Genetically encoded guidance of cell fate transitions via endoribonuclease mediated microRNA sensors of cell state

Introduction:: Precise guidance of cell state transitions via genetic engineering will have profound implications for a variety of applications including differentiation of pluripotent stem cells for uses in cellular therapies, regenerative medicine, and human organoid models. To effectively guide cells towards desired lineages, an important challenge is to recapitulate developmental processes whereby cells undergo a series of differentiation steps, each one taken when the cells are ready. To address this challenge, we introduced a rationally designed genetic platform to sense cell state and conditionally activate expression of transcription factors that automatically guide sequential transitions to desired cell types. Via our sensor/actuator genetic platform, endogenous cell-state specific miRNAs regulate translation of constitutively expressed CRISPR-specific endoribonucleases (ERNs), which in turn, regulate translation of constitutively expressed transcription factors. Building upon this, we developed and characterized high miRNA senor mediated by ERN, allowing us to obtain durable conditional expression.

Materials and Methods:: HEK293FT were purchased from Thermo Fisher and were grown in DMEM with 4.5g/L glucose, L-glutamine, and sodium pyruvate (Cellgro) supplemented with 10% characterized FBS (HyClone). The PGP1 hiPSCs were a kind gift from George Church (Harvard University, USA) and can be obtained from Coriell (NJ, USA). The Alstem 11 hiPSCs were purchased from AlSTEM (CA, USA). Mammalian cell transfections were performed using FuGene HD (Promega) and Lipofectamine Stem Transfection reagent following manufacturer’s protocol. Flow cytometry data reported in this study was collected on a BD LSR Fortessa. Data analysis was carried out using CytoFlow package developed in the MATLAB environment.

Results, Conclusions, and Discussions::
First, we demonstrated a genetically encoded ERN based high miRNA sensors platform for sensing cell state in HEK293FT cells using transiently expressed miRNA. Next, we optimized circuit stoichiometries using single-cell analyses resulting in significantly improved sensor performances. We then demonstrated the use of a high miRNA sensor/actuator to automatically guide stepwise human induced pluripotent stem cell (hiPSC) differentiation. Specifically, we generated hiPSC lines that enables Doxycycline induction of efficient differentiation from hiPSCs to mesoderm cells then to endothelial cells. We also embed in this cell line a high miRNA sensor regulating conditional expression of a specific transcription factor to automatically guide the resulting endothelial cells towards the hematopoietic lineage. Our platform provides a customizable approach to monitor cell state transitions in physiological and pathological conditions and efficiently rewire cell state transitions by rationally designed genetic programs.




Acknowledgements (Optional): : This work is supported by Wellcome Foundation LEAP HOPE project.

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