Isolation, Culture And Plurilineage Induction Of The Thawed Human Mesenchymal Stem Cells

In addition to hematopoietic stem cells, there are also some mesenchymal stem cells (MSCs) in bone marrow. MSCs can be served as a cell feeder layer to support survival, proliferation and differentiation of hematopoietic stem cells in the bone marrow. Also, ex vivo experiments have demonstrated that they have active self-proliferative and multi-differentiative capacities into various cell types of mesenchymal tissues, such as osteoblasts, chondrocytes and adipocytes. Recent researches have detected that the cultured cells also have the potential of differentiating into neural cells, which suggests that intrinsic genomic mechanisms of commitment, lineage restriction, and cell fate are mutable. The marrow cells are easily accessible, overcoming the risks of obtaining neural stem cells from the brain, and provide a renewable population. Autologous transplantation overcomes the ethical and immunologic concerns associated with the use of fetal tissue. Moreover, MSCs grow rapidly in culture, precluding the need for immortalization, and can differentiate with use of a simple protocol. In the meantime, MSCs can be transduced and express ectogenic genes to cure the target cells. Therefore, MSCs have been considered as prospective adult stem cells in tissue engineering, cell transplantation, gene therapy and clinic use.The long-term cultivation of MSCs may be failed for many factors, such as genotypic drift, senescence, transformation, phenotypic instability, contamination or incubator failure. The failure of cultivation will result in absence of MSCs for experimental and clinical use. Therefore, it is necessary to cryopreservate MSCs as cell seeds.In our report, Human bone marrow-derived mesenchymal stem cells (hMSCs) were isolated and cultured. We investigate its growth pattern and differentiation potentials of hMSCs into chondrocytes, adipocytes and neurocytes. For inquisition of ex vivo expansion and pluripotential differentiation of cryopreservated hMSCs into chondrocytes, adipocytes and neurocytes, cryopreservated hMSCs were thawed and passaged for more than 15generations. Thawed hMSCs could be subcultivated for many passages without noticeable loss of viability and capability of differentiation. It was verified that thawed hMSCs have the similar growth and differentiation characteristics to pre-cryopreservated cells even if they are propagated for many passages. Thawed hMSCs at passage 15 were induced to chondrocytes, adipocytes and neurocytes under corresponding conditioned induction medium, and induced cells were observed for their morphology and expressions of type II collagen, triglyceride, Nestin and neuron-specific enolase(NSE). Thawed hMSCs differentiated into chondrocytes after exposure to transforming growth factor - 3 i(TGF- 3 i), insulin-like growth factor -I (IGF-I) and Vitamin C(Vc), and uniformly changed from a spindle-like fibroblastic appearance into a polygonal shape at 14 days. The induced cells were heterochromatic to toluidine blue and expressed cartilage matrix- procollagenal (II) mRNA. hMSCs induced with adipogenical induction medium consisting of dexamethasone (DMSO), 3-isobutyl-l-methylxanthine, indomethacin and insulin-like growth factor -I (IGF-I) had lipid vacuoles accumulation after 3 days of induction, which showed that hMSCs differentiated into adipocytes by displaying a perinuclear accumlation of lipid vacuoles, as detected by Oil Red O. In the meantime, the thawed hMSCs were also induced into neurocytes. Nestin and NSE, special surface markers associated with neural cells at different stage were expressed. The morpha of the induced cells changed after induction with a combination of 3 -mercaptoethanol, dimethyl sulfoxide (DMSO), butylated hydroxyanisole, hydrocortisone, IGF-I and All-trans retinoic acid (ATRA). The retractile cell bodies extended long processes that terminated at typical growth cones and filopodia. As above results, thawed hMSCs are a population of pluripotential cells that have potentials of differentiating into chondrogenic, adipogenic and neural lineages. Therefore, hMSCs may constitute an abundant and accessible cellular reservoir for treatment of various diseases, which may also save much time and culture materials. It suggested that thawed hMSCs could indeed be used as prospective adult stem cells in tissue engineering, cell transplantation and gene therapy.