Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers

详细信息    查看全文

• 作者：Daniel Gallego-Perez ; PhD^a ; ^d ; ^f ; ¹ ; Jose J. Otero ; MD ; PhD^b ; ^c ; ^f ; ¹ ; ^{jose.otero@osumc.edu" class="auth_mail" title="E-mail the corresponding author} ; Catherine Czeisler ; PhD^b ; ^f ; Junyu Ma ; PhD^a ; Cristina Ortiz ; BSc^b ; ^f ; Patrick Gygli ; PhD^b ; ^f ; Fay Patsy Catacutan ; BSc^b ; ^f ; Hamza Numan Gokozan ; MD^b ; ^f ; Aaron Cowgill^b ; ^f ; Thomas Sherwood ; PhD^c ; Subhadip Ghatak ; PhD^d ; ^f ; Veysi Malkoc ; PhD^a ; Xi Zhao ; PhD^a ; Wei-Ching Liao ; PhD^a ; Surya Gnyawali ; PhD^d ; ^f ; Xinmei Wang ; PhD^a ; Andrew F. Adler ; PhD^e ; ² ; Kam Leong ; PhD^e ; ³ ; Brian Wulff ; PhD^b ; ^f ; Traci A. Wilgus ; PhD^b ; ^f ; Candice Askwith ; PhD^c ; Savita Khanna ; PhD^d ; ^f ; Cameron Rink ; PhD^d ; ^f ; Chandan K. Sen ; PhD^d ; ^f ; ^{sen.16@osu.edu" class="auth_mail" title="E-mail the corresponding author} ; L. James Lee ; PhD^a ; ^f ; ^{lee.31@osu.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Induced neuron ; Nuclear reprogramming ; Transfection ; Nanochannel electroporation
• 刊名：Nanomedicine: Nanotechnology, Biology and Medicine
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：12
• 期：2
• 页码：399-409
• 全文大小：1942 K

文摘

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.

c_2">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.