Technologies enabling robust closed-loop manufacturing of human pluripotent stem cell-derived cardiomyocytes

PROJECT SUMMARY Cell therapy is a rapidly growing field, with Chimeric Antigen Receptor T (CART) cells and stem cell-based therapies showing remarkable efficacy in treating hematologic cancers and regenerating tissue for conditions like Parkinson’s Disease, blindness, type 1 diabetes, and heart failure. However, manufacturing cellular therapies is complex, resulting in high costs and variable product quality. This study aims to address these challenges by developing feedback-controlled closed-loop processes for differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs). Despite advancements in hPSC-CM differentiation processes over the past decade, enabling their adoption in various applications, including human development studies, disease modeling, drug screening, and clinical trials for heart failure treatment, significant variability exists between batches and cell lines during manufacturing. This variability becomes more pronounced with the transition from 2D to 3D platforms. Building on preliminary data comparing successful and failed hPSC differentiation batches that identified genetic and epigenetic features predictive of CM generation, notably variations in WNT and MAPK signaling in cardiac progenitor cells (CPCs), this study aims to develop control strategies to enhance differentiation reproducibility across various conditions. This project's overarching goal is to improve the robustness and reproducibility of hPSC differentiation into CMs through feedback-controlled closed-loop manufacturing processes. Leveraging single-cell transcriptomics and epigenomics, our project will gain a deep understanding of differentiation failure modes, enabling early identification and correction of process deviations. The proposed aims are: Aim 1: Identify 2D CM differentiation failure modes through integrated single-cell transcriptomics and epigenomics. Single-cell RNA sequencing (scRNAseq) and transposase-accessible chromatin sequencing (ATACseq) performed throughout CM differentiation runs and analysis of cell trajectories will identify key points of deviation from differentiation and predict mechanism leading to off-target cell-type generation. Aim 2: Develop 2D closed-loop CM differentiation processes and evaluate their robustness compared to standard protocols. We will build genetic and epigenetic sensor hPSC lines to monitor gene expression and chromatin accessibility at the single-cell level, test modulation of cardiac developmental pathways to rescue low efficiency CM batches, and compare the robustness of closed-loop processes to standard protocols. Aim 3: Develop scalable, 3D closed-loop CM differentiation processes and assess process variability. Utilizing a 3D bioreactors for CM manufacturing, we will compare failure modes in 3D to 2D differentiation, develop cell sensors and processes to modulate developmental signaling pathways, and increase the reliability of CM production in scalable manufacturing platforms.