title: High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.
abstract: The generation of induced pluripotent stem (iPS) cells presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. Although several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPS cells. The rapid expansion of minor reprogrammed cells in the heterogeneous population can also obscure investigation of relevant transition processes. Understanding the biological mechanisms essential for successful iPS cell generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that in mouse embryonic fibroblasts, reprogramming follows an orderly sequence of stage transitions, marked by changes in the cell-surface markers CD44 and ICAM1, and a Nanog-enhanced green fluorescent protein (Nanog-eGFP) reporter. RNA-sequencing analysis of these populations demonstrates two waves of pluripotency gene upregulation, and unexpectedly, transient upregulation of several epidermis-related genes, demonstrating that reprogramming is not simply the reversal of the normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and the improved understanding of the reprogramming process will lead to new reprogramming strategies.
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK.
authors.forename: James
authors.initials: J
authors.lastname: O'Malley
authors.forename: Stavroula
authors.initials: S
authors.lastname: Skylaki
authors.forename: Kumiko A
authors.initials: KA
authors.lastname: Iwabuchi
authors.forename: Eleni
authors.initials: E
authors.lastname: Chantzoura
authors.forename: Tyson
authors.initials: T
authors.lastname: Ruetz
authors.forename: Anna
authors.initials: A
authors.lastname: Johnsson
authors.forename: Simon R
authors.initials: SR
authors.lastname: Tomlinson
authors.forename: Sten
authors.initials: S
authors.lastname: Linnarsson
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kaji
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology*; D004817:Epidermis / Q000378:metabolism; D005347:Fibroblasts; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017423:Sequence Analysis, RNA; D059010:Single-Cell Analysis; D015854:Up-Regulation / Q000235:genetics
chemical_list: D015415:Biomarkers; C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors; C090509:Icam1 protein, mouse; D018799:Intercellular Adhesion Molecule-1