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

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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.