Perspectives of Mesenchymal Stem Cell-Based Neuroregeneration

    

    

    Figure 1: Current approaches to the mesenchymal stem cell-based neuroregeneration (see text for details).
    
    

Undifferentiated mesenchymal stem cells

One of the earliest neurotransplantations of mesenchymal stem cells into experimental animals was performed in 1998 by Azizi and collaborators. Human bone marrow-derived MSCs were directly injected into the striatum of rat brain. Only 20% of the infused cells had engrafted, but there was no evidence of an inflammatory response or rejection. After infusion into the brain, the human MSCs lost their immunoreactivity. The cells migrated from the site of injection to successive areas of the brain [7]. In another experiment, mesenchymal stem cells implanted to embryonic rat brain ventricles differentiated in vivo in a regionally and temporally specific manner. Whereas donor cells localized near to the subventricular zone continued to express nestin, a marker of neural precursors, the cells in the neocortex and midbrain expressed mature neuronal markers like Microtubule-Associated Protein Tau (MAPT) or Microtubule- Associated Protein 2 (MAP-2) [8]. On the other hand, Tondreau and colleagues found that more than 80% of mesenchymal stem cells cultured in vitro constitutively expressed nestin and �-III tubulin. Moreover, MSCs cultured in non-differentiating medium after fifth passage started to express mature neuronal or glial markers as: Tyrosine Hydroxylase (TH), Microtubule-Associated Protein 2 (MAP-2) and Glial Fibrillary Acidic Protein (GFAP) [9]. It must be remembered that MSCs derived from different sources hold distinct differentiation potential. For example, the in vitro expanded UCB-derived MSCs harbor a small unique population of cells that possesses huge inherent neurogenic potential and differentiate using simple protocol of neuronal induction. These �pluripotent progenitors� generate cells expressing neural progenitor markers and are responsible for the immediate neuronal differentiation in vivo [3,10].

The huge advantage of MSCs is that they can bypass the bloodbrain barrier [11]. When injected systemically they migrate into the various brain areas, home precisely to the site of injury, in particular to hypoxic, apoptotic or inflamed parts and easily integrate into the nervous tissue. MSCs are known to secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities for damaged tissues. To date, the experimental and clinical evidence showed no �danger signal� in tissues after MSCs injection. Thus, MSCs have been widely tested as a promising therapeutic tool in animal models of neurological diseases, such as multiple sclerosis (MS), Huntington�s disease (HD), Amyotrophic Lateral Sclerosis (ALS) as well as in traumatic brain injury [12-15].

Differentiated mesenchymal stem cells

The in vitro pre-differentiation of MSCs into neural lineage can be achieved by culturing them in the induction media. Such media consist of a cocktail of growth factors and small molecules that drive cells to the neural fate. In early experiments, Sanchez-Ramos and collaborators demonstrated that human and mouse bone marrowderived MSCs can be induced to differentiate into neural cells under special culture conditions. MSCs cultured in the presence of epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) expressed nestin, GFAP and neuron-specific nuclear protein (NeuN) [16]. The differentiation to neural lineage is more effective when human and mouse bone marrow-derived MSCs were co-cultured with rat foetal mesencephalic or striatal cells [16]. However, it has not been shown conclusively that mature neurons with signalling capacity can be generated from MSCs [17]. The role of differentiation media composition and cell culture substrates in in vitro pre-differentiation of MSCs into neural lineage was studied by Kim et al. [18]. The tested culture media contained basic fibroblast growth factor (bFGF), Nerve Growth Factor (NGF) and retinal acid (RA). The cell culture substrates coated with laminin, gelatin, collagen and fibronectin were examined. Combination of RA and bFGF together with fibronectin-coated dishes provoke pre- neuronal differentiation in 40% MSCs [18]. According to Kaka and co-authors other molecules important for neural lineage differentiation are dimethyl sulfoxide (DMSO), platelet-derived growth factor (PDGF) and heregulin (HRG), followed by triiodothyronine (T3) [19]. Numerous strategies have been employed to achieve MSC-derived neural phenotypes from different cell sources (extensively reviewed in [3]). It is suggested that origin and biological potential of the human mesenchymal stem cells is essential for the study on inducing them to neuronal differentiation. Nevertheless, the choice of the best source for treating neurodegeneration still remains unclear [3]. The most important advantage of pre-differentiation into neural lineage may be concern for safety, as some studies have noted formation of tumors in undifferentiated murine MSCs cultures, whereas studies on human MSCs transformation are insufficient.

