title: Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion.
abstract: mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA-related toxicity we combined nonmodified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28 [OSKMNL]) with immune evasion mRNAs (E3, K3, and B18R [EKB]) from vaccinia virus. Additionally, we included mature, double-stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|2 BioNTech RNA Pharmaceuticals GmbH , Mainz, Germany .
authors.forename: Marco Alexander
authors.initials: MA
authors.lastname: Poleganov
authors.affiliation: 3 Stemgent, Cambridge, Massachusetts.
authors.forename: Sarah
authors.initials: S
authors.lastname: Eminli
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .
authors.forename: Tim
authors.initials: T
authors.lastname: Beissert
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|4 III. Department for Internal Medicine, Johannes Gutenberg University , Mainz, Germany .
authors.forename: Stephanie
authors.initials: S
authors.lastname: Herz
authors.affiliation: 3 Stemgent, Cambridge, Massachusetts.
authors.forename: Jung-Il
authors.initials: JI
authors.lastname: Moon
authors.affiliation: 5 Whitehead Institute for Biomedical Research , Cambridge, Massachusetts.
authors.forename: Johanna
authors.initials: J
authors.lastname: Goldmann
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|2 BioNTech RNA Pharmaceuticals GmbH , Mainz, Germany .
authors.forename: Arianne
authors.initials: A
authors.lastname: Beyer
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|4 III. Department for Internal Medicine, Johannes Gutenberg University , Mainz, Germany .
authors.forename: Rosario
authors.initials: R
authors.lastname: Heck
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .
authors.forename: Isabell
authors.initials: I
authors.lastname: Burkhart
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|4 III. Department for Internal Medicine, Johannes Gutenberg University , Mainz, Germany .
authors.forename: Diana
authors.initials: D
authors.lastname: Barea Roldan
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Türeci
authors.affiliation: 3 Stemgent, Cambridge, Massachusetts.
authors.forename: Kevin
authors.initials: K
authors.lastname: Yi
authors.affiliation: 3 Stemgent, Cambridge, Massachusetts.
authors.forename: Brad
authors.initials: B
authors.lastname: Hamilton
authors.affiliation: 1 TRON-Translational Oncology at the University Medical Center of the Johannes Gutenberg University Mainz , Mainz, Germany .|2 BioNTech RNA Pharmaceuticals GmbH , Mainz, Germany .|4 III. Department for Internal Medicine, Johannes Gutenberg University , Mainz, Germany .
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D000067470:Cellular Reprogramming Techniques; D066026:Endothelial Progenitor Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D057131:Immune Evasion; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D035683:MicroRNAs / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D035683:MicroRNAs; D012333:RNA, Messenger