Induction Of Osteoblasts Differentiated By Mouse Parthenogenetic Embryonic Stem Cells

In the field of bone tissue engineering research, the source of osteogenic cells is the key factor that constrains its research and application. Different types of stem cells including bone marrow stromal stem cells and adipose-derived stem cells have been applied as seed cells. However, there is no satisfying seed cell available for bone tissue engineering. Therefore it is essential to find alternative seed cell for this research field.Objective:1. Verifying totipotency of KM×129/SV mouse parthenogenetic embryonic stem cells and demonstrating their directional differentiation potential into osteoblasts; 2. Establishing the culture conditions to induce parthenogenetic embryonic stem cells differentiate into osteoblasts and identifying the induced osteoblasts; 3. Observing the adhesion and growth of induced cells on three-dimensional scaffold.Methods:Parthenogenetic embryonic stem cells were cultured in vitro, and the totipotency of parthenogenetic embryonic stem cells was tested by different methods, includeding alkaline phosphatase staining, immunohistochemistry of totipotent surface antigen SSEA-1, telomerase and totipotent transcriptional factors, RT-PCR to detect totipotent transcriptional factors and parthenogenetic embryonic stem cells differentiation ability in vivo. Then osteoblasts inducing factors such as dexamethasone,β-glycerophosphate and vitamin C were added to the culture medium to induce parthenogenetic embryonic stem cells to differentiate into osteoblasts directly. Various methods were used to test the induced osteoblasts, such as RT-PCR to observe osteoblasts marker genes, transmission electron microscopy, alizarin red and von Kossa staining to measure the characteristic of osteoblasts. Confocal laser scanning microscope and scanning electron microscope were used to observe the adhesion and growth of induced parthenogenetic embryonic stem cells seeded on coral blocks acting as three-dimensional scaffold.Results:The results showed that:1.the KM×129/SV mouse parthenogenetic embryonic stem cells could be cultured and kept stable proliferation ability in vitro; alkaline phosphatase staining was positive; totipotent surface antigen of SSEA-1, and totipotent transcriptional factors such as Nanog, Oct4 and Sox2 were expressed; telomerase activity was highly expressed; the parthenogenetic embryonic stem cells were undifferentiated. The parthenogenetic embryonic stem cells could be differentiated into three germ layers cell types in vivo with totipotency. 2.The morphology of cells were significantly changed during the inducing process; the osteoblast marker genes such as collagen I and osteocalcin were expressed after twenty-eighth days with induction; alizarin red staining and von Kossa staining were all positive on the twenty-eighth days after induction, calcium nodules were formed. When parthenogenetic embryonic stem cells were induced, numbers of mitochondrial and Golgi bodies were increased, and the higher electron density mineralizations were formed in induced cells.3. The parthenogenetic stem cells been induced for seven days were with normal activity seeded on coral material, which could stable proliferate and well adhere with coral material tested by confocal laser scanning microscope and scanning electron microscope.Conclusions:The results showed that the KM×129/SV mouse parthenogenetic stem cells could be cultured steadily and passaged in vitro, with the totipotency similar to embryonic stem cells. These cells could be induced into osteoblasts through direct induction method. The parthenogenetic stem cells induced after seven days were with normal activity seeded on coral material, and could stable proliferate and well adhere with coral material. This experiment provided us an ideal kind of seed cells for bone tissue engineering, we believe that with further research we will find more optimal inducing conditions which can improve the efficiency of inducing parthenogenetic embryonic stem cells into osteoblasts and promote the application of this method into the further clinical treatment.