The Study On DMD Patient Specific IPSCs Induction And HESCs Differentiate Into Definitive Endoderm Cells

Pluripotent stem cell (PSCs),including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),represent a novel tool for gene editing ang gene therapy. after gene correcting,human pluripotent stem cells could be used for certain genetic diseases by autologous transplantation.It is a way to combine pluripotent stem cells with gene editing and gene therapy, we have been always focued on Duchenne-Muscular Dystrophy (DMD) and Hemophilia A. As the target cells for gene therapy, the problem is how to produce iPS.In this present study,part Ⅰ provide a way to generate the iPS cells from urine sample of a DMD patient. Part Ⅱ using ET5,which is successfully targeted the reconstructive human coagulation factor Ⅷ (hF Ⅷ) gene, hF Ⅷ-BDDAK39, into the rDNA locus of hES cell line H9by using a human rDNA targeting vector (pHrneo),we differentiate ET5into the definitive endoderm cells, that will be another way to generate hepatic cells.Part I:Generate the iPS cells from urine sample of a DMD patient.Objective:Induced patient-specific iPSC of DMD patient.Methods:1.Collect urine sample of a DMD patient with clinical diagnosis of the deletion mutation of exon50in DMD gene,maintain the urine cells.2.By the retrovirus-mediated transfection,we translate four transtranscription factors, Oct4, Sox2,Klf4and c-myc into urine cells,then almost20days later,there appare iPS cells clones.3. The iPSC clones were characterized by pluripotent cell markers, alkaline phosphatase staining, karyotype and EB differentiation analysis.Results:1.Following the methods above, we can generate iPSC-like clones from urine cells.2.The iPSC-like clones present pluripotent cell markers positively, through multiple passages,this clone retained iPS homogeneous features and no chromosomal abnormalities are found after sequential passages.3.In vitro EB differentiation analysis show that these derived iPSC-like clones remain the abilities to differentiate into different germ layers.Conclusion:By retrovirus system,we can generate iPSC from urine sample of DMD patient.Part Ⅱ:Differentiate ET5into the definitive endoderm cellsObjective:To establish the technology of in vitro differentiation of hESCs into DECs.Methods:1.Combine BMP4,Activin A,chir99021differentiate ET5into DEC.2.The RT-PCR detecte the expression of the specificity marker gene from the differentiation of ET5at day two,three and five.3. Flow cytometry analyses the expression of the specific surface marker.Results:1.With high level BMP4, Activin A,and chir99021in the differentiation medium, epithelial-like cell appare at day2.The RT- PCR can detect the expression of the specific markers gene Sox17, FoxA2after RNA extraction of the differentiated definitive endoderm cells.3.Flow cytometry analyses the expression of the specific surface marker,the positive rate of c-kit is50.60%、and CXCR4is39.01%.Conclusion:We have established the technology of differentiation of hESCs into DEC in vitro, by which the characteristics of hESC-derived DEC express specific markers gene Sox17, FoxA2and the specific surface marker c-kit and CXCR4...