Mesenchymal stem cells as a multimodal treatment for nervous system diseases

Abstract Neurological disorders are a massive challenge for modern medicine. Apart from the fact that this group of diseases is the second leading cause of death worldwide, the majority of patients have no access to any possible effective and standardized treatment after being diagnosed, leaving them and their families helpless. This is the reason why such great emphasis is being placed on the development of new, more effective methods to treat neurological patients. Regenerative medicine opens new therapeutic approaches in neurology, including the use of cell‐based therapies. In this review, we focus on summarizing one of the cell sources that can be applied as a multimodal treatment tool to overcome the complex issue of neurodegeneration—mesenchymal stem cells (MSCs). Apart from the highly proven safety of this approach, beneficial effects connected to this type of treatment have been observed. This review presents modes of action of MSCs, explained on the basis of data from vast in vitro and preclinical studies, and we summarize the effects of using these cells in clinical trial settings. Finally, we stress what improvements have already been made to clarify the exact mechanism of MSCs action, and we discuss potential ways to improve the introduction of MSC‐based therapies in clinics. In summary, we propose that more insightful and methodical optimization, by combining careful preparation and administration, can enable use of multimodal MSCs as an effective, tailored cell therapy suited to specific neurological disorders.


| INTRODUCTION
Injury of the nervous system leads to a cascade of events that eventually ends with neuronal loss and acute or chronic dysfunction. Such Over several years, myths related to the lack of neurogenesis de novo and lymphatic drainage in the nervous system as well as regarding the immune-privileged state of the nervous system have been debunked. 4,5 Thanks to recent research, the current view of the central nervous system includes a system that is relatively able to recover. As many diseases and injuries of the nervous system are still untreatable or not efficiently curable by standard medical and pharmaceutical practices, alternatives featuring regenerative medicine might overcome existing barriers. 6 Transplantation of cells and tissues into the nervous system, which was first performed in the 1980s, aims to promote regeneration through direct replacement of lost cells. 7 Obtained fetal tissues and implants derived from various sources of neural progenitor cells (NPCs) and neurons still evoke debate around ethical and safety issues. 8 Apart from the source of new neurons, NPCs have also been shown to have immunomodulatory characteristics. 9 Nevertheless, discontinuation of the need for treatment with immunosuppressive drugs that comes with allogeneic treatment diminishes or even removes the positive effects of therapies. 10 Recently, autologous transplantation strategies featuring iPSC technology have appeared. 11 However, clinical translation of this approach is far from realized because the tumorigenic and long-term immunogenic potentials of these cells have not been tested.
Strategies for treating diseases and injuries of nervous system appoint a less direct but still beneficial source of cells for transplant to cure such yet incurable diseases-mesenchymal stem cells (MSCs).
They can be easily obtained from tissues, expanded ex vivo, and transplanted in an autologous or allogeneic manner. 12 Due to their immunomodulative properties, MSCs can resolve inflammation triggered by injury or degeneration. 13 Via their secretome, they can support the survival of neurons and affect the regeneration of tissue loss by influencing local neurogenic niches. 14 In this review, we introduce unique MSC characteristics valuable for the repair of the nervous system in various diseases based on in vitro and preclinical studies. Taking into consideration the clinical application of MSCs, this review is focused only on the properties of human MSCs from the three most common sources: bone marrow (BM), Wharton's jelly (WJ), and adipose tissue (AT).
Clinical studies will be reviewed, focusing on their safety and efficacy. We will also explore discrepancies between clinical studies and suggest potential ways to enhance the effectiveness of MSC therapies.
This narrative review was prepared based on publications found in the PubMed database using the following keywords: MSCs, nervous system, neurodegeneration, and neurological diseases (or each disease specifically, eg. PD, AD, epilepsy, and SCI). For clinical trials, the name of each neurological disease and the term "mesenchymal stem cells" were used as key words, adding "clinical trial" as a filter in the PubMed database. Additionally, clinical trials were filtered from the ClinicalTrials.gov database.

