The Establishment Of Integration-free Phenylketonuria Patient-specific Induced Pluripotent Stem Cells And Osteogenic Differentiation From Embryoid Bodies Osteogenic

Background: The biological characteristics of induced pluripotent stem cells(iPSCs)have provided new ideas and methods for the study of many diseases.iPSCs can self-renewal strongly,grows rapidly,and be directed to differentiate into specific tissues.It can provide cells with different differentiation stages,and provide research targets for different diseases.The iPSCs cells are reprogrammed by the somatic cell of the patient which carry the pathogenic genes and specific genetic background of related diseases,so it was individual specific.iPSCs have great significance for drugs screening studying the pathogenesis of rare diseases such as Turner syndrome and phenylketonuria.Objective: Established the method of obtaining and identifying induced pluripotent stem cells from urine;obtain induced pluripotent stem cells of phenylketonuria patients and identify the pluripotency.Established the model of osteogenesis from induced pluripotent stem cells and compare the osteogenesis of Turner syndrome patients with normal.Methods: Obtain urine cells from normal volunteers and phenylketonuria patients.After culturing and amplification,the non-integration plasmid containing transcription factors was introduced into cells by electrotransfection,and induced pluripotent stem cells were purified;alkaline phosphatase staining,real-time quantitative PCR detection of pluripotent gene expression,test for absence of the reprogramming vectors,RT-qPCR,embryoid bodies formation,bisulfite promoter sequencing,flow cytometry and other experiments were performed to identify iPSCs pluripotent in normal and disease.The induction of iPSCs cells into the mesoderm of the embryoid body was performed to induce osteogenesis,after induce osteogenesis of the pluripotent stem cells in the Turner syndrome patient and normal people,alizarin red staining were performed,and the osteogenesis difference between the two groups was compared by RT-qPCR.Results: Urine cells of the normal people and the patients with phenylketonuria were obtained,and several induced pluripotent stem cells were obtained by purification;alkaline phosphatase staining,real-time quantitative detection of pluripotent gene expression,test for absence of the reprogramming vectors,RT-qPCR for markers of pluripotency,embryoid body formation,bisulfite promoter sequencing,flow cytometry proved iPSCs are pluripotent.Alizarin red staining showed a large number of mineralized nodules,and real-time quantitative expression of the osteogenic gene in osteogenesis of iPSCs;induced pluripotent stem cells induced by Turner syndrome and normal persons were stained with alizarin red after induction of osteoblasts;RT-qPCR of in osteogenic markers comparison showed that there was no difference between two groups.Conclusion: We mastered the non-integrated,serum-free and feeder-free reprogramming culture method for obtaining UiPSCs from urine;we got normal and phenylketonuria patients' iPS cell lines from urine,provided a disease models for the research,the drug screening and pathogenic mechanism of phenylketonuria.We established the model of osteogenesis from induced pluripotent stem cells and compared the difference between Turner syndrome and normal people in osteogenic of iPSCs,and it is proved that there are not differences of osteoporosis.Complications such as osteoporosis,arthritis,may be caused by osteoclasts or hormone levels.