Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Author:

Zhou Ping,Shi Jia-Min,Song Jing-E,Han Yu,Li Hong-Jiao,Song Ya-Meng,Feng Fang,Wang Jian-Lin,Zhang Rui,Lan FengORCID

Abstract

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

Funder

the National Natural Science Foundation of China

Gansu Province Science Foundation for Youths

Young Elite Scientist Sponsorship Program by CSA

Chengguan District Science and Technology Project

Fundamental Research Funds for the Central Universities

Lanzhou University Hospital of Stomatology Research Support Fund

Publisher

Springer Science and Business Media LLC

Subject

Cell Biology,Biochemistry, Genetics and Molecular Biology (miscellaneous),Molecular Medicine,Medicine (miscellaneous)

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