Large Scale Nanochannel Electroporation (NEP) for Cell Reprogramming

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DESCRIPTION (provided by applicant): 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.