Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

et al. (2011) Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network. PLoS ONE 6(8): e24351. doi:10.1371/journal.pone.0024351 Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network Nancy Mah 0 Ying Wang 0 Mei-Chih Liao 0 Alessandro Prigione 0 Justyna Jozefczuk 0 Bjo rn Lichtner 0 Katharina Wolfrum 0 Manuela Haltmeier 0 Max Flo ttmann 0 Martin Schaefer 0 Alexander Hahn 0 Ralf Mrowka 0 Edda Klipp 0 Miguel A. Andrade-Navarro 0 James Adjaye 0 Jo rg D. Hoheisel, Deutsches Krebsforschungszentrum, Germany 0 1 Computational Biology and Data Mining Group, Max Delbru ck Center for Molecular Medicine , Berlin, Germany , 2 Max Planck Institute for Molecular Genetics, Molecular Embryology and Aging Group , Dresden, Germany, 3 Genomatix Software GmbH, M u nchen, Germany, 4 Theoretical Biophysics , Humboldt University , Berlin, Germany, 5 Experimental Nephrology , Internal Medicine III, Universita tsklinikum Jena, Friedrich-Schiller-Universita t Jena , Jena, Germany , 6 The Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University , Riyadh , Saudi Arabia Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal. - Funding: This work was funded by German Ministry of Education and Research (BMBF; http://www.bmbf.de/), within the Medical Systems Biology Project: DrugiPS (No. FKZ 0315398A-F). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Human embryonic stem (ES) cell research has been fuelled by the potential of using their regenerative properties in cell replacement therapies. To date, only three clinical trials using embryonic stem cell therapy have been approved by the U.S. Food and Drug Administration (FDA) for spinal cord injury patients [1]) and two forms of macular degeneration (ClinicalTrials.gov Identifiers NCT01345006 and NCT01344993). Scientific, ethical and regulatory issues exclude the widespread use of embryonic stem cells as therapeutic transplantation material. In contrast, induced pluripotent stem (iPS) cells offer advantages over ES cells. iPS cells can be derived from somatic cells, such as fibroblasts, thus bypassing the need for blastocystderived ES cells. Furthermore, because iPS cells are derived from the patients own cells, they are thought to represent a renewable and immunologically compatible cell source for cell replacement therapy, though recent publications have questioned the validity of this general assumption [2,3,4], highlighting the need to investigate differences between iPS and ES cells. Since the landmark discovery that somatic cells can be reprogrammed to an embryonic-like state to create iPS cells by over-expressing a combination of four core transcription factors, consisting of OCT4, SOX2, with either KLF4 and c-MYC (OSKM) or LIN28 and NANOG (OSLN) [5,6], many variations of the induction protocol have been developed, including the replacement of some of the core factors by others (Nr5a2, Esrrb, Prmt5 [7,8,9]) or chemicals (PD0325901, A-83-01, E-616452, AMI-5, kenpaullone [10,11,12,13,14]), and different methods of delivery into cells, such as non-integrating adenoviruses, episomalbased plasmids, protein delivery, and transfection of in vitro generated mRNAs [15,16,17,18]. Despite the abundance of publications on the derivation of iPS cells, we still have a limited knowledge on how the core factors induce pluripotency at the molecular level [17,19,20,21,22]. To gain insights into this, we profiled transcriptional changes occurring during the early (24, 48 and 72 h post-transduction) stages of reprogramming of somatic human fibroblasts (HFF1), employing the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC). We observed activated expression of a number of pluripotency-associated genes at these early time points. Finally, we assessed the effect of the reprogramming protocol on reactive oxygen species (ROS) levels, induced DNA damage, activation of p53 and senescence. Based on these findings, we propose three complementary strategies for enhancing the efficiency of reprogramming based on initiating pluripotency amplification pathways, pre-selecting cells expressing pluripotency-associated cell surface antigens, and transiently suppressing innate immune response triggered by the perturbation of cells by the exogenous reprogramming factors. Transcriptional changes accompanying retroviral transduction of the reprogramming factors- OSKM into HFF1 cells In order to gain molecular insights into the processes operative during the early stages of reprogramming, we profiled genomewide transcriptional changes in HFF1 cells at 24, 48, and 72 h post-transduction of OSKM encoding viruses. The transcriptomes of these cells were compared to two HFF1-derived iPS cell lines (iPS2, iPS4) and the ES cell lines (H1, H9) as references of pluripotency. We detected exogenous protein expression of the OSKM factors as early as 24 h with successive increases at 48 and 72 h (Figure 1A). Of the reprogramming factors, endogenous forms of KLF4, and c-MYC could be detected on the microarrays (Figure 1B) and distinguished from exogenous transcripts, based on transcribed 39UTR regions. Expression of endogenous OCT4/ POU5F1 could not be differentiated from its exogenous counterpart, as the Illumina probe is located exclusively within the coding region of this gene. Endogenously expressed SOX2 was not detected at these time points. The transcriptomes of viral transduced cells b (...truncated)