The Study On Human Embryonic Pluripotent Stem Cells Differentiating Into Osteoblasts

Bone defects caused by diseases and trauma always present a dilemma for orthopaedic surgeons. Modern Tissue engineering is a new discipline developed rapidly in recent years .Its influence is not merely of some tissue defects in plastic surgery and other treatment of common diseases. With the potential of stem cell differentiation further study is bound to human health plagued some major diseases, such as Cancer, diabetes treatment propose new ways different from traditional. Recently, There are many troubles in the repair of the defect by using autografts, allografts, or xenografts. The bone marrow stromal cells have been applicated generally as seed cells of bone tissue engineering. But in clinical research, we found that the cells come from bone marrow was limited in quantity and could cause some complications in harvesting from the bone marrow. Accordingly, the key problem that tissue engineering will resolve is to choose seed cells which have rich resources and manufactured easily.Stem cells are a kind of original cells with self-renewal ability and differentiation potential, which can be directionally induced to various targeted tissue cell types, such as the cells of osteogenic, chondrogenic, adipogenic, and nerve cells under specific micro-environment.Stem cell is classified into two kinds, sub-categories-embryonic stem cell and adult stem cell. It is reported that during the long-term culture in vitro, the human embryonic pluripotent stem cells always keeps the ability of multiple differential potential. The human embryonic pluripotent stem cells have the advantage such as rich resources, convenience of extraction, more proliferation potential, it also haves relatively low immunogenicity, so human embryonic pluripotent stem cells have a better prospect for clinical application, which provide ideal autogenous source of seed cells for bone tissue engineering,in order to meet the need for the cell quantities and quality in the treatment of bone deficiency by means of tissue-engineering technology.Therefor, we focused on human embryonic pluripotent stem cells, established and optimized the experimental method and conditions for inducing the human embryonic pluripotent stem cells to osteoblasts. By means of Von kossa staining, RT-PCR, immunocytochemistry and so on, we detected and assessed the induced cells to evaluate the effect of directed differentiation. In order to investigate the differentiation potential of human embryonic pluripotent stem cells induced to osteoblasts and the underlying possible mechanism of differentiation in vitro. Our study will provide experimental basis for the use of human embryonic pluripotent stem cells as seed cell in the cell transplantation therapy for bone defects.1.Separation, amplification and identification of human embryonic pluripotent stem cells(1)Separation and amplification of human embryonic pluripotent stem cells: Take the fetal liver (8¹8weeks)under aseptic condition, then digest the erythrocyte with typeâ…¡collagenase and finally get the human embryonic pluripotent stem cells by adherent method. Cultured in the high glucose DMEM medium with 10%FBS, that is in a incubator with 37℃a nd 5%CO₂. The results showed that part of the separated cells got attached after 24 hours. Through subculture, the cells grew in parallel or as whirlpool, and rich of cytoplasm. (2)Detection of the cell surface markers: The results showed that the target cells showed positive expression for CD166, CD73 and CD105, but did not express the HLA-DR. It confirms that the cells we have got were mesenchymal stem cells.2.Induced differentiation of human embryonic pluripotent stem cells into osteoblasts Take the P3 cells from logarithmic phase, digest them with separation midium, adjust the cell density to 5×104¹Ã—10⁵/ml, inoculate in collagen coated 6-well plates, and culture them in 37℃with 5% CO2 and 95% air. When the cells adhered and grew to occupation of 80% of the bottom of the culture dish, add inducing medium to induce the differentiation. Human embryonic pluripotent stem cells with osteogenic medium (1×10^-7mol/L dexame thasone, 50mg/L vitamin C, 0.01mol/Lβ-glycerophosphate, 10% FBS,high glucose DMEM medium) were induced into osteoblasts. Using Von Kossa staining detected the formation of calcium nodus, they stained dark brown black. Immunofluorescence assay confirmed the expression of typeâ… collagen and BGP is positive and the cells were examined by RT-PCR for the expression of osteopontin genes.In summary, human embryonic pluripotent stem cells can be induced into osteoblasts by the above-mentioned agents in vitro, demonstrating the potential of human embryonic pluripotent stem cells in differentiation to osteoblasts. These results not only provided new experimental evidence for the induced differentiation of stem cells, but also offered important theoretical and experimental basis for treatment of bone defects. With the biological characteristics and mechanism of differentiation deeply studies, human embryonic pluripotent stem cells will lay the foundation for cell replacement therapy, gene therapy and organ transplant.