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High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.

TitleHigh-resolution analysis with novel cell-surface markers identifies routes to iPS cells.
Publication TypeJournal Article
Year of Publication2013
AuthorsO'Malley J, Skylaki S, Iwabuchi KA, Chantzoura E, Ruetz T, Johnsson A, Tomlinson SR, Linnarsson S, Kaji K
Date Published2013 Jul 4
KeywordsAnimals, Antigens, CD44, Biological Markers, Epidermis, Fibroblasts, Flow Cytometry, Gene Expression Profiling, Genes, Reporter, Induced Pluripotent Stem Cells, Intercellular Adhesion Molecule-1, Mice, Nuclear Reprogramming, Sequence Analysis, RNA, Single-Cell Analysis, Up-Regulation

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.

Alternate JournalNature
PubMed ID23728301
PubMed Central IDPMC3743022
Grant List261075 / / European Research Council / International
/ / Medical Research Council / United Kingdom