Abstract: Developing effective therapies for serious neurological insults remains a major challenge for biomedical research. Despite intense efforts, the ability to promote functional recovery after contusion injuries, ischemic insults, or the onset of neurodegenerative diseases in the brain and spinal cord remains very limited even while the need for such therapies is increasing with an aging population. Recent studies suggest that cellular therapies utilizing mesenchymal stem cells (MSCs) may provide a functional benefit in a wide range of neurological insults. MSCs derived from a variety of tissue sources have been therapeutically evaluated in animal models of stroke, spinal cord injury, and multiple sclerosis. In each situation, treatment with MSCs results in substantial functional benefit and these pre-clinical studies have led to the initiation of a number of clinical trials worldwide in neural repair.
Cell based therapies are emerging as innovative approaches for the treatment of neurological disorders that currently lack effective therapies. Critical to the success of cell therapies is the selection and mode of delivery of cell populations. A leading candidate population for neurological applications is mesenchymal stem cells (MSCs). Here we briefly review the data supporting the clinical application of MSCs in three distinct neural insults and review the current thinking on the mechanisms of action of MSCs in the setting of neural injury. Several lines of evidence support the hypothesis that transplanted MSCs modulate both the host immune response to injury as well as direct neural stem cells and progenitors to differentiate along lineages that support rather than inhibit regeneration. It seems likely that the therapeutic efficacy of MSCs is a consequence of their capacity to localize to areas of insult and release a broad spectrum of trophic factors that guide endogenous neural cell repair. The ability to identify the spectrum of signals involved in neural repair will allow for engineering of MSCs that will potentially enhance the host’s capacities to promote functional recovery.
Origins and Potential of MSCs
Adult mesenchymal stem cells can be isolated from a range of tissues including bone marrow or bone marrow aspirates, fat, and other somatic tissues (Caplan, 2007). The multiple sources of MSCs and their relative ease of isolation have resulted in them becoming a preferred therapeutic cell population in a variety of therapeutic settings (Caplan, 2007). Our understanding of the biology of MSCs has developed rapidly as a result of the ability to grow them in vitro while maintaining their capacity to give rise to multiple cell lineages as well as direct their differentiation down specific pathways (Lennon and Caplan, 2006). The most common MSC differentiation pathways are along mesodermal lineages to form muscle, bone, cartilage, fat, and tendon (Pittenger et al., 1999) and these can be modulated depending upon local cues. There is considerable evidence suggesting that MSCs are also capable of differentiating into non-mesenchymal lineages including endothelial cells (Oswald et al., 2004) and neural cells (Jiang et al., 2002) although in the case of neural cells, it is unclear whether this reflects trans-differentiation, ectopic marker expression, or cell fusion (Rutenberg et al., 2004).
The capacity to engineer specific cell types from MSCs has facilitated their use in rebuilding damaged mesenchymal tissues; however, the utility of MSCs is not restricted to cell or tissue replacement. Mesenchymal stem cells also release signals that modulate host tissue responses. For example, MSCs have a strong immunosuppressive effect on the host immune system and alter the relative level of pro- and anti-inflammatory cytokine expression by T cells. Similarly, through the release of trophic factors MSCs are capable of enhancing the endogenous repair potential of many tissues. The augmentation of host MSC responses by the treatment with exogenous MSCs has emerged as a therapeutic approach to a range of tissue insults. Currently clinical trials are ongoing to use MSCs in the treatment of graft-versus-host disease, heart failure, stroke, spinal cord injury, and multiple sclerosis.
Neural Applications of MSCs
Common features of neural insults
Regardless of the precise mechanism of damage, insults to the adult CNS provoke a similar range of responses in the damaged tissue. For example, ischemia resulting from blood vessel occlusion or damage leads to functional deficits reflecting local death of neurons, demyelination following oligodendrocyte loss, and stimulation of a glial scar generated by astrocyte hypertrophy and proliferation (Beck et al., 2008). Likewise contusion or penetrating injuries to the brain or spinal cord generate a similar spectrum of responses with neuronal cell death and demyelination spreading from the original site of injury. Neurodegenerative diseases including Alzheimer’s disease and multiple sclerosis also provoke similar responses since neuronal cell death in Alzheimer’s disease, which is correlated with the deposition of beta amyloid plaques, generates a local inflammatory response and glial scar formation (Yan et al., 2009). In multiple sclerosis patients, the infiltrating activated immune cells attack oligodendrocytes, the myelinating cells of the CNS, and generate focal demyelinated lesions or plaques in which naked axons are surrounded by reactive astrocytes (Stadelmann et al., 2008). The demyelinated axons fail to conduct electrical signals with resulting functional deficits (Waxman, 1991).
