Programmed Differentiation Circuits for Organoids using Meso-Microfluidics

我们提出了一个新的平台，利用合成生物学遗传电路和微/介观流体 仪器来快速推进类器官领域。具体地说，我们将通过基因工程改造自我- 将人类多能干细胞组织成共同发育的肝脏和胰腺类器官 具有血管形成和其它成熟特性，例如成人水平的白蛋白产生。的 将合成生物学应用于类器官发育（可编程类器官）提供了一个令人兴奋的新 对细胞状态的活细胞传感器进行编码的类器官的工程化和测试以及嵌入的机会 表达细胞类型和细胞状态特异性转录因子的电路。我们将设计新的微流体 和介观流体平台，以实现低成本和高通量的可编程微流体的开发和测试。 类器官我们的假设是，hiPSC衍生的肝脏和胰腺的共同发育提供了细胞-细胞间相互作用。 有助于血管化的相互作用和类器官发育中的其他重要因素， 导致成熟器官形成。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， 肝脏类器官发展，共同发展肝脏和胰腺类器官，并创造遗传电路，导致 成熟类器官形成。我们将使用合成分类器遗传电路来评估细胞状态的变化， 真实的时间，并产生相关的蛋白质输出，用于以细胞类型特异性的方式驱动分化。这 这些电路将能够自主产生新的和改进的类器官，包括成熟的类器官。 肝类器官和共同发育的肝/胰腺类器官。确定的精确时空性质 细胞状态转换和驱动分化的相关转录因子不仅对 创造新的和有效的类器官发育计划，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方面。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的影响。比如说， 共同开发匹配的类器官系统的能力将使得能够进行患者特异性药物开发（例如， 癌症治疗），这是更准确的比昂贵和有争议的替代品，如使用 人源化小鼠。这项工作还将支持生产成熟的类器官和器官移植的长期目标。 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.