| MSCs AND THEIR PROPERTIES
MSCs, which possess self-renewal potential and multipotent properties, can be found in neonatal and adult tissues. These adherent, fibroblast-like cells were first isolated from BM in 1970 by Friedenstein et al. 15 Over the years, these cells have been called by many names, such as mesenchymal stromal stem cells, multipotent adult progenitor cells, medicinal signaling cells, and mesenchymal progenitor cells (MPCs). Currently, MSCs is the most common terminology, but is sometimes used interchangeably with mesenchymal stromal stem cells to underline their origin from the nonhematopoietic compartment of BM. In addition, MPCs are occasionally presented as a distinct population. 16 Apart from BM, MSCs have also been identified in AT, 17 umbilical cord blood, 18 the umbilical cord lining, 19 subendothelial layers, 20 the perivascular zone, 21 WJ, 22 dental pulp, 23 synovial fluid 24 and the synovial membrane, 25 amniotic fluid, 26 fetal liver 27 and even urine 28 or endometrium. 29 Recently, pericytes with MSC-like characteristics were also found in the brain. 30 Independent of the tissue source, the isolated cells need to express common characteristics to be defined as MSCs. As no single marker has been specified for these cells yet, analysis of a set of surface antigens needs to be performed. According to the International Society for Cellular Therapy gold standard, MSCs need to be positive for CD73, CD90 and CD105 (all >95%) and negative for CD34, CD45, CD11b/integrin alpha M or CD14, CD79 alpha or CD19, and HLA class II (all <2%). 31 The status of HLA class II can change upon cell stimulation but the expression of costimulatory molecules, such as CD40, CD80, CD86, CD134, and CD142, cannot be changed. 32,33 Moreover, the multipotent character of MSCs needs to be proven by their differentiation into adipocytes, chondrocytes, and osteocytes when cultured in vitro. 31 Some studies suggest that MSCs are also capable of transdifferentiating in vitro to cells outside mesenchymal lineages, such as neural and glial cells, cardiomyocytes, skeletal myocytes, hepatocytes, and endothelial cells; however, these studies have been questioned by recent findings. 34,35 As a distinct entity from the multipotency understood as differentiation potential per se, the term functional multipotency has been coined. 36 This characteristic refers to the ability of different types of stem cells to exert pleiotropic influence on injured tissue to support the maintenance of homeostasis, which remains crucial during development but also during tissue repair after injury. Interestingly, studies have shown that sustaining the stemness of MSCs by incorporating specially adjusted scaffolds can highly augment the therapeutic

Significance statement
This concise review summarizes the results of preclinical and clinical trials in neurological diseases of different etiologies. This review focuses on possible mechanisms of action of mesenchymal stem cells (MSCs) but also discusses approaches to augment their effects. A summary of the properties of MSCs reveals their broad therapeutic potential, which can orchestrate regenerative processes after neural injuries. potential of MSCs in treating spinal cord injuries. 37 Such results should encourage abandonment of the uncertain approach of using transdifferentiated cells.
The most key and characteristic feature of MSCs is their broad secretome, which can influence tissue regeneration. It has been shown that MSCs can produce many immunomodulatory, proangiogenic, tissue remodeling, antiapoptotic, growth, and trophic factors that can support survival of host cells, reconstruction of injured tissue and activation and differentiation of local progenitors. 14 However, depending on the source of MSCs, they can differ in their properties. [38][39][40]

| Differences between sources
Although MSCs from various sources share common characteristics, some differences can be found between them. These variations in MSC populations may reflect particular regional properties of the niches from which they originate. 41 MSC features are also susceptible to variations between cell culture conditions and isolation protocols.
It has been shown that MSCs obtained from the same patient can vary in their properties between isolations. Additionally, discrepancies between different subpopulations of BM-derived MSCs (BM-MSCs) have been shown. 42 In the case of Wharton's jelly-derived MSCs (WJ-MSCs), depending on the chosen isolation method (isolated enzymatically by collagenase, trypsin, or hyaluronidase, or by extraction directly from explants), cells can slightly differ in features such as the expression of pluripotency markers and cell proliferation rates. 43 However, in some reports, it was noted that autologous MSCs differ from those obtained from healthy donors and that such differences can influence the final outcomes of therapies. 44 Amable