Two major themes emerge from analyses of these different types of CNS insult. One is that they all result in the loss of neurons and oligodendrocytes concomitant with the generation of reactive astrocytes and the formation of a glial scar. Second, a significant component of the overall damage to the CNS is a result of either primary or secondary inflammatory attack on the host tissue. This suggests that therapeutic strategies targeted at promoting the genesis of neurons and oligodendrocytes while suppressing reactive astrocyte responses and modulating pro- to anti-inflammatory immune responses would provide an ideal approach to treating a spectrum of neural insults. The current literature provides strong support for the hypothesis that MSCs are uniquely suited to fulfill these roles through the release of bioactive trophic factors (Caplan, 2007).
MSCs in stroke
Stoke is the third leading cause of neurological injury in the USA and can be caused by the occlusion of small vessels in the brain that result in a localized loss of blood supply and subsequent neuronal death. This neuronal loss triggers a cascade of events including inflammatory response that leads to a spreading of the affected area. Current therapies for ischemic insults include relief of the vessel blockage through treatment with tissue plasminogen activator (tPA) that, while releasing blood flow, may stimulate further injury on reperfusion. A standard animal model of stroke involves occlusion of the middle cerebral artery (MCAO). Treatment of these rodents with MSCs delivered either directly into the brain or intravenously resulted in a significant reduction of the extent of the damaged area and improved neurological outcome (Li and Chopp, 2009).
The basis of MSC-induced functional improvement is not well understood. It has been proposed that MSCs regulate the levels of cell death through the release of trophic factors as well as alter the gap junction coupling between astrocytes that allows these cells to respond more effectively to control damage (Li and Chopp, 2009). Recent studies suggest that MSCs may also locally increase the levels of tPA in astrocytes around the stroke lesion and that this increases neuroprotection and enhances neurite outgrowth (Xin et al., 2010).
MSCs in spinal cord injury
Spinal cord injuries result in long-term functional deficits as a result of the failure of severed adult CNS neurons to regrow long distances, connect to their original targets, and restore circuitry. Several factors are thought to contribute to the lack of regeneration of spinal cord axons. These include a reduction in the intrinsic growth capacity of adult CNS projection neurons, the presence of inhibitory cues derived from damaged CNS myelin, and the formation of a glial scar by local astrocytes in response to inflammatory stimuli (Fitch and Silver, 2008). Attempts to negate any single inhibitory mechanism have not resulted in a significant enhancement of axonal regeneration, suggesting that multiple approaches will be required to generate functional recovery. This hypothesis has recently received strong support from the use of combinatorial therapies directed at intrinsic and environmental regulators of regeneration (Kadoya et al., 2009). Remarkably, treatment with MSCs appears to enhance functional recovery in the absence of combinatorial treatments. The underlying mechanisms responsible for MSC-stimulated spinal cord regeneration are currently unclear. Studies with other stem cell populations suggest that they antagonize the negative effects of immune cells (Busch et al., 2010) while MSCs appear to release trophic factors that promote axonal regeneration and may also enhance the survival of damaged neurons (Cho et al., 2009).
MSCs in multiple sclerosis
Perhaps the most advanced application for MSCs in the neurological clinical arena is in multiple sclerosis (MS). Multiple sclerosis is an inflammatory disease of the CNS characterized by extensive mononuclear cell infiltration and demyelination. MS is generally considered to be a T-cell mediated disease based on local inflammation, response to immune modulation or immunosuppression (Stuve et al., 2006; Perini et al., 2007), and the genetic association with the major histocompatibility complex (Haines et al., 1996). The best characterized model of MS is experimental allergic encephalomyelitis (EAE) (Martin, 1997) induced by immunization of susceptible host animals with specific myelin proteins. This animal model has formed the basis for the development of therapeutic approaches to MS. The ultimate goal of such therapies is the restoration of function. Long-term functional recovery requires regulation of the pathogenic process, which may be modulated by MSCs. For example, in EAE, treatment with mouse MSCs reduces disease burden (Gerdoni et al., 2007; Kassis et al., 2008). While animal models are essential for identifying potential therapeutic approaches, the development of MSC-based clinical programs requires demonstration that human MSCs have similar functional properties.
We and others have shown that human MSCs have similar disease regulatory characteristics to murine cells (Aggarwal and Pittenger, 2005; Zhang et al., 2005; Bai et al., 2007; Bai et al., 2009). Injection of bone marrow derived human MSCs into animals with either chronic or relapsing remitting EAE resulted in a rapid reduction in functional deficits and led to long term recovery (Figure 1). This recovery was correlated with the migration of the transplanted MSCs into the CNS and their accumulation in regions of demyelination. Histological characterization of the demyelinated lesions in the spinal cord of control and MSC treated animals showed a number of significant changes. For example, the extent of astrogliosis was reduced in the presence of MSCs, and the number of oligodendrocytes and their progenitors was substantially increased. Treated animals demonstrated a significant reduction in the size of the lesions and a dramatic increase in the number of myelinated axons. Together these studies suggest a localized effect of the MSCs on the cell fate influenced by host endogenous neural stem cells or progenitors in the area of lesions.
Consistent with the notion that MSCs alter neural cell fate in EAE, culture studies indicate that neural stem cells (or neurospheres) grown in the presence of MSCs generate more neurons and oligodendrocytes while giving rise to fewer astrocytes than controls. This effect is seen in neurospheres derived from both developing and adult animals. More importantly, neurospheres derived from EAE animals also show a pronounced potential to generate neurons and oligodendrocytes when grown from animals treated with MSCs.
Mechanisms of Action of MSCs
It seems likely that common molecular mechanisms underlie the therapeutic benefit of MSCs in the different neurological conditions. Although currently there is not a clear understanding of the detailed mechanisms by which MSCs mediate neural recovery, several possibilities exist. First, MSCs that infiltrate lesion areas may differentiate directly into neural cells. Early studies suggested that MSCs injected into the lateral ventricles of developing animals differentiated into astrocytes and other neural cell types (Woodbury et al., 2000; Deng et al., 2001). This seems unlikely to account for the histological changes seen in the EAE studies, however, since when labeled MSCs were injected into naïve or EAE animals no evidence of them adopting a neural fate was detected based on their expression of neuronal (TuJ1) or glial (GFAP, CC1) antigens. Furthermore, even when grown in highly neuralizing conditions in vitro, the proportion of “neuralized” MSC progeny remains relatively small and their functional properties are not well known (Alexanian, 2007; Bai et al., 2007). One likely explanation for the appearance of GFAP+ MSCs in other models of neural damage is cell fusion (Terada et al., 2002; Weimann et al., 2003). Indeed, intravenously injected bone marrow-derived cells are known to fuse with hepatocytes in liver, Purkinje neurons in the cerebellum, and cardiac muscle in the heart. However, the notion of MSC transdifferentiation into non-mesenchymal phenotypes is poorly supported by the current data.
The rapid and sustained functional recovery seen in animals with EAE after treatment with MSCs suggests that these cells alter several aspects of disease progression. First, treatment with MSCs suppressed T-lymphocyte activities thereby exerting an immunoregulatory capacity (Di Nicola et al., 2002; Gerdoni et al., 2007; Nauta and Fibbe, 2007). The cytokine profile of spleen was biased away from TH1 pro-inflammatory signals such as interferon gamma, IL-17, and IL-2 and towards Th2 anti-inflammatory signals such as IL-4 and IL-5. Though the mechanisms mediating such effects are still only partially understood, it is likely that they involve both cell-to-cell contact and soluble factors. Second, endogenous neural stem or progenitor cells are activated by MSCs (Munoz et al., 2005). Neural stem cells exist in the developing and adult mammalian nervous system including that of humans. They are capable of undergoing expansion and differentiation into neurons, astrocytes, and oligodendrocytes in vitro (Reynolds and Weiss, 1992) and after transplantation in vivo (Svendsen et al., 1997). Although their restricted locations in the brain may limit their clinical effectiveness, stimulation by MSCs may enhance their response and facilitate endogenous CNS repair. In the EAE studies, it is likely that the recovery of myelination is a reflection of suppression of the autoimmune responses in combination with induced proliferation or enhanced differentiation of endogenous progenitor cells. Consistent with this hypothesis, an increase in the density of NG2+ cells and oligodendrocytes was seen in MSC-treated animals presumably reflecting the release of multiple bioactive factors by MSCs (Caplan and Dennis, 2006). Indeed earlier studies have suggested that bone marrow stromal cells can promote neurogenesis in the hippocampus (Munoz et al., 2005). Whether the functional improvement seen in EAE reflects remyelination or neuroprotection derived from oligodendrocyte precursor cells (OPCs) is currently unclear. A similar combination of anti-inflammatory signals and modulation of neural cell fate may underlie the efficacy of MSCs in stroke and spinal cord injury.
Conclusions and Future Directions
In conclusion, MSCs are emerging as an effective therapeutic approach to a wide range of neural insults. Studies in demyelinating diseases have highlighted the importance of both the immunoregulatory actions of MSCs and their neuromodulatory properties. These properties are a reflection of several unique characteristics of MSCs. Specifically, these cells “home” to areas of insults, they release a wide range of trophic signals that influence surrounding tissues and they have immunosuppressive properties that allow their long term survival in non-immunocompatible hosts. While MSCs are currently being utilized in the setting of a number of neural injuries, in the future their potential may be enhanced by more effective targeting to injury sites as well as augmenting or enhancing the spectrum of trophic factors they deliver. Such studies offer a new perspective for the treatment of demyelinating diseases such as MS and neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease.
(Corresponding author: Robert H. Miller, Ph.D., Center for Translational Neurosciences, Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.)
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