title: Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.
abstract: Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24-48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 10(12)-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ramunas
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Eduard
authors.initials: E
authors.lastname: Yakubov
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Brady
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Stéphane Y
authors.initials: SY
authors.lastname: Corbel
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Holbrook
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Moritz
authors.initials: M
authors.lastname: Brandt
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Stein
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Juan G
authors.initials: JG
authors.lastname: Santiago
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Cooke
authors.affiliation: *Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California, USA; Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California, USA; SpectraCell Laboratories, Inc., Houston, Texas, USA; and Department of Mechanical Engineering, Stanford University, Stanford, California, USA hblau@stanford.edu.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Blau
mesh_terms: D015153:Blotting, Western; D002455:Cell Division; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D005333:Fetus / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; C509186:TERT protein, human; D019098:Telomerase
keywords: nucleoside modified mRNA; proliferative capacity; senescence; telomerase