| MSCs IN NEURODEGENERATION AND BRAIN INJURY-PRECLINICAL TRIALS
The previously mentioned features of MSCs make them a perfect tool in cellular therapies for pathological processes in the nervous system driven by excessive inflammation and neurodegeneration. MSCs secrete a variety of factors, including neurotrophic factors. 50 MSCs from different sources can also differentiate into neuronal lineages by forming primary neurospheres; however, only WJ-MSCs and BM-MSCs could form secondary neurospheres. 51 Moreover, differentiated WJ-MSCs secreted more neurotrophic factors than BM-MSCs and AT-MSCs. 51

| Routes of MSC transplantation
Preclinical studies have established ways of MSC implementation via intravenous, intra-arterial, intrathecal, intranasal, intraperitoneal, intraspinal, intracerebroventricular, intracerebral, or direct administration to particular structures. The route of administration is important because it determines the number of successfully grafted cells in the injured site, which can be correlated with therapeutic outcome. 52,53 Additionally, taking into consideration that neurological disorders may not be localized, indirect administration, for example, intrathecal injection, may be of great importance.
To understand the pros and cons of each route of administration, an invaluable tool is cell tracking. Various methods have been developed for intravital imaging. 54 Studies have shown that although the most feasible method of MSC transplantation is through intravenous injection, in such conditions, most of the cells become trapped in the lungs. 55 Nevertheless, such entrapped MSCs can release microvesicles and immunomodulative factors and affect the overall state of the patient by modulating peripheral immune cells. 56 Moreover, cell tracking can visualize MSC migratory potential.

| Active migration of MSCs toward injury
Once administered indirectly, MSCs need to actively migrate to the injury region. Active migration of MSCs is possible due to the expression of receptors and cell adhesion molecules. Pivotal roles are played by receptors, integrins, selectins and proteolytic enzymes. 54 One of the pathways crucial for MSC migration is the METR/HIF-1/CXCR4 pathway. 57 It was shown that preconditioning MSCs with stroke patients' sera enhanced the METR/HIF-1/CXCR4 pathway and increased the migratory potential of MSCs, which translated into improved recovery in a transient middle cerebral artery occlusion (tMCAO) stroke model in rats. 57 Important chemoattractants that can enhance MSCs to regions of injury in the brain are MCP-1 and stromal cell-derived factor-1 (SDF-1). In a study by Lee et al., it was shown in the MCAO rat model of stroke that MCP-1 and SDF-1 have both region-and time-dependent differential expression, which directs intravenously injected MSCs to migrate either to the cortex 1 day after injury or to the striatum in later days. 58 That MSC migration dependent on CXCR4 receptor expression was also shown in elegant in vitro studies with microfluidics systems. 59

