Collaborative Research: RECODE: Directing and Controlling Cardiac Differentiation Through Cellular and Microenvironmental Manipulation and Application of Machine-Learning

美国心脏病死亡率正在上升，特别是心力衰竭患者的患病率不断上升。天然人类心脏组织的有限可用性阻碍了研究、药物发现和临床心脏再生努力。干细胞来源的心脏组织治疗具有非常高的潜力，以实现临床有意义的结果。然而，细胞类型的异质混合物的产生是基于细胞的心脏治疗的关键障碍。通过采用发育生物学，组织工程和机器学习，通过定向分化编码（RECODE）研究可再生细胞和类器官为克服这一障碍奠定了基础，并开发了方法和设计方法，以产生理解和治疗心脏病所需的功能细胞类型。该项目将支持来自亚拉巴马州立大学--一所历史悠久的黑人大学--的本科生参加奥本大学的暑期研究体验。尽管我们对人类诱导多能干细胞和心脏发育生物学的理解取得了重大进展，但我们从足够数量的多能干细胞中产生特定心脏细胞亚型的能力仍然有限。人诱导多能干细胞的心脏分化已被分解为从多能性到中胚层到心脏祖细胞到第一和第二心脏区域的逐步过程。然而，这种进展以不同的速率发生，需要不同的浓度，持续时间和暴露于关键细胞信号分子的时间，并产生不同浓度的心肌细胞。了解群体动态和给定细胞将朝向成为一种细胞类型而不是另一种细胞类型的概率对于做出预测和指导决策以实现所需的最终细胞类型或细胞类型的混合物是必要的。该RECODE项目的目标是建立一个配对的实验过程和指导混合模型，利用分化心肌细胞的实时测量结果来预测正在进行的心脏分化的结果和应进行调整以实现预期结果的过程参数。该项目工作将（1）将创新的机器学习工具和心脏发育阶段结合起来，挖掘单细胞RNA测序数据，以确定在这些情况下控制细胞命运的关键发育决策和杠杆，（2）在3D中进行定向心脏分化，以解决传统2D分析中不存在的复杂自分泌，旁分泌，细胞-细胞和细胞-基质相互作用，（3）采用心肌细胞亚型特异性荧光报告基因实时定量分化结果，以及（4）开发过程控制分析平台，其将分化结果数据与实验定义的输入参数整合，所述输入参数能够在-该RECODE奖由土木，机械，和制造业创新和工程生物学和健康集群在化学，生物工程，环境，该奖项反映了NSF的法定使命，并通过使用基金会的智力价值进行评估，更广泛的影响审查标准。

US mortality rates from heart disease are increasing, driven particularly by the increasing prevalence of patients with heart failure. Limited availability of native human cardiac tissue impedes research, drug discovery, and clinical cardiac regeneration efforts. Treatment with stem cell-derived cardiac tissues has exceptionally high potential to achieve clinically meaningful outcomes. However, the generation of a heterogeneous mixture of cell types is a critical barrier to cell-based cardiac therapy. By employing developmental biology, tissue engineering, and machine learning, this Reproducible Cells and Organoids via Directed-Differentiation Encoding (RECODE)research builds the foundation for overcoming this obstacle and develops methodologies and design approaches to produce functional cell types needed in understanding and treating heart disease. This project will support undergraduate students from Alabama State University – a historically black university – to participate in summer research experiences at Auburn University.Despite significant advances in our understanding of human induced pluripotent stem cell and cardiac development biology, our ability to generate specific cardiac cell subtypes from pluripotent stem cells in sufficient quantities remains limited. Cardiac differentiation of human induced pluripotent stem cells has been broken down into a stepwise process from pluripotency to mesoderm to cardiac progenitors to first and second heart fields. However, this progression occurs at differing rates, require differing concentrations, durations, and timing of exposure to key cell signaling molecules, and yield varying concentrations of cardiomyocytes. Understanding the population dynamics and probabilities that a given cell will move towards becoming one cell type versus another is necessary for making predictions and directing decisions to achieve a desired final cell type or a mixture of cell types. The goal of this RECODE project is to establish a paired experimental process and guiding hybrid model utilizing real-time measurements from differentiating cardiomyocytes to predict both the outcome of ongoing cardiac differentiation and the process parameters that should be adjusted to achieve the desired result. The project work will (1) marry innovative machine learning tools and cardiac developmental stages to mine single cell RNA sequencing data to identify key developmental decisions and levers that control cell fate at these instances, (2) perform directed cardiac differentiation in 3D to address complex autocrine, paracrine, cell-cell and cell-matrix interactions that are absent in conventional 2D assays, (3) employ cardiac cell subtype-specific fluorescent reporter to quantify differentiation outcome in real-time, and (4) develop a process control analytical platform that integrates differentiation outcome data with experimentally-defined input parameters that can enable generation of specific composition of cardiac cell subtypes on-demand using robustly validated and reproducible differentiation design rules.This RECODE award is co-funded by the Mechanics and Engineering Materials Cluster in the Division of Civil, Mechanical, and Manufacturing Innovation and the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.