EAGER: Biomanufacturing: Engineering Cell-Intrinsic Control of Cardiomyocyte Differentiation in Human Pluripotent Stem Cells

人类多能干细胞（human pluripotent stem cells, hPSCs）的发现以及将其导向特定细胞类型的能力的最新科学进展，为人类发育和疾病的研究、药物筛选和安全性分析以及基于细胞的再生治疗提供了革命性的潜力。然而，为了实现高效人造血干细胞的这一前景，需要高效的生物制造平台来稳定地产生大量高质量的人造血干细胞及其衍生物。传统的生物制造策略采用生物反应器来控制细胞的环境，提供指导细胞命运的外部信号。在这里，研究人员提出了一种新的内在范式来控制hPSC的命运，利用分子生物学的最新进展来编程hPSC，使其在缺乏外部信号的情况下发育成心肌细胞。这个项目的成功完成将提供一个更深入的基本理解，即人造血干细胞如何决定变成更特化的细胞，并提供一个新的策略，通过工程细胞从干细胞生物制造特化细胞。该项目将开发一种控制人类多能干细胞（hPSC）分化的新模式，利用细胞对细胞状态的内在感知和发育信号通路的控制。研究人员将利用最近的研究进展来了解典型Wnt信号如何驱动hPSC向中胚层的转移，以及随后对心脏中胚层和功能性心肌细胞的转移，从而设计出能够感知其分化状态并驱动信号以诱导所需分化轨迹的细胞。研究人员将鉴定中胚层和心脏中胚层特异性启动子元件，并利用这些元件激活Wnt通路调节剂的表达，生成在缺乏外部信号的情况下分化为功能性心肌细胞的hPSC系。然后，研究人员将测试一个假设，即细胞对分化的内在控制能够比外在控制更强大和可扩展的心肌细胞生物制造，通过小分子的应用，在缩小规模用于高通量筛选应用和扩大规模用于生产适合临床评估的细胞。该项目更广泛的影响包括为心肌细胞以外的体细胞谱系的生物制造提供工程hPSC系的蓝图。教育和外展活动也将在研究生和本科生水平的干细胞工程师，并将提供外展到K-12学生，K-12教师，本科生，以及干细胞科学和生物制造方面的公众。

PI: Palecek, Sean P. Proposal Number: 1547225Recent scientific advances in the discovery of human pluripotent stem cells (hPSCs) and the ability to direct hPSCs to specialized cell types offer the potential to revolutionize the study of human development and disease, drug screening and safety profiling, and cell-based regenerative therapies. To realize this promise of hPSCs, however, efficient biomanufacturing platforms are needed to robustly generate large quantities of high quality hPSCs and their derivatives. Traditional biomanufacturing strategies employ bioreactors to control the environment of the cell, providing extrinsic signals that direct cell fates. Here, the investigators propose a novel intrinsic paradigm for control of hPSC fate, using recent advances in molecular biology to program an hPSC to develop into a heart muscle cell in the absence of external cues. Successful completion of this project will provide a deeper fundamental understanding of how hPSCs make decisions to turn into more specialized cells and provide a new strategy to biomanufacture specialized cells from stem cells by engineering the cell. This project will develop a novel paradigm for controlling human pluripotent stem cell (hPSC) differentiation, using cell intrinsic sensing of cell state and control of developmental signaling pathways. The investigators will employ recent advances in understanding how canonical Wnt signaling drives hPSC commitment to mesoderm and subsequent specification to cardiac mesoderm and functional cardiomyocytes to design cells that sense their differentiation status and drive signaling to elicit desired differentiation trajectories. The investigators will identify mesoderm and cardiac mesoderm-specific promoter elements and use these to activate expression of Wnt pathway modulators, generating hPSC lines that differentiate to functional cardiomyocytes in the absence of extrinsic signaling. Then, the investigators will test the hypothesis that cell intrinsic control of differentiation enables more robust and scalable biomanufacturing of cardiomyocytes than extrinsic control via application of small molecules during scale down for high throughput screening application and scale up for production of cells suitable for clinical evaluation. The broader impacts of this project include providing a blueprint for engineering hPSC lines for biomanufacturing of somatic lineages beyond cardiomyocytes. Education and outreach activities will also stem cell engineers at the graduate and undergraduate levels, and will provide outreach to K-12 students, K-12 teachers, undergraduate students, and the general public on aspects of stem cell science and biomanufacturing.