A focus on allogeneic mesenchymal stromal cells as a versatile therapeutic tool for treating multiple sclerosis

Multiple sclerosis (MS) is a central nervous system (CNS) chronic illness with autoimmune, inflammatory, and neurodegenerative effects characterized by neurological disorder and axonal loss signs due to myelin sheath autoimmune T cell attacks. Existing drugs, including disease-modifying drugs (DMD), help decrease the intensity and frequency of MS attacks, inflammatory conditions, and CNS protection from axonal damage. As they cannot improve axonal repair and show side effects, new therapeutic options are required. In this regard, due to their neuroprotection properties, immunomodulatory effects, and the ability to differentiate into neurons, the transplantation of mesenchymal stromal cells (MSCs) can be used for MS therapy. The use of adipose-derived MSCs (AdMSCs) or autologous bone marrow MSCs (BMSCs) has demonstrated unexpected effects including the invasive and painful isolation method, inadequate amounts of bone marrow (BM) stem cells, the anti-inflammatory impact reduction of AdMSCs that are isolated from fat patients, and the cell number and differentiation potential decrease with an increase in the age of BMSCs donor. Researchers have been trying to search for alternate tissue sources for MSCs, especially fetal annexes, which could offer a novel therapeutic choice for MS therapy due to the limitation of low cell yield and invasive collection methods of autologous MSCs. The transplantation of MSCs for MS treatment is discussed in this review. Finally, it is suggested that allogeneic sources of MSCs are an appealing alternative to autologous MSCs and could hence be a potential novel solution to MS therapy.


Introduction
MS is one of the most prevalent central nervous system (CNS)-influencing autoimmune and inflammatory neurological diseases. It induces neural fiber myelin destruction, which results in severe neurological symptoms and causes social and economic impairment in young patients, mainly between the ages of 20 and 40 [1].
The clinical signs and symptoms of MS rely on where the plaques or neural lesions are positioned and generally include tingling and numbness, imbalance of sensory, cognitive and vision impairment, fatigue, sleep and balance disorder, pains and spasms, and sexual problems [2].
Immune cell infiltration and their secretions cause inflammation in the central nervous tissues, the white and gray matter, and MS damage. Many reviews have reported that the interfering of CD4 T (T helper) cells and acquired immune reply arises from the interaction of T cells with antigen-presenting cells (APCs) that playing a primary role in the initiation and progressing of the disorder. B cells, macrophages, microglial cells (resident macrophages of the CNS), and dendritic cells (DC) are among these APCs [3].
There is still no definitive and complete treatment strategy for MS since MS's cause is not fully understood. Treatment for MS requires a comprehensive treatment strategy. This versatility includes nutrition, rehabilitation, and medication through using of disease-modifying drugs )DMD(, including interferon-ß, fingolimod, dimethyl fumarate, and glatiramer acetate. These medications decrease the intensity and frequency of MS attacks, reduce impairment, and moderate the disease stage, while they have different side effects [4]. Stem cells (SCs) can prevent tissue degeneration as part of the natural regenerative systems of the body and regenerating the damaged tissues. They have a minimal undesirable response after injection, and most of them are safe for sick persons.
Mesenchymal stromal cells (MSCs) with self-renewal, differentiation ability, and various functional properties, such as neural differentiation potency, neurotrophic and neuroprotective ability, and anti-inflammatory impact, suggest several new tissue regeneration mechanisms used in multiple clinical trials [5].
According to the International Society of Cell Therapy (ISCT), three minimal measures for identifying hMSC include: MSCs are adherent to plastic if cultured in standard settings. Positive selection markers of MSCs include expressing CD73, CD90, and CD105. In contrast, negative selection markers of MSCs include no expression of hematopoietic markers, including B cell (CD19 or CD79a), monocyte/macrophage (CD14 or CD11b), endothelial and Pan-leukocyte marker (CD45), hematopoietic (CD34), and human leukocyte antigen -DR (HLA-DR). MSCs can in vitro differentiate into trilineage differentiation, chondrocytes osteoblasts, and adipocyte s [6].
Based on ISCT criteria, the isolation of MSCs has generated nonclonal, heterogeneous cultures of SCs, which contain SCs with various multipotent characteristics, committed progenitors, together with differentiated cells. The nature and functions of hMSCs are still unclear; however, sources of putative hMSCs for cell therapy are currently nonclonal stromal cultures derived from BM and alternative tissues while some reports underscore their efficacy for treating different diseases [7].
To date, many surprising data were received mostly via autologous human adipose-derived mesenchymal stromal cells (hAdMSCs) or human bone marrowderived mesenchymal stromal cells (hBMSCs) in experimental animals and clinical trials [4,[8][9][10]. However, their clinical usage was restricted due to the low yield of SCs and the invasive collecting methods. As a result, these limitations have driven researchers to look for alternate sources of tissue for MSCs, including fetal annexes, which could be an innovative clinical choice for treating MS.
Here, experimental and clinical trials offer a description of MSC transplantation in MS therapy. Besides, an appealing alternative to autologous MSCs is suggested to be allogeneic sources of MSCs, which display similar qualities to autologous MSCs and are not invasive for isolation, and thus could be a potential novel solution to MS therapy [11][12][13].

