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

RECODE：人类诱导多能干细胞定向分化为腔室特异性心肌细胞的单细胞水平编程

• 批准号: 2225300
• 负责人: Sean Palecek
• 金额: $ 150万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2225300&HistoricalAwards=false
• 关键词: RECODE; Single; Cell; Level; Programming

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.

干细胞具有生成体内任何细胞类型的潜力。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代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、专门的细胞类型。 现有的方法通常使用外部因素，如药物，蛋白质，材料支架和机械力。 然而，这种方法经常受到群体中细胞对信号反应不同的困扰。这导致转化为所需的特化细胞类型的效率低下。 编程他们的遗传密码，以指导他们通过正常的心肌发育被提出作为指导干细胞分化的策略。 在这种情况下，混合物中的每个细胞都可以独立地发展，并且仍然实现所需的分化。 该项目将创造新的方法，从心脏的每个腔室产生细胞。如果成功，这将推动对各种人类心脏病的研究。 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外联模块。与高中学生和教师的联系，提供本科生研究机会，并在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，该团队开发了定义的小分子介导的方法来调节经典Wnt信号传导。这导致诱导多能干细胞（iPSC）定向为各种心血管细胞类型，包括心肌细胞（CM）。这些努力有助于阐明Wnt信号传导的基本机制基础，并产生了一系列非天然转录因子和相关遗传结构，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，表达水平的线索变化和信号产生的时间来调节iPSCs中的Wnt和视黄酸（RA）信号。该项目将测试以下假设：通过开发和整合工程可编程超结构，精确和特异性单细胞水平控制Wnt信号传导，将实现对CM分化的单细胞水平控制，而对RA信号传导的控制将促进室特异性（心房和心室）CM的分化。 该项目将通过构建房性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。 如果成功的话，这种单细胞水平的合成生物学控制分化的方法可能会为iPSCs分化为其他细胞和组织类型建立蓝图。该奖项反映了NSF的法定使命，并已被视为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，