Cell reprogramming holds great promise for a number of medical and biological applications, including regenerative/reparative medicine and cellular disease models. Significant progress has been made in this field since the introduction of induced pluripotent stem cells (iPSCs) and the subsequent development of directed nuclear reprogramming (i.e., transdifferentiation) approaches. However, nuclear reprogramming technologies have not been used to date to treat patients due to several obstacles, among them, the high heterogeneity and sometimes inherent unpredictability of the reprogrammed cell population, which is largely due in part to the inability to control the quantity and combination o the transfected reprogramming factors (DNA- or mRNA- based). A number of transfection methods, biological (e.g., viral vectors), chemical (e.g., lipoplexes, polycations) and physical (e.g., microinjection, electroporation), have been developed;however, the great majority of these techniques, with the exception of microinjection, are based on stochastic processes that lead to random cell transfection with significant cell-to-cell variations. Microinjection on the oter hand is only compatible with relatively large cells, and has low yields. New technologies capable of delivering reprogramming factors in a controlled (i.e., timing and dosage), safe, and efficient manner, at the single cell level, are clearly needed in this field for successful transition from te lab bench to the clinic. Our recently developed nanochannel-based electroporation (NEP) technology meets these criteria, thus potentially making it a powerful tool for this purpose. Here we propose to build upon our unique expertise on NEP to develop a more robust and versatile 3D system that could be implemented in a wide range of cell reprogramming applications. We will first implement modeling and micro/nanoscale technologies to develop an optimum 3D NEP platform that can support sequential transfection of large cell numbers (?106), and then we will test this platform using induced pluripotency and direct neuronal transdifferentiation as nuclear reprogramming models. Finally, we will use our NEP technology to methodically study a number of aspects of the cell reprogramming process that cannot be addressed using conventional transfection technologies.

Public Health Relevance

Our recently developed 2D nanochannel electroporation (NEP) technology is capable of highly controlled delivery of charged chemical/biochemical agents (e.g., DNA/RNA, drugs) to a small cell population (up to 300 cells) at the single cell level with efficiencies of nearly ~100%, and negligible cell damage. Here we propose to build upon this premise to develop a robust and versatile 3D NEP system that can transfect a large cell population (>1x106 cells). In this proposal we will be specifically targeting the cell reprogramming field, in which although great breakthroughs have been made recently, full realization of its potential is still hampered by the inability to generate reprogrammed cells with uniform and predictable properties because the currently available transfection technologies are random/stochastic in nature.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Exploratory/Developmental Grants (R21)
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Gene and Drug Delivery Systems Study Section (GDD)
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Hunziker, Rosemarie
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Ohio State University
Engineering (All Types)
Biomed Engr/Col Engr/Engr Sta
United States
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Gallego-Perez, Daniel; Pal, Durba; Ghatak, Subhadip et al. (2017) Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nat Nanotechnol 12:974-979
Kaya, Behiye; Goceri, Evgin; Becker, Aline et al. (2017) Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma. PLoS One 12:e0170991
Gallego-Perez, Daniel; Chang, Lingqian; Shi, Junfeng et al. (2016) On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance. Nano Lett 16:5326-32
Gallego-Perez, Daniel; Otero, Jose J; Czeisler, Catherine et al. (2016) Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers. Nanomedicine 12:399-409
Gokozan, Hamza Numan; Baig, Faisal; Corcoran, Sarah et al. (2016) Area postrema undergoes dynamic postnatal changes in mice and humans. J Comp Neurol 524:1259-69
Chang, Lingqian; Gallego-Perez, Daniel; Chiang, Chi-Ling et al. (2016) Controllable Large-Scale Transfection of Primary Mammalian Cardiomyocytes on a Nanochannel Array Platform. Small 12:5971-5980