Mesenchymal stromal cells (MSCs)
Mesenchymal stem cells possess a broad range of diversities and consist of several sub-populations; hence it is better to call them "mesenchymal stromal cells." These are nonhematopoietic, spindle-shaped, and self-renewable cells that are easily accessible and cultural, along with the ability to be expandable in vitro with exceptional genomic stability. They could be harvested from many tissues, including the placenta, BM, adipose, umbilical cord, peripheral blood, endometrium, Wharton's jelly, menstrual blood, muscle, decidual, and other tissues in vitro [14].
Inflammatory cytokines, including chemokines, are elevated in damaging tissue. For example, chemokine upregulation such as CCL19, SDF-1 (stem cell-derived factor 1), CCL21, and CXCL10 stimulate the expression of their receptors, including CXCR4, CXCR3, CXCR6, CCR, and CCR10 on MSCs. The rise of MSCs receptors induces movement in the gradient of chemotactic cytokines [15].
Besides, MSCs, as the body's natural pharmacies, have various regenerative effects due to their various microenvironments and cellular niches, differentiate and secrete diverse growth factors despite their similar phenotype and morphology [16].
The proposed action mechanisms of MSCs in neurological diseases are demonstrated in Fig. 1 [19]. B-cell proliferation is repressed by MSCs, not by inducing apoptosis, but by stopping the B cell-division cycle (in the G0/G1 phase) [20].
MSCs prevent the differentiation of B cell into plasma cell and the development of antibodies; also, they inhibit B cell differentiation with a decrease in immunoglobulin (IgM, IgG, and IgA) production. Therefore, an effective mechanism of MSCs is the production and secretion of soluble factors as a paracrine leader for B cell suppression [21].
Corcione et al. [20] showed that the expression of chemokine receptors, including CXCR4, CXCR5, and CCR7, on B cell surface was downregulated by co-culture with MSCs, and decreasing them was paralleled by a reduction of B cell chemotaxis in reply to CXCL13, CXCL12, and CCL19 and the ligands of CXCR5, CXCR4, and CCR7, respectivel y [22]. Furthermore, MSC interfered with B cell function in several ways, such as chemotaxis, differentiation to antibodyproducing plasma cells, and proliferation. According to the successful use and efficiency of anti-CD20 drugs that significantly target B cells, therapies that target B cells, such as MSCs injection in MS patients, can be promising [22,23].

The impact of MSCs on T cells
Recently, another function of MSCs has been shown that can moderate the function of many activated T cells. Earlier investigations show that MSCs inhibit T cells in a contact-dependent way with soluble factors and their two cell adhesion proteins ( VCAM-1 and ICAM-1) [21]. MSCs could hinder T CD8 and CD4 proliferation and inhibit T lymphocytes derived from other species [15]. Some influences of MSCs on T cells include: To delay T cell proliferation at the damage location, MSCs release hepatocyte growth factor (HGF), galectin 1, semaphoring 3A, and prostaglandin E2 (PGE2) [24]. MSCs stimulate differentiation of T cells into CD4+ CD25+ Treg in the vicinity of immature DCs [25].
MSCs have a dual effect with indolamine-2,3-dioxygenase (IDO) and that produces in an inflammatory condition with the presence of interferon-gamma (IFN-γ) on their surface, such as antiproliferative impacts on T cells and persuading impacts on Treg cells proliferation [26]. MSCs modify immune responses and keeping homeostasis by releasing many cytokines by own or stimulating secreting immune cytokines [27].
MSCs constrain naive T cell differentiation into Th17 cells. The recently identified CD4 + T h 17 subsets create IL-17 and have been involved in several autoimmunity types as an essential member of progressing disease [28].

