title: Fluorescent labeling and visualization of human induced pluripotent stem cells with the use of transcription activator-like effector nucleases.
abstract: BACKGROUND
  The visualization of induced pluripotent stem (iPS) cells with the use of fluorescent techniques is useful for the in vivo evaluation of iPS-derived functional cells following differentiation and distribution of the transplanted cells. The data obtained from the fluorescently labeled iPS cells would lead to amelioration and validation of protocols directing the differentiation of iPS cells into functional cells. In this study, we established enhanced green fluorescent protein (EGFP)-labeled iPS cells to enable their easy visualization.
  
  
  METHODS
  Human iPS cells were transfected with (a) 2 transcription activator-like effector nuclease (TALEN) vectors targeted to the adeno-associated virus integration site 1 (AAVS1) locus and (b) the targeting vector carrying the homology arms, EGFP gene, and a drug-selection marker.
  
  
  RESULTS
  Several puromycin-resistant clones were obtained after transfection of the targeting vector and corresponding TALEN-expressing vectors. EGFP expression in these clones was observed with the use of a fluorescent microscope. Clones were examined for specific recombination, which revealed precise targeting at the AAVS1 locus. Furthermore, EGFP protein expression was sustained after directed differentiation into a hepatic lineage.
  
  
  CONCLUSIONS
  We were successful in evaluating the behavior of iPS-derived hepatic cells. The data suggest that genomic knock-in at the AAVS1 locus is suitable for long-term observation of iPS-derived cells. Manipulation of the iPS genome can also be applied for the purification of hepatic cells during iPS cell differentiation by introducing the fluorescent protein under the regulation of a hepatic cell-specific promoter. Another application involves gene correction of iPS cells from patients with hepatic disease for regenerative medicine.
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Sekine
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Takebe
authors.affiliation: Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, Yokohama, Japan. Electronic address: zoukisai@yokohama-cu.ac.jp.
authors.forename: H
authors.initials: H
authors.lastname: Taniguchi
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D058948:Cell Tracking / Q000379:methods*; D002478:Cells, Cultured; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D014162:Transfection / Q000379:methods*
chemical_list: D015415:Biomarkers; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases