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ORIGINAL RESEARCH ARTICLE

Manipulation of OCT4 Levels in Human Embryonic Stem Cells Results in Induction of Differential Cell Types

Ryan T. Rodriguez^*,1, J. Matthew Velkey, Carolyn Lutzko, Rina Seerke^*, Donald B. Kohn, K. Sue O’Shea and Meri T. Firpo^*,3,2

^* Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, California 94143; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109; and Division of Research Immunology/BMT Children’s Hospital Los Angeles, Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, California 90027

To whom requests for reprints should be addressed at ³ 2001 6th Street SE, Minneapolis, MN 55455. E-mail: firpo001{at}umn.edu

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.

Key Words: stem cell • self-renewal • differentiation • knockdown • overexpression

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