| Popis: |
Background: Derivation of the osteoblast-like cell from human pluripotent stem cell becomes a hot topic in bone tissue engineering. Although many improvements have been achieved in this field, low induction efficiency because of the non-directed differentiation process hampers their application in bone regeneration. We think lack of detailed understanding on the osteogenic differentiation process should be the main reason.Methods: Monolayer cultured human embryonic stem cells and human induced pluripotent stem cells were inducted in traditional serum-containing osteogenic medium for 35 days. Except for traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied cell counting, cell telomerase activity, cell cycle and quantitative expression of runt-related transcription factor 2 as essential indicators to analyze the cell type changes during the differentiation process. Results: The population of differentiated cells are quite heterogenous throughout 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type changes and tumorigenicity of obtained cells. Moreover, nuclear staining should be a recommended method to evaluate the cell number, because, it is still a great challenge to dissociate cells with varying differentiation times into single cells with high survival rate. Finally, a dynamic map was made to integrated analysis of these results, and the cell types at defined stages of osteogenic differentiation of human pluripotent stem cells was concluded. Conclusions: This study lay foundation to improve the in vitro osteogenic differentiation efficiency of human pluripotent stem cells by supplementing functional compounds at each stage according to a time-frame, then establish a step-wised induction system in the future. |