The effect of MSCs on APCs
Previous studies have demonstrated that hMSCs affect APC, especially DCs, as potent APCs, by reducing HLA-DR expression and costimulatory molecules (CD80, CD86) [30]. Surprisingly, MSCs delay the maturation of DCs by reducing IL-12 secretion and increasing Tumor necrosis factor-inducible gene 6 protein (TSG-6), IL-10, and IL-6 production by inhibiting the activation of nuclear factor-kappa B (NF-κB) cell signaling cascade and mitogen-activated protein kinase (MAP kinase) [31]. NSCs and MSCs can express toll-like receptors (TLRs) and stimulate indoleamine 2, 3-dioxygenase 1 (IDO1) production in the immune cells. IDO1 is an immunosuppressive molecule that regulates the proliferation, differentiation, and migration of activated cells, while human MSCs stimulate through TLR ligands; further, they secret CXCL10, IL-8, and IL-6 molecule s [22]. Macrophages as DCs show similarly affected by MSCs and a sudden change to M2 activated macrophages with secreting an anti-inflammatory cytokine and decrease APC activity being showed.
Beyth et al. [32] exhibit that while hMSCs can raise superantigen-induced purified T lymphocytes activation, the addition of antigen-presenting cells, including APCs (either dendritic or monocytes cells) to the cultures, prevents the responses of T lymphocytes. MSCs exert an immunomodulatory and immunosuppressive role in the responses of immune cells by secreting soluble paracrine factors, as shown in Fig. 3.

The effect of MSCs on neurons
MSCs have mesodermal lineage, but interestingly, multiple investigations have revealed to transdifferentiate these cells into ectodermal and endodermal lineages, particularly neurons and glial-like cells [34]. MSCs can also induce glial cells to produce neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) that could act for some of the neurodegenerative actions by influencing precursors and astrocytes through nuclear factor erythroid 2-related factor 2 (Nrf2) or p38MAPK [35].
MSCs transplantation has been demonstrated to enhance neurological functional recovery in MS preclinical animal models, including experimental autoimmune encephalomyelitis (EAE) mice [36] and non-immune CNS including stroke models [37].
Kim et al. [38] used lipopolysaccharide-induced inflammation models in vitro and in vivo to examine the protective influence of MSCs on the dopaminergic system by antiinflammatory effects. Human MSCs therapy notably declined nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) Fig. 3 Immunomodulatory and immunosuppressive role of MSCs in the responses of immune cells, adapted from reference [33]. Reproduced by permission of Elsevier production, inducible NO synthase mRNA expression, TNFα, and lipopolysaccharide-induced microglial activation than the treatment group of lipopolysaccharide-only. In mesencephalic dopaminergic neurons and microglia co-cultures, hMSCs injection significantly decreased the loss of tyrosine hydroxylase-immunopositive (TH-ip) cells [39]. In Fig. 4, the neuroprotective and neurorestorative roles of MSCs are depicted. MSCs release various neurotrophic factors that induce endogenous neurogenesis, promote axonal remyelination, decrease apoptosis, and inhibit microglial activation and astrocyte proliferation.

The effect of MSCs on inflammation
Inflammation of the CNS is one of the main pathogenic pathways in MS. Inflammation and hypoxia are vital signs of damaged tissue that can activate paracrine patterns of MSCs, which are mostly mediated via VEGF, insulin-like growth factor 1 (IGF-1), HGF, and fibroblast growth factor 2 (FGF2). Hence, when a tissue is damaged, inflammatory chemokines, including SDF-1 (CXCL12), CXCL10, CCL19, and CCL21, are increased in that tissue [40]. Increased chemokines stimulate the expression of their receptors, including CXCR4, CXCR3, CXCR10, CCR, and CCR6 on MSC, and this increase of cell surface receptors is capable of promoting immigration on the side of the chemokines gradient [41].

Autologous MSCs administration in patients with MS
Bone marrow-MSCs (BMSCs) Currently, BMSCs are the most regularly utilized MSCs in clinical trials. However, BMSCs have special restrictions, such as the invasive and painful isolating method used for collecting, inadequate amounts of SCs in the adult bone marrow (BM); approximately 0.001-0.01% of SC population overall, and decrease of SCs differentiation behavior and numbers with increasing the donor's age [43].
Before they get into cellular aging and stop proliferation, BMSCs could be passaged in vitro a restricted number of times. These cells are not dead and could be retained for months in this non-proliferative stage [44]. The senescence state touches molecular and functional patterns of BMSCs are touched. This state was primarily explained by Leonard Hayflick in the 1960s [45]. Senescence molecular pathways, such as DNA damage, are triggered by increasing oxidative stress and cyclindependent kinase inhibitor p16INK4a, reducing the telomeres or modified telomeric structures epigenetic modifications [46].
Many intrathecal injection studies have been used BMSCs, which have shown relatively beneficial effects in patients with advanced MS [47]. In an investigation by Bonab et al. on 22 patients with advanced MS, intrathecal injection of BMSCs showed no side effects after 1 year of follow-up, while a considerable improvement or stabilization in the progressive MS disorder course was observed in patients after the first year of MSCstransplantation [48].

