Generation Of Human Induced Pluripotent Stem (iPS) Cells Line

Objectives:1) Mastered technique for generating human iPS cells by lentivirus-mediated delivery of foreign genes into somatic cells.2) Generated CCD-derived iPS cell lines, which will be potentially employed to investigate the regulatory mechanisms on controlling the fates of iPS cells and perform the directed differentiation.Methods:1) Lentiviral vector with Oct4, Sox2, C-myc and Klf4 identified by enzyme digestion and PCR(1)Identification of lentiviral vector with Oct4, Sox2, C-myc or Klf4 by enzyme digestion:the vectors were cut by BamHⅠ/KpnⅠand EcoRⅠ/EcoRⅤ, respectively.(2) Identification of lentiviral vector with Oct4, Sox2, C-myc and Klf4 by PCR: primers (table1, specific for Oct4, Klf4, c-Myc and Sox2, respectively) were employed to amplify Oct4, Klf4, c-Myc and Sox2 from lentiviral vector with Oct4, Sox2, C-myc or Klf4 gene, respectively.2) Production and titer datermination of lentivirus carrying Oct4, Sox2, C-myc and Klf4 genesAccording to standard protocol from Roche, lentivirus carring Oct4, Sox2, C-myc and Klf4 genes were produced, followed by datermining the titer of lentivirus harboring Oct4, Sox2, C-myc or Klf4 gene, respectively.3) Reprogramming CCD cells to iPS cells by the 4 genes of Oct4, Klf4, c-Myc and Sox2 though Ientivirus-mediated gene transferCCD cells were infected by lentiviruses harboring Oct4, Sox2, C-myc or Klf4 gene,24 hrs after infection, the medium was replaced by hES cell medium. Change the medium every day until the colonies become big enough to be picked up about 20 days after infection.4) Identification of human iPS cell line(1) Morphology under microscopy(2) Detect alkaline phosphatase (AP) activity(3) Immunocytochemistry (ICC) was used to detect the expression of hES cell marker genes(4) Total RNA was isolated, and RT-PCR was used to detect the expression of hES cell marker genes(5) The karyotype analysis of CCD cell-derived hiPS cells(6) EB-based differentiation of CCD cell-derived hiPS cells(7) Teratoma formation by transplanting CCD cell-derived hiPS cells into SCID miceResults:1) Lentiviral vector with Oct4,Sox2,C-myc and Klf4 indetified by enzyme digestion and PCRLentiviral vectors were confirmed to be right by enzyme digestion and PCR.2) Production and titer datermination of lentivirus carrying Oct4, Sox2, C-myc or Klf4 geneViruses were successfully produced and the titers were beyond 1×106 IU/ml.3) Reprogramming CCD cells to iPS cells by the 4 genes of Oct4, Klf4, c-Myc and Sox2 though lentivirus-mediated gene transfer 48-72 hours after infection, green fluorescence was observed under fluorescence microscopy, the morphogoly of CCD cells changed 7-8 days after infection, about 20 days, we picked colonies.4) Identification of human iPS cell line(1) One line of iPS cells had morphological characteristics of hES cells and alkaline phosphatase activity proved positive(2) One line of iPS cells expressed hES cell-specific marker genes(3) One line of iPS cells expressed hES cell-specific markers:SSAE-4(+), TRA-1-60(+), TRA-1-81(+), Oct4(+), SSAE-1(-)(4) Karyotype of CCD cell-derived hiPS cells was normal(5) CCD cell-derived hiPS cells formed embryoid bodies (EBs) in vitro, and EBs expressed the specific marker genes of three germ layers(6) Teratomas formation is carrying on.Conclusion:1) Mastered technique for reprogramming CCD cells to iPS cells by lentivirus-mediated gene transfer2) CCD cell-derived hiPS cells line had some charactistics of hES cells, suggesting that hiPS cell line was generated preliminarily...