Primary Study On Osteogenic Differentiation Of HPSCs Promoted By BFP-1 Peptide

Objective:Investigate the expression of osteogenic markers during the osteogenic differentiation of human pluripotent stem cells(hPSCs);Investigate the effect of BFP1-1 on the osteogenic differentiation of human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hi PSCs);Investigate the effect of BFP-1 peptide on the expression of bone related markers in hESCs.It provides important clues for the construction of high efficiency induced osteogenic differentiation system of hPSCs.Methods:First of all,hESCs were induced to differentiate into osteoblasts.The osteogenic differentiation process of hESCs was analyzed by quantitative detection of telomerase activity,quantitative RT-PCR,immunofluorescence and flow cytometry at0,3,7,14,21,28 and 35 days,respectively.Then,hi PSCs were induced to differentiate into osteoblasts with 0?g·m L^-1?5?g·m L^-1?20?g·m^-1BFP-1 were added into medium.After cultured for 14 days,alizarin red staining was used to observe the calcium deposition in different groups.After selecting the optimal concentration,hi PSCs and hESCs were induced to differentiate into osteoblasts.With 20?g·m L^-1BFP-1 as the control,the experimental group was treated with 20?g·m^-1 BFP-1 at 0-7 days,7-14days,14-21 days,21-28 days and 0-28 days,respectively.The effects of BFP-1 on the osteogenic differentiation efficiency of hPSCs were evaluated by RT-PCR,immunofluorescence and alizarin red staining at 0,7,14,21 and 28 days.Finally,in the osteogenic differentiation process without BFP-1 as the control,the experimental group was added 20?g·m L^-1BFP-1 at 7-21 days and 0-28 days respectively.RT-PCR,immunofluorescence and alizarin red staining were used to evaluate the effect of BFP-1 on the osteogenic differentiation efficiency of hESCs at 0,21 and 28 days after osteogenic differentiation.Results:(1)Through this study,the dynamic expression map of related markers in the process of osteogenic differentiation of hESCs was preliminarily drawn.Mesenchymal like cells were obtained at 7 days of osteogenic differentiation,pre-osteoblast like cells were obtained at 14-21 days,and osteoblasts were induced at 28-35 days.(2)20?g·m L^-1BFP-1 can significantly improve the osteogenic differentiation efficiency of hi PSCs.With BFP-1 added on 7-14 days and 14-21 days,it can improve the osteogenic differentiation efficiency of hPSCs,and this effect is especially obvious in the osteogenic differentiation process of hESCs.(3)BFP-1 added at 7-21 days can significantly improve the osteogenic differentiation efficiency of hESCs.Conclusions:The experimental results showed that on the basis of preliminary understanding of the osteogenic differentiation process of hESCs,the addition of BFP-1 in 7-21 days could significantly improve the efficiency of osteogenic differentiation of hESCs,which provided an important clue for us to construct a system of efficient osteogenic differentiation induced by hPSCs.