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Stem cells STEM CELLS Stem Cells’ Definition: Stem cells are defined as ‘immature’ or undifferentiated cells which are found in multicellular organisms. They are able to produce identical daughter cells (clonogenic), divide indefinitely (self-renewing), and differentiate into multiple cell lineages (potent) giving different cell types with different functions (Robey, 2000). Stem cells remain uncommitted until they receive a signal to develop into specialized cell. They may remain dormant or quiescent for prolonged periods until they are exposed to a particular stimulus. A stem cell is able to produce at least one and often many types of highly-differentiated cell. They serve as a repair system being able to divide without limit to replenish other cells. When stem cells divide, each new cell has the potential to either remain as a stem cell or become another cell type with new special functions, such as blood cells, nerve cells, or heart cells (Little et al., 2006). Stem Cells Properties: Stems cells are those that have 3 general properties: (1) They are capable of dividing and renewing themselves for long periods (selfrenewal). (2) They are unspecialized. (3 They can differentiate into multiple types of functionally mature specialized cells (Multipotency) (Blau et al., 2001). Stem cells (1) Self-renewal: Stem cells have the capacity for self-renewal (undergoing cycles of mitotic division while maintaining the same undifferentiated state), which ensures that stem cell reserves are not exhausted throughout time (He et al., 2009). For stem cells to maintain themselves in tissue and at the same time provide cells to maintain the differentiated function of that tissue, stem cells fate is controlled by two general modes of cell division. The first mechanism is asymmetric cell division such that it produces one differentiated daughter cell and one cell that is still a stem cell. This allows maintaining a constant number of stem cells, which is generally sufficient under physiological conditions. The second mechanism, symmetric cell division is a highly regulated process in which a stem cell gives rise to daughter cells that have a finite probability of either becoming stem cells or differentiated cells. This leads to an expansion of the stem cell pool, a condition required after tissue injury or in diseased conditions causing loss of differentiated cells (Yu et al., 2006). The mechanisms that regulate these division processes are complex and may be a combination of both mechanisms depending on the cellular environment or biological needs (Pattern et al., 2000). (2) Stem cell potency: The ability to differentiate into specialized cell types and be able to give rise to any mature cell type is referred to as potency. Potency of the stem cell specifies the differentiation potential i.e., the potential to differentiate into different cell types. According to potency stem cells can be classified into: pluripotent and multipotent (Hima Bindu & Srilath, 2011). totipotent, Stem cells Totipotent stem cells: They are cells that can generate full functional organism, as well as the placenta and other supporting tissues (embryonic as well as extra-embryonic tissues). The only totipotent cells are the fertilized egg and the cells produced by the first few divisions of the fertilized egg as they can give rise to any type of cell: cells of the trophoblast and cells of the 3 germ layers (endoderm, mesoderm, and ectoderm), all of which are necessary for complete embryonic development (Sorapop et al., 2006). Pluripotent stem cells: They are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three embryonic germ layers (ectoderm, mesoderm and endoderm) but not the whole organism (can not give rise to trophoblasts). They are classified into: embryonic stem cells (ESCs), embryonic germ cells, embryonic carcinoma cells and multipotent adult progenitor cell from bone marrow (Muller-Sieburg et al., 2002). Multipotent stem cells: Stem cells that have a more retricted ability to give rise to cells of different lineages within a single germ layer are considered multipotent (adult stem cells) .They can differentiate into a number of cells, but only those of a closely related family of cells. These are true stem cells but can only differentiate into a limited number of types. They include haemopoietic stem cells, neuronal stem cells and mesenchymal stem cells (Krause et al., 2001). Stem cells Unipotent stem cells: Tissue-specific progenitor stem cells or unipotent stem cells can differentiate and give rise to only one cell type, their own. Unipotentiality of cells means, only able to generate one cell type but have the property of self-renewal, which distinguishes them from non-stem cells. Unipotent stem cells include the stem cells in the gut epithelium, the skin, and the seminiferous epithelium of the testis. Most epithelial tissues self-renew throughout adult life due to the presence of unipotent progenitor cells (Blanpain et al., 2007). Induced pluripotent stem cells: Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that can be produced from adult somatic cells by genetic reprograming or forced introduction of genes. iPSCs are epigenetically reprogrammed to lose tissuespecific features and gain pluripotency. Similar to hESCs, they can theoretically differentiate into any type of cells ( Patel & Yang, 2010). In 2006, Takahashi and Yamanaka introduced the concept of induced pluripotent stem cells by generating stem cells by using a combination of 4 reprogramming factors, including Oct4 (Octamer binding transcription factor-4), Sox2 (Sex determining region Y)-box 2, Klf4 (Kruppel Like Factor-4), and c-Myc and were demonstrated both self-renewing and differentiating like ESCs (Takahashi & Yamanaka, 2006). In 2007, they reported that a similar approach was applicable for human fibroblasts and human iPSCs can be generated (Takahashi et al., 2007). James Thomson's group also reported the generation of human iPSC using a different combination of factors. Since then, a number of different reprogramming factors and methods have been established (Yu et al., 2007). Stem cells Initially iPSCs were generated using either retroviruses or lentiviruses, which might cause insertional mutagenesis and thus could perhaps even lead to adverse effects like those seen in some attempts at gene therapy (Hacein-Bey-Abina et al., 2003). In addition, retroviruses may make iPSCs immunogenic (Zhao et al., 2011). Thus, for the purpose of cell transplantation therapy many ways to generate integration-free iPSCs have been reported. These methods include plasmid (Okita et al., 2008), adenovirus (Stadtfeld et al., 2008), synthesized RNAs (Warren et al., 2010), and proteins (Kim et al., 2009). Figure (1): Reprogramming of adult stem cells into iPS cells (Meregalli et al., 2011). Patient-derived iPSCs have been shown to be useful