The discovery of MSCs In 1867, German pathologist Cohnheim suggested the possible existence of stem cells of nonhematopoietic tissues in bone marrow when he was researching wound healing. He injected animals with nonsoluble dye aniline, thus the cells contain the dye will show up at the distal part of the injured tissue. Besides inflammatory cells, these cells included fibroblasts, which were later thought to be derived from the bone marrow, and thus were speculated with functions other than hematogenesis[9]. In 1869, Goujon found red marrow gained the function of osteogenesis after autotransplantation, which was supporting the former speculations. In the middle of 1970, Friedenstein et al[10] first reported that a small part of cells in bone marrow could differentiate into osteoid or cartilage-liked clusters during culturing, which was named fibroblast colony-forming unit (CFU-F) or marrow stromal fibroblasts. With the development of research, CFU-F is found to have supporting and inducing functions for hematopoietic cells of bone marrow[11]. Since it derives from bone marrow stroma, it is named bone marrow stromal cells. These cells can form osteoid or cartilage-liked tissue when implanted into the abdominal cavity or kidney capsule, so they are also called bone marrow osteogenic stem cells[12]. Fridenstein et al not only proved the existence of these stem cells, but also developed an easy and possible way of separation and culture in vitro. Until today, this is still a typical approach, and has been widely used.   In 1988, Owen et al [13] considered bone marrow stromal cell system was developing with the hematopoietic stem cells, and it had the ability of self-renewal and differentiating into cell clusters of all kinds of connective tissues, thus introducing the concept of stromal cell system. So MSCs are multipotent stromal cells and they can differentiate into a variety of cell types[14], including: osteoblasts[15], chondrocytes[16], and adipocytes.   In long-term cultures of bone marrow tissue in vitro, stromal cell system mainly includes the following kinds of anchorage-dependent cells: fibroblast-like cells, macrophages, adipocytes, and endothelial cells, in which fibroblast-like cells are thought to be multipotent stromal stem cells. Mark and Ajumdar et al, using gradient centrifugation, were able to harvested high purity of fibroblast-like anchorage-dependent cells which took up only 0.001%-0.01% of the total amount of cells, in the layer with the density of 1.077/cm3. These cells showed up all the features of formal experiments concerning stem cells carried out by Fridenstein et al. Since they all eventually differentiated into cells of mesenchymal system, Mark et al renamed them as MSCs. However, these fibroblast-like mesenchymal cells are different from real fibroblasts. In a research comparing the two kinds of cells, MSCs could differentiate into specific connective tissue while fibroblasts separated from bone marrow could not in the same inducing condition.   MSCs separation and culture Bone marrow is the most important source of MSCs. In separation and culture of MSCs from the bone marrow, the usual steps are to separate mononuclear cells of low density, and inoculate the mononuclear cell of low density in the culture media containing selected fetal bovine serum. The anchorage-dependent cells are considered as the primary MSCs in vitro. Subculture of MSCs shows dramatic but different potential of proliferation. Some cells can proliferate for more than 15 subcultures while some stop proliferation after four doublings. This phenomenon could be caused by several factors, such as the process of getting bone marrow, the small amounts of MSCs in bone marrow (only 2-5 MSCs in 1×106 mononuclear cells in bone marrow), and the ages and health conditions of the donors[17-18]. However, too much subculture could impair or damage the cell functions, mainly shown as the obvious signs of aging or apoptosis[19].   Methods of separation Nowadays, there are mainly three methods of separating MSCs. (1) Density gradient centrifugation method: According to the different density between MSC and other cells, cells from bone marrow can be separated into three layers using Percoll separation solution. The layer with low density is the layer containing MSCs, which can be used for culture and harvest of the single kind of cells[17]. (2) Fluorescence activated cell sorting method: According to the small volume of MSCs, also the lack of granules, it can be separated using this method. Zohar et al [18] separated S cells with small volume, less granules in cytoplasm from periosteum of fetal rats, which could differentiate into chondrocytes and smooth muscle cells of bone. (3) Specific monoclonal antibody separation: Since some stromal precursor cells express surface marks such as CD105, stro-1, the specific monoclonal antibody combined with magnetic separation technology can be used to separate some kinds of cells[20]. Based on present researches, there is still a lack of specific marks in MSCs of bone marrow, so density gradient centrifugation method is widely used for the easy access of equipment and reagent, and the simple composition of separated cells.   Culture media The culture media of MSCs are various that Dulbecco’s modified Eagle’s medium, Ham’s nutrient mixture F-12 and RPMI 1640 have all been used. Liu et al[21] used low glucose Dulbecco’s modified Eagle’s medium with different concentrations of fetal bovine serum as the culture medium, and found that the different concentrations of serum greatly affected the purity of MSCs. When the concentration of serum was 10%, the purity of MSCs could reach as high as 95%. While, when the concentration increased to 20%, HLA-DR+ cells would rise accordingly to 36.1%, which indicated that some cells had differentiated into fibroblasts, probably because high concentration of serum could enhance the differentiation of MSCs. Ai et al [22] found that the concentrations of fetal bovine serum at 5%-10% and the culture time within 4 to 24 hours are the best for the growth of MSCs. Fu et al [23] used a specific culture media MesencultTM, which is suitable for MSCs’ growth, and it is beneficial for MSCs’ proliferation while depressing differentiation, so the cells all kept undifferentiated within eight generations.   Surface antigen of MSCs MSCs express many kinds of surface antigen, but none of them is specific. It expresses the surface marks of mesenchymal cells, endothelial cells and epithelial cells. As a result of the composition of culture medium, the density of cell inoculation and oxygen pressure may all affect the phenotypes of cells. Therefore, there are various relevant reports[24]. However, no matter what method of culture is used, the phenotypes of some cells still remain consistent, such as not expressing CD45 and CD34, which are surface marks of hematopoietic cells, expressing SH2 (CD105), SH3 (CD73) and SH4 (CD73). Up to now, the phenotypes of marrow mesenchymal progenitor cells (before culture) still have not been confirmed yet[20].   The discovery of surface antigens of MSC was mainly through flow cytometry. These antigens can also be found in mesenchymal cells, endothelial cells and epithelial cells, but not in hematopoietic stem cells. At present, there are several CD antigens including CD29, CD44, CD59, CD71, CD90, CD105, CD120a, CD124, CD166, which all express in MSCs; while the surface marks of hematopoietic stem cells include CD3, CD11a, CD14, CD19, CD28, CD33, CD34, CD38, CD45, CD56, CD117, which are negative in MSCs. Also, antigens closely related to allograft rejection such as: HLA-DR, B7-1 (CD80), B7-2 (CD86), CD40 and CD40L are all negative[25].   At present, the surface antigens used to identify cells are mostly chosen from the above antigens. The popular positive marks are CD29, CD44, CD90, CD105, SH2, SH3, STRO-1, negatives are CD14, CD19, CD34, CD45, and allograft rejection associated antigen: HLA-DR, B7-1 (CD80), B7-2 (CD86), CD40, CD40L. Besides, MSCs also express SB-10, a kind of mixed antigens[26]. The results of cytochemistry showed that ANAE and PAS are strongly positive in these cells, while SB and ACP are negative, indicating MSCs have special metabolic characters. Also, approximately 5% of the cells are positive in ALP, indicating that there are different stages of stem cells which are differentiated into osteoblasts.   Inducing and differentiation of MSCs Inducing in vitro MSCs have the potential of self-renewal and multi-directional differentiation as a kind of stem cells. In order to inducing the MSCs into bone tissue, Dexamethasone, β-glycerol-phosphoric acid and ascorbate should be contained in the culture medium. The MSCs will gradually form clusters, and the expression of alkaline phosphatase will increase. In this kind of culture condition, evidence of calcium deposition can be found after 1 week, and the amount can keep on increasing in the whole culture process[27]. It has been showed that 1,25-(OH)2-D3, transforming growth factor-β, interleukin(IL)-6, hyaluronic acid and bone morphogenetic protein-2 all can induce the differentiation of MSCs into bone tissue[28]. The adding of bone morphogenetic protein-1 from cartilages and integrin also can promoting the differentiation[29]. The extent of differentiation of MSCs into cartilages can be told by testing the expression of collagen II in culturing cells. The use of 1-methyl-3-isobutyl xanthine, dexamethasone, insulin and indomethacin can induce MSCs differentiate into adipocytes, with accumulation of lipid vesicles in the cells. These cells can express peroxisome proliferator-activated receptor γ2, lipoprotein lipase and fatty acid-binding protein αP2. Many inducing factors can induce more than 95% of MSCs differentiating into adipocytes, and those induced adipocytes can grow well in vitro for at least 3 months, also, the basic culture medium, cell density, growth factors and cell factors all affect the differentiations of MSCs.   Proliferation and differentiation in vivo Up to now, it is still not so clear about the specific environment for MSCs to direct differentiation in vivo, only animal experiments are available. It has been reported that after proliferation of canine MSCs in vitro, they are adhered on biological materials, and with the secretion of bone stroma, they can differentiate into bones, which can be used to repair the defects of femoral shafts. After the MSCs of rabbits proliferation in vitro, it can reproduce bones and cartilages with the carriers, which can be used to repair cartilage injuries, and tendons as well[30]. When human MSCs of bone marrow is injected to athymic mouse subcutaneously, after 1 month, many kinds of tissues, including bone, cartilage, fat, skeletal muscle and tendons, differentiating from human MSCs, will appear in injection cites.   Cytokine impact on MSCs proliferation Li et al [31] used MTT to observe the impacts of cytokines to MSCs proliferation. They found that interferon-γ, tumor necrosis factor-α, stem cell factor and insulin-like growth factor could dramatically stimulate the proliferation of cells, while IL-4, IL-1 and basic fibroblast growth factor had no effect on cell growth. Among the former factors, interferon-γ and tumor necrosis factor-α were most effective, which can indicate the important value of MSCs in tissue repair.   Clinical application of MSCs Recent researches have reveal that: (1) The terminal differentiation of MSCs might exceed the borders of germinal layer, meaning that cells traditionally stemming from middle embryonic layer and then differentiating into mesenchymal cells, would instead transform into parenchymal cells, such as cardiac muscular cells, nervous cells, etc.   (2) The karyotype of MSCs remains normal within 12 generations, and telomerase keeps active, meaning that MSCs has a strong ability to proliferate in vitro, but are not eternally alive as malignant tumor cells.   (3) MSCs can be harvested from autografts of bone marrow, thus the induced tissue can avoid problems such as tissue matching and immunological rejection when transplanting. In that way, it is possible to use the MSCs as the seeds of tissue engineering, to repair many kinds of injury and defects of tissues, therefore making the repair and restore of tissues and organs possible.   Animal experiments have proven that, through proliferation and inducing in vitro, bone marrow MSCs can differentiate into needed cells, which are then implanted in the injured tissues, and the outcomes are good. The most popular used approach is to proliferate the MSCs in vitro, and then the cells are combined with scaffolds to form the cell-material complex, and then the complexes are implanted in the defects[32-33]. All the above researches have shown a promising future for application of bone marrow MSCs in the treatment of bone and cartilage injury.   The degenerative changes of some organs may result in some related diseases, such as amyotrophy, myodystrophy[33]. Experiments have proven that MSCs could differentiate into muscular cells both in vitro and in vivo. So hopefully, they would become regenerating muscle tissues, and thus cure amyotrophy and myodystrophy.   The recent research reveals that MSCs are susceptible to transfection and expression of exogenous genes, thus being considered as a great gene carrier. Researchers also considered that, for diseases caused by gene mutation, they can be treated by introducing normal genes into patients’ MSCs, and the cells proliferating in vitro and then being injected back to the patients[34].   The hematopoietic function of MSCs has been widely admitted. As a part of bone marrow stromal cells, MSCs act the main cells making up the hemopoietic microenvironment, and also play an important role in hematopoietic regulation. It has been revealed by research that, MSCs cultured in vitro are able to excrete cytokines such as IL-6, IL-7, IL-8, IL-11, stem cell growth factor, megakaryocyte colony stimulating factor, Fit-3 conjugant stromal cell-derived growth factor, etc. Among them, most cytokines can stimulate the proliferation and differentiation of hematopoietic cells. Meanwhile, MSCs have the ability of differentiating into stromal cells in certain conditions, which can solve the hematopoietic dysfunction caused by deficiency of stromal cells after injury[35].   