Senior Research Associate Portal Biotechnologies, Massachusetts, United States
Abstract: The ability to generate diverse functional cell types rapidly and efficiently from induced pluripotent stem cells (iPSCs) holds transformative potential for disease modeling, drug discovery, and regenerative medicine. Protocols have been derived to differentiate iPSCs into several different cell types; however, these methods come with limitations such as low efficiency, variability between batches, and long production times. Herein we describe a novel mechanoporation technology that allows for cytosolic delivery of cargos (e.g. nucleic acids, proteins, peptides) by means of rendering cell membranes temporarily permeable through mechanical stress. The technology has demonstrated compatibility across multiple cell types and materials, while having minimal effects on endogenous gene expression. Our approach is scalable, reproducible, and minimizes some of the safety concerns associated with other delivery methods. Our results show that mechanical delivery of mRNA encoding Neurogenin 2 (NGN2) can enable differentiation of iPSCs into neuronal precursors via increased expression of early neuronal markers and a decrease in pluripotency markers. These data illustrate the potential to overcome current limitations associated with differentiation while maintaining a viability of 75% at a RNA expression level of over 70%. Additionally, we show that our technology enables delivery of circular RNA, antibodies, and other impermeable cargos, such as DNA-encoded libraries, which can be used for diverse cell engineering and drug discovery applications. At a larger scale for clinical processes that meet GMP requirements, we have demonstrated delivery of over 1 billion cells per minute. By continuing to simplify stem cell modification, we aim to further unlock the biological potential of iPSCs while simultaneously reducing manufacturing complexity and safety concerns associated with other delivery modalities.