tools for drug development and modeling of diseases. Scientists hope to use them in transplantation medicine. Patient-specific iPSCs can be used to recapitulate phenotypes of not only monogenic diseases but also late-onset polygenic diseases, such as Parkinson's disease (Devine et al., 2011), Alzheimer's disease (Israel et al., Stem cells 2012), and schizophrenia (Brennand et al., 2011). Excitement surrounds the potential for application of these cells to both analysis of disease mechanisms and investigation of potential new treatments. Somatic cells derived from iPSCs, particularly cardiac myocytes and hepatocytes, could also be used for toxicology testing as an alternative to existing approaches (Yamanaka, 2009). In addition, iPSCs can be used in animal biotechnology and genetic engineering. This allows for the generation of disease models and the production of useful substances, such as enzymes, that are deficient in patients with genetic diseases. One of the most striking applications of iPSCs was reported by Nakauchi and coworkers, who generated a rat pancreas in a mouse, by microinjecting rat iPSCs into mouse blastocysts deficient in a gene essential for pancreas development. In the future, it might become possible to generate organs for human transplantation using a similar strategy (Kobayashi et al., 2010). Stem Cells Origin: (1) Embryonic Stem Cells (ESC): Embryonic stem cells derived from the inner cell mass of the blastocyst (5- to 7day-old embryo) possess two important characteristics: self-renewal and pluripotency. ES cell lines are sometimes referred to as immortal due to their ability to keep dividing or self-renewing over many generations. Derivation of human pluripotent embryonic stem cell lines and recent advances in hESC biology has created great interest in the field of stem cell-based engineering, but there is Stem cells ethical debates surrounding their use (Rippon & Bishop, 2004). Figure (2): Differentiation potentiality of human embryonic stem cell lines (Meregalli et al., 2011). (2) Fetal Stem Cells: Fetal stem cells are primitive cell types found in the organs of fetus (approximately 10 weeks of gestation). Fetal stem cells can be isolated from fetal blood and bone marrow as well as from other fetal tissues, including liver and kidney. Fetal stem cells are generally tissue-specific, generating the mature cell types within the particular tissue or organ they are found in. This type of stem cell is currently grouped into an adult stem cell (Guillot et al., 2006). Stem cells (3) Cord Blood Stem Cells: Blood from the umbilical cord contains multipotent stem cells that are genetically identical to the newborn. The umbilical cord blood is often banked, or stored, for possible future use of stem cell therapy (Lee et al., 2004). (4) Adult Stem Cells: Adult stem cells are undifferentiated cells that occur in a developed organism in the post natal state and have two properties: the ability to divide and renew themselves and to be specialized to give cell types of the tissue of origin. Adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin. They are found both in children, as well as adults. Their main role is thought to be maintenance and repair of tissues (Tuan et al., 2003). Adult stem cells are classified by the source tissue or types of cells from which they are derived into: bone marrow stem cells, peripheral blood stem cells, heart and lung stem cells, intestinal and liver stem cells, pancreatic stem cells, kidney stem cells, central nervous system stem cells, skin stem cells, skeletal muscle stem cells, dental pulp derived stem cells, and adipose tissue stem cells (Toma et al., 2001). Bone marrow stem cells: The bone marrow (BM) contains three kinds of stem cells: endothelial stem cells, hematopoietic stem cells and mesenchymal stem cells (bone marrow stromal cells). 1- Endothelial stem cells: Endothelial progenitor cells (EPCs) are present in BM and blood. They have the ability to differentiate into endothelial cells, the cells Stem cells that line the inner surfaces of blood vessels throughout the body. They participate in new vessel formation (neovascularization) in physiological states as wound healing and in pathological states as tumor angiogenesis (Folkman, 1998). 2- Hematopoietic stem cells (HSCs): HSCs are present in bone marrow, peripheral blood and umbilical cord blood. They give rise to all the types of blood cells through the process of haematopoiesis. They give rise to both the myeloid and lymphoid lineages of blood cells. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, and megakaryocytes or platelets. Lymphoid cells include T cells, B cells, and natural killer cells (Kondo et al., 2003). 3- Mesenchymal stem cells: MSC refer to a postnatal, multipotent and self-renewing precursor derived from an original embryonic MSC. They function to maintain the turnover of skeletal tissues in homeostasis or tissue repair during adulthood. MSCs progress through discrete stages of differentiation in an orderly manner to give rise to functionally and phenotypically mature tissues, including bone, smooth muscle, tendons and cartilage (Bianco et al., 2001). Stem cells Stem Cell Niche: Stem cell niche is defined as the cellular and molecular microenvironments that regulate stem cell function, provides signals to stem cells in the form of secreted and cell surface molecules to control the rate of stem cell proliferation, determine the fate of stem cell daughters, and protect stem cells from exhaustion or death. This includes control of the balance between quiescence, self-renewal, and differentiation, as well as the engagement of specific programs in response to stress (Xie & Spradling, 2000). Within the human body, stem-cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding micro-environment actively signals to stem cells to promote either self-renewal or differentiation to form new tissues. Several factors are important to regulate stem-cell characteristics within the niche: cell–cell interactions between stem cells, as well as interactions between stem cells and neighbouring differentiated cells, interactions between stem cells and adhesion molecules, the oxygen tension, extracellular matrix components, cytokines, growth factors and the physicochemical nature of the environment including pH, ionic strength and metabolites (O’Brien & Bilder 2013). Stem cells Figure (3): Composition of the niche. Stem cell niches are complex, heterotypic, dynamic structures, which include different cellular components, secreted factors, immunological control, ECM, physical parameters and metabolic control (Lane et al., 2014). Stem cells Mesenchymal Stem Cells: 1- Defining mesenchymal stem cells: Stem cells are classically defined by their multipotency and selfrenewal. They are derived from mesodermal germ layer. Their function is to maintain the turnover of skeletal tissues in homeostasis or tissue repair during adulthood (Chanda et al., 2010). MSCs were firstly identified in the 1960s as colony-forming unit fibroblasts (CFU-Fs) by Friedenstein and coworkers who observed that the bone marrow is a source of stem cells for mesenchymal tissue. They described these cells as mononuclear nonphagocytic cells with fibroblast-like phenotype and colongenic potential capable of adhering to the culture surface in a monolayer culture (Friedenstein et al., 1968). Friedenstein was the first investigator to demonstrate the ability of MSCs to differentiate into mesoderm-derived tissue. During the 1980s, MSCs were shown to differentiate into osteoblasts, chondrocytes, and adipocytes (Piersma et al., 1985). In the 1990s, MSCs were shown to differentiate into a myogenic phenotype (Wakitani et al., 1995). In 1999, Kopen et al. described the capacity of MSCs to transdifferentiate into ectoderm-derived tissue. 2- Characterization of Mesenchymal Stem Cells: MSCs express a large number of adhesion molecules, extracellular matrix proteins, cytokine and growth factor receptors, associated with their function and cell interactions within the bone marrow stroma (Deans and& Moseley, 2000). The International Society for Cell Therapy proposed criteria for MSCs that comprise: (1) adherence to plastic in standard culture conditions; (2) expression of Stem cells the surface molecules CD73, CD90, and CD105 in the absence of CD34 (the primitive hematopoietic stem cell marker), CD45 (a marker of all hematopoietic cells), HLA-DR, CD14 or CD11b (an immune cell marker), CD79a, or CD19 surface molecules as assessed by fluorescence-activated cell sorter analysis; and (3) a capacity for differentiation into mesodermal cells such as osteoblasts, adipose cells, cartilage cells or skeletal muscle cells under standard differentiation conditions in vitro (Dominici et al, 2006). The typical CD markers of bone marrow-derived MSCs are, CD105, CD73CD90, CD45−, CD34− and HLA-DR− (Hagmann et al., 2013). These criteria were established to standardize human MSC isolation but may not apply uniformly to other species. For example, murine MSCs have been shown to differ in behavior and marker expression when compared with human MSCs. It is believed that certain in vivo surface markers may no longer be expressed after explantation when MSCs are isolated and expanded in culture, although new markers are acquired during expansion (Peister et al., 2004). MSCs Multilineage Differentiation Potential: Differentiation of MSCs is regulated by genetic events, involving transcription factors. Differentiation to a particular phenotype pathway can be controlled by some regulatory genes which can induce progenitor cells’ differentiation to a specific lineage. The microenvironment has the strongest influence on the maturation and differentiation of MSCs. However, growth factors, induction chemicals, cell-to-cell communication, physical factors and cell structure were found to have an effect (Ding et al., 2011). Stem cells 1. Mesoderm Differentiation: Theoretically, mesodermal differentiation is easily attainable for MSCs because they are from same embryonic origin. For osteogenesis, MSCs in the presence of dexamethasone, 𝛽-glycerophosphate, and ascorbic acid express alkaline phosphatase and calcium accumulation, a morphology consistent with osteogenic differentiation. Osteogenic differentiation of MSCs is a complex process controlled by multiple signaling pathways and transcription factors. Runt related transcription factor 2 (Runx2) and Caveolin-1 are considered key regulators of osteogenic differentiation. Bone morphogenetic proteins (BMPs), especially BMP-2, BMP-6, and BMP-9, strongly promote osteogenesis in MSCs. BMP-2 induces the p300 mediated acetylation of Runx2, a master osteogenic gene, which results in enhanced Runx2 transactivating capability. The acetylation is specific to histone deacetylases 4 and 5, which, by deacetylating Runx2, promote its subsequent degradation by Smurf1 and Smurf2, and E3 ubiquitin ligases (Hwang et al., 2009). Core binding factor alpha-1/osteoblast-specific factor2 (cbfa1/osf2) and Wnt signaling are also involved in osteogenic differentiation of MSCs (Gaur et al., 2005). In adipogenesis, dexamethasone and isobutyl-methylxanthine (IBMX) and indomethacin (IM) have been used for induction of differentiation. Peroxisome proliferator-activated receptors-𝛾2 (PPAR𝛾2), CCAAT/enhancer binding protein (C/EBP), and retinoic C receptor have been implicated in adipogenesis. Phosphatidylinositol 3-kinase (PI3K) activated by Epac leads to the activation of protein kinase B (PKB)/cAMP response element-binding protein (CREB) signaling and the upregulation of PPAR𝛾 expression, which in turn activate the transcription of adipogenicgenes (Ding et al., 2011). Stem cells In chondrogenesis, transforming growth factor (TGF)-𝛽1 and 𝛽2 are reported to be involved. Differentiation of MSCs into cartilage is characterized by upregulation of cartilage specific genes, collagen type II, IX, aggrecan, and biosynthesis of collagen and proteoglycans. The emerging results suggested the possible roles of Wnt/𝛽catenin in determining differentiation commitment of mesenchymal cells between osteogenesis and chondrogenesis (Huang et al., 2004). A recent report suggested that miR-449a regulates the chondrogenesis of human MSCs through direct targeting of Lymphoid Enhancer-Binding Factor-1 (Paik et al., 2012). Elevated 𝛽catenin signaling induces Runx2, resulting in osteoblast differentiation, whereas reduced 𝛽-catenin signaling has the opposite effect on gene expression, inducing chondrogenesis (Day et al., 2005). Figure (4): Molecular regulation of MSC differentiation. Wnt signaling and TGF𝛽 induce intracellular signaling and regulate differentiation of MSCs (Williams & Hare, 2011). Stem cells 2. Ectoderm Differentiation: In vitro neuronal differentiation of MSCs can be induced by DMSO, butylated hydroxyanisole (BHA), 𝛽-mercaptoethanol, KCL, forskolin, and hydrocortisone (Ding et al., 2011). Moreover, Notch-1 and protein kinase A (PKA) pathways are found to be involved in neuronal differentiation. In presence of other stimulatory factors, downregulation of caveolin-1 promotes the neuronal differentiation of MSCs by modulating the Notch signaling pathway (Wang et al., 2012). 3. Endoderm Differentiation: In liver differentiation, hepatocyte growth factor and oncostatin M were used for induction to obtain cuboid cells which expressed appropriate markers (𝛼-fetoprotein, glucose 6-phosphatase, tyrosine aminotransferase, and CK-18) and albumin production in vitro. Recent studies identified methods to develop pancreatic islet 𝛽-cell differentiation from adult stem cells. The resulting cells showed specific features, high insulin-1 mRNA content, and synthesis of insulin and nestin (Bhandari et al., 2010). Murine adipose tissuederived mesenchymal stem cells can also differentiate to endoderm islet cells (expressing Sox17, Foxa2, GATA-4, and CK-19) with high efficiency then to pancreatic endoderm (Pancreatic and duodenal homeobox 1[Pdx-1], Ngn2, Neurogenic differentiation [NeuroD], paired box-4 [PAX4], and Glut-2), and finally to pancreatic hormone-expressing (insulin, glucagon, and somatostatin) cells (Chandra et al., 2009). Stem cells Figure (4): MSCs multilineage differentiation potential. Adapted from Caplan & Bruder (2001). Stem cells 3- Sources of Mesenchymal Stem Cells: MSCs can be derived from many tissue sources: BM, adipose tissue, synovial tissue, lung tissue, umbilical cord blood, and peripheral blood. Despite sharing similar characteristics, these MSCs from different sources differ in their differentiation potential and gene expression profile (Baksh et al., 2007). MSCs have been isolated from nearly every tissue type (brain, spleen, liver, kidney, lung, BM, muscle, thymus, aorta, vena cava, pancreas) of adult mice, which suggests that MSCs may reside in all postnatal organs (da Silva et al., 2006). MSCs have been reported to constitute about 0.01%–0.001% of the bone marrow mononuclear population. Stem cells harbored in the bone marrow are considered to have the highest multilineage potential. These cells can be isolated from marrow aspirates of the superior iliac crest, femur, and tibia. For this purpose, marrow cells are usually enriched for mononuclear cells with Ficoll or Percol and then plated on culture plastic vessels in order to prepare adherent cell populations. It has recently been demonstrated that late plastic adherent MSCs possess higher osteogenic potential (Eslaminejad et al., 2006). Adipose tissue as well as birth-associated tissues, including umbilical cord and dental pulp has been found to contain MSC-like population. The presence of cells with multipotent diff erentiation capacity in adipose tissue is promising due to the ease of accessibility of adipose tissue and its abundance in the body. Adipose tissue can be an appropriate substitute for marrow in regenerative medicine and tissue engineering . Adipose-derived stromal cells (ADSCs) can be derived from adipose collected by liposuction and lipectomy. ADSCs are able to maintain proliferation potential as well as diff erentiation capacity even in older people. By now, many studies conducted on animal models have confirmed the regenerative potential of ADSCs in bone defects (Bunnell et al., 2008). Stem cells The umbilical cord from a newborn baby contains two arteries and a vein covered with mucus connective tissue rich in hyaluronic acid, referred to as Wharton’s jelly. According to studies, MSC-like cells can be derived from various components of this cord. For example, blood from an umbilical cord is a rich source for pluripotent cells referred to as umbilical-cord-blood-derived MSCs (UCB-MSCs). These cells are quite similar to marrow derived MSCs and have osteogenic potential in an optimized culture (Hutson et al., 2005). Several stem cell types in dental tissue have been reported including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) (Nakamura et al., 2009). 4- Immunology of Mesenchymal Stem Cells: Human MSCs express moderate levels of human leukocyte antigen (HLA) major histocompatibility complex class I, lack major histocompatibility complex class II expression, and do not express costimulatory molecules B7 and CD40 ligand (Tse et al., 2003). Tolerance of MSCs as an allogeneic transplant is due to this unique immunophenotype coupled with powerful immunosuppressive activity via cell-cell contact with target immune cells and secretion of soluble factors, such as nitric oxide, indoleamine 2,3-dioxygnease, and heme oxygenase1. MSCs produce an immunomodulatory effect by interacting with both innate and adaptive immune cells (Figure 5) (Ren et al., 2008). The innate immune cells (neutrophils, dendritic cells, natural killer cells, eosinophils, mast cells, and macrophages) are responsible for a nonspecific defense to infection, and MSCs have been shown to suppress most of these inflammatory cells. Neutrophils are one of the first cells to respond to an infection, and an Stem cells important process in their response to inflammatory mediators is the respiratory burst, characterized by large oxygen consumption and production of reactive oxygen species. MSCs have shown to dampen the respiratory burst process by releasing interleukin (IL)-6. MSCs have been shown to inhibit the differentiation of immature monocytes into dendritic cells which play an important role in antigen presentation to naïve T cells. Additionally, cocultures of MSCs and dendritic cells inhibit the production of tumor necrosis factor- 𝛼, a potent inflammatory molecule (Aggarwal & Pittenger, 2005). Natural killer cells are important innate cells in the defense against viral organisms and in tumor defense by their secretion of cytokines and cytolysis. MSCs cultured with freshly isolated resting natural killer cells have been shown to inhibit IL-2– induced proliferation and decrease secretion of interferon (IFN- 𝛾) by 80%. However, IL-2–activated natural killer cells can lyse autologous and allogeneic MSCs. The adaptive immune system, composed of T and B lymphocytes, is capable of generating specific immune responses to pathogens with the production of memory cells. Once a T cell is activated by a foreign antigen binding to a specific T-cell receptor, the T cell proliferates and releases cytokines (Uccelli et al., 2008). T cells exist as CD8+ cytotoxic T cells that induce death of target cells or CD4 + helper T cells that modulate the other immune cells. MSCs have been shown to suppress T-cell proliferation in a mixed lymphocyte culture. Proliferation of cytotoxic and helper T cells are suppressed via soluble factors released by MSCs, such as hepatocyte growth factor (HGF) and transforming growth factor (TGF) 𝛽1. When MSCs are present during naïve T-cell differentiation to CD4+ T-helper cells, there is a marked decrease in the production of interferon and an increase in the production of IL-4. MSCs are suggested to alter naïve T cells from a Stem cells proinflammatory state (heavy production of interferon- ) to an anti-inflammatory state (greater production of IL-4) (Aggarwal & Pittenger, 2005). It has been demonstrated that MSCs stop the B-cell cycle at the G0/G1 stage and inhibit their diff erentiation into plasma cells (Corcione et al., 2006). Ramasamy et al. (2007) have indicated that BMSCs are able to inhibit dendritic cell (DC) diff erentiation and prevent them from entering into the cell cycle. DCs are able to efficiently present antigens to lymphocytes. Figure (5): MSC interactions with immune cells. MSCs are immunoprivileged cells that inhibit both innate (neutrophils, dendritic cells, natural killer cells) and adaptive (T cells and B cells) immune cells. INF, interferon; TNF, tumor necrosis factor. (Williams & Hare, 2011). Stem cells 5- Delivery of