General Characteristics of MSCs: MSCs, especially connective tissue-derived cells (bone, cartilage, fat, tendon, stroma), differentiate into many tissue cells (heart, liver, pancreas, nervous system cells) and/or synthesize soluble factors that contribute to tissue/organ regeneration. MSCs expand in vitro by protecting their stem cell properties and support hematopoiesis; when they are transplanted they are not rejected by immunosuppressive properties. Thus, these cells have possible applications in many clinical areas [7].

MSCs are the main cells of connective tissue. These cells were first identified in 1976 by Friedenstein [8], who, using fetal calf serum, showed that cell colonies are adhesive and morphologically resemble fibroblasts in bone marrow culture. These cells can differentiate into bone, fat, and cartilage cells. Previously, these cells were called colony-forming unit fibroblasts (CFU-F) and bone marrow stromal fibroblasts, but they are now known as mesenchymal stem/stromal cells.

Bone marrow, one of the richest sources of stem cells in organisms, hosts hematopoietic, endothelial, and mesenchymal stem/progenitor cells originating from the mesoderm [9]. Different studies indicate that an average of 2 to 100 MSCs have been identified in the 1 × 10⁶ mononuclear cells (MNCs) in bone marrow aspiration. MSCs can be isolated from many tissues other than bone marrow. Enzymatic methods are used for the isolation of MSCs in solid tissues. MSCs can be isolated and expanded because of the adhesion properties from bone/periosteum, muscle tissue, dental pulp and maxillofacial tissues, liver, lipoaspiration materials, umbilical cord blood, cord stroma, placenta, amniotic fluid, and synovial fluid, and even stimulation through the peripheral blood. They have many properties such as adherence to plastic tissue culture dishes, showing fibroblastoid morphology, differentiation, and surface markers, and these features are independent of the tissues they were obtained from. These properties are substantially similar, but the differentiation capacity and functional characteristics may show some changes according to origin of tissue type [10].

In cultures with a heterogeneous cell population, MSCs can be distinguished from other cells by their cell surface molecules, which are different from other hematopoietic stem cells (HSC) and/or tissue-specific antigens. In contrast, being involved in adhesion, cell-cell and cell-extracellular matrix are known to be expressed high. However, a specific antigen has not been described for MSCs [11].

Molecular cell surface properties of bone marrow-derived and cultured MSCs have been studied in detail by flow cytometry. According to this technique, the hematopoietic antigen (e.g., CD45, CD34, CD14, CD11b, HLA class II, CD79α, CD19) positivity rate must not exceed 2 % in the cell population. However, stroma-associated antigens (CD73, CD90, CD29, CD44) must be positive.

According to the International Society for Cellular Therapy (ISCT) criteria, for the antigens CD105 (endoglin), CD73 (ecto-5′-nucleotidase), and CD90 (Thy-1), more than 90 % must be positive in an MSC population [10].

MSCs, which are found in various tissues as support cells, synthesize a large number of bioactive molecules, cytokines, chemokines, enzymes, and extracellular matrix proteins in relation to these features. MSCs release growth factors required for hematopoietic cells in bone marrow such as macrophage colony-stimulating factor (M-CSF), Flt-ligand, and stem cell factor (SCF). Furthermore, not only interleukin-6 (IL-6), IL-7, IL-8, IL-11, IL-12, IL-14, and IL-15 cytokines but also MSCs synthesize chemokines, including stromal-derived factor-1alpha (SDF-1alpha)/CXCL12 and monocyte chemoattractant protein (MCP)-1 [12].

MSCs transplanted into the injured area of the spinal cord have been shown to reduce apoptosis in neuronal cells [13]. Other studies have shown that neurotrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor-β (NGF-β) are released by the MSCs that increase the life of the neuronal cells [14–16]. In addition, in vitro cocultures obtained from 25 different bone marrow MSCs and neuronal stem cells obtained from the cerebral cortex of mice showed that increased survival and migration properties have been observed for neuronal cells [17].