| MSCs act through immunomodulation
MSCs are mostly recognized as immunomodulatory cells that can balance inflammation in the tissue environment by upregulating antiinflammatory signaling and decreasing pro-inflammatory signaling and thus regulate immunological cells such as lymphocytes, macrophages or microglia and astrocytes. 60,61 MSCs can influence inflammation by secreting soluble factors or direct cell-cell contact. MSCs constitutively or upon stimulation secrete indoleamine 2,3-dioxygenase, transforming growth factor beta, hepatocyte growth factor, IL-6 and IL-10, prostaglandin E2, heme oxygenase 1 and soluble HLA-G5. 13 Inflammation is an integral part of pathological processes that emerge in the nervous system. Depending on the mechanism of injury or neurodegeneration, the innate or adaptive system plays a more important role. Due to their migratory potential, MSCs can migrate to the site of injury and, due to their immunomodulatory properties, decrease inflammation. Excessive inflammation in the brain is the most devastating force causing degeneration of the central nervous system. 62,63 Acute injuries due to ischemia (hypoxia-ischemia encephalopathy [HIE] and stroke), mechanical-driven trauma (such as TBI) or progressive neurodegeneration lead to moderate activation of microglia followed by activation of astrocytes, the main sources of inflammatory cytokines. 64 If this state persists, damage to the bloodbrain barrier (BBB) occurs, and intensified inflammation appears due to the migration of peripheral immune cells (lymphocytes and monocytes). 65 Such processes suppress neurogenesis and endogenous repair. 66 Thus, MSCs could be a perfect tool in brain injuries, as they can modulate the inflammatory state. It has been shown that MSCs can attenuate microgliosis and astrogliosis in rats with induced HIE, SCI, or epilepsy. [67][68][69] MSCs can also suppress the proliferation and differentiation of B lymphocytes. 70 Moreover, transplanted MSCs can switch activated M1-phenotype microglia to the regenerative M2 phenotype. [71][72][73] Additionally, in an AD model of APP/PS1 double transgenic mice, transplantation of MSCs led to reduced β-amyloid (Aβ) peptide deposition by microglia but without secretion of proinflammatory factors. [73][74][75] MSCs have also been shown to improve BBB integrity in a rat TBI model. 76 This effect was mainly mediated by the activity of metalloproteinase inhibitor 3 (TIMP-3) released by MSCs. Several growth factors are secreted by MSCs: brain-derived neurotrophic factor (BDNF), nerve growth factor, insulin-like growth factor 1, glial cell-line-derived neurotrophic factor (GDNF) and VEGF. [79][80][81] The neuroprotective function of transplanted MSCs is based on a reduction in neuronal sensitivity to glutamate receptor ligands and altered gene expression, suggesting a link between the therapeutic effects of MSCs and the activation of cell plasticity in damaged nervous structures. 82 Experimental models proved that the MSC secretome promotes axonal growth and neuroprotection and minimizes cavity formation in SCI. 83,84 Neurotrophic and neurotropic effects of MSCs were also clearly presented in some elegant ex vivo studies employing adult rat dorsal root ganglia organotypic cultures. 37,85 Interestingly, this type of culture can be used to decipher other effects of MSCs on injured tissue, for example, immunomodulation. 37 Lu et al. investigated the nature of chronic scars and their ability to block axon growth. Chronically injured spinal cord axons can regenerate through the gliotic scar in the presence of local growth-stimulating factors. 86 MSCs may provide a source of growth factors to enhance axonal elongation across spinal cord lesions and minimize cavity formation in SCI. 68 In some neurological disorders, such as PD, ALS, and stroke, gene therapies are proposed as a method of treatment. 90 One of the challenges of such approaches is the delivery of genes of interest, especially in nonfocal neurodegenerations. MSCs can thus serve as carriers for genes whose expression is needed in specific neurological disorders: GDNF (PD and ALS), 91,92 VEGF (PD), 93 GDNF and VEGF (ALS), 94 BDNF (Huntington's disease [HD], SCI, and stroke), 95-97 conserved dopamine neurotrophic factor, 98 and PlGF (stroke). 99 It has been shown that modified cells have a more pronounced therapeutic effect than unmodified MSCs. This result stems from the fact that despite the desired alterations, modified MSCs maintain the rest of their characteristics, such as a capacity for immunomodulation. However, more studies are needed to determine whether transient delivery of such growth factors is sufficient or repeated transplantation is needed depending on the treated disease. In contrast, MSCs can also be used to decrease undesirable genes to promote repair. One study has shown that modification of MSCs with lentiviral RNAi downregulating adenosine kinase, the major adenosine-removing enzyme, may be beneficial for treating epilepsy. Indeed, transplantation of such modified MSCs resulted in a decrease in seizures, and this effect was strictly connected to elevated levels of adenosine. 100 Moreover, modified MSCs transplanted directly into the injury site demonstrated a better ability to promote neuron survival and decrease damage than unmodified MSCs. 101 Additionally, taking into consideration the impact of miRNAs in the regeneration of tissue, MSCs can serve as carriers of different miRNAs to the nervous system. 102,103 However, in some cases, a decrease in miRNAs can be beneficial. For example, in the case of SCI, suppression of miR-383 enhances the therapeutic potential of MSCs in SCI. 104 There have also been studies with an established cell line, SB623

| MSCs support regeneration through their neurotrophic activity
hBM-MSCs (SanBio Inc.), which overexpresses the NOTCH 1 intracellular domain. These cells transplanted in preclinical trials have ameliorated damage in models of PD or after TBI, 105,106 probably due to enhanced neuropoietic and proangiogenic activity. 107,108 Moreover, in a TBI model, transplanted SB623 cells formed "biobridges" between the neurogenic niche and the site of injury. 106 This effect has been correlated with metalloproteinase-9 (MMP-9) activity. Functional formation of these biobridges can play an important role not only in TBI but also in ischemic injuries in the brain. 109 These results can partially explain the regenerative potential of SB623 cells in clinical trials with stroke patients. 110 Currently, a clinical trial is underway in which SB623 cells are being applied to treat patients with TBI (STEMTRA, NCT02416492).
MSCs have been shown to differentiate into many types of cells from the nervous system, including dopamine neurons, 111  MSCs, seem to be the key to modifying the environment to pursue regeneration ( Figure 1). The multimodal activity of transplanted MSCs in models of neurodegenerative diseases has led not only to a pathophysiological view of the course of disease but also, in some cases, to a therapeutic effect, such as better cognitive outcomes in models of AD, better motor activity in models of PD and ALS or a decrease in the amount and severity of recurrent seizures in epilepsy. 119 Understanding which conditions are crucial to boost efficiency by optimizing the route, time and number of administrations of MSC transplantations in preclinical models will improve our knowledge and enhance translation into clinical trials.
In most of the studies, bacterial tests were performed. 123,125,127,130,[133][134][135][136][137][138]140,141 In the majority of studies, karyotype analysis was also carried out. 123,125,128,130,132,[136][137][138]140 Additionally, the differentiation potential of MSCs was assessed in a few clinical studies. 125,130,131,136,138,140,142 Only in a minority of studies parallel testing in animals was conducted. 143,144 In some clinical trials, out of 12 stroke patients after treatment. 135 However, because of the lack of a control group in this study, the authors cannot exclude spontaneous recovery as a cause for changes in infarct size. Nevertheless, they also noted a significant correlation between neuroimaging results and mean changes in the National Institutes of Health Stroke Scale score.
In a study by Lee   Currently, there are two ongoing clinical trials concerning the application of autologous MSCs in stroke, one of which is a randomized, controlled, and observer-blinded trial. The detailed methodology of these studies has already been published. 146,147 In clinical trials concerning patients with SCI, Park  Only some studies have implemented repeated transplantation of MSCs. 123,126,128,132,140,141 In other studies, deterioration of the clinical status of patients was noted, which may have been due to the single MSC administration. 131 As shown above, MSCs ameliorate functional deficits in several central nervous system diseases in both experimental animal models and in the clinic. Therapeutic mechanisms may include neuroprotective effects, immunomodulation, tissue remodeling, and activation of local progenitors. Therefore, MSCs prepare the environment for axonal ingrowth, stimulate angiogenesis, and result in functional recovery.

| DISCUSSION AND FUTURE REMARKS
Growing knowledge about MSC regenerative potential raises great hopes for applying them in the clinic. However, there is still space for improvement, and more preclinical studies have to be performed to evaluate cell culture conditions, the potential for neuronal priming, and the timing and route of administration to obtain the best improvements for patients. Another emerging issue is related to the necessity of side-to-side clinical and preclinical studies. Such an approach has been implemented in only a minority of clinical studies. 143,144 This type of scientific approach is of necessary to not only identify potential improvements but also ensure solid scientific methods are being employed to explain observed phenomena.
It is also worth mentioning that the discrepancy between preclinical