mesenchymal stem cells: The routes of MSC administration are classified into two categories: systemic and topical. There are two types of topical administration: intralesional injection (e.g., intracranial, intracerebral, and subcutaneous) and local vascular injection (e.g., superior vena cava, mesenteric blood vessels, coronary artery). Compared with systemic administration, topical administration routes may have a common advantage in that MSCs arrive directly at the target tissue with little loss during migration (Kim et al., 2014). Types of systemic administration include intravenous (IV), intraperitoneal (IP), intra-arterial (IA) and intracardiac (IC). IV is the most common method in preclinical and clinical settings because of its convenience. However, MSCs administered via this route are more easily trapped in small lung capillaries because of their larger size and expression of cell adhesion molecules. Lung entrapment of MSCs decreases the number of MSCs delivered to target tissues and can result in ineffectual treatment. However, some reports have shown that MSCs delivered via IV injection have protective effects in various animal models even when lung entrapment occurs (Makela et al., 2015). Administration routes determine the microenvironments that MSCs first encounter after entering the patient’s body, thus influencing their differentiation, immunogenicity, and survival (Ishikane et al., 2010). Stem cells 6- Migration and Homing of Mesenchymal Stem Cells: One of the most important features of MSCs is their ability to reach damaged tissue in response to a correct combination of signaling molecules from the injured tissue and corresponding receptors on the MSCs themselves. Therapeutic efficacy of MSC depends on efficient mobilization from their bone marrow niches and trafficking through the circulation to the injured or stressed tissue (Ponte et al., 2007). At the injury site, MSCs could possibly help with repair in two ways: (1) they diff erentiate to tissue cells in order to restore lost morphology as well as function, and (2) MSCs secrete a wide spectrum of bioactive factors that help to create a repair environment by possessing antiapoptotic eff ects, immunoregulatory function, and the stimulation of endothelial progenitor cell proliferation (Granero-Molt´o et al., 2009). Factors Regulating MSC Homing to Damaged or Tumor Tissues: Oxygen Level: Hypoxic tissues express genes that increase cell survival under hypoxic conditions and re-establish the vasculature for oxygen delivery production of (Semenza, chemotactic 2000). factors In addition, implicated hypoxia in cell induces the migration, differentiation and new bone formation. The platelets, inflammatory cells and macrophages arriving at the site of injury secrete cytokines and growth factors, including IL-1 to IL-6, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP). This cellular response leads to the invasion of MSCs, which differentiate into tissue specific cells to complete the repair (Rui et al., 2012). Stem cells Chemokines (Chemotactic Cytokines): Chemokines are small proteins (8–10 KDa) with a capacity appropriate for the migration for creating a chemical environment of lymphocytes, neutrophils, and other immune cells towards inflammation, angiogenesis, and the organogenesis site. They include: 1- Stromal factors cell-derived is stimulating stromal factor-1 (SDF-1): cell-derived factor factor/CXC ligand 12 One 1 of the best-investigated (SDF-1)/pre-B (CXCL12), cell considered a growthmaster regulator of CXC receptor 4 (CXCR4)-positive stem and progenitor cells. SDF-1 signals through its G CXC chemokine receptor-4 protein-coupled trans-membrane receptor (CXCR4). When transfected at sites of ischemic injury, this factor modulates cell differentiation into mature reparative cells. SDF-1/CXCR4 signalling is a key factor in bone marrow stromal cell migration. Therefore, improving the CXCR4 ligand in bone marrow stromal cells should promote their proliferation and migration (Wynn et al., 2004). SDF-1 and other cytokines associate and interact. Shinohara et al demonstrated that SDF-1 and monocyte chemo-attractant protein 3 (MCP3) jointly regulate the homing of MSCs from the systemic circulation in fracture repair. The proportion of cells expressing SDF-1 and MCP-3 was significantly greater in transduced than non-transduced MSCs. Therefore, the homing of MSCs from the systemic circulation is involved in fracture repair via an SDF-1–MCP-3 pathway. The two factors act synergistically. Another study found that by promoting the expression of BMP-2, SDF-1 Stem cells increased MSC migration and differentiation in promoting fracture healing (Granero-Moltó et al., 2009). SDF-1 signaling is important for maintaining postnatal tissue homeostasis, such as cellular inflammatory and immune response, blood homeostasis, and bone remodeling (Yu et al., 2003). At the level of cell function, the rearrangements binding and of SDF-1 integrin to activation, CXCR4 leads eventually to cytoskeleton resulting in the directional migration of CXCR4-expressing cells towards high gradients of SDF-1 (Dar et al., 2006). 2- Transforming chemotactic factor growth for factor bone β (TGF-β): marrow-derived TGF-β MSCs. is It an effective promotes the proliferation of MSCs, preosteoblasts, chondrocytes and osteoblasts and induces collagen, osteopontin, osteonectin, proteoglycans, alkaline phosphatase and other extracellular proteins (Mendelson et al., 2011). 3-Platelet derived growth factor: Blood PDGF is strongly chemotactic for inflammatory cells and has a strong stimulatory effect on MSCs and osteoblast proliferation and migration. From the early to middle stages of bone healing, PDGF promotes the effects of mesenchymal cells in cartilage and bone formation in development. The combined application of PDGF and BMP accelerates bone defect repair, but it is still uncertain whether PDGF can be used for the clinical treatment of fractures (Kodama et al., 2012). Vasculogenesis: It is an important aspect of tissue repair (Kumar et al, 2010). VEGF stimulates MSC mobilization and recruitment to ischemic or damaged tissues although MSCs do not express receptors for VEGF. In Stem cells 2007, Ball et al reported VEGF-A acts through platelet derived growth factor (PDGF) receptors, which determines the vascular cell fate of MSC. Although chemokine signaling initiates homing of MSCs, it still requires multiple steps for their passage through the blood vessel and extravasate at the site of injury or inflammation. Evidence suggests that the MSCs follow somewhat similar mechanisms utilized by the immune cells to migrate to inflammatory sites. Endothelial transmigration is mediated by active interactions between the molecules (glycoprotein or glycolipids, PSGL-1, Lselectin, integrins, GPCR, LFA-1, Mac-1) expressed on leukocyte surface and their receptors (E-selectin, P-selectin, PNAd, MAdCAM, VCAM-1, ICAMs) on