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Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts

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Item Type:Article
Title:Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts
Creators Name:Ilia, K. and Shakiba, N. and Bingham, T. and Jones, R.D. and Kaminski, M.M. and Aravera, E. and Bruno, S. and Palacios, S. and Weiss, R. and Collins, J.J. and Del Vecchio, D. and Schlaeger, T.M.
Abstract:Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.
Keywords:Cell Differentiation, Cultured Cells, Cellular Reprogramming, Fibroblasts, Induced Pluripotent Stem Cells, Transcription Factors
Source:Science Advances
Publisher:American Association for the Advancement of Science
Page Range:eadg8495
Date:December 2023
Official Publication:https://doi.org/10.1126/sciadv.adg8495
PubMed:View item in PubMed

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