Differentiation of cryopreserved umbilical cord mesenchymal stem cells into osteoblasts

doi:10.3969/j.issn.2095-4344.2013.36.009

Abstract

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells are considered as novel seed cells in bone tissue engineering. Cryopreservation is an effective method for storing cells for a long time.
OBJECTIVE: To explore whether umbilical cord mesenchymal stem cells of cryopreservation could be induced to differentiated into osteoblasts.
METHODS: Mesenchymal stem cells were isolated from the Wharton’s jelly of human umbilical cord tissue by the tissue explant adherent method. Morphology of primitive cells was observed by inverted microscopy. Immunophenotypes and cell cycle of umbilical cord mesenchymal stem cells were measured using flow cytometry. After frozen storage for 6 months, the second passage of umbilical cord mesenchymal stem cells was thawed and subcultured to passage 12. Upon induction with osteogenic inductive medium, the osteogenic ability of passage 12 of umbilical cord mesenchymal stem cell was evaluated by alkaline phosphatase activity, the immunofluorescent analysis of osteocalcin and bone sialoprotein and the assay of alizarin red staining separately.
RESULTS AND CONCLUSION: Primary umbilical cord mesenchymal stem cells displayed a typical fibroblast-like morphology. Flow cytometry showed that the cultured cells expressed high levels of the mesenchymal stem cells surface markers CD73, CD105 and CD90, but did not express hematopoietic cells surface markers CD34 and CD45. The survival rate of umbilical cord mesenchymal stem cells after resuscitation was 90%. The cell cycle analysis indicated that 75% of the cells of passage 8 were in G₀/G₁phase and 25% in S+G₂M phase. Passage 12 cells treated with osteogenic inductive medium displayed a higher alkaline phosphatase activity compared with control cells (P < 0.01). Moreover, the cells, induced in osteogenic inductive medium, were positive for osteocalcin and bone sialoprotein staining and formed the mineralized nodules. Umbilical cord mesenchymal stem cells still maintain their biological characteristics after cryopreservation, and can be induced into osteoblasts with osteogenic inductive medium.

Key words: stem cells, cell differentiation, osteoblasts, osteocalcin

CLC Number:

R394.2

Chen Yan, Pan Li-jie, Yuan Jie, Li Tian-xia. Differentiation of cryopreserved umbilical cord mesenchymal stem cells into osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(36): 6436-6442.

Figures/Tables 7

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