the endothelium. Once at the site of tissue damage MSCs have to extravasate (diapedesis), which involves transient disassembly of the endothelial junctions and penetration through the underlying basement membrane (Luster et al, 2005). Expression of Receptors and Adhesion Molecules: Homing is in a significant part dependent on the chemokine receptor, CXCR4, and its binding partner, stromalderived factor-1 CXCL12. Wynn et al demonstrated that CXCR4 is resent on a subpopulation of MSCs, which aid in CXCL12-dependent migration and homing. Aside from CXCR4, freshly isolated BM MSCs and cultured MSCs also express CCR1, CCR4, CCR7, CCR10, CCR9, CXCR5, and CXCR6 which are also involved in MSC migration (Honczarenko et al., 2006). Integrins are another family of cell surface molecules involved in migration of variety of cells and are expressed on adipose-derived MSC-like cells. integrin ligands such as VCAM and ICAM are also expressed on MSCs (Krampera et al., 2006). Stem cells Culture Conditions of MSCs 1-Passage number: It has been shown that with higher passage number, the engraftment efficiency of MSCs decreased. Rombouts et al. had performed a time course experiment; they showed that freshly isolated MSCs had a better efficiency of homing compared to cultured cells (Rombouts & Ploemacher, 2003). 2- Homing receptors:The CXCR4 chemokine receptor that recognizes SDF-1α is highly expressed on bone marrow MSCs, but is lost upon culturing. However, when MSCs are cultured with cytokines (such as HGF, SCF, IL-3, and IL-6), and under hypoxic conditions, CXCR4 expression can be reestablished (Shi et al., 2007). 3-Culture confluence: Matrix metalloproteases (MMPs have been demonstrated to play a role in MSC migration. Expression of MMPs in MSCs is influenced by factors such as hypoxia and increased culture confluence (De Becker et al., 2007). 7- Biological properties supporting clinical use of mesenchymal stem cells in therapy: MSCs have been known as promising tool for therapeutic purpose based on their several advantages including selfrenewal, extensive in vitro expansion, immunomodulation property, engraftment capacity, multi-lineages differentiation potential including few ethical concerns as compared to embryonic stem cells. Moreover, increasing evidences have been shown that MSCs can be isolated from various cell types including adipose tissue, dental pulp, peripheral blood, placenta and umbilical cord. These unique biological properties of MSCs highlight great potential in several applications such as regenerative medicine, tissue engineering and cell-based therapy (Wei et al., 2013). Moreover, MSCs can secrete a number of bioactive molecules that affect to biological changing of other cells or known as paracrine effect. These paracrine Stem cells effects are categorized into six main activities as immunomodulation, anti-apoptosis, angiogenesis, supporting the growth and differentiation local stem and progenitor cells, anti-scarring and chemoattraction (Meirelles Lda et al., 2009). Several studies have shown that MSCs secreted a variety of angiogenic factors including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), placental growth factor (PIGF), monocyte chemoattractant protein 1 (MCP1) and interleukin 6 (IL-6). These paracrine factors are shown to promote local angiogenesis that is important for tissue repair process. Additionally, MSCs secreted large amounts of chemokines which play a role in recruitment of leukocytes to the site of injury and further initiating the immune response. MSCs can limit the area of tissue injury by their anti-apoptosis activity. VEGF, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Stanniocalcin-1, transforming growth factor beta (TGF-β), bFGF and granulocyte-macrophage colony-stimulating growth factor (GMCSF) were found to reduce apoptosis of the normal tissues around the injured tissues. Anti-scarring or anti-fibrotic is a one activity of paracrine factors secreted by MSCs. Moreover, MSCs could support the growth of haematopoietic stem cells in vitro via secretion of paracrine factors including stem cell factor (SCF), leukemia inhibitory factor (LIF), IL-6 and macrophage colonystimulating factor (M-CSF) (Newman et al., 2009). Finally, MSCs possess immunomodulatory effects on both the innate and adaptive immune systems by secreting a number of paracrine factors including secreted prostaglandin E2 (PGE-2), TGF-, HGF, indoleamine 2,3-dioxygenase (IDO), LIF, MCSF, PGE-2, IL-6, IDO, TGF-β and PGE-2. These factors affect various biological activities of the immune cells such as suppression of T cell proliferation, enhancement of anti-inflammatory cytokines secretion, inhibition of dendritic cell maturation and inhibition of NK cell proliferation. Taken together, all these activities are believed to Stem cells involve the therapeutic potency of MSCs that make them interesting for cell-based therapy (Yi & Song, 2012). 8-Potential Use of Mesenchymal Stem Cells in Therapy: The potential of MSC therapy involving their unique characteristics has been demonstrated in various in vivo disease models and has shown encouraging results for possible clinical use. In a clinical setting, MSCs are now being explored in trials for various conditions, including orthopedic injuries, graft transplantation versus (BMT), host disease cardiovascular (GVHD) diseases, following autoimmune bone marrow diseases, and liver diseases. Furthermore, genetic modification of MSCs to overexpress antitumor genes has provided prospects for use as anticancer therapy in clinical settings (Kim & Cho 2013). BMT and GVHD: HSCT has been widely used over the past several decades to treat patients with various malignant and nonmalignant diseases. However, the procedure remains complicated by regimen-related toxicity, engraftment failure, and GVHD. Preconditioning regimens, such as chemotherapy and/or radiotherapy, may damage the bone marrow and lead to a diminished engraftment of stem cells. MSCs are an attractive therapeutic approach during or after transplantation as their transplantation can minimize the toxicity of the conditioning regimens while inducing hematopoietic engraftment and decrease the incidence and severity of GHVD (Tabbara et al., 2002). GVHD is a severe inflammatory condition that results from immune-mediated attack of recipient tissues by donor T cells during BMT. The clinical efficacy of MSCs in acute GVHD (aGVHD) was first observed in a 9-year-old boy with steroid-resistant Stem cells grade IV aGVHD. The patient, who was unresponsive to other therapies, showed a complete response after receiving haploidentical third-party MSCs (Le Blanc et al., 2004). Following this pilot study, MSC treatment has been studied extensively in steroid-refractory GVHD. Based on these properties, MSCs have been further developed into an FDA-approved commercialized "off-the-shelf" product known as Prochymal (Osiris