the oligodendrocytic markers galactocerebroside (GalC) and myelin basic protein (MBP), concomitant with a significant decrease in the expression of the astrocytic marker, GFAP was observed. Subsequent analysis aimed to determine whether these MSC-derived effects were instructive (stimulation of oligodendrocytic differentiation) or selective (providing conditions favourable to cells already committed to an oligodendrocytic fate and/or sub-optimal conditions for those committed to astrocytic/neuronal fates) in nature. Specifically, rates of cell proliferation and cell death within different subpopulations of the neuroprogenitors were investigated (Rivera et al., 2006). It was concluded that the acquirement of an oligodendrocyte phenotype by neural stem cells occurred independently of rates of cell proliferation and cell death in the different progenitor subpopulations, suggesting that MSC-derived soluble factors were functioning by an instructive mechanism. It is known that MSCs demonstrate neural antigen expression following transplantation in vivo. It may therefore be appropriate to also consider media conditioned by neural antigen-positive MSCs. Although MSCs are largely negative for such neural antigens under standard growth conditions, removal of serum from culture medium can induce their expression (Wislet-Gendebien et al., 2004). Mouse embryonic neural progenitors showed increased expression of GFAP during culture in CM, and decreased expression of neuronal (Tuj1) and oligodendrocytic (O4) markers (WisletGendebien et al., 2003). Furthermore, a significant decrease in the rate of cell death of the GFAP-positive subpopulation was recorded after the 48 h, but no change in cell proliferation was observed. In an attempt to identify possible factors responsible for influencing the astrocytic differentiation, the nestin-positive MSCs were found showed to exhibit up-regulation of the biologically-active form of bone morphogenetic protein 4 (BMP4) at both the mRNA and protein levels (MSCs that were negative for nestin expression did not express active BMP4). This indicated that BMP-4 was, at least in part, causative of astrocyte differentiation by the embryonic neuroprogenitor. A more recent in vitro study showed the effects of co-culture with MSCs or following treatment with MSC-CM, revealing MSC-mediated protection of neural cells against death or damage (Tate et al., 2010). Targeted immunological analysis confirmed the presence of several known cytokines and NTFs in the MSC-CM. 5. Conclusion Several proof-of-principle cell culture and animal studies suggest that the use MSCs represents a promising therapeutic route for the treatment neurological and neurodegenerative disorders. Research reviewed herein demonstrates the remarkable capacity of MSCs to migrate, adapt, engraft and survive in neural microenvironments, as well as secrete a wide complement of trophic factors and ECM molecules. A limited number of human clinical trials have also taken place, with some promising results (Venkataramana et al., 2010). However, many questions require attention before more widespread application can be considered in humans. These include the nature of cellular material (e.g. bone marrow aspirates versus more homogeneous cultures of MSCs), method of transplantation (e.g. systemic versus direct administration into the damaged nervous tissue), timescales and duration of treatment, appropriate numbers of cells for transplantation, and what constitutes a suitable passage number for introduced cells (Charriere et al., 2010). Without question, the long-term safety of transplanted MSCs will need to be evaluated. These questions are considered by Muir and Kalladka in relation to stroke therapy in this issue.

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There is also scope to exploit the natural characteristics of MSCs further. For example, by using MSCs as tailor-made delivery vehicles for specific active molecules to sites of neural damage (Meyerrose et al., 2010). There have already been several efforts to genetically engineer MSCs to over-express neurotrophic factors and cytokines, such as HGF, FGF, NT3 and BDNF (GlavaskiJoksimovic et al., 2010; Zhang et al., 2010; Zhao et al., 2006). MSCs have been used to deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in an attempt to treat brain tumours in rats (Choi et al., 2011). Preliminary results are promising, with high rates of transfection efficiency reported, and enhanced efficacy of MSCs in mediating functional recovery from neural injury. There may also be potential to use MSCs as a source of cell-free therapeutic cocktails for systemic or direct administration into affected individuals. 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