The Study Of Stepwise Induction Differention Of Human Induced Pluripotent Stem Cells Into Osteoblast Like Cells

Bone tissue is an important part of human body to exercise movement,support,protection and other functions.However,bone defects with different types caused by trauma,infection,tumor and congenital diseases are very common and have become a difficult point in clinical treatment.Similarly,the defect of jaw bone has always been a major problem in oral surgery and implant repair.At present,in order to solve the problems of bone source,rejection reaction and mechanical requirements,bone tissue regeneration engineering has become an effective way to solve the problem of bone defect.Among them,the key factors in bone tissue engineering are osteoblasts,which are the basic cells for bone formation.So,how to obtain sufficient mature osteoblasts is the key of bone tissue engineering in basic research and clinical applications.Human induced pluripotent stem cells(hiPSCs)have the same pluripotency as human embryonic stem cells(hESCs),and can proliferate indefinitely and keep the ability of differentiating into cellular derivations of all three primary germ layers.Moreover,hiPSCs are obtained by reprogramming human body cells,which avoids the ethical problems of hESCs,and have a broad application prospect in the fields of tissue development,drug screening and cell therapy.Therefore,this paper mainly studied how to efficiently induce hiPSCs to differentiate into osteoblast like cells,so as to provide enough high-quality seed cells for bone tissue engineering.In view of the incongruity of initiation conditions of osteogenic differention of hiPSCs,the optimal suspension culture time of embryoid bodies(EBs)method and the optimal initial differentiation density of monolayer method were explored.Firstly,the expression of related to mesoderm,neural crest,mesenchymal and osteogenic genes in EBs derived from hiPSCs and suspension culture for 3-10 days was examined by RT-PCR.Then,EBs with varying times in suspension(3,5,7 and 10 days)were attached onto gelatine surfaces,and were induced osteoblastic differentiation for 14 days.Finally,their efficiency of osteogenic differentiation was determined by RT-PCR,alkaline phosphatase assay,alizarin red assay and immunofluorescence assay.The results showed that the the optimal suspension culture time of EBs were 3-5 days.Moreover,the hiPSCs with initiation densities of 20%,50%,80% and 100% on the surface of Matrigel were induced osteogenic differentiation for 7 days,14 days,21 days and 28 days by monolayer method.The efficiency of osteogenic differentiation was analyzed by the above methods.The results showed that the density of 80% was suitable for monolayer method.The above results laid a foundation for further exploration of the directed osteogenic differentiation system of hiPSCs.Finally,the related compounds such as CHIR99021,CYC,FGF2,SB431542,LY294002 and BMP4,were selected to construct the differentiation system of hiPSCs into osteoblast like cells through mesoderm and ectoderm in vitro.These compounds are agonists or inhibitors of signaling pathways that play an important role in the differentiation of embryo into mesoderm and ectoderm based on literature review.Meanwhile,the induction time of mesodermal differentiation of hiPSCs was divided into 2 days(D0-D2 phase)and 5 days(D3-D5 phase)based on the experience of inducting hPSCs differentiation into cardiomyocytes through mesoderm and the study of stepwise differentiation of hPSCs into osteoblasts.The hPSCs were cultured on the surface of Matrigel up to 80%.Above compounds were used to induce differentiation of mesoderm for 2 days,and the expression related to mesoderm and osteoblast cells was detected by RT-PCR.Subsequently,osteogenic induction medium was used to induce differentiation for another 14 days and the osteogenic differentiation efficiency was detected.The results showed that CHIR99021 and SB431542 were considered to be the most effective compounds for D0-D2 phase.On the basis of determining the conditions of D0-D2 phase,the above compounds were used to induce differentiation for 3 days respectively.By detecting the expression related to mesoderm,neural crest,mesenchymal and osteoblast cells by RT-PCR and analyzing the efficiency after 14 days of continuous osteogenic differentiation,BMP4 was considered to be the most effective compound for D3-D5 phase.In the later stage,it will be explored that the differentiation of mesoderm and ectoderm into mesenchymal,and then into osteoblasts by small compounds,so as to realize the osteogenic differentiation of hiPSCs with high efficiency.It has great significance to construct the induction system of directed osteogenic differentiation of hiPSCs with defined components.