One of the most advantageous in vivo applications of MSCs is that immunogenicity of these cells is low and has inhibitory properties on the immune system. Besides these immune-regulating properties, they exert anti-inflammatory effects. They also prevent alloreactive T-cell activation and further reactions, inhibit activated B lymphocytes, and stimulate regulatory T cells. Thus, an immunosuppressive effect was shown resulting from the inhibiting lymphocyte proliferation in animals and humans. A dose-dependent stimulating effect probability is shown on the immune system in some circumstances [18]. In addition, the immunomodulatory effects of MSCs, anti-apoptotic factors, and angiogenic and antifibrotic properties are also available. These features help explain their role in wound healing [19]. MSCs also prevent scar tissue formation with their antifibrotic property in spinal cord injury and inhibit the axonal regeneration [19].

The most interesting feature of MSCs, especially for applications in regenerative medicine, is the potential to differentiate into a variety of cell types in appropriate microenvironments, including connective tissue. Several researchers have stimulated in vitro osteogenic, adipogenic, chondrogenic, and myogenic differentiation with appropriate signals and demonstrated generated hematopoietic stroma. In the field of regenerative medicine, “stem cell plasticity” is the ability of a cell to differentiate into tissues other than that from which it originated [10]. Myoblast cell [20] and hepatic [21], cardiac [22], renal [23], and neural cell [24] differentiations have been shown in the studies with bone marrow-derived stem cells.

MSCs migrate to sites of inflammation in the body and differentiate to damaged cells and are helpful in healing wounds. The same property is realized for transplanted MSCs in the damaged area. Rat bone marrow stromal cells transplanted to spinal cord injury patients differentiated into neurons [25] and astrocytes [26], and functional improvement was documented based on BBB behavioral rating scoring [27].

Effective mechanisms of MSCs for improvement in damage, differentiation to mature functional cells, cell function recovery after damaged cell-MSC fusion, cell-cell and cell-extracellular matrix relationship with MSCs, release of the soluble factors (growth factors, cytokines, chemokines, etc.), paracrine factors, and the enzyme or immunomodulatory, anti-inflammatory, anti-apoptotic, and angiogenic effects can be considered [28].

Migration is required of MSCs from their niche into the damaged tissues. The signal for migration of cells has been shown to come from the damaged tissue microenvironments. Soluble factors like SDF-1/CXCL12, MCP-1, and complement C3 fraction released in the damaged area are known to have important roles in migration [29].

In spinal cord injury, a major problem is damage to cells forming the layer of myelin (oligodendrocytes and Schwann cells) and the transmission of pulses in the myelin sheath surrounding axons [30]. In studies, MSCs have been shown to prevent myelin loss and to promote myelination in spinal cord injury. Akiyama et al. [31] showed that bone marrow stromal cells differentiate into cells that express myelin and a myelin layer reformed around demyelinated axons [31]. In this study, the pulse transmission rate of the demyelinated axons was increased compared with the control group, as determined by in vitro electrophysiological methods. These results were supported in other studies carried out by Akiyama. Bone marrow-derived stromal cells differentiated especially into Schwann cells have been proven in another study [32].

Clinical benefits were obtained with MSC infusion or tissue implantation in in vivo applications, but the fate of these cells after infusion, migration to damaged tissue and placement or accumulation in that area, proliferation, and differentiation features have not been studied in detail. In most studies, after cell infusion, difficulties are encountered in determining the presence of cells in tissue or blood.

Demonstration of different sex cells of the donor or use of species-specific markers (e.g., beta-2-microglobulin for human cells in animal tissues) are among the methods applied for the purpose of tracking MSCs in tissue. Tagging cells with fluorescent dye is one of the easiest methods of tissue tracking [33]. Temporary fluorescent markers such as CFSE, DiL, and DiO can also be used for short-term experimental studies [34]. Genetic marking with green fluorescent protein (GFP) is the preferred method because it is permanent. In vivo tracking of luciferase-labeled MSCs with a bioluminescence device and labeling with superparamagnetic iron oxide nanoparticles and imaging in vivo under magnetic resonance are other commonly used methods [35].

The advantages of MSCs in clinical practice are as follows: