Research On Induction Of Hemophilia A Patient-specific IPS Cells From Urine-Derived Cells

Hemophilia A is an X-linked recessive genetic disease. Male patients have an X chromosome with a mutated gene, resulting in structural defects of FVIII or decreased FVIII levels, which leads to clinical manifestations of bleeding of varying severity. Given the special characteristics of this disease, the cells used in induction iPS cells should obtained trauma minimized. In this study, I successfully induced and identified hemophilia A patient-specific iPS cells from renal tubular epithelial cells in the urine. These iPS cells can be differentiated into endothelial cells, which provides a theoretical basis for future cellar and gene therapy. In addition, this study also made some initially exploring of non-viral method for inducing iPS cells, in order to improve the safety of gene therapy.Part Ⅰ:Induction and identification of hemophilia A patient-specific iPS cellObjective:To induce a cell line of the intron1inversion and a cell line of point mutation in exon14of hemophilia A patient-specific iPS cells by retroviral methods and verify their treatment potential.Methods:(1) Isolate and culture tubular epithelial cells from the urine of patients with hemophilia A;(2) Use the method of retroviral to transfected four kinds of reprogramming factors Oct4, Sox2, c-Myc and Klf4into renal tubular epithelial cells of patients with hemophilia A, to obtain hemophilia A patients eventually specific iPS cells;(3) iPS cells obtained were identified by cell morphology, alkaline phosphatase staining, the pluripotent stem cell surface marker staining, Karyotype analysis and differentiation of three germ layers by teratoma in vivo;(4) By the method of co-culturing with OP9cells of mouse, iPS cells were differentiated into endothelial cells.Results:(1) Successfully isolated renal tubular epithelial cells from the urine of patients, and the renal tubular epithelial cells can be subcultured;(2) Successfully induced and identified two hemophilia A patient-specific iPS cells, the iPS cells express induced pluripotent stem cell surface markers, and can form teratomas in nude mice to differentiate into three germ layers;(3) Successfully differentiated iPS cells into endothelial cells, providing a theoretical basis for future cellar and gene therapy for hemophilia A.Conclusion:Hemophilia A patient-specific iPS cells can be obtained from the urine cells, and can be differentiated into endothelial cells.Part Ⅱ:Preliminary exploration of a non-viral method to induce iPS cellObjective:Try to establish a non-viral method for inducing iPS cells from urine cells by nucleofection of oriP/EBNA1episomal vectors.Methods:(1) Isolate and culture tubular epithelial cells from the urine of patients with hemophilia A;(2) By nucleofection, deliver different combinations of oriP/EBNA1episomal vectors with different reprogramming factors in to urine cells, and observe the changes cells;(3) Identification of the cells induced.Results:(1) Successfully isolated renal tubular epithelial cells from the urine of patients, and the renal tubular epithelial cells can be subcultured;(2) Screened out the best program of nucleofection, optimal combination of episomal vectors, and the best medium;(3) During the induction, there emerges some clones with similar morphology with iPS cell clones, but in the subsequent culturing, these clones occurred differentiation;(4) These differentiated cells showed a nervous system cells-like morphology, and identified as neuronal cell by immunofluorescence.Conclusion:By the method of nucleofection of oriP/EBNA1episomal vectors, reprogramming occurred to urine cells, but it is easy to differentiate into neural cells.