Therapeutics Inc., Columbia, MD, USA), which is derived from the bone marrow of healthy adult donors. Prochymal was used in a randomized prospective study to treat patients directly after diagnosis of GVHD (Wu et al., 2011). While studies on the use of MSCs for treatment of aGVHD have yielded promising results, the therapeutic efficacy of MSCs in chronic GVHD (cGVHD) is less clear because of the paucity of studies. While some studies indicated efficacy of MSCs, even in cGVHD, others suggested that MSCs are less effective in cGVHD than aGVHD. In studies of MSC therapy in both aGVHD and cGVHD patients, the response rates were higher in aGVHD than cGVHD patients. In addition, the infusion of MSCs following HSCT could prevent the development of aGVHD, while the development cGVHD remained unaffected ( Kim & Cho, 2013). Neurodegenerative diseases: Amylotrophic lateral sclerosis: MSCs have the ability to differentiate into neurons (Wang et al., 2010). The first MSCs transplantation for neurodegenerative disorder was conducted in acid sphingomyelinase mouse model. After the injection of MSCs, there was a decrease in disease abnormalities and improvement in the overall survivability of the mouse. Based on this experiment, a study was designed to ascertain the potency of MSC transplantation into amylotrophic lateral sclerosis (ALS), a neurodegenerative disease that particularly degenerate the motor neurons and disturb muscle functionality (Mazzini et al., 2003). The MSCs were isolated Stem cells from the bone marrow of patients and then injected into the spinal cord of the same patients, followed by tracking of MSCs using MRI at 3 and 6 months. As a result, neither structural changes in the spinal cord nor abnormal cells proliferation was observed. However, the patients were suffering from mild adverse effects, i.e. intercostal pain irradiation and leg sensory dysesthesia which were reversed in few weeks duration. In another study, the AD-MSCs were genetically modified to express GDNF and then transplanted in rat model of ALS which improved the pathological phenotype and increased the number of neuromuscular connections (Suzuki et al., 2008). Parkinson's disease: Parkinson's disease (PD) is a neurodegenerative disorder, characterized by substantial loss of dopaminergic neurons. The MSCs enhanced tyrosine hydroxylase level after transplantation in PD mice model. MSCs by secretion of trophic factors like vascular endothelial growth factor (VEGF), FGF-2, EGF, neurotrophin-3 (NT3), HGF and BDNF contribute to neuroprotection without differentiating into neurocytes (Wang et al., 2010). Other strategies are being adopted like genetic modifications of hMSCs, which induce the secretions of specific factors or increase the dopamine (DA) cell differentiation. BM-MSCs were transduced with lentivirus carrying LMX1a gene and the resulted cells were similar to mesodiencephalic neurons with high DA cell differentiation. Research group from the university hospital of Tubingen in Germany first time delivered MSCs through nose to treat neurodegenerative patients. The experiments were performed on Parkinson diseased rat with nasal administration of BM-MSCs. After 4.5 months of administration, MSCs were found in different brain regions like hippocampus, cerebral, brain stem, olfactory lobe and cortex, suggesting that MSCs could survive and proliferate in vivo successfully. Additionally, it was observed that this type of administration increased the level of tyrosine hydroxylase and decreased the toxin Stem cells 6-hydroxydopamine in the lesions of ipsilateral striatum and substantia nigra. This delivery method of MSCs administration could change the face of MSCs transplantation in future (Danielyan et al., 2014). Alzheimer disease: Alzheimer disease (AD) is one of the most common neurodegenerative diseases. Its common symptoms are dementia, memory loss and intellectual disabilities. Till now no treatment has been established to stop or slow down the progression of AD. Recently, researchers are in the search to reduce the neuropathological deficits by using stem cell therapy in AD animal model. It was demonstrated that human AD-MSCs modulate the inflammatory environment, particularly by activating the alternate microglia which increases the expression of Aβ-degradation enzymes and decreases the expression of pro-inflammatory cytokines (Ma et al., 2013). Furthermore, it was observed that MSCs modulate the inflammatory environment of AD and inadequacy of regulatory T-cells (Tregs) and later on it was reported that they could modulate microglia activation. It was previously demonstrated that human UCB-MSCs activate Tregs which in turn regulated microglia activation and increased the neuronal survival in AD mice model (Yang et al., 2013). Most recently, it was evidenced that MSCs enhanced the cell autophagy pathway, causing to clear the amyloid plaque and increased the neuronal survivability both in vitro and in vivo (Shin et al., 2014). Autoimmune diseases: MSCs are also used in immune disorders because MSCs have the capacity of regulating immune responses. After revealing the facts that human BM-MSCs could protect the haematopoietic precursor from inflammatory damage, other hMSCs can be used for the treatment of autoimmune diseases (Riordan et al., 2007). Stem cells Rheumatoid arthritis: Rheumatoid arthritis (RA) is a joint inflammatory disease which is caused due to loss of immunological self-tolerance. In preclinical studies on animal models, MSCs were found helpful in the disease recovery and decreasing the disease progression. The injections of human AD-MSCs into DBA/1 mice model resulted in the elevation of inflammatory response in the animal. Following the injections of AD-MSCs, the Th1/Th17 antigen-specific cells expansion took place due to which the levels of inflammatory chemokines and cytokines reduced, whereas this treatment increased the secretion of IL-10. Along with its antiinflammatory function, IL-10 is an important factor in the activation of Tregs that controls self-reactive T-cells and motivates peripheral tolerance in vivo (Wehrens et al., 2013). Similar to this, human BM-MSCs demonstrated the same results in the collagen-induced arthritis model in DBA/1 mice. These studies suggest that MSCs can improve the RA pathogenesis in DBA/1 mice model by activating Treg cells and suppressing the production of inflammatory cytokines. However, some contradictions were reported in adjuvant-induced and spontaneous arthritis model, showing that MSCs were only effective if administered at the onset of disease. On exposure to inflammatory microenvironment, MSCs lost their immunoregulatory properties (Papadopoulou et al., 2012). Type 1 diabetes: Type 1 diabetes is an autoimmune disease caused by the destruction of β-cells due the production of auto antibody directed against these cells. As a result, the quantity of insulin production reduces to a level which is not sufficient to control the blood glucose. MSCs can differentiate into insulin producing cells and have the capacity to regulate the immunomodulatory effects. For the first time, nestin positive cells were isolated from rat pancreatic islets and differentiated into pancreatic endocrine cells. Nestin positive cells were isolated from human pancreas and transplanted to diabetic nonobese diabetic/severe Stem cells combined immunodeficiency (NOD-SCID) mice, which helped in the improvement of hyperglycaemic condition (Zulewski et al., 2001). However, these studies were found controversial and it was suggested that besides pancreatic tissues, other tissues can be used as an alternative for MSCs isolation to treat type 1 diabetes. Human BM-MSCs were found effective in differentiating into glucose competent pancreatic endocrine cells in vitro as well as in vivo. It was demonstrated that UCBMSCs behave like human ESCs, following similar steps to form the differentiated β-cells (Prabakar et al., 2012). Unsal et al.