RECODE: Single Cell-Level Programming of Human Induced Pluripotent Stem Cell Directed Differentiation to Chamber-Specific Cardiomyocytes

干细胞有可能产生体内任何类型的细胞。它们是研究人类发育和疾病、测试药物安全性和有效性以及替换患者受损组织的潜在细胞来源。实现干细胞前景的一个障碍是难以将它们转化为统一的、特化的细胞类型。现有的方法一般采用药物、蛋白质、材料支架和机械力等外部因素。然而，这种方法经常受到人群中对信号做出不同反应的细胞的困扰。这将导致向所需的特化单元格类型的低效转换。对它们的遗传密码进行编程，引导它们通过正常的心肌发育，被认为是指导干细胞分化的一种策略。在这种情况下，混合物中的每个细胞都可以独立进行，并且仍然可以实现所需的分化。这个项目将创造新的方法从心脏的每个腔室中产生细胞。如果成功，这将推动研究各种人类心脏疾病的努力。还将开发合成生物学和干细胞生物制造方面的创新教育和推广模块。向高中学生和教师伸出援手，提供本科生研究机会，并在这两个机构培训研究生，将支持生物制造劳动力的发展。在之前的工作中，该团队开发了明确的小分子介导方法来调节典型的Wnt信号传导。这导致诱导多能干细胞（iPSCs）向包括心肌细胞（CMs）在内的各种心血管细胞类型发展。这些努力有助于阐明Wnt信号传导的基本机制基础，并产生了一系列非自然转录因子和同源遗传结构，以实现细胞中的转录编程。在这项工作中获得的专业知识将用于尝试通过控制反馈、表达水平的提示变化和信号产生的时间来调节iPSCs中的Wnt和维甲酸（RA）信号。该项目将测试这样一个假设，即通过开发和整合工程可编程上层结构来精确和特异性地控制Wnt信号传导，将使单细胞水平上控制CM分化，而控制RA信号传导将促进向室特异性（心房和心室）CM的分化。该项目将通过构建心房和室性心律失常的疾病模型来利用CM分化的进展，用于疾病建模和药物测试应用。如果成功，这种单细胞水平合成生物学控制分化的方法可能为多能干细胞向其他细胞和组织类型的分化建立蓝图。该RECODE项目由化学、生物工程、环境和运输系统部门的细胞和生化工程项目以及分子和细胞生物学部门的系统和合成生物学项目共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Stem cells have the potential to generate any cell type found in the body. They are a potential source of cells to study human development and disease, test drugs for safety and efficacy, and replace damaged tissue in patients. A roadblock to realizing the promise of stem cells is the difficulty of turning them into a uniform, specialized cell type. Existing methods generally use external factors such as drugs, proteins, material scaffolds and mechanical forces. However, this approach is often plagued by cells in the population responding to the signals differently. This results in inefficient conversion to the desired specialized cell type. Programming their genetic code to guide them through normal heart muscle development is proposed as a strategy to direct stem cell differentiation. In this scenario, every cell in the mixture can progress independently and still achieve the desired differentiation. This project will create new methods to generate cells from each chamber of the heart. If successful, this will advance efforts to study various human heart diseases. Innovative education and outreach modules in synthetic biology and stem cell biomanufacturing will also be developed. Outreach to high school students and teachers, providing undergraduate research opportunities, and training graduate students at both institutions will support biomanufacturing workforce development. In prior work, the team developed defined, small molecule-mediated approaches to regulate canonical Wnt signaling. This resulted in the direction of induced pluripotent stem cells (iPSCs) to a variety of cardiovascular cell types, including cardiomyocytes (CMs). These efforts helped elucidate the fundamental mechanistic basis of Wnt signaling and generated a collection of non-natural transcription factors and cognate genetic architectures to enable transcriptional programming in cells. The expertise gained in that work will be used to attempt to regulate Wnt and retinoic acid (RA) signaling in iPSCs through controlled feedback, cued variation in expression levels, and timing of signal production. The project will test the hypothesis that precise and specific single cell-level control of Wnt signaling via the development and integration of engineered programable superstructures will enable single-cell level control over CM differentiation while control over RA signaling will facilitate differentiation to chamber-specific (atrial and ventricular) CMs. This project will leverage advances in CM differentiation by constructing disease models of atrial and ventricular arrhythmias for disease modeling and drug testing applications. If successful, this approach for single cell-level synthetic biology control of differentiation might establish a blueprint for differentiation of iPSCs to other cell and tissue types.This RECODE project is jointly funded by the Cellular and Biochemical Engineering Program in the Division of Chemical, Bioengineering, Environmental, and Transport Systems and the Systems and Synthetic Biology Program in the Division of Molecular and Cellular Biology.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.