Ilia Katherine, Shakiba Nika, Bingham Trevor, Jones Ross D, Kaminski Michael M, Aravera Eliezer, Bruno Simone, Palacios Sebastian, Weiss Ron, Collins James J, Del Vecchio Domitilla, Schlaeger Thorsten M
Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA.
Institute for Medical Engineering and Science, MIT, Cambridge, MA, USA.
bioRxiv. 2023 Jan 25:2023.01.25.525529. doi: 10.1101/2023.01.25.525529.
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. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.
将人类成纤维细胞重编程为诱导多能干细胞（iPSC）的效率较低，转录因子（TF）轨迹的异质性导致细胞状态不同。然而，TF动态对重编程效率的影响仍不清楚。在这里，我们确定了核心多能性TF——OCT4的成功重编程轨迹，并设计了一种基因控制器，可高精度地执行此类轨迹。通过将产生广泛OCT4轨迹的基因回路与活细胞成像相结合，我们以克隆分辨率跟踪OCT4轨迹，发现集落形成需要独特的恒定OCT4轨迹。然后，我们开发了一种合成基因回路，该回路可在确定的轨迹周围产生紧密的OCT4分布，并且在重编程效率方面优于其他对OCT4调节不太准确的回路。我们的合成生物学方法可推广用于识别和执行细胞命运编程应用中的TF动态。