Researches in recent years have shown that MSCs also played a remarkable role in immune system, for example, negative modulation of unrelated donor can support the proliferation of recipient’s hematopoietic stem cells in vitro, without activating reactive lymphocyte against donor antigens, meaning that stromal stem cells can down regulate allogeneic immune response, therefore alleviate GVHD[36]. Remberger et al [37] discovered that MSCs could have an initial effect on GVHD since patients survived for a longer time and fewer patients died of acute GVHD as compared to the control group.   (4) How do the MSCs run in curing disease? Atsuta et al [38] discovered that Fas/Fas-L-induced MM apoptosis plays a crucial role in the MSCs-based inhibition of multiple myeloma growth. Bergfeld et al [39] found that the IL-6/STAT3 pathway have been shown to play a central role in control the interaction between MSCs and tumor cells. Hypoxic MSCs with an increased ability can migrate toward tumors through the upregulation of chemokine receptors, such as CXCR4 and CX3CR1[40]. MSCs have inherent tumor-trophic migratory properties, which allow them to serve as vehicles for delivering effective and targeted therapy to isolated tumors and metastatic disease[41]. In addition, Xia et al [42] discovered that MSCs not only act as a cell carrier, but also allow the replication of CRAd, significantly enhancing the oncolytic effect and resulting in the augmented tumor inhibition efficiency. However, these may just be only a small part of the action mechanism of MSCs. So, more in-depth studies are needed.   (5) However, the clinical application of MSCs will bring many problems. Not merely is it hard to get enough bone marrow MSCs (BMSCs) expanded for Cell transplantation, but also the growth and differentiation of BMSCs are not an easy thing. Hagmann et al [43] discovered that the expression of typical MSC markers depending on the media applied. And it is still unknown which MSC culture is the best in terms of the safety and growth of MSCs. Yamout et al [44] discovered that 30% of patients failed to grow an adequate number of BMSCs (< 2×106) despite repeated bone marrow aspirations reflecting an inherent deficiency of such cells in the bone marrow of those patients. Why the successful MSC transplantation is so difficult? And whether to get enough MSCs means a successful MSC transplantation? Maybe the quality of MSCs is more important than the quantity. Siegel et al [45] found that high-clonogenic BMSCs were smaller, divided more rapidly and more frequent in BMSC preparations from younger, female donors. Therefore, in order to improve curative effect, the younger, female donors are a better choice. Unfortunately, to improve the quantity and quality of MSCs may be not the biggest challenge for the clinical application of MSCs. The MSC transplantation has some side effects and possible complications including headache[46] and neuropathic pain[47] after parenchymal injection of BMSCs. However, compared with the following those would not be a problem. Negative effects of MSC treatment may result in an increase in invasive fungal infections[37]. In a clinical trial using MSCs to prevent GVHD in patients with hematologic malignancies, MSCs reduced the development of GVHD, but the relapse rate among patients was higher than that in the control group[48]. In general, due to their immunosuppressive actions, MSCs are known to favor tumor growth[49] and metastasis[50]. Studies have reported that MSCs may exert antitumorigenic effects in vitro and in a model of Kaposi’s sarcoma[51-52]. Since MSCs are supposed to be the progenitor cells of Ewing sarcomas[53], osteoblasts, and fibroblasts, the possibility exists that MSCs may have contributed to the secondary malignancy in these patients. Shalapour et al [54] discovered that leukemia cells and MSC have genetic aberrations in common, and that they are clonally related. The endogenous expression of IL-6 and CCL5 by MSCs, have been shown to increase the growth and metastasis of breast cancer cells, respectively[55-56]. However, there have not been significant subsequent reports on the promotion of tumor growth by MSCs in recent years. Some MSCs are able to escape from dividing spontaneously, and these have undergone tumorigenic transformation generating TMC[57]. To use BMSCs in clinical trials, more clinical researches will be needed."