Differentiation of cryopreserved umbilical cord mesenchymal stem cells into osteoblasts

Chen Yan; Pan Li-jie; Yuan Jie; Li Tian-xia

doi:10.3969/j.issn.2095-4344.2013.36.009

Abstract：BACKGROUND:Human umbilical cord mesenchymal stem cel s are considered as novel seed cel s in bone tissue engineering. Cryopreservation is an effective method for storing cel s for a long time. <br> OBJECTIVE:To explore whether umbilical cord mesenchymal stem cel s of cryopreservation could be induced to differentiated into osteoblasts. <br> METHODS:Mesenchymal stem cel s were isolated from the Wharton’s jel y of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cel s was observed by inverted microscopy. Immunophenotypes and cel cycle of umbilical cord mesenchymal stem cel s were measured using flow cytometry. After frozen storage for 6 months, the second passage of umbilical cord mesenchymal stem cel s was thawed and subcultured to passage 12. Upon induction with osteogenic inductive medium, the osteogenic ability of passage 12 of umbilical cord mesenchymal stem cel was evaluated by alkaline phosphatase activity, the immunofluorescent analysis of osteocalcin and bone sialoprotein and the assay of alizarin red staining separately. <br> RESULTS AND CONCLUSION:Primary umbilical cord mesenchymal stem cel s displayed a typical fibroblast-like morphology. Flow cytometry showed that the cultured cel s expressed high levels of the mesenchymal stem cel s surface markers CD73, CD105 and CD90, but did not express hematopoietic cel s surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cel s after resuscitation was 90%. The cel cycle analysis indicated that 75%of the cel s of passage 8 were in G 0/G 1 phase and 25%in S+G 2 M phase. Passage 12 cel s treated with osteogenic inductive medium displayed a higher alkaline phosphatase activity compared with control cel s (P<0.01). Moreover, the cel s, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Umbilical cord mesenchymal stem cel s stil maintain their biological characteristics after cryopreservation, and can be induced into osteoblasts with osteogenic inductive medium.