title: Generation of human induced pluripotent stem cells using non-synthetic mRNA.
abstract: Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), University Leipzig, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Rohani
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), University Leipzig, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Fabian
authors.affiliation: Translational Centre for Regenerative Medicine (TRM), University Leipzig, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Holland
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), University Leipzig, Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Naaldijk
authors.affiliation: Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Dressel
authors.affiliation: Interdisciplinary Centre for Bioinformatics (IZBI), Leipzig University, Leipzig, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Löffler-Wirth
authors.affiliation: Interdisciplinary Centre for Bioinformatics (IZBI), Leipzig University, Leipzig, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Binder
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Arnold
authors.affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), University Leipzig, Germany; Loughborough University, Wolfson School of Mechanical and Manufacturing Engineering, Centre for Biological Engineering, Loughborough, United Kingdom. Electronic address: Stolzing@gmail.com.
authors.forename: A
authors.initials: A
authors.lastname: Stolzing
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology; D052816:Foreskin / Q000166:cytology; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D016898:Interferon-alpha / Q000032:analysis; D007371:Interferon-gamma / Q000032:analysis; D059785:Karyotype; D008297:Male; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D013724:Teratoma / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D016898:Interferon-alpha; D012333:RNA, Messenger; D014157:Transcription Factors; D007371:Interferon-gamma
keywords: Induced pluripotent stem cell; Reprogramming; mRNA