Adipose-derived MSCs (AdMSCs)
Human adipose tissue is a plentiful and available source of isolating MSCs, especially for autologous SCs therapy in enough numbers with high yield (more than BM) and a minimally invasive lipectomy and discomfort method. The immunomodulatory, differentiation behaviors, and secretion profile of AdMSCs, including granulocytemacrophage colony-stimulating factor (GM-CSF), TNFα, IL-8, and IL-6, are equal to BMSCs. In contrast, AdMSCs are independent of age and have identical genetic stability, safety profile, viability and differentiation ability, migration, and lodging patterns in young and elderly patients in contrast to BMSCs, and are beneficial for immune-related diseases, including graft versus host disorder (GvHD), MS, and rheumatic disease [49].
Recent studies suggest that murine AdMSCs inhibited T-cells expansion via cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) molecules and also suppressed lipopolysaccharide (LPS)-induced DC maturation [10]. Andez et al. [50] performed a phase I/II, triple blinded, placebo-controlled, and randomized experiment with low-dose (1 × 10 6 cells/kg) or high-dose (4 × 10 6 cells/kg) autologous AdMSCs. They tracked the work for 12 months, showing that AdMSCs are harmless and viable in secondary progressive MS (SPMS) patients. Therefore, AdMSCs inhibit inflammation of the nervous system (NS) and increase functional improvement from traumatic brain hurt through neural stem cells.
Allogeneic mesenchymal stromal cell transplantation in MS Some significant benefits of using allogeneic stem cells in MS treatment include eliminating the need for patient tissue for cell isolation, along with the time needs for cell proliferation. Therefore, after autologous MSCs isolation, the appropriate number of cells is not created for the injection to patients [51].
Some patients' cells, such as the older people, do not answer well to in vitro expansion methods, resulting in insufficient patient injection numbers. Mazzanti et al. [52] research showed the differences in MSCs cytokine profiles between healthy donors and MS patients, especially for IP10 chemokine, bound to CXCR3 reported attracting monocytes, T cells, and NK cells. Therefore, although MSCs isolated from both cell types represent the same proliferation, differentiation, toll-like receptor (TLR) marker, immunosuppressive behavior, phenotype, prevention of DC differentiation, and activation, the use of allogenic MSCs for autoimmune disorders can be more beneficial than autologous [53].
Allogeneic MSCs can be derived from perinatal origins, including umbilical cord, amniotic fluid or membrane, chorionic membrane, Wharton's jelly, and placental decidua. Investigations showed significant advantages of the mentioned sources compared to adult sources, such as BMSCs [54].
A variety of clinical phase trials using hMSCs to treat MS have already been reported which are presented in Table 1.

Human Wharton's jelly MSCs (WJMSCs)
The umbilical cord matrix or Wharton's jelly is a type of tissue surrounding the umbilical blood vessels. Some investigations show that MSCs obtained from other tissues could offer theoretical benefits over BMSCs. WJMSCs show primitive nature, multi-lineage potency, immunomodulatory ability, minor immunogenic behavior, secretion of neurotrophic factors and anti-inflammatory molecules, facility of isolation, high reproduction, and without ethical concerns [55].
In the EAE model, injection of hWJMSCs-derived oligodendrocyte progenitor cells into the brain decreased the disease's symptoms and significantly increased remyelination [13]. Donders et al. [56] showed that hWJMSC inhibited activated T cells proliferation with paracrine factors such as IDO1 and contact-dependent manner both in the EAE model rats and in vitro. Therefore, hWJMSChas trophic support behaviors and effectively moderate immune system cell functioning.

