Programmed Differentiation Circuits for Organoids using Meso-Microfluidics

我们提出了一个利用合成生物遗传电路和微/介观流体的新平台 仪器设备，以迅速推进有机化合物领域。具体地说，我们将通过基因工程自我改造 将人类多能干细胞的组织组织成共同发育的肝和胰腺类器官 具有血管形成和其他成熟特性，如成人水平的白蛋白产生。这个 合成生物学在有机化合物开发中的应用(可编程有机化合物)提供了一种令人兴奋的新方法 设计和测试编码活细胞状态传感器的有机体和嵌入的机会 表达细胞类型和细胞状态特定转录因子的回路。我们将设计新型微流控技术 和介流平台，以实现低成本和高吞吐量的可编程开发和测试 有机化合物。我们的假设是，HiPSC来源的肝和胰腺的共同发育提供了细胞-细胞 在器官发育过程中有助于血管形成和其他重要因素的相互作用 导致成熟器官的形成。为了验证我们的假设，我们将对活细胞传感器进行基因编码，以监测 肝脏类器官发育，共同发育肝脏和胰腺类器官，并创建遗传电路，导致 成熟的类器官形成。我们将使用合成分类器遗传电路来评估细胞状态的变化 实时，并产生相关的蛋白质输出，以特定细胞类型的方式驱动分化。这 这些电路将能够自主产生新的和改进版本的有机化合物，包括成熟的 肝脏类器官和共同发育的肝/胰腺类器官。确定了宇宙的精确时空性质 细胞状态转换和相关转录因子驱动分化不仅是必不可少的 创建新的和有效的有机化合物开发计划，但也将提供重要的科学见解 了解差异化和共同发展的细微之处。成功实现我们的目标 将在基因治疗、药物测试和个性化医疗领域产生广泛影响。例如, 共同开发匹配的有机系统的能力将使患者特定的药物开发(例如 癌症治疗)，这比昂贵和有争议的替代方案更准确，例如使用 人性化的老鼠。这项工作还将支持生产成熟的有机体和器官的长期目标 适合移植的系统。

We propose a new platform that leverages synthetic biology genetic circuits and micro/mesofluidic instrumentation to rapidly advance the field of organoids. Specifically, we will genetically engineer self- organizing tissues from human pluripotent stem cells into co-developed liver and pancreas organoids possessing vascularization and other mature properties, such as adult level albumin production. The application of synthetic biology to organoid development (programmable organoids) provides an exciting new opportunity for engineering and testing of organoids encoding live cell sensors of cell state and for embedding circuits that express cell-type and cell-state specific transcription factors. We will engineer novel microfluidic and mesofluidic platforms to enable low cost and high throughput development and testing of programmable organoids. Our hypothesis is that co-development of hiPSC-derived liver and pancreas provides cell-cell interactions that contribute to vascularization and other important elements in organoid development that will lead to mature organ formation. To test our hypothesis, we will genetically encode live-cell sensors to monitor liver organoid develop, co-develop liver and pancreas organoids, and create genetic circuits that lead to mature organoid formation. We will use synthetic classifier genetic circuits that evaluate changes in cell state in real time, and generate relevant protein outputs for driving differentiation in a cell-type specific manner. This these circuits will enable autonomous generation of new and improved versions of organoids, including mature liver organoids and co-developed liver/pancreas organoids. Determining the precise spatiotemporal nature of cell state transitions and the relevant transcription factors to drive differentiation is not only essential for creating new and effective organoid developmental programs, but will also provide important scientific insights to understanding the fine aspects of differentiation and co-development. Successful achievement of our aims will have a broad impact in the areas of gene therapy, drug testing, and personalized medicine. For example, the ability to co-develop matched organoid systems will enable patient-specific drug development (e.g. for cancer therapy) that is more accurate than expensive and controversial alternatives, such as the use of humanized mice. This work will also support the long-term goal of producing mature organoids and organ systems suitable for transplantation.