afimoxifene and Teratoma

Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain embryonic stem-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific downregulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFβ3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the nonsteroidal anti-inflammatory drug Nabumetone and the anticancer drug 4-hydroxytamoxifen can generate iPSCs without Sox2. Nabumetone could also produce iPSCs in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPSCs. In summary, we report a new concept of combining genomics and computational chemical biology to identify new drugs useful for iPSC generation. This hypothesis-driven approach provides an alternative to shot-gun screening and accelerates understanding of molecular mechanisms underlying iPSC induction.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Butanones; CCN Intercellular Signaling Proteins; Computational Biology; Cyclooxygenase 2; Drug Discovery; Embryonic Stem Cells; Female; Fibroblasts; Fluorescent Antibody Technique; Gene Silencing; Genetic Vectors; Induced Pluripotent Stem Cells; Kruppel-Like Factor 4; Mice; Mice, Nude; Nabumetone; Oligonucleotide Array Sequence Analysis; Pregnancy; Proto-Oncogene Proteins; Retroviridae; RNA, Small Interfering; SOXB1 Transcription Factors; Tamoxifen; Teratoma; Transfection; Transforming Growth Factor beta3

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Enzyme Induction; Estradiol; Estrogen Antagonists; Fulvestrant; Humans; Integrases; Kinetics; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Point Mutation; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; Teratoma; Tumor Cells, Cultured; Viral Proteins