Human placental MSCs (PLMSCs)
The placenta's fetal side is three parts, including the amnion, chorion, and umbilical cord, and the maternal side includes the decidua basalis of the uterus.
Researches have represented that perinatal resources of MSCs, including chorionic plate (CP), amniotic membrane (AM), decidua, and umbilical cord (UC), have benefits over adult MSCs. It seems that decidua-derived mesenchymal stem cells (DD-MSCs) have a significant role as immune modulators in the placenta, capable of modifying lymphocyte behavior and preventing immune reactions [59].
In recent studies, PLMSCs have shown self-renewal capacity and multi-lineage differentiation, no ethical problems, ease accessibly, abundant, and strongly powerfully immunosuppressive behavior isolated from discarding pregnancy products without invasive procedures. In comparison, the extra-embryonic placental tissue is an excellent mass and is readily processed relative to the BM or adipose tissues and creates high quantities of MSCs [60].
Clark et al. reported that PLMSCs-derived extracellular vesicles improve myelin regeneration in EAE models. The injection of PLMSCs significantly decreased the mean clinical score, inflammatory process, and neural demyelination in EAE mice of MS. Moreover, the concentration of IL-23, as a pro-inflammatory cytokine, was reduced. Additionally, the concentration of IL-27, as an anti-inflammatory cytokine, was enhanced in the serum. IL-23 and IL-27 cytokines are associated with excellent and poor MS disease prognosis, respectively [61].
The placental MSC-derived extracellular vesicles that facilitate myelin regeneration were investigated by Clark et al. in an animal MS model. Their in vitro data showed that by promoting OPCs to change into myelinating mature oligodendroglia or oligodendrocyte cells, placental MSC-derived extracellular vesicles facilitate myelin regeneration. These results confirm that the secretion of EVs mediates the action process of PMSCs. So, EVs derived from PMSC are a practical option for multiple sclerosis cellular-based therapies [62].

Human umbilical cord MSCs (hUC-MSCs)
Human umbilical cord MSCs are heterogeneous cell populations, such as 1 and 2 type in culture, and have differential filaments (vimentin and cytokeratin). They are potential cells for cell therapy because of their collection without pain or invasive method, higher and faster proliferation, differentiation abilities, and more vital immune tolerance due to lower HLA-1 expression than other MSCs, especially BMSCs. Also, they differentiate into vast cell types, such as trilineage differentiation, muscle, cardiomyocyte, astrocyte, neuron, and oligodendrocyte.
BMSCs can easily differentiate from adipocyte cells, whereas they have less potency in forming chondrogenic and osteogenic cells than hUC-MSC. Moreover, hUC-MSCs can express immature neuronal markers [63].
Furthermore, an enhancement of HGF factor was seen in the UC-MSCs-treated group, which can have played an important role in improving MS disease. It is necessary to mention that HGF is a multi-functional mediator for the regeneration of tissue with its ability to induce matrix invasion, cell motility, and mitogenesis. In vivo and in vitro experiments have shown that hUC-MSCs have not been transformed into tumor-associated fibroblasts, rendering them safer than BMSCs [64].

Challenges in MSC-based therapy
The most vital dangers of MSCs are fibrosis, proinflammation, and tumorigenicity which is considered the leading risk factor of MSCs in clinical applications. MSCs can migrate to cancer environments through several chemokines, including CXCR4, CXCL7, CXCL6, and CXCL5, along with inflammatory molecules, such as TNF-α, IFN-γ, and IL-6 and growth factors like plateletderived growth factor (PDGF) and HGF [65].
MSCs have been linked to dual effects of progression and suppression on tumor growth, which is also observed in the angiogenic process. Neo-angiogenesis is possible probably due to settling into the walls of tumor vessels after engraftment, enhancing tumor proliferation due to the pro-angiogenic profiles stimulation, and differentiate into pericytes and endothelial cells in vessels. Additionally, MSCs probably stimulate hypoxia-related gene expression and epithelial-mesenchymal transition (EMT) in tumor cells and elevate cancer cell invasion.
In contrast, in other tumor-based studies, MSCs have been indicated to suppress cancer progress by activating programmed cell death in endothelial cells [66].
Furthermore, the therapeutic promise of MSCs in inflammatory and injured tissues probably rely on numerous features, such as the stem cells' quantity, the passage number of transplanted cells, the injection time and method, cell engraftment frequency, cryopreserved duration of MSCs, type, stage, the disease's involved cells, the protocol for in vitro culture, such as using of fetal bovine serum (FBS) or essential supplements, the proper cells' dose, induction factors, oxygen concentrations, and mechanical stimuli [67].

Conclusions and future insights
The potency of MSCs, such as MS, has attracted significant attention for systemic transplantation of these cells to treat CNS disorders. Although most proposals persist on autologous MSCs injection for use in autoimmune disorders, and especially MS, this study concluded that, due to relative safety, uncomplicated manufacturing procedure, the ability to isolate readily, low immunogenicity, independent to the donors' age, less ethical considerations, secretion of relevant factors, less invasive than autologous stem cells, and easy expansion and in vitro manipulation to reach the pureness and essential amounts for transplantation, allogeneic transplantation could be considered a superior option for MS treatment.