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Nanomedicine. 2016 Feb;12(2):399-409. doi: 10.1016/j.nano.2015.11.015. Epub 2015 Dec 19.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Author information

1
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.
2
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.
3
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.
4
Department of Chemical and Biomolecular Engineering, College of Engineering, The Ohio State University, Columbus, OH.
5
Department of Neuroscience, College of Medicine, The Ohio State University, Columbus, OH.
6
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.
7
Department of Biomedical Engineering, Duke University, Durham, NC.
8
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.
9
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.

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.

FROM THE CLINICAL EDITOR:

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.

KEYWORDS:

Induced neuron; Nanochannel electroporation; Nuclear reprogramming; Transfection

PMID:
26711960
PMCID:
PMC5161095
DOI:
10.1016/j.nano.2015.11.015
[Indexed for MEDLINE]
Free PMC Article

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