The Establishment Of The Platform For Human Amniotic Fluid Cell Culturing And Retrovirus Packaging For Inducing Pluripotent Stem Cells

Gene therapy, as a new method for curing disease, has a promising prospect. Along with the development of molecular pathology, molecular biology and other subjects in biomedical field, diseases which gene therapy aims at are from single gene inheritance diseases to those severely threatening human health, such as tumor, viral diseases and cardiovascular diseases.For in vitro gene therapy, it's important to choose proper target cells. Stem cells have the potential to self renew and differentiate into many cell types, so they are the ideal target cells in gene therapy. In 2006, a new technology emerges that transfers some transcription factors to the somatic cells to induce these cells to the pluripotent state. This new technology makes it possible to utilize the patients' somatic cells that are induced to the pluripotent stem cells in autoallergic gene therapy.Our lab plans to use the amniotic fluid cells from the infants who have genetic diseases to be induced by retrovirus. In this way, we can establish different kinds of disease-specific iPS cell lines, then these cell lines and our lab's human ribosomal DNA targeting vector can be used together in the gene therapy research. This study is the preparation, and aims at establishing the platform for human amniotic fluid cell culturing and retrovirus packaging for induction.The experiment results show that after getting the human amniotic fluid, through inoculation and cultivation, cells adhered to grow and after 6 days the cell cluster appeared, then the adherent epithelial-like cells and long fibroblast-like cells were mechanically removed, leaving the short and spindle-like cells. The cells were infected by lentivirus encoding Slc7al receptor, thus these cells could also express Slc7a1. In this way, we got the cells suitable for induction. At the same time, in order to get the retrovirus for induction, we successfully utilized Plat-E to package the retrovirus vectors encoding exogenous inducing factors. For detecting the infection ability, pMX-GFP retrovirus, as a control, infected the human amniotic fluid cells expressing Slc7a1. After 48h, GFP could be objected in the cells, showing that retrovirus packaged by Plat-E could infect the human amniotic fluid cells expressing Slc7a1.This study successfully established the purification and screening technical conditions of human amniotic fluid cells, and the platform for retrovirus packaging by Plat-E, providing a basis for the later research.