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

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 integrated 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 step-wise induction system in the future.

UPLC-ESI-Q-TOF-MSE-based metabolomics analysis of Acer mono sap and evaluation of osteogenic activity in mouse osteoblast cells

“…The regenerative capacity of bone tissues in the large area of bone damage is complicated and very hard to treat due to the limitations in the clinics. 26 Upon recognition of organic or inorganic compounds, the osteocytes act as mechanical sensors to repair the damage caused in the bone and improve bone function. 27 During the repair mechanisms, the chemokines, cytokines, and various growth factors are involved and behave in a paracrine manner to enhance bone repair.…”

Multi-layered in vitro 3D-bone model via combination of osteogenic cell sheets with electrospun membrane interlayer

“…In fact, terminal differentiation frequently occurs concurrently with the cessation of proliferation and permanent exit from the division cycle. 49 In this sense, culturing the final constructions with differentiation medium for 10-days suggests that the 3rd week of osteogenic differentiation has been reached, as the cells within the construct had previously been exposed to the osteogenic media for 10-days. This period of osteogenic differentiation is the late differentiation phase, in which mineralization is slowly but steadily completed and bone-specific proteins are generated.…”