Institute of Biomedical Engineering (BME), University of Toronto, Toronto, ON, Canada.
Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Mol Syst Biol. 2022 Nov;18(11):e10886. doi: 10.15252/msb.202110886.
During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.
在发育过程中，细胞状态的转变是通过细胞类型和剂量特异性的分子调控因子的身份变化来协调的。能够在人类多能干细胞（hPSC）中合理设计这种转变将在再生医学中实现许多应用。在此，我们报告了合成基因电路的产生，该电路可以使用内源性 miRNA（分类器）的类似 AND 的逻辑集成来检测所需的细胞状态，并在检测到后使用 miRNA 介导的输出微调技术（miSFITs）产生精细调整的输出蛋白水平。具体来说，我们使用模型指导的 miRNA 选择和电路优化方法创建了一个“hPSC ON”电路。该电路表现出强大的 PSC 特异性检测和分级输出蛋白产生。接下来，我们使用经验方法创建了一个“hPSC-Off”电路。该电路用于以特定状态和精细调整的方式调节内源性 BMP4 的分泌，以控制分化的 hPSC 的组成。我们的工作为 hPSC 中所需生理因素的定制细胞状态特异性控制提供了一个平台，为 hPSC 衍生组织中的细胞组成编程奠定了基础。