(2015) showed that MSCs when transplanted together with islets cells into streptozotocin treated diabetic rat model enhance the survival rate of engrafted islets and are found beneficial for treating non-insulin-dependent patients in type 1 diabetes. Cardiovascular diseases: For myocardial repair, cardiac cells transplantation is a new strategy which is now applied in animal models. MSCs are considered as good source for cardiomyocytes differentiation. However, in vivo occurrence of cardiomyocytes differentiation is very rare and in vitro differentiation is found effective only from young cell sources (Ramkisoensing et al., 2011). MSCs trans-differentiated into cardiomyocytes with cocktail of growth factors were used to treat myocardial infarction and heart failure secondary to left ventricular injury. The systematic injection of BM-MSCs into diseased rodent models partially recompensed the infarcted myocardium (Nagaya et al., 2005). Roura et al. (2012) reported that UCB-MSCs retained for several weeks in acute myocardial infarction mice, proliferated early and then differentiated into endothelial lineage. Stem cells Liver diseases: MSCs have been used to treat cirrhosis in a limited number of trials. Cirrhosis is a chronic liver disease characterized by progressive hepatic fibrosis and loss of hepatic structure with formation of regenerative nodules. Liver transplantation is often the only option in advanced stage patients; however, it is limited by lack of donors, surgical complications, and rejection. MSCs have the potential to be used for the treatment of liver diseases due to their regenerative potential and immunomodulatory properties (Ren et al., 2012). The MSC secretion profile also represents an attractive property, as MSCs are known to secrete several anti-fibrotic molecules such as hepatocyte growth factor (HGF) (Berardis et al., 2014). Furthermore, MSC therapy could provide minimally invasive procedures with relatively few complications, as compared to liver transplantation. In a phase I trial, four patients suffering from end-stage liver cirrhosis were treated with autologous MSCs and showed improved quality of life with no side effects during follow-up (Mohamadnejad et al., 2007). In another phase I to II clinical trial, eight patients with end-stage liver diseases received autologous MSCs. MSC administration was well tolerated and improved liver functions. Thus, MSC therapy is safe, feasible, and applicable in end-stage liver disease (Kharaziha et al., 2009). Cancer: MSCs are emerging as vehicles for cancer gene therapy due to their inherent migratory abilities toward tumors. Whether MSCs themselves have antitumor effects is still controversial as some studies have suggested that even unmodified MSCs inhibit tumor growth and angiogenesis (Otsu et al., 2009), while others report that MSCs promote tumorigenesis and metastasis (Karnoub et al., 2007). Nonetheless, MSCs have been genetically modified to overexpress various Stem cells anticancer genes, such as ILs, IFNs, prodrugs, oncolytic viruses, antiangiogenic agents, proapoptotic proteins, and growth factor antagonists, for targeted treatment of different cancer types. The lack of safety mechanisms following MSC administration has delayed the application of engineered MSCs in clinical settings. Recently, a safety system to allow control of the growth and survival of MSCs has been developed. The safety mechanism is a suicide system based on an inducible caspase-9 protein that is activated using a specific chemical inducer of dimerization (CID). Exposure to CID induced directed MSC killing within 24 hours. The development of such safety mechanisms and their incorporation into MSC therapy may allow extensive use of genetically engineered MSCs to treat cancer patients in clinical settings ( Kim and Cho, 2013). Bone fracture: The osteogenic potential of MSCs has already been verified. MSCs have been shown to readily differentiate down an osteogenic pathway in response to chemical signals. MSCs have been shown to be the primary source for endochondral bone formation, and as such are ideal for bone repair constructs. In a study of human MSCs it was found that mechanical strain alone could induce osteogenic differentiation (Sumanasinghe et al., 2006). Two approaches have been used for cell delivery: bone marrow aspiration and direct introduction at the lesion or expansion ex vivo before implantation. Percutaneous autologous bone marrow grafting has been shown to be an effective treatment for tibial diaphyseal nonunion in one study. The efficacy is influenced by the amount of progenitor cells in the harvested graft (Hernigou et al., 2005). Stem cells The combination of mesenchymal stem cells, platelet rich plasma, and synthetic bone substitute was found to be more effective in inducing new bone formation (osteogenesis) than the use of platelet rich plasma combined with synthetic bone substitute and the use of synthetic bone substitute alone (Kitaori et al., 2009). Genetic manipulation of MSCs can be achieved by transduction using viral vectors such as the adenovirus (Ad) or transfection by nonviral vectors such as liposomes. Many investigators have tried to regenerate bone by transfecting MSCs with the BMP gene. For example, Lieberman et al. (1999) have indicated that autologous BMSCs expressing Ad-BMP2 can considerably promote segmental femoral defects in rat models when compared with BMSCs expressing Ad-LacZ. It has been shown that Ad-Runx2-MSCs transplanted in murine calvarial defects produce more bone tissue compared to MSCs (Zhao et al., 2007). Recent studies have focused on simultaneous application of BMPs and RUNX2. When these two factors were entered into an immortal MSCs line and injected into mice, considerable bony ossicle with marrow cavity was observed (compared to the application of cells that expressed Ad-BMP2) (Yang et al., 2003).