Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
Institute for Medical Engineering and Science, MIT, Cambridge, MA 02139, USA.
Sci Adv. 2023 Dec;9(48):eadg8495. doi: 10.1126/sciadv.adg8495. Epub 2023 Nov 29.
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.
将人类成纤维细胞重编程为诱导多能干细胞（iPSCs）的效率很低，转录因子（TF）轨迹的异质性导致了不同的细胞状态。然而，TF 动力学对重编程效率的影响仍不清楚。我们开发了一种系统，可以准确地报告活细胞中的 OCT4 蛋白水平，并利用它来揭示成功重编程过程中的 OCT4 轨迹。我们的系统包括一个合成遗传回路，该回路利用噪声产生广泛的 OCT4 轨迹，并利用 microRNA 靶向内源性 OCT4 来设置细胞内总 OCT4 蛋白水平。通过将 OCT4 融合到荧光蛋白上，我们能够通过活细胞成像以克隆分辨率跟踪 OCT4 轨迹。我们发现，超生理稳定的 OCT4 水平对于高效的 iPSC 集落形成是必需的，但不是充分的。我们的合成遗传电路设计和高通量活细胞成像管道可用于研究其他细胞命运编程应用中的 TF 动力学。
