title: piggyBac Transposon Mediated Reprogramming and Flow Cytometry Analysis of CD44 and ICAM1 Cell-Surface Marker Changes.
abstract: Generation of iPSCs is inefficient and the molecular mechanisms underlying reprogramming are not well understood. While several studies have demonstrated that reprogramming is not entirely a random process and contains predictable stepwise changes, varying degrees of cellular heterogeneity that arise in different reprogramming systems can obscure the process. Among several reprogramming systems available, delivery of polycistronic reprogramming factor expression cassettes with piggyBac transposon into mouse embryonic fibroblasts (MEFs) is one of the simplest and most robust reprogramming approaches that provide a low background of partially reprogrammed cells. Using two novel cell surface markers, ICAM1 and CD44, clear cell population changes undergoing reprogramming can be observed over a time course upon induction of the reprogramming factors. Consequently, this technique allows for easy identification of factors that enhance or delay reprogramming, and can be a useful strategy in elucidating key mechanisms for efficient generation of iPSCs.
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, EH16 4UU, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Brightwell
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh, EH16 4UU, UK. Keisuke.Kaji@ed.ac.uk.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kaji
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000032:analysis*; D002469:Cell Separation; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D017930:Genes, Reporter; D016259:Genes, myc; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics; D018960:Hyaluronan Receptors / Q000032:analysis*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000502:physiology
chemical_list: D000954:Antigens, Surface; D004251:DNA Transposable Elements; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins; C489428:Myc protein, mouse; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D018799:Intercellular Adhesion Molecule-1; D049452:Green Fluorescent Proteins
keywords: CD44; Flow cytometry; ICAM1 (CD54); Induced pluripotent stem cells (iPSCs); Mouse embryonic fibroblasts (MEF); Nanog-GFP; Reprogramming; piggyBac transposon