title: Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.
abstract: Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: Steven C
authors.initials: SC
authors.lastname: Tsai
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: David F
authors.initials: DF
authors.lastname: Chang
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: Chang-Mu
authors.initials: CM
authors.lastname: Hong
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: Ping
authors.initials: P
authors.lastname: Xia
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: Dinithi
authors.initials: D
authors.lastname: Senadheera
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Department of Pediatrics, Children's Hospital Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and.
authors.forename: Lisa
authors.initials: L
authors.lastname: Trump
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California;
authors.forename: Suparna
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Division of Research Immunology and Bone Marrow Transplantation, Department of Pediatrics, The Saban Research Institute of Children's Hospital Los Angeles, University of Southern California, Los Angeles, California; Division of Experimental Hematology and Cancer Biology, Department of Pediatrics, Children's Hospital Research Foundation, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; and Division of Regenerative Medicine and Cellular Therapies, Hoxworth Blood Center, University of Cincinnati, Cincinnati, Ohio carolyn.lutzko@cchmc.org.
authors.forename: Carolyn
authors.initials: C
authors.lastname: Lutzko
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis*; D039904:Pluripotent Stem Cells; D020033:Protein Isoforms / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger
keywords: OCT4 isoforms; OCT4A; human embryonic stem cells; pluripotency; self-renewal