title: ONSL and OSKM cocktails act synergistically in reprogramming human somatic cells into induced pluripotent stem cells.
abstract: The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of >2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France.
authors: Laura
authors: L
authors: Jung
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France Present Address: Aix Marseille Université, INSERM, GMGF UMR_F 910, 13385 Marseille, France.
authors: Philippe
authors: P
authors: Tropel
authors: Vectalys SAS CanalBiotech II, 3 rue des Satellites, Toulouse 31400, France.
authors: Yohann
authors: Y
authors: Moal
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France Service de Biologie de la Reproduction, Centre Hospitalier Universitaire, Strasbourg F-67000, France.
authors: Marius
authors: M
authors: Teletin
authors: Service de Génétique, Centre Hospitalier de Mulhouse 20, rue du Dr Laennec, Mulhouse Cedex 68070, France.
authors: Eric
authors: E
authors: Jeandidier
authors: Vectalys SAS CanalBiotech II, 3 rue des Satellites, Toulouse 31400, France.
authors: Régis
authors: R
authors: Gayon
authors: Service de Génétique, Centre Hospitalier de Mulhouse 20, rue du Dr Laennec, Mulhouse Cedex 68070, France.
authors: Christian
authors: C
authors: Himmelspach
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France.
authors: Fiona
authors: F
authors: Bello
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France.
authors: Cécile
authors: C
authors: André
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France.
authors: Adeline
authors: A
authors: Tosch
authors: Department of Molecular Cell Biology, Molecular Cell Differentiation, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany Department of Clinical Neurophysiology, University of Goettingen, Goettingen, Germany Genome and Stem Cell Center, GENKOK, Erciyes University, Kayseri, Turkey.
authors: Ahmed
authors: A
authors: Mansouri
authors: Service de Chirurgie Plastique Reconstructrice et Esthétique, Chirurgie B - Hôpital Civil, Strasbourg F-67000, France.
authors: Catherine
authors: C
authors: Bruant-Rodier
authors: Vectalys SAS CanalBiotech II, 3 rue des Satellites, Toulouse 31400, France.
authors: Pascale
authors: P
authors: Bouillé
authors: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR1704, Université de Strasbourg, Illkirch 67404, France Centre Hospitalier Universitaire, Strasbourg F-67000, France viville@igbmc.fr.
authors: Stéphane
authors: S
authors: Viville
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D020405:Dermis / Q000166:cytology / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C000715187:Lin28A protein, human; C489427:MYC protein, human; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors
keywords: OSKM, ONSL cocktails; human iPSC cells; lentivirus; reprogramming; retrovirus