Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.
Katherine IliaNika ShakibaTrevor BinghamRoss D JonesMichael M KaminskiEliezer AraveraSimone BrunoSebastian PalaciosRon WeissJames J CollinsDomitilla Del VecchioThorsten M SchlaegerPublished in: Science advances (2023)
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.
Keyphrases
- optical coherence tomography
- induced pluripotent stem cells
- diabetic retinopathy
- depressive symptoms
- optic nerve
- single cell
- high throughput
- transcription factor
- endothelial cells
- genome wide
- high resolution
- protein protein
- cell fate
- gene expression
- quantum dots
- emergency department
- mesenchymal stem cells
- small molecule
- cell proliferation
- amino acid
- air pollution
- binding protein
- mass spectrometry
- cell therapy
- bone marrow
- extracellular matrix
- cancer therapy
- living cells
- drug induced
- pluripotent stem cells