Chracterization And Differentiation Of Porcine Amniotic Fluidstem Cells

Amniotic Fluid Stem(AFS)cells a pluripotent stem cells which exist in amniotic fluid and express Oct4. These cells show many charming features, such as robust proliferation potention, pluripotent ability and low immunogenicity, and have wildly promise in the fields of biology and medicine. This study isolated the porcine AFS cells from amniotic fluid in different development stage embryos of landrace crossbred, and investigated the relationship of age of embryos to the adhere-rate and activity of porcine AFS cells, and further optimized the cultrue condition of porcine AFS cells. Further more, characterization of porcine AFS cells using PT-PCR, Flow-cytometry, immunofluorescence and differentiation in vivo and vitro,. These results has provided useful experience for the basic research and application of amniotic fluid stem cells of large animals.1. Establishment the culture system to isolation of porcine AFS cellsCollected porcine AFS cells through centrifugation from amniotic fluid which derived from fetus,and cultured these cells in specific AFS cells medium. Using several kinds of basic medium and different concentration of FBS, the optimized culture system was selected. Besides, probed the effect of fetus age to the adhere-rate of cells and cell activity. The result suggested thatα-MEM plus 10% FBS is the best culture condition for porcine AFS cells, and the effect of fetus age to the adhere-rate of cells and cell active is visible. The AFS cells derived from amniotic fluid of 40d-70d fetus prefer to adhere, and also has great proliferated potency.2. Characterization of porcine AFS cellsThe growth curve and cell cycle of porcine AFS cells was detected, and characterized the biological features of which using PT-PCR, immunofluorescence and flow cytometry porcine. The result showed that porcine AFS cells displayed robust proliferated potential in vitro culture, and 3 days later cell accessed to exponential growth, after 6 days, the number of cells reached culmination. The result of flow cytometry revealed that porcine AFS cells had normal karyocyte and high proportion of which stayed in the G1 and S stage. Furthermore, porcine AFS cells expressed many marker genes of ES cells, such as Oct4,Nanog,SSEA-4,Tra-60,Tra-81 and CD117 plus crucial MHC antigen HLA-ABC, but not for SSEA1. Besides these cells also expressed mesenchymal stem cells markers CD44, CD90 and CD166, and none of hematopoietic stem cells markers CD45 and CD 34.3. Induction of porcine AFS cells to differentiateFormed embryoid bodies (EBs) through suspended culture in vitro, and followed detection of EBs using AP staining and RT-PCR, and induced to differentiate into adipocytes,neurocytes,premeiotic germ-like cells and myocardial cells. Furthermore, porcine AFS cells were injected into nude mouse and detected the differentiated capability of which in vivo. The results diplayed that porcine AFS cells had the ability of Ebs formation in vitro and the EBs were positive for AP staining and expressed many markers of three germ layers. Furthermore, porcine AFS cells could be induced to differentiate into adipocytes,neurocytes,premeiotic germ-like cells and myocardial cells in vitro and expressed specific genes in target cells. After injected porcine AFS cells into subcutaneous of nude mouse, 6-8 weeks later, none of teratoma was appeared in the body, which suggested that porcine AFS cells have not the risk of tumorigenicity.