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

Nancy Mah,Martin Schaefer,Björn Lichtner,Alessandro Prigione,Edda Klipp,Mei-Chih Liao,Miguel A Andrade-Navarro,Ying Wang,Katharina Wolfrum,Manuela Haltmeier,Ralf Mrowka,James Adjaye,Alexander Hahn,Justyna Jozefczuk,Max Flöttmann,Jörg D Hoheisel

doi:10.1371/journal.pone.0024351

Abstract

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.

Highlights

Human embryonic stem (ES) cell research has been fuelled by the potential of using their regenerative properties in cell replacement therapies
Since the landmark discovery that somatic cells can be reprogrammed to an embryonic-like state to create induced pluripotent stem (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]
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

Summary

Introduction

Human embryonic stem (ES) cell research has been fuelled by the potential of using their regenerative properties in cell replacement therapies. 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]

Methods

Results

Conclusion