Genetically modified mesenchymal stem cells

Combining the benefits of undifferentiated MSCs transplantation with blood-brain barrier bypassing and drug or therapeutic gene delivery may be realized with genetically engineered MSCs. Ryu and collaborators in their study [20] evaluated the therapeutic effects for CNS disorders using human bone marrow-derived mesenchymal stem cells, engineered to secrete interferon-�, as delivery vehicles. MSCs injected intravenously preferentially migrate to the sites of inflammation and may therefore be used for tissue directed immunosuppression, or delivery of therapeutic molecules to the injuried site. Another potent strategy for suppression of the inflammatory processes throughout the CNS may be mesenchymal stem cells genetically modified with NTPDase (nucleoside triphosphate diphosphohydrolase, CD39) gene. Elevated concentrations of extracellular ATP in the local microenvironment of the injury mark the damaged site and contribute to the promotion of primary immune response [21]. NTPDases capable of ATP cleavage contribute to the decrease in ATP concentration, thus terminating the prolonged inflammatory response. MSCs engineered to express therapeutic enzyme could effectively combine the high enzymatic activity with its local delivery to maintain the low concentration of ATP (see also [22]).

Some other experiments used mesenchymal stem cells genetically engineered to over-express brain- derived neurotrophic factor or nerve growth factor. The modified cells were injected into the striatum of YAC 128 mouse model of Huntington�s disease. It effected in supporting the injured neurons and suppression of the neurodegenerative processes [15,23]. Sadan and colleagues have modified bone marrow-derived mesenchymal stem cells into neurotrophic factor-secreting cells, thus combining stem cell-based regenerative therapy with the NTF-based neuroprotection. The engineered MSCs injected to the rat model of Parkinson�s disease induced regeneration in the damaged striatal dopaminergic nerves. In these experiments, the neurotrophic factor-secreting cells were more effective than unmodified MSCs [24].

Mesenchymal stem cells have the potential as drugs or other molecules carriers to treat patients with neural diseases and neuropathologies for which limited treatment options exist. It seems that genetically manipulated MSCs provide attractive platforms with lesion-targeting capability for the sustained production of therapeutic proteins in vivo. When considering the limitations of current methods of drug delivery to the brain, MSCs have the potential to become a safe cellular delivery vehicle. Progress along these cell lines has been made in rodent models of neurodegenerative disorders and ischemic stroke (described in [4]).

Clinical and experimental trials in CNS disorders

Mesenchymal stem cells have numerous advantages of potential clinical importance, that make them ideal candidates for stem cellbased therapy. There have been several clinical studies using MSCs during recent years, some of them focused on the central nervous system disorders [25]. There are also several ongoing clinical trials with genetically modified MSCs as delivery vehicles for the treatment of number of CNS pathologies including brain ischemia, amyotrophic lateral sclerosis and multiple sclerosis [11]. The representative clinical and experimental trials using MSCs were collected in Table 1.

  

    
Disease 
    
Cells 
    
Route of delivery 
    
References 
  
  

    
Traumatic Brain Injury 
    
MSCs 
    
intravenous 
    
NCT01649700 
  
  

    
Parkinson Disease 
    
MSCs 
    
intravenous 
    
NCT01446614 
  
  

    
Multiple Sclerosis 
    
MSCs 
    
intravenous 
    
NCT01377870 
  
  

    
MSC-derived����������������� neural progenitors 
    
intravenous 
    
NCT01933802 
  
  

    
Ischemic stroke 
    
MSCs 
    
intravenous 
    
NCT01468064 
  
  

    
Amyotrophic Lateral Sclerosis 
    
MSCs 
    
intrathecal 
    
NCT02116634 
  
  

    
Neurotrophic factors- secreting mesenchymal stromal cells 
    
intramuscular, intrathecal 
    
NCT02017912 
  

Table 1: The representative clinical trials using MSCs [compiled from: 3,11,25,26].