title: Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.
abstract: Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage.
  
  
  BACKGROUND
  In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH; Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gallego-Perez
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH. Electronic address: jose.otero@osumc.edu.
authors.forename: Jose J
authors.initials: JJ
authors.lastname: Otero
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Catherine
authors.initials: C
authors.lastname: Czeisler
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
authors.forename: Junyu
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ortiz
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gygli
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Fay Patsy
authors.initials: FP
authors.lastname: Catacutan
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Hamza Numan
authors.initials: HN
authors.lastname: Gokozan
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Aaron
authors.initials: A
authors.lastname: Cowgill
authors.affiliation: Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, OH.
authors.forename: Thomas
authors.initials: T
authors.lastname: Sherwood
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Subhadip
authors.initials: S
authors.lastname: Ghatak
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
authors.forename: Veysi
authors.initials: V
authors.lastname: Malkoc
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
authors.forename: Wei-Ching
authors.initials: WC
authors.lastname: Liao
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Surya
authors.initials: S
authors.lastname: Gnyawali
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
authors.forename: Xinmei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, NC.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Adler
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, NC.
authors.forename: Kam
authors.initials: K
authors.lastname: Leong
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Brian
authors.initials: B
authors.lastname: Wulff
authors.affiliation: Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Traci A
authors.initials: TA
authors.lastname: Wilgus
authors.affiliation: Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, OH.
authors.forename: Candice
authors.initials: C
authors.lastname: Askwith
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Savita
authors.initials: S
authors.lastname: Khanna
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH.
authors.forename: Cameron
authors.initials: C
authors.lastname: Rink
authors.affiliation: Department of Surgery, College of Medicine, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH. Electronic address: sen.16@osu.edu.
authors.forename: Chandan K
authors.initials: CK
authors.lastname: Sen
authors.affiliation: Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH; Center for Regenerative Medicine and Cell-Based Therapies (CRMCBT), The Ohio State University, Columbus, OH. Electronic address: lee.31@osu.edu.
authors.forename: L James
authors.initials: LJ
authors.lastname: Lee
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D002460:Cell Line; D065150:Cellular Reprogramming; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D018274:Electroporation / Q000379:methods; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D050809:POU Domain Factors / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D015854:Up-Regulation
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D050809:POU Domain Factors; D014157:Transcription Factors; C499317:Ascl1 protein, mouse; C489726:Myt1 protein, mouse; C074066:Pou3f2 protein, mouse
keywords: Induced neuron; Nanochannel electroporation; Nuclear